The GAL expression system is the most frequently used induction technique in the yeast Saccharomyces cerevisiae. Here we report a simple but powerful genetic circuit for use with the GAL induction system. Briefly, an artificial positive feedback circuit was incorporated into the GAL regulatory network. We selected green fluorescent protein (GFP) as a reporter of GAL1 induction, and designed a strain that expressed a constitutively active Gal3 mutant protein (Gal3c) under control of the GAL10 promoter. In the resulting strain, GAL1 and GAL10 promoters regulate the expression of GFP and GAL3c, respectively. Because Gal3c sequesters the Gal80 repressor away from the Gal4 transcriptional activator in the same manner as the galactose-bound Gal3, the expressed Gal3c protein provokes further expression of GFP and Gal3c, yielding further enhancement of GAL induction. Thus, this GAL3c-mediated positive feedback circuit permits substantially enriched induction of a target gene at extremely low concentrations, or even in the absence, of galactose, while maintaining the strict glucose-mediated repression of the target.Keywords: GAL induction system; Gal3c; galactose; positive feedback circuit; protein production; Saccharomyces cerevisiae;

•Microbial conversion of biomass can be applied to produce bio-based polymers.•Metabolic engineering expands the type of biomonomers produced by fermentation.•Various fermentative products can serve as building blocks of bio-based polymers.•The synthesis of bio-based polymers from biomonomers remains limited.The worldwide market for plastics is rapidly growing, and plastics polymers are typically produced from petroleum-based chemicals. The overdependence on petroleum-based chemicals for polymer production raises economic and environmental sustainability concerns. Recent progress in metabolic engineering has expanded fermentation products from existing aliphatic acids or alcohols to include aromatic compounds. This diversity provides an opportunity to expand the development and industrial uses of high-performance bio-based polymers. However, most of the biomonomers are produced from edible sugars or starches that compete directly with food and feed uses. The present review focuses on recent progress in the microbial conversion of biomass into bio-based polymers, in which fermentative products from renewable feedstocks serve as biomonomers for the synthesis of bio-based polymers. In particular, the production of biomonomers from inedible lignocellulosic feedstocks by metabolically engineered microorganisms and the synthesis of bio-based engineered plastics from the biological resources are discussed.Download high-res image (241KB)Download full-size image

•Metabolically engineered C. glutamicum displays beta-xylosidase.•A superior lysine producer was constructed by metabolic engineering.•Engineered strains assimilated xylose and xylooligosaccharides.•Diaminopentane was produced from xylooligosaccharides.Xylooligosaccharide-assimilating Corynebacterium glutamicum strains were constructed using metabolic engineering and cell surface display techniques. First, C. glutamicum was metabolically engineered to create lysine-producing strains. Beta-xylosidase BSU17580 derived from Bacillus subtilis was then expressed on the C. glutamicum cell surface using PorH anchor protein, and enzymes involved in the xylose assimilation pathway were also expressed. Metabolic engineering had no effect on the activity of beta-xylosidase. The engineered strains efficiently consumed xylooligosaccharides and produced 12.4 mM of lysine from 11.9 g/L of xylooligosaccharides as the carbon source. Finally, co-expression of lysine decarboxylase enabled production of 11.6 mM of 1,5-diaminopentane (cadaverine) from 13 g/L of consumed xylooligosaccharides.Download high-res image (145KB)Download full-size image

•Metabolically engineered Escherichia coli is treated as a microbial chassis.•Pathway engineering to generate E. coli chassis is summarized.•Strategies for accumulating 5 important metabolic intermediates are highlighted.The present work reviews literature describing the re-design of the metabolic pathways of a microbial host using sophisticated genetic tools, yielding strains for producing value-added chemicals including fuels, building-block chemicals, pharmaceuticals, and derivatives. This work employed Escherichia coli, a well-studied microorganism that has been successfully engineered to produce various chemicals. E. coli has several advantages compared with other microorganisms, including robustness, and handling. To achieve efficient productivities of target compounds, an engineered E. coli should accumulate metabolic precursors of target compounds. Multiple researchers have reported the use of pathway engineering to generate strains capable of accumulating various metabolic precursors, including pyruvate, acetyl-CoA, malonyl-CoA, mevalonate and shikimate. The aim of this review provides a promising guideline for designing E. coli strains capable of producing a variety of useful chemicals. Herein, the present work reviews their common and unique strategies, treating metabolically engineered E. coli as a “microbial chassis”.

•Filamentous fungi are capable of organic acid fermentation and protein production.•Recent advances in genetic engineering may be suited for application in fungi.•Future insights of metabolic engineering based on latest technology are discussed.•This review presents current and potential applications of fungi in bioproduction.Filamentous fungi exhibit versatile abilities, including organic acid fermentation, protein production, and secondary metabolism, amongst others, and thus have applications in the medical and food industries. Previous genomic analyses of several filamentous fungi revealed their further potential as host microorganisms for bioproduction. Recent advancements in molecular genetics, marker recycling, and genome editing could be used to alter transformation and metabolism, based on optimized design carbolated with computer science. In this review, we detail the current applications of filamentous fungi and describe modern molecular genetic tools that could be used to expand the role of these microorganisms in bioproduction. The present review shed light on the possibility of filamentous fungi as host microorganisms in the field of bioproduction in the future.

•Expansion of the yeast metabolism was demonstrated.•Bacterial phosphoenol pyruvate carboxylase increased in the isobutabol titer 1.45-fold.•Introduction of Entner–Doudoroff (ED) pathway had no effect to isobutanol bio-production.•Further tracer and metabolome analyses revealed that ED pathway was successfully constructed in yeast.•Activity of the ED pathway, however, was too weak to improve isobutanol biosynthesis.Bacterial phosphoenol pyruvate carboxylase (PPC) and enzymes in the Entner–Doudoroff (ED) pathway were heterologously expressed in Saccharomyces cerevisiae to improve the NADPH supply required for the bio-production of chemicals such as isobutanol. The heterologous expression of PPC from Synechocystis sp. PCC6803 increased in the isobutabol titer 1.45-fold (93.2 ± 1.6 mg/L) in metabolically engineered S. cerevisiae strains producing isobutanol. This result suggested that the pyruvate and NADPH supply for isobutanol biosynthesis was activated by PPC overexpression. On the other hand, the expression of two enzymes organizing the ED pathway (6-phosphogluconate dehydratase [6PGD] and 2-dehydro-3-deoxy-phosphogluconate aldolase [KDPGA]) had no effect to isobutabol bio-production. Further analysis, however, revealed that additional expression of 6PGD and KDPGA improved the growth rate of S. cerevisiae strain BY4742 gnd1Δ. A 13C-labeling experiment using [1−13C] glucose also suggested that metabolic flow levels in the ED pathway increased slightly with the additional expression. These results showed that the ED pathway was successfully constructed in S. cerevisiae, even though activity of the pathway was too weak to improve isobutanol biosynthesis.

Simultaneous saccharification and fermentation (SSF) of d-lactic acid was performed using brown rice as both a substrate and a nutrient source. An engineered Lactobacillus plantarum NCIMB 8826 strain, in which the ʟ-lactate dehydrogenase gene was disrupted, produced 97.7 g/L d-lactic acid from 20% (w/v) brown rice without any nutrient supplementation. However, a significant amount of glucose remained unconsumed and the yield of lactic acid was as low as 0.75 (g/g-glucose contained in brown rice). Interestingly, the glucose consumption was significantly improved by adapting L. plantarum cells to the low-pH condition during the early stage of SSF (8–17 h). As a result, 117.1 g/L d-lactic acid was produced with a high yield of 0.93 and an optical purity of 99.6% after 144 h of fermentation. SSF experiments were repeatedly performed for ten times and d-lactic acid was stably produced using recycled cells (118.4–129.8 g/L). On average, d-lactic acid was produced with a volumetric productivity of 2.18 g/L/h over 48 h.

•Acetate addition increases total lipid and phospholipid content in Chlamydomonas sp.•Lipid from Chlamydomonas sp. JSC4 is highly heterogeneous.•F. heterosporum lipase has a broad range of water tolerance.•An interaction between water and methanol exists for the whole-cell biocatalyst.•High initial methanol consumption rate is crucial for high final FAME yield.Lipid from Chlamydomonas sp. JSC4 was used as a feedstock for biodiesel production. The lipid was found to contain high amounts of phospholipids and free fatty acid in addition to the triglycerides. Two enzymatic methods for the efficient conversion of the heterogenous lipid to fatty acid methyl esters (FAME) were carried out. The method using either a lipase cocktail containing Candida cylindracea lipase and Thermomyces lanuginosus lipase combination (m I) or immobilized Fusarium heterosporum lipase-expressing Aspergillus oryzae whole-cells (m II) were both successful. However, the method using lipase cocktail showed 30.8% relative stability after the fourth batch, whereas the whole-cell biocatalyst showed 98.1%. Although the whole-cell biocatalyst tolerated a wide range of water content, an exploration of the effect of water-methanol interaction on the biocatalytic process showed that 24% water and 7:1 methanol to oil ratio is more favorable for FAME production. A higher initial methanol consumption rate facilitated a more stable system with the whole-cell biocatalyst, producing over 97% FAME in 32 h. The efficient conversion of a highly heterogenous substrate in the presence of high amounts of water could be an effective technique for the enzymatic conversion of microalgal lipids.Download high-res image (100KB)Download full-size image

Clustered regularly interspaced short palindromic repeats (CRISPR)-mediated manipulation of genomic information is becoming more versatile by combining nuclease-deficient CRISPR systems with a wide variety of effectors including base-editing deaminases, transcriptional regulators, and epigenetic modifiers. The programmable binding ability of CRISPR systems is essential when the systems are employed as targeting domains to recruit the effectors to specific genomic loci. The discovery of a variety of Cas9 orthologs and engineered variants enables high-fidelity genome editing and a wider selection of genomic targets, and CRISPR-mediated deaminases enable more precise and predictable genome editing compared with CRISPR nuclease-based editing. Finally, combining transcriptional regulators with CRISPR systems can control expression of specific genes in a genome. Some applications and future challenges of CRISPR-derived tools are also discussed.

We demonstrate metabolic enzyme ligation using a transpeptidase (Staphylococcal sortase A) in the microbial cytoplasm for the redirection of metabolic flux through metabolic channeling. Here, sortase A expression was controlled by the lac promoter to trigger metabolic channeling by the addition of isopropyl-β-d-thiogalactopyranoside (IPTG). We tested covalent linking of pyruvate-formate lyase and phosphate acetyltransferase by sortase A-mediated ligation and evaluated the production of acetate. The time point of addition of IPTG was not critical for facilitating metabolic enzyme ligation, and acetate production increased upon expression of sortase A. These results show that sortase A-mediated enzyme ligation enhances an acetate-producing flux in E. coli. We have validated that sortase A-mediated enzyme ligation offers a metabolic channeling approach to redirect a central flux to a desired flux.Keywords: enzyme ligation; Escherichia coli; metabolic engineering; sortase A

Protocatechuic acid (3,4-dihydroxybenzoic acid; PCA) serves as a building block for polymers and pharmaceuticals. In this study, the biosynthetic pathway for PCA from glucose was engineered in Corynebacterium glutamicum. The pathway to PCA-employed elements of the chorismate pathway by using chorismate-pyruvate lyase (CPL) and 4-hydroxybenzoate hydroxylase (4-HBA hydroxylase). As C. glutamicum has the potential to synthesize the aromatic amino acid intermediate chorismate and possesses 4-HBA hydroxylase, we focused on expressing Escherichia coli CPL in a phenylalanine-producing strain of C. glutamicum ATCC21420. To secrete PCA, the gene (ubiC) encoding CPL from E. coli was expressed in C. glutamicum ATCC 21420 (strain F(UbiC)). The formation of 28.8 mg/L of extracellular 4-HBA (36 h) and 213 ± 29 mg/L of extracellular PCA (80 h) was obtained by the C. glutamicum strain F(UbiC) from glucose. The strain ATCC21420 was also found to produce extracellular PCA. PCA fermentation was performed using C. glutamicum strain F(UbiC) in a bioreactor at the optimized pH of 7.5. C. glutamicum F(UbiC) produced 615 ± 2.1 mg/L of PCA from 50 g/L of glucose after 72 h. Further, fed-batch fermentation of PCA by C. glutamicum F(UbiC) was performed with feedings of glucose every 24 h. The maximum production of PCA (1140.0 ± 11.6 mg/L) was achieved when 117.0 g/L of glucose was added over 96 h of fed-batch fermentation.

Xylitol, a value-added polyol deriving from d-xylose, is widely used in both the food and pharmaceutical industries. Despite extensive studies aiming to streamline the production of xylitol, the manufacturing cost of this product remains high while demand is constantly growing worldwide. Biotechnological production of xylitol from lignocellulosic waste may constitute an advantageous and sustainable option to address this issue. However, to date, there have been few reports of biomass conversion to xylitol. In the present study, xylitol was directly produced from rice straw hydrolysate using a recombinant Saccharomyces cerevisiae YPH499 strain expressing cytosolic xylose reductase (XR), along with β-glucosidase (BGL), xylosidase (XYL), and xylanase (XYN) enzymes (co-)displayed on the cell surface; xylitol production by this strain did not require addition of any commercial enzymes. All of these enzymes contributed to the consolidated bioprocessing (CBP) of the lignocellulosic hydrolysate to xylitol to produce 5.8 g/L xylitol with 79.5 % of theoretical yield from xylose contained in the biomass. Furthermore, nanofiltration of the rice straw hydrolysate provided removal of fermentation inhibitors while simultaneously increasing sugar concentrations, facilitating high concentration xylitol production (37.9 g/L) in the CBP. This study is the first report (to our knowledge) of the combination of cell surface engineering approach and membrane separation technology for xylitol production, which could be extended to further industrial applications.

To find a novel host for the production of 4-vinylphenol (4VPh) by screening Streptomyces species.The conversion of p-coumaric acid (pHCA) to 4VPh in Streptomyces mobaraense was evaluated using a medium containing pHCA. S. mobaraense readily assimilated pHCA after 24 h of cultivation to produce 4VPh. A phenolic acid decarboxylase, derived from S. mobaraense (SmPAD), was purified following heterologous expression in Escherichia coli. SmPAD was evaluated under various conditions, and the enzyme’s kcat/Km value was 0.54 mM−1 s−1. Using intergenetic conjugation, a gene from Rhodobacter sphaeroides encoding a tyrosine ammonia lyase, which catalyzes the conversion of l-tyrosine to p-coumaric acid, was introduced into S. mobaraense. The resulting S. mobaraense transformant produced 273 mg 4VPh l−1 from 10 g glucose l−1.A novel strain suitable for the production of 4VPh and potentially other aromatic compounds was isolated.

Lignocellulosic hydrolysates contain compounds that inhibit microbial growth and fermentation, thereby decreasing the productivity of biofuel and biochemical production. In particular, the heterocyclic aldehyde furfural is one of the most toxic compounds found in these hydrolysates. We previously demonstrated that Corynebacterium glutamicum converts furfural into the less toxic compounds furfuryl alcohol and 2-furoic acid. To date, however, the genes involved in these oxidation and reduction reactions have not been identified in the C. glutamicum genome. Here, we show that Cgl0331 (designated FudC) is mainly responsible for the reduction of furfural into furfuryl alcohol in C. glutamicum. Deletion of the gene encoding FudC markedly diminished the in vivo reduction of furfural to furfuryl alcohol. Purified His-tagged FudC protein from Escherichia coli was also shown to convert furfural into furfuryl alcohol in an in vitro reaction utilizing NADPH, but not NADH, as a cofactor. Kinetic measurements demonstrated that FudC has a high affinity for furfural but has a narrow substrate range for other aldehydes compared to the protein responsible for furfural reduction in E. coli.

The metabolic state of microflora (mixed microbial cultures) in microbial fuel cells (MFCs) is currently unclear. Metabolomic analyses were conducted of microflora growing on the anodic electrodes of MFCs operated at pH 7.0, 5.5, or 4.0 and utilizing starch as the major carbon substrate. A much higher current was produced at pH 7.0 than at pH 5.5 and 4.0, correlating with an increased population ratio of Geobacter species to the total bacteria growing on the electrode. Most intracellular metabolites related to the tricarboxylic acid (TCA) cycle were present at a higher level at pH 7.0 than at pH 5.5 and 4.0, and the levels of metabolites correlated well with the obtained current densities. A high intracellular adenosine triphosphate (ATP)/adenosine diphosphate (ADP) ratio at pH 7.0, compared to at pH 5.5 and 4.0, likewise supported current production. Overall, the metabolomic analyses demonstrated that activation of the TCA cycle and increased ATP generation are critical parameters for electricity generation by microflora.

We engineered efficient 2,3-butanediol (23BD) production from cellobiose using Bacillus subtilis. First, we found that B. subtilis harboring an empty vector could produce 23BD from cellobiose. However, productivity using cellobiose as a carbon source was lower than that when using glucose. This lower productivity was improved by adding purified beta-glucosidase from Thermobifida fusca YX (Tfu_0937) in the fermentation. Encouraged by these findings, we found that hydrolysis of cellobiose to glucose was an important reaction of 23BD biosynthesis in B. subtilis using cellobiose. Hence, we created efficient 23BD production from cellobiose using exogenous Tfu_0937-expressing B. subtilis. Using the engineered strain, 21.2 g L−1 of 23BD was produced after 72 h of cultivation. The productivity and yield were 0.294 g L−1 h−1 and 0.35 g 23BD/g cellobiose, respectively. We successfully demonstrated efficient 23BD production from cellobiose by using BGL-expressing B. subtilis.

•Recombinant yeast capable of utilizing lignocellulose has been developed.•Efficiency of lignocellulose hydrolysis has been improved.•Robustness of yeast has been improved using rational and evolutionary strategies.•Advanced molecular biology tools are critical for effective biomass breakdown.Conferring biomass hydrolysis activity on yeast through genetic engineering has paved the way for the development of groundbreaking processes for producing liquid fuels and commodity chemicals from lignocellulosic biomass. However, the overproduction and misfolding of heterologous and endogenous proteins can trigger cellular stress, increasing the metabolic burden and retarding growth. Improving the efficiency of lignocellulosic breakdown requires engineering of yeast secretory pathway based on system-wide metabolic analysis as well as DNA constructs for enhanced cellulase gene expression with advanced molecular biology tools. Also, yeast is subjected to severe stress due to toxic compounds generated during lignocellulose pretreatment in consolidated saccharification and fermentation processes. The prospect for development of robust yeast strains makes combining evolutionary and rational engineering strategies.

Recombinant yeast strains that display heterologous amylolytic enzymes on their cell surface via the glycosylphosphatidylinositol (GPI)-anchoring system are considered as promising biocatalysts for direct ethanol production from starchy materials. For the effective hydrolysis of these materials, the ratio optimization of multienzyme activity displayed on the cell surface is important. In this study, we have presented a ratio control system of multienzymes displayed on the yeast cell surface by using different GPI-anchoring domains. The novel gene cassettes for the cell-surface display of Streptococcus bovis α-amylase and Rhizopus oryzae glucoamylase were constructed using the Saccharomyces cerevisiae SED1 promoter and two different GPI-anchoring regions derived from Saccharomyces cerevisiae SED1 or SAG1. These gene cassettes were integrated into the Saccharomyces cerevisiae genome in different combinations. Then, the cell-surface α-amylase and glucoamylase activities and ethanol productivity of these recombinant strains were evaluated. The combinations of the gene cassettes of these enzymes affected the ratio of cell-surface α-amylase and glucoamylase activities and ethanol productivity of the recombinant strains. The highest ethanol productivity from raw starch was achieved by the strain harboring one α-amylase gene cassette carrying the SED1-anchoring region and two glucoamylase gene cassettes carrying the SED1-anchoring region (BY-AASS/GASS/GASS). This strain yielded 22.5 ± 0.6 g/L of ethanol from 100 g/L of raw starch in 120 h of fermentation.

Recent increasing attention to environmental issues and the shortage of oil resources have spurred political and industrial interest in the development of environmental friendly and cost-effective processes for the production of bio-based chemicals from renewable resources. Thus, microbial production of commercially important chemicals is viewed as a desirable way to replace current petrochemical production. Corynebacterium glutamicum, a Gram-positive soil bacterium, is one of the most important industrial microorganisms as a platform for the production of various amino acids. Recent research has explored the use of C. glutamicum as a potential cell factory for producing organic acids such as lactate and succinate, both of which are commercially important bulk chemicals. Here, we summarize current understanding in this field and recent metabolic engineering efforts to develop C. glutamicum strains that efficiently produce l- and d-lactate, and succinate from renewable resources.

Phospholipids (PLs) containing specific polar head groups and fatty acids, artificially synthesized from a complex mixture of natural PLs, have considerable industrial applications. The biocatalytic approaches to synthesizing structured PLs are of great interest because the enzymes used show high selectivity and performance under mild conditions, leading to the generation of products that cannot easily be obtained by chemical catalysis. Although the limited supply of phospholipases (e.g., phospholipase D) has thus far been an obstacle to the widespread use of enzymatic processing, recent advances in enzyme preparation have opened up various applications for PL modification. In this review, attempts to increase the productivity and utility of microbial phospholipases and lipases are presented. We also summarize recent developments in enzyme-catalyzed modification of PLs, focusing particularly on the relevant reactions, bioreactor design, and novel proof-of-concept experiments.

Cold-adapted β-1,3-xylanase (P.t.Xyn26A) from the psychrotrophic bacterium, Psychroflexus torquis, was expressed as a fusion protein with tandem repeats of the N-terminal domain of Protein S from Myxocuccus xanthus (ProS2) in Escherichia coli. After cell lysis in phosphate buffer, most of the ProS2-P.t.Xyn26A was located in the insoluble fraction and aggregated during purification. Arginine hydrochloride (ArgHCl) efficiently solubilized the ProS2-P.t.Xyn26A. The solubilized ProS2-P.t.Xyn26A was purified using immobilized metal affinity chromatography (IMAC) with 500 mM ArgHCl. After cleavage of ProS2-P.t.Xyn26A by human rhinovirus 3C protease, we confirmed that recombinant P.t.Xyn26A maintained its native fold. This is the first report of the expression of a cold-adapted enzyme fused with a ProS2 tag under IMAC purification using a high concentration of ArgHCl. These insights into the expression and purification should be useful during the handling of cold-adapted enzymes.

We demonstrated direct utilization of xylooligosaccharides using β-xylosidase-displaying Escherichia coli. After screening active β-xylosidases, BSU17580 from Bacillus subtilis or Tfu1616 from Thermobifida fusca YX, were successfully displayed on the E. coli cell surface using Blc or HdeD as anchor proteins, and these transformants directly assimilated xylooligosaccharides as a carbon source. The final OD 600 in minimal medium containing 2% xylooligosaccharides was 1.09 (after 12 h of cultivation) and 1.30 (after 40 h of cultivation). We then constructed an E. coli strain displaying both β-glucosidase and β-xylosidase. β-glucosidase- and β-xylosidase-displaying E. coli was successfully grown on a 1% cellobiose and 1% xylooligosaccharides mixture, and the OD 600 was 1.76 after 10 h of cultivation, which was higher and reached faster than that grown on a glucose/xylose mixture (1.20 after 30 h of cultivation).Keywords: cell-surface display; cellobiose; E. coli; xylooligosaccharides; β-glucosidase; β-xylosidase;

We report an eco-friendly, one-pot, room-temperature method for the rapid synthesis of electrocatalytically active Au@Pt (50 nm) bimetallic nanoparticles via a tryptophan (Trp) mediated supramolecular interface in an aqueous environment. Our results demonstrate a simple universal approach for high shell–metal loading where a pre-stabilized tryptophan polymerized-Au core serves as a template to facilitate subsequent deposition of Pt. We observed that the amine-stabilized poly-Trp bi-layer has an enhancing effect on the electrocatalytic potential of Au@Pt NPs by the virtue of an amine stabilized interface, thereby enhancing the HER activity over glassy carbon electrodes. Several characterization techniques were used to confirm the inherent core–shell morphology of the resulting Au@Pt NPs. This Trp mediated facile green synthesis strategy has the potential to synthesize an array of Au-core containing bimetallic nanoparticles with enhanced catalytic activity and stable structure integration.

A novel green one-pot approach for surface modification of graphene oxide into organosulfur modified graphene nanosheets (OS-GNS) is being reported. Alliin (garlic phytochemical) mediated organothiol linkages over OS-GNS facilitated one-step attachment of pre-synthesized gold nanoparticles. In absence of alliin treatment, no Au NP attachment was observed.

To develop cost-effective systems for d-lactate production, here, the effect of high-cell density cultivation of metabolically engineered Lactobacillus plantarum on d-lactate production was evaluated. A xylose-assimilating strain of L. plantarum was anaerobically cultured with mixed sugars (glucose and xylose) as substrates. Compared to undiluted nutrient-rich de Man, Rogosa, and Sharpe (MRS) medium, d-lactate production by cultivating in 10-fold diluted MRS (0.1 MRS) medium or normal saline solution was 89.7 and 81.3 %, respectively. Notably, the xylose consumption rate was comparable in the three cultures, whereas the glucose consumption rate decreased by 18.3 and 26.1 % in 0.1 MRS medium and normal saline solution, respectively, resulting in a reduction of the d-lactate production rate. The d-lactate productivity in high-cell density cultivation was proportional to the initial cell concentrations. The use of a two-step cultivation process involving growing and resting cells in a single bioreactor revealed that the ratio of the glucose and xylose consumption rates (based on grams consumed) in resting cell conditions was 1.88, whereas that in growing conditions was 2.58. Cultivation of L. plantarum in growing conditions for 24 h produced 73.2 g/l d-lactate with the yield of 0.90 g/g, whereas cells cultivation under resting cell conditions in a saline solution for 24 h produced 68.7 g/l d-lactate with the yield of 0.93 g/g. In total, 141.9 g/l d-lactate was produced after 48 h cultivation, a value that represents the highest reported concentration of d-lactate produced from mixed sugars to date. Our findings contribute to the cost-effective, large-scale production of d-lactate.

Oxygenic photosynthetic microorganisms such as cyanobacteria and microalgae have attracted attention as feedstocks for next-generation biofuels. To date, however, there have been no reports on efficient bioethanol production from cyanobacterial glycogen by yeast fermentation. Additionally, multiple pretreatment and enzymatic hydrolysis steps of polysaccharides are required for conventional ethanol production from agricultural crops and microalgae. Here, we investigate direct ethanol production from Arthrospira (Spirulina) platensis, a fast-growing halophilic cyanobacterium that accumulates large amounts of glycogen, using lysozyme and a recombinant amylase-expressing yeast strain to eliminate the need for biomass pretreatment and amylase hydrolysis. In the direct conversion process from A. platensis to ethanol, 6.5 g L−1 (ethanol productivity of 1.08 g per L per day) of ethanol was produced. The total ethanol yield based on glycogen consumption was 86% of theoretical yield, which to our knowledge, is the highest yield of bioethanol from an oxygenic photosynthetic microorganism. The present findings indicate that A. platensis is a remarkable carbohydrate feedstock in the form of glycogen, which is a promising material for the production of bioethanol and various other commercially valuable chemicals.

Sidewall modification of multiwalled carbon nanotubes (abbreviated as MWCNTs) was achieved using Allivum sativum (garlic) extract by an acid-free green process. These organosulfur modified-MWCNTs were then decorated with gold nanoparticles and examined by transmission electron microscopy. The presence of organosulfurs over the modified nanotube surface was confirmed. Nanotube surface modification and subsequent presence of thiols as an active linker was confirmed by Raman spectroscopy, Fourier transform infrared spectroscopy, energy dispersive X-ray and X-ray photoelectron spectroscopy. In the absence of these organosulfurs (thiols), no gold nanoparticle attachment was observed. Both small (1–8 nm) and large (12–20 nm) gold nanoparticles were found to decorate the modified nanotube surface suggesting coalescence among nanoparticles.

A facile, eco-friendly, room-temperature method for rapid one-pot synthesis of Au@Pd bimetallic nanoparticles exhibiting a core–shell morphology (∼60 nm) has been developed based on the successive reduction of Au(III) and Pd(II) precursors with tryptophan (Trp) in an aqueous environment. The unique supramolecular chemistry arising due to the hydrogen bonded indole group layer over the Au core seemed critical in the formation Pd shell. The core–shell morphology and surface analysis of the resulting Au@Pd nanoparticles were confirmed by aberration corrected scanning transmission electron microscopy followed by X-ray photoelectron spectroscopy. The formation of the core (Au) and shell (Pd) was also confirmed by Energy Dispersive X-Ray elemental scanning analysis. The resulting Au, Pd and Au@Pd nanoparticles were also analysed by UV-Vis spectroscopy, X-ray diffraction and Dynamic Light Scattering. Our results suggest a simple coordination mechanism where the pre-stabilized poly-Trp Au core serves as a template to facilitate the subsequent reduction of Pd(II) via active carboxyl groups. This study effectively demonstrates for the first time that core–shell nanoparticle synthesis (reduction and stabilization) can be effectively achieved by simple amino acids like Trp in an aqueous reaction mixture.

In the present study, we established a genetic system for manipulating the oleaginous heterotrophic microalgae Aurantiochytrium sp. KRS101, using cycloheximide resistance as the selectable marker. The gene encoding ribosomal protein L44 (RPL44) of Aurantiochytrium sp. KRS101 was first identified and characterized. Proline 56 was replaced with glutamine, affording cycloheximide resistance to strains encoding the mutant protein. This resistance served as a novel selection marker. The gene encoding the Δ12-fatty acid desaturase of Mortierella alpina, used as a reporter, was successfully introduced into chromosomal DNA of Aurantiochytrium sp. KRS101 via 18S rDNA-targeted homologous recombination. Enzymatic conversion of oleic acid (C18:1) to linoleic acid (C18:2) was detected in transformants but not in the wild-type strain.

Here, we demonstrate display of beta-glucosidase (BGL) on the surface of Schizosaccharomyces pombe cells using novel anchor proteins. A total of four candidate anchor proteins (SPBC21D10.06c, SPBC947.04, SPBC19C7.05, and SPBC359.04c) were selected from among almost all of S. pombe membrane proteins. The C-terminus of each anchor protein was genetically fused to the N-terminus of BGL, and the fusion protein was expressed using S. pombe as a host. The highest cell surface-associated BGL activity (107 U/105 cells was achieved with SPBC359.04c serving as the anchor, followed by SPBC947.04 (44 U/105 cells) and SPBC21D10.06c (38 U/105 cells). S. pombe displaying BGL with SPBC359.04c as an anchor showed the highest growth on 2 % cellobiose (10.7 × 107 cells/mL after 41 h of cultivation from an initial density of 0.1 × 107 cells/mL). Additionally, culturing BGL-displaying S. pombe in medium containing cellobiose as the sole carbon source did not affect protein expression, and ethanol fermentation from cellobiose was successfully demonstrated using BGL-displaying S. pombe. This is the first report describing a cell surface display system for the functionalization of S. pombe.

Agricultural residues comprising lignocellulosic materials are excellent sources of pentose sugar, which can be converted to ethanol as fuel. Ethanol production via consolidated bioprocessing requires a suitable microorganism to withstand the harsh fermentation environment of high temperature, high ethanol concentration, and exposure to inhibitors. We genetically enhanced an industrial Saccharomyces cerevisiae strain, sun049, enabling it to uptake xylose as the sole carbon source at high fermentation temperature. This strain was able to produce 13.9 g/l ethanol from 50 g/l xylose at 38 °C. To better understand the xylose consumption ability during long-term, high-temperature conditions, we compared by transcriptomics two fermentation conditions: high temperature (38 °C) and control temperature (30 °C) during the first 12 h of fermentation. This is the first long-term, time-based transcriptomics approach, and it allowed us to discover the role of heat-responsive genes when xylose is the sole carbon source. The results suggest that genes related to amino acid, cell wall, and ribosomal protein synthesis are down-regulated under heat stress. To allow cell stability and continuous xylose uptake in order to produce ethanol, hexose transporter HXT5, heat shock proteins, ubiquitin proteins, and proteolysis were all induced at high temperature. We also speculate that the strong relationship between high temperature and increased xylitol accumulation represents the cell’s mechanism to protect itself from heat degradation.

The biochemical properties of a putative β-1,3-xylanase from the hyperthermophilic eubacterium Thermotoga neapolitana DSM 4359 were determined from a recombinant protein (TnXyn26A) expressed in Escherichia coli. This enzyme showed specific hydrolytic activity against β-1,3-xylan and released β-1,3-xylobiose and β-1,3-xylotriose as main products. It displayed maximum activity at 85 °C during a 10-min incubation, and its activity half-life was 23.9 h at 85 °C. Enzyme activity was stable in the pH range 3–10, with pH 6.5 being optimal. Enzyme activity was significantly inhibited by the presence of N-bromosuccinimide (NBS). The insoluble β-1,3-xylan Km value was 10.35 mg/ml and the kcat value was 588.24 s−1. The observed high thermostability and catalytic efficiency of TnXyn26A is both industrially desirable and also aids an understanding of the chemistry of its hydrolytic reaction.

We constructed beta-glucosidase (BGL)-displaying Corynebacterium glutamicum, and direct l-lysine fermentation from cellobiose was demonstrated. After screening active BGLs, Sde1394, which is a BGL from Saccharophagus degradans, was successfully displayed on the C. glutamicum cell surface using porin as an anchor protein, and cellobiose was directly assimilated as a carbon source. The optical density at 600 nm of BGL-displaying C. glutamicum grown on cellobiose as a carbon source reached 23.5 after 48 h of cultivation, which was almost the same as that of glucose after 24 h of cultivation. Finally, Sde1394-displaying C. glutamicum produced 1.08 g/l of l-lysine from 20 g/l of cellobiose after 4 days of cultivation, which was about threefold higher than the amount of produced l-lysine using BGL-secretory C. glutamicum strains (0.38 g/l after 5 days of cultivation). This is the first report on amino acid production using cellobiose as a carbon source by BGL-expressing C. glutamicum.

We produced organic acids, including lactate and succinate, directly from soluble starch under anaerobic conditions using high cell-density cultures of Corynebacterium glutamicum displaying α-amylase (AmyA) from Streptococcus bovis 148 on the cell surface. Notably, reactions performed under anaerobic conditions at 35 and 40°C, which are higher than the optimal growth temperature of 30°C, showed 32% and 19%, respectively, higher productivity of the organic acids lactate, succinate, and acetate compared to that at 30°C. However, α-amylase was not stably anchored and released into the medium from the cell surface during reactions at these higher temperatures, as demonstrated by the 61% and 85% decreases in activity, respectively, from baseline, compared to the only 8% decrease at 30°C. The AmyA-displaying C. glutamicum cells retained their starch-degrading capacity during five 10 h reaction cycles at 30°C, producing 107.8 g/l of total organic acids, including 88.9 g/l lactate and 14.0 g/l succinate. The applicability of cell surface-engineering technology for the production of organic acids from biomass by high cell-density cultures of C. glutamicum under anaerobic conditions was demonstrated.

In this study, we demonstrate the one-step production of cadaverine (1,5-diaminopentane) from cellobiose using an Escherichia coli strain displaying β-glucosidase (BGL) on its cell surface. L-lysine decarboxylase (CadA) derived from E. coli and BGL from Thermobifida fusca YX (Tfu0937) fused to the anchor protein Blc from E. coli were co-expressed using E. coli as a host. The expression of CadA was confirmed by Western blotting and BGL activity on the cell surface was evaluated using pNPG as a substrate. Growth on cellobiose as the sole carbon source was also achieved. The OD600 value of the BGL and CadA co-expressing strain was 8.0 after 48 h cultivation, which is higher than that obtained by growth on glucose (5.4 after 48 h cultivation). The engineered strain produced cadaverine from cellobiose more effectively than from glucose: 6.1 mM after 48 h from 28 g/L of consumed cellobiose, vs. 3.3 mM from 20 g/L of consumed glucose.

One of the most important projects in the post-genome-era is the systemic identification of biological network. The almost of studies for network identification focused on the improvement of computational efficiency in large-scale network inference of complex system with cyclic relations and few attempted have been done for answering practical problem occurred in real biological systems. In this study, we focused to evaluate inferring performance of our previously proposed method for inferring biological network on simple network motifs.We evaluated the network inferring accuracy and efficiency of our previously proposed network inferring algorithm, by using 6 kinds of repeated appearance of highly significant network motifs in the regulatory network of E. coli proposed by Shen-Orr et al and Herrgård et al, and 2 kinds of network motif in S. cerevisiae proposed by Lee et. al. As a result, our method could reconstruct about 40% of interactions in network motif from time-series data set. Moreover the introduction of time-series data of one-factor disrupted model could remarkably improved the performance of network inference.The results of network inference examination of E. coli network motif shows that our network inferring algorithm was able to apply to typical topology of biological network. A continuous examination of inferring well established network motif in biology would strengthen the applicability of our algorithm to the realistic biological network.

We screened for high-activity endoglucanase (EG) as a first step toward the creation of cellulose-assimilating Streptomyces lividans transformants. EGs derived from Thermobifida fusca YX, Tfu0901, and S. lividans, cellulase B (CelB), were successfully expressed. Genes encoding Tfu0901 or CelB were introduced into S. lividans using the integrative vector pTYM18 and the high-copy-number vector pUC702, and EG activity was detected in the supernatant of each transformant. To achieve coexpression of EG and transglutaminase, the transglutaminase gene was introduced into EG-secreting S. lividans using pUC702. S. lividans coexpressing EG and transglutaminase effectively assimilated phosphoric acid swollen cellulose. The yield of Streptomyces cinnamoneus transglutaminase in the culture supernatant was 7.2 mg/L, which was 18 times higher than that of the control strain. To demonstrate the versatility of our system, we also created an EG-producing S. lividans transformant capable of coexpressing endoxylanase. The EG-secreting S. lividans transformants constructed here can be used to produce other useful compounds through cellulose fermentation.

Flow cytometry enables comparative quantification, population analysis, and high-throughput screening of agonist-mediated G-protein-coupled receptor (GPCR) signaling in genetically engineered yeasts. By using flow cytometry, we found that transformation of yeast cells with a low plasmid number is critical both for the construction of large screening libraries and for stable signal transmission in cell ensembles. Based on these findings, we constructed an engineered yeast strain for the improved identification of signal promotion by Gαi-specific human GPCRs using flow cytometry.

Here we present a successful transplantation of the GAL genetic regulatory circuitry into the G-protein signaling pathway in yeast. The GAL regulon represents a strictly regulated transcriptional mechanism that we have transplanted into yeast to create a highly robust induction system to assist the detection of on–off switching in G-protein signaling. In our system, we engineered yeast to drive the positive GAL regulatory gene in response to agonist-promoted G-protein signaling and to induce transcription of a green fluorescent protein (GFP) reporter gene under the control of the GAL structural gene promoter. Consequently, in response to agonist stimulation of G-protein-coupled receptors (GPCRs), the engineered yeast achieved more than a 150-fold increase in reporter intensity in up to 98% of cells, as determined by flow cytometric sorting. Surprisingly, agonist-stimulated induction of the GFP reporter gene was higher than that by galactose. Our approach to boost reporter gene induction could be applicable in establishing more efficient yeast-based flow cytometric screening systems for agonistic ligands for heterogeneous GPCRs.

Glutathione is a valuable tri-peptide that is widely used in the pharmaceutical, food, and cosmetic industries. Glutathione is produced industrially by fermentation using Saccharomyces cerevisiae, and supplementation of fermentation with several amino acids can increase intracellular GSH content. More recently, however, focus has been given to protein as a resource for biofuel and fine chemical production. We demonstrate that expression of a protease on the cell surface of S. cerevisiae enables the direct use of keratin and soy protein as a source of amino acids and that these substrates enhanced intracellular GSH content. Furthermore, fermentation using soy protein also enhanced cell concentration. GSH fermentation from keratin and to a greater extent from soy protein using protease-displaying yeast yielded greater GSH productivity compared to GSH fermentation with amino acid supplementation. This protease-displaying yeast is potentially applicable to a variety of processes for the bio-production of value-added chemicals from proteinaceous biomass resources.

Glutathione (GSH) is a valuable tri-peptide that is widely used in the pharmaceutical, food, and cosmetic industries. Glutathione is produced industrially by fermentation using Saccharomyces cerevisiae. In this study, we demonstrated that engineering in sulfate assimilation metabolism can significantly improve GSH production. The intracellular GSH content of MET14 and MET16 over-expressing strains increased up to 1.2 and 1.4-fold higher than that of the parental strain, respectively, whereas those of APA1 and MET3 over-expressing strains decreased. Especially, in the MET16 over-expressing strain, the volumetric GSH concentration was up to 1.7-fold higher than that of the parental strain as a result of the synergetic effect of the increases in the cell concentration and the intracellular GSH content. Additionally, combinatorial mutant strains that had been engineered to contain both the sulfur and the GSH synthetic metabolism synergistically increased the GSH production. External addition of cysteine to S. cerevisiae is well known as a way to increase the intracellular GSH content; however, it results a decrease in cell growth. This study showed that the engineering of sulfur metabolism in S. cerevisiae proves more valuable than addition of cysteine as a way to boost GSH production due to the increases in both the intracellular GSH content and the cell growth.

To improve the ability of recombinant Saccharomyces cerevisiae strains to utilize the hemicellulose components of lignocellulosic feedstocks, the efficiency of xylose conversion to ethanol needs to be increased. In the present study, xylose-fermenting, haploid, yeast cells of the opposite mating type were hybridized to produce a diploid strain harboring two sets of xylose-assimilating genes encoding xylose reductase, xylitol dehydrogenase, and xylulokinase. The hybrid strain MN8140XX showed a 1.3- and 1.9-fold improvement in ethanol production compared to its parent strains MT8-1X405 and NBRC1440X, respectively. The rate of xylose consumption and ethanol production was also improved by the hybridization. This study revealed that the resulting improvements in fermentation ability arose due to chromosome doubling as well as the increase in the copy number of xylose assimilation genes. Moreover, compared to the parent strain, the MN8140XX strain exhibited higher ethanol production under elevated temperatures (38 °C) and acidic conditions (pH 3.8). Thus, the simple hybridization technique facilitated an increase in the xylose fermentation activity.

Yeasts are promising hosts for industrial bio-refinery applications. In yeast cell surface displays, functional proteins, such as cellulases or lipases, are genetically fused to an anchor protein and expressed on the cell surface. Saccharomyces cerevisiae is the most commonly used yeast for cell surface display. Engineered yeasts have been utilized for a variety of applications, such as bioethanol production, chemicals synthesis, adsorption of environmental pollutants, and protein evolution. Here, we summarize recent developments in yeast cell surface display techniques for bio-refinery applications, including methods using hosts such as Pichia pastoris, Yarrowia lipolytica, and S. cerevisiae, focusing on the characteristics of anchor proteins and applications.

We have constructed a filamentous fungus Aspergillus oryzae that secretes a llama variable heavy-chain antibody fragment (VHH) that binds specifically to epidermal growth factor receptor (EGFR) in a culture medium. A major improvement in yield was achieved by fusing the VHH with a Taka-amylase A signal sequence (sTAA) and a segment of 28 amino acids from the N-terminal region of Rhizopus oryzae lipase (N28). The yields of secreted, immunologically active anti-EGFR VHH reached 73.8 mg/1 in a Sakaguchi flask. The VHH fragments were released from the sTAA or N28 proteins by an indigenous A. oryzae protease during cultivation. The purified recombinant VHH fragment was specifically recognized and could bind to the EGFR with a high affinity.

A novel extracellular glutathione fermentation method using engineered Saccharomyces cerevisiae was developed by following three steps. First, a platform host strain lacking the glutathione degradation protein and glutathione uptake protein was constructed. This strain improved the extracellular glutathione productivity by up to 3.2-fold compared to the parental strain. Second, the ATP-dependent permease Adp1 was identified as a novel glutathione export ABC protein (Gxa1) in S. cerevisiae based on the homology of the protein sequence with that of the known human glutathione export ABC protein (ABCG2). Overexpression of this GXA1 gene improved the extracellular glutathione production by up to 2.3-fold compared to the platform host strain. Finally, combinatorial overexpression of the GXA1 gene and the genes involved in glutathione synthesis in the platform host strain increased the extracellular glutathione production by up to 17.1-fold compared to the parental strain. Overall, the metabolic engineering of the glutathione synthesis, degradation, and transport increased the total (extracellular + intracellular) glutathione production. The extracellular glutathione fermentation method developed in this study has the potential to overcome the limitations of the present intracellular glutathione fermentation process in yeast.

Recently, genetic engineering efforts have been made to develop recombinant Saccharomyces cerevisiae strains able to utilize xylose, an inexpensive and abundant carbon source. However, their construction and selection processes are limited by the speed and expenses of the existing testing methods, thus a rapid and equally precise method will significantly increase the number of tested strains. Here, near infrared (NIR) spectroscopy is proposed as a successful alternative method for screening recombinant xylose-fermenting S. cerevisiae. Supernatant samples of fermentation solutions from one diploid and three haploid recombinant strains were collected along the fermentation process. NIR spectra of the diluted supernatant provided effective differentiation of strains consistent with their phenotypic and genotypic features. This result could be used as a feedback for multicomponent analysis, in order to develop regression model for quantification of consumed glucose and xylose, produced ethanol, glycerol, and xylitol. Robust partial least-squares regression models with high prediction accuracy that are effective with any strain were achieved for all components when the modeling was performed with combined data of all strains, achieving 0.21–1.49 g/L of standard error of prediction with calibration, prediction, limit of detection and limit of quantification in the range of 1.0–4.5 and 3.0–13.4 g/L, respectively.

G-protein-coupled receptors (GPCRs) are considered as important targets for drug discovery. The yeast Saccharomyces cerevisiae is an attractive host for high-throughput screening of agonistic ligands for human GPCRs because it can simplify the complicated signaling pathways that are present in mammalian cell lines. Unfortunately, many human GPCRs induce only partial signal activation in yeast cells depending on their coupling efficiency with yeast G-proteins. This problem often results in unsatisfactory detection sensitivity, thereby resulting in a limitation to yeast-based detection systems. Here we introduce a new highly sensitive detection method that provides robust agonist detection of human GPCRs. Our strategy is designed to invoke feedback activation of signals within yeast G-protein signaling pathways. Briefly, agonist stimulation of human GPCRs triggers expression of an artificial signal activator that amplifies signaling. We chose human somatostatin receptor subtype 5 (hSSTR5) as a model of a human GPCR. Investigation of the response of hSSTR5-expressing yeast to various concentrations of somatostatin demonstrated that feedback activation of the signal can successfully improve the detection limit and the maximum level of signaling. This novel approach will enhance the usefulness of yeast-based screening of agonistic ligands for a variety of human GPCRs.

In order to achieve efficient d-lactic acid fermentation from a mixture of xylose and glucose, the xylose-assimilating xylAB operon from Lactobacillus pentosus (PXylAB) was introduced into an l-lactate dehydrogenase gene (ldhL1)-deficient Lactobacillus plantarum (ΔldhL1-xpk1::tkt-Δxpk2) strain in which the phosphoketolase 1 gene (xpk1) was replaced with the transketolase gene (tkt) from Lactococcus lactis, and the phosphoketolase 2 (xpk2) gene was deleted. Two copies of xylAB introduced into the genome significantly improved the xylose fermentation ability, raising it to the same level as that of ΔldhL1-xpk1::tkt-Δxpk2 harboring a xylAB operon-expressing plasmid. Using the two-copy xylAB integrated strain, successful homo-d-lactic acid production was achieved from a mixture of 25 g/l xylose and 75 g/l glucose without carbon catabolite repression. After 36-h cultivation, 74.2 g/l of lactic acid was produced with a high yield (0.78 g per gram of consumed sugar) and an optical purity of d-lactic acid of 99.5%. Finally, we successfully demonstrated homo-d-lactic acid fermentation from a mixture of three kinds of sugar: glucose, xylose, and arabinose. This is the first report that describes homo-d-lactic acid fermentation from mixed sugars without carbon catabolite repression using the xylose-assimilating pathway integrated into lactic acid bacteria.

Glutathione is a valuable tri-peptide that is widely used in the pharmaceutical, food, and cosmetic industries. Glutathione is produced industrially by fermentation using Saccharomyces cerevisiae. We demonstrated that expression of amylase genes in glutathione-producing S. cerevisiae enables direct use of starch as a carbon source, thus eliminating the Crabtree effect that is caused by excess glucose. Consequently, cell growth and glutathione productivity were significantly improved. This approach is potentially applicable to a variety of fermentative processes for production of value-added chemicals under aerobic conditions.

The enzymatic process presents an advantage of producing specified phospholipids that rarely exist in nature. In this study, we investigated the regiospecific modification of phosphatidylcholine (PC) in the sn-1 position using immobilized Rhizopus oryzae. In a reaction mixture containing egg yolk PC and exogenous lauric acid (LA) in n-hexane, lipase-producing R. oryzae cells immobilized within biomass support particles (BSPs) showed a much higher transesterification activity than lipase powders. To improve the product yield, several parameters including substrate ratio and reaction time were investigated, resulting in the incorporation of 44.2% LA into the product PC after a 48-h reaction. The analysis of the molecular structure showed that a large proportion of exogenous LA (>90%) was incorporated in the sn-1 position of the enzymatically modified PC. Moreover, the BSP-immobilized R. oryzae maintained its activity for more than 12 batch cycles. The presented results, therefore, suggest the applicability of BSP-immobilized R. oryzae as a whole-cell biocatalyst for the regiospecific modification of phospholipids.

We demonstrate glutamate production from β-glucan using endoglucanase (EG)-expressing Corynebacterium glutamicum. The signal sequence torA derived from Escherichia coli K12, which belongs to the Tat pathway, was suitable for secreting EG of Clostridium thermocellum using C. glutamicum as a host. Using the torA signal sequence, endoglucanase from Clostridium cellulovorans 743B was successfully expressed, and the secreted EG produced 123 mg of reducing sugar from 5 g of β-glucan at 30 °C for 72 h, which is the optimal condition for C. glutamicum growth. Subsequently, glutamate fermentation from β-glucan was carried out with the addition of Aspergillus aculeatus β-glucosidase produced by recombinant Aspergillus oryzae. Using EG-secreting C. glutamicum, 178 mg/l of glutamate was produced from 15 g of β-glucan. This is the first report of glutamate fermentation from β-glucan using endoglucanase-secreting C. glutamicum.

Recombinant yeast strains highly tolerant to formic acid during xylose fermentation were constructed. Microarray analysis of xylose-fermenting Saccharomyces cerevisiae strain overexpressing endogenous xylulokinase in addition to xylose reductase and xylitol dehydrogenase from Pichia stipitis revealed that upregulation of formate dehydrogenase genes (FDH1 and FDH2) was one of the most prominent transcriptional events against excess formic acid. The quantification of formic acid in medium indicated that the innate activity of FDH was too weak to detoxify formic acid. To reinforce the capability for formic acid breakdown, the FDH1 gene was additionally overexpressed in the xylose-metabolizing recombinant yeast. This modification allowed the yeast to rapidly decompose excess formic acid. The yield and final ethanol concentration in the presence of 20 mM formic acid is as essentially same as that of control. The fermentation profile also indicated that the production of xylitol and glycerol, major by-products in xylose fermentation, was not affected by the upregulation of FDH activity.

We developed a novel enzymatic glutathione (GSH) production system using Saccharomyces cerevisiae as a whole-cell biocatalyst, and improved its GSH productivity by metabolic engineering. We demonstrated that the metabolic engineering of GSH pathway and ATP regeneration can significantly improve GSH productivity by up to 1.7-fold higher compared with the parental strain, respectively. Furthermore, the combination of both improvements in GSH pathway and ATP regeneration is more effective (2.6-fold) than either improvement individually for GSH enzymatic production using yeast. The improved whole-cell biocatalyst indicates its great potential for applications to other kinds of ATP-dependent bioproduction.

In order to achieve efficient homo L-lactic acid fermentation from xylose, we first carried out addition of xylose assimilation ability to Lactococcus lactis IL 1403 by introducing a plasmid carrying the xylRAB genes from L. lactis IO-1 (pXylRAB). Then modification of xylose assimilation pathway was carried out. L. lactis has two pathways for xylose assimilation called the phosphoketolase pathway (PK pathway) that produces both lactic acid and acetic acid and the pentose phosphate pathway (PP pathway) that produces only lactic acid as a final product. Thus a mutant strain that disrupted its phosphokeolase gene (ptk) was constructed. The Δptk mutant harboring pXylRAB lacked the PK pathway and produced predominantly lactic acid from xylose via the PP pathway, although its fermentation rate slightly decreased. Further introduction of the transketolase gene (tkt) to disrupted ptk locus led restoration of fermentation rate and this was attributed to enhancement of the PP pathway. As a result, ptk::tkt strain harboring pXylRAB produced 50.1 g/l of L-lactic acid from xylose with a high optical purity of 99.6% and a high yield of 1.58 (moles per mole xylose consumed) that is close to theoretical value of 1.67 from xylose.

A novel HER2-targeted carrier was developed using bionanocapsules (BNCs). Bionanocapsules (BNCs) are 100-nm hollow nanoparticles composed of the l-protein of hepatitis B virus surface antigen. An affibody of HER2 was genetically displayed on the BNC surface (ZHER2-BNC). For the investigation of binding affinity, ZHER2-BNC was incubated with the cancer cell lines SK-BR-3 (HER2 positive), and MDA-MB-231 (HER2 negative). For analysis of HER2 targeting specificity, ZHER2-BNC or ZWT-BNC (without affibody) was incubated with both SK-BR-3 and MDA-MB-231 cells by time lapse and concentration. For the delivery of encapsulated molecules (calcein), fluorescence of ZHER2-BNC mixed with liposomes was also compared with that of ZWT-BNC and nude liposomes by incubation with SK-BR-3 cells. As a result, ZHER2-BNC-liposome complex demonstrated the delivery to HER2-expressing cells (SK-BR-3) with a high degree of specificity. This indicates that genetically engineered BNCs are promising carrier for cancer treatment.

Yeast, such as Saccharomyces cerevisiae or Kluyveromyces lactis is appropriate strain for ethanol production or some useful compounds production. Cellulases expressing yeast can ferment ethanol from cellulosic materials; however, the productivity should be increase more and more. To improve and engineer the productivity, the target gene(s) were introduced into yeast genome. Generally, using genetic engineering, increasing integrated gene numbers are increased, the expressed protein ability such as enzymatic activities are also increased. In this mini-review, we focused on the effect of integrated gene copy number and the polyploidy on the productivity such as enzymatic activity and/or product yield.

Lactic acid (LA) is an important and versatile chemical that can be produced from renewable resources such as biomass. LA is used in the food, pharmaceutical, and polymers industries and is produced by microorganism fermentation; however, most microorganisms cannot directly utilize biomass such as starchy materials and cellulose. Here, we summarize LA production using several kinds of genetically modified microorganisms, such as LA bacteria, Escherichia coli, Corynebacterium glutamicum, and yeast. Using gene manipulation and metabolic engineering, the yield and optical purity of LA produced from biomass has been significantly improved. In this review, the drawbacks as well as improvements of LA production by fermentation is discussed.

The cell surface engineering system, in which functional proteins are genetically displayed on microbial cell surfaces, has recently become a powerful tool for applied biotechnology. Here, we report on the surfactant modification of surface-displayed lipase to improve its performance for enzymatic synthesis reactions. The lipase activities of the surfactant-modified yeast displaying Rhizopus oryzae lipase (ROL) were evaluated in both aqueous and nonaqueous systems. Despite the similar lipase activities of control and surfactant-modified cells in aqueous media, the treatment with nonionic surfactants increased the specific lipase activity of the ROL-displaying yeast in n-hexane. In particular, the Tween 20-modified cells increased the cell surface hydrophobicity significantly among a series of Tween surfactants tested, resulting in 8–30 times higher specific activity in organic solvents with relatively high log P values. The developed cells were successfully used for the enzymatic synthesis of phospholipids and fatty acid methyl esters in n-hexane, whereas the nontreated cells produced a significantly low yield. Our results thus indicate that surfactant modification of the cell surface can enhance the potential of the surface-displayed lipase for bioconversion.

A novel cell surface display system in Aspergillus oryzae was established by using a chitin-binding module (CBM) from Saccharomyces cerevisiae as an anchor protein. CBM was fused to the N or C terminus of green fluorescent protein (GFP) and the fusion proteins (GFP-CBM and CBM-GFP) were expressed using A. oryzae as a host. Western blotting and fluorescence microscopy analysis showed that both GFP-CBM and CBM-GFP were successfully expressed on the cell surface. In addition, cell surface display of triacylglycerol lipase from A. oryzae (tglA), while retaining its activity, was also successfully demonstrated using CBM as an anchor protein. The activity of tglA was significantly higher when tglA was fused to the C terminus than N terminus of CBM. Together, these results show that CBM used as a first anchor protein enables the fusion of both the N and/or C terminus of a target protein.

We successfully demonstrated batch ethanol fermentation repeated ten times from raw starch with high ethanol productivity. We constructed a yeast diploid strain coexpressing the maltose transporter AGT1, α-amylase, and glucoamylase. The introduction of AGT1 allows maltose and maltotriose fermentation as well as the improvement of amylase activities. We also found that α-amylase activity during fermentation was retained by the addition of 10 mM calcium ion and that the highest α-amylase activity was 9.26 U/ml during repeated fermentation. The highest ethanol productivity was 2.22 g/l/h at the fourth batch, and after ten cycles, ethanol productivity of more than 1.43 g/l/h was retained, as was α-amylase activity at 6.43 U/ml.

A yeast with the xylose isomerase (XI) pathway was constructed by the multicopy integration of XI overexpression cassettes into the genome of the Saccharomyces cerevisiae MT8-1 strain. The resulting yeast strain successfully produced ethanol from both xylose as the sole carbon source and a mixed sugar, consisting of xylose and glucose, without any adaptation procedure. Ethanol yields in the fermentation from xylose and mixed sugar were 61.9% and 62.2% of the theoretical carbon recovery, respectively. Knockout of GRE3, a gene encoding nonspecific aldose reductase, of the host yeast strain improved the fermentation profile. Not only specific ethanol production rates but also xylose consumption rates was improved more than twice that of xylose-metabolizing yeast with the XI pathway using GRE3 active yeast as the host strain. In addition, it was demonstrated that xylitol in the medium exhibits a concentration-dependent inhibition effect on the ethanol production from xylose with the yeast harboring the XI-based xylose metabolic pathway. From our findings, the combination of XI-pathway integration and GRE3 knockout could be result in a consolidated xylose assimilation pathway and increased ethanol productivity.

We have developed a novel cell surface display in Corynebacterium glutamicum using porin proteins as anchor proteins. Porins are localized at C. glutamicum mycolic acid layer and exist as a hexamer. We used α-amylase from Streptococcus bovis 148 (AmyA) as a model protein to be displayed on the C. glutamicum cell surface. AmyA was fused to the C terminus of the porins PorB, PorC, or PorH. Expression vectors using fused proteins under the control of the cspB promoter were constructed and introduced into the C. glutamicum Cm strain. Immunostaining microscopy and flow cytometric analysis revealed that PorB-AmyA, PorC-AmyA, and PorH-AmyA were displayed on the C. glutamicum cell surface. AmyA activity was only detected in the cell fraction of C. glutamicum cells that displayed AmyA fused to PorB, PorC or PorH and AmyA activity was not detected in the supernatants of C. glutamicum culture broths after 72 h cultivation. Thus, we have demonstrated that C. glutamicum porins are very efficient anchor proteins for protein display in C. glutamicum.

Insect cell expression systems are widely used to produce active recombinant proteins. Here, we have developed a high-level expression vector containing a selectable marker for continuous production of recombinant proteins in insect cells. The plasmid, pXIHAbla, developed in this study, established a polyclonal cell line 8 days shorter than pXINSECT-DEST38 and pBmAneo. In addition, pXIHAbla exhibited an approximately fivefold higher average enhanced GFP expression level and approximately a twofold higher bionanocapsule secretion level than pXINSECT-DEST38. Using this plasmid, insect cells that highly express active proteins have been easily established.

Here, we demonstrated the one-step production of cadaverine from starch using a Corynebacterium glutamicum strain coexpressing Streptococcus bovis 148 α-amylase (AmyA) and Escherichia coli K-12 lysine decarboxylase (CadA). We constructed the E. coli–C. glutamicum shuttle vector, which produces CadA under the control of the high constitutive expression (HCE) promoter, and transformed this vector into C. glutamicum CSS secreting AmyA. The engineered C. glutamicum expressed both CadA and AmyA, which retained their activity. We performed cadaverine fermentation using 50 g/l soluble starch as the sole carbon source without pyridoxal-5’-phosphate, which is the coenzyme for CadA. C. glutamicum coexpressing AmyA and CadA successfully produced cadaverine from soluble starch and the yield of cadaverine was 23.4 mM after 21 h. CadA expression levels under the control of the HCE promoter were assumed to be sufficient to convert l-lysine to cadaverine, as there was no accumulation of l-lysine in the culture medium during fermentation. Thus, we demonstrated that C. glutamicum has great potential to produce cadaverine from biomass resources.

The introduction of several kinds of genes into the yeast chromosome is a powerful tool in many fields from fundamental study to industrial application. Here, we describe a general strategy for one-step gene integration and a marker recycling method. Forty base pairs of a short sequence derived from a region adjacent to the HIS3 locus were placed between cell surface displaying β-glucosidase (BGL) and URA3 marker genes. HIS3 deletion and BGL–URA3 fragment integration were achieved via a PCR fragment consisting of the BGL–URA3 fragment attached to homology sequences flanked by the HIS3 targeting locus. The obtained his3::URA3 disruptants were plated on a 5-FOA plate to select for the URA3 deletion due to repeated sequences at both sides of URA3 gene. In all selected colonies, BGL genes were integrated at the targeted HIS3 locus and URA3 was completely deleted. In addition, introduced BGL was efficiently expressed, and the transformants fermented cellobiose to ethanol effectively. As our strategy creates next transformation markers continuously together with gene integration, this method can serve as a simple and powerful tool for multiple genetic manipulations in yeast engineering.

Previously, a Saccharomyces cerevisiae strain was engineered for xylose assimilation by the constitutive overexpression of the Orpinomyces xylose isomerase, the S. cerevisiae xylulokinase, and the Pichia stipitis SUT1 sugar transporter genes. The recombinant strain exhibited growth on xylose, under aerobic conditions, with a specific growth rate of 0.025 h−1, while ethanol production from xylose was achieved anaerobically. In the present study, the developed recombinant yeast was adapted for enhanced growth on xylose by serial transfer in xylose-containing minimal medium under aerobic conditions. After repeated batch cultivations, a strain was isolated which grew with a specific growth rate of 0.133 h−1. The adapted strain could ferment 20 g l−1 of xylose to ethanol with a yield of 0.37 g g−1 and production rate of 0.026 g l−1 h−1. Raising the fermentation temperature from 30°C to 35°C resulted in a substantial increase in the ethanol yield (0.43 g g−1) and production rate (0.07 g l−1 h−1) as well as a significant reduction in the xylitol yield. By the addition of a sugar complexing agent, such as sodium tetraborate, significant improvement in ethanol production and reduction in xylitol accumulation was achieved. Furthermore, ethanol production from xylose and a mixture of glucose and xylose was also demonstrated in complex medium containing yeast extract, peptone, and borate with a considerably high yield of 0.48 g g−1.

The cDNA sequence of the gene for xylose isomerase from the rumen fungus Orpinomyces was elucidated by rapid amplification of cDNA ends. The 1,314-nucleotide gene was cloned and expressed constitutively in Saccharomyces cerevisiae. The deduced polypeptide sequence encoded a protein of 437 amino acids which showed the highest similarity to the family II xylose isomerases. Further, characterization revealed that the recombinant enzyme was a homodimer with a subunit of molecular mass 49 kDa. Cell extract of the recombinant strain exhibited high specific xylose isomerase activity. The pH optimum of the enzyme was 7.5, while the low temperature optimum at 37°C was the property that differed significantly from the majority of the reported thermophilic xylose isomerases. In addition to the xylose isomerase gene, the overexpression of the S. cerevisiae endogenous xylulokinase gene and the Pichia stipitis SUT1 gene for sugar transporter in the recombinant yeast facilitated the efficient production of ethanol from xylose.

A Candida antarctica lipase B (CALB)-displaying yeast whole-cell biocatalyst was constructed with the integration of the CALB cell-surface display expression cassette in the yeast genome and cell fusion by mating. Lipase hydrolytic activity of the yeast whole-cell biocatalyst subsequently increased, in both a- and α-type yeast cells, with the number of copies of the CALB cell-surface display expression cassette introduced, and reached 43.6 and 32.2 U/g-dry cell at 168 h cultivation, respectively. The lipase hydrolytic activity of whole cells in diploid yeast cells containing eight copies of the CALB cell-surface expression cassette reached 117 U/g-dry cell, and this value is approximately ninefold higher than that of the previously reported haploid CALB cell-surface displaying yeast using a multi-copy plasmid (Tanino et al. Appl. Microbial Biotechnol 75:1319–1325, 2007). This improved novel CALB-displaying yeast whole-cell biocatalyst could repeatedly catalyze the polyester, polybutylene adipate, synthesis reaction, using adipic acid and 1, 4-butandiol as the monomer molecules, four times in succession. This is the first report of the polymer synthesis using enzyme displaying yeast as the catalyst. The ratios of cyclic compounds in the polybutylene adipates synthesized with the CALB-displaying yeast whole-cells were lower than that in the polybutylene adipate synthesized with conventional metal catalysis. From these results, it appears that the use of CALB-displaying yeast cells could be useful for the polyester synthesis reaction, with reduced by-product production.

In this paper, we provide the first report of utilizing recombinant fungal whole cells in enzymatic biodiesel production. Aspergillus oryzae, transformed with a heterologous lipase-encoding gene from Fusarium heterosporum, produced fully processed and active forms of recombinant F. heterosporum lipase (FHL). Cell immobilization within porous biomass support particles enabled the convenient usage of FHL-producing A. oryzae as a whole-cell biocatalyst for lipase-catalyzed methanolysis. The addition of 5% water to the reaction mixture was effective in both preventing the lipase inactivation by methanol and facilitating the acyl migration in partial glycerides, resulting in the final methyl ester content of 94% even in the tenth batch cycle. A comparative study showed that FHL-producing A. oryzae attained a higher final methyl ester content and higher lipase stability than Rhizopus oryzae, the previously developed whole-cell biocatalyst. Although both FHL and R. oryzae lipase exhibit 1,3-regiospecificity towards triglyceride, R. oryzae accumulated a much higher amount of sn−2 isomers of partial glycerides, whereas FHL-producing A. oryzae maintained a low level of the sn−2 isomers. This is probably because FHL efficiently facilitates the acyl migration from the sn−2 to the sn−1(3) position in partial glycerides. These findings indicate that the newly developed FHL-producing A. oryzae is an effective whole-cell biocatalyst for enzymatic biodiesel production.

A novel cell-surface display system was constructed in Aspergillus oryzae. Each of the five genes encoding the putative cell-wall-localized protein from the A. oryzae genome was cloned and these cell-surface anchor functions were examined by fusion to the C-terminal of the green fluorescent protein (GFP). Using the MP1 and CWP proteins as anchor proteins, GFP signals were strongly observed on the cell surface of recombinant A. oryzae. When these proteins were used as anchor proteins for cell-surface display of β-glucosidase from A. oryzae, enzyme activity was detected on the cell surface. In particular, β-glucosidase activity of recombinant A. oryzae using MP1, a putative glycosylphosphatidylinositol (GPI) anchor protein was higher than CWP. Based on these results, it was concluded that the MP1 protein can act as a GPI-anchor protein in A. oryzae, and the proposed cell-surface display system using MP1 allows for the display of heterogeneous and endogenous proteins.

To develop a new approach for improving heterologous protein production in Aspergillus oryzae, we focused on the functional role of the N-terminal region of Rhizopus oryzae lipase (ROL). Several N-terminal deletion variants of ROL were expressed in A. oryzae. Interestingly, a segment of 28 amino acids from the C-terminal region of the propeptide (N28) was found to be critical for secretion of ROL into the culture medium. To further investigate the role of N28, the ROL secretory process was visualized in vivo using ROL–green fluorescent protein (GFP) fusion proteins. In cells producing ROL with N28, fluorescence observations showed that the fusion proteins are transported through endoplasmic reticulum (ER), Golgi, and cell wall, which is one of the typical secretory processes in a eukaryotic cell. Because the expression of the mature ROL–GFP fusion protein induced fluorescence accumulation without its translocation into the ER, N28 is considered to play a crucial role in protein transport. When N28 was inserted between the secretion signal and GFP, fluorescence observations showed that GFP, which is originally a cytoplasmic protein, was efficiently translocated into the ER of A. oryzae, resulting in an enhanced secretion of mature GFP after proteolytic cleavage of N28. These findings suggest that N28 facilitates protein translocation into ER and can be a promising candidate for improving heterologous protein production in A. oryzae.

Corynebacterium glutamicum is an important microorganism in the industrial production of amino acids. We engineered a strain of C. glutamicum that secretes α-amylase from Streptococcus bovis 148 (AmyA) for the efficient utilization of raw starch. Among the promoters and signal sequences tested, those of cspB from C. glutamicum possessed the highest expression level. The fusion gene was introduced into the homoserine dehydrogenase gene locus on the chromosome by homologous recombination. L-Lysine fermentation was conducted using C. glutamicum secreting AmyA in the growth medium containing 50 g/l of raw corn starch as the sole carbon source at various temperatures in the range 30 to 40°C. Efficient L-lysine production and raw starch degradation were achieved at 34 and 37°C, respectively. The α-amylase activity using raw corn starch was more than 2.5 times higher than that using glucose as the sole carbon source during L-lysine fermentation. AmyA expression under the control of cspB promoter was assumed to be induced when raw starch was used as the sole carbon source. These results indicate that efficient simultaneous saccharification and fermentation of raw corn starch to L-lysine were achieved by C. glutamicum secreting AmyA using the cspB promoter and signal sequence.

We constructed a high-throughput screening (HTS) system for target cells based on the detection of protein–protein interactions by flow cytometric sorting due to the improvement in the yeast cell surface display system. Interaction model proteins, which are the ZZ domain derived from Staphylococcus aureus and the Fc part of human immunoglobulin G (IgG), were displayed on the yeast cell surface. We achieved a rapid and enhanced expression of these proteins as a result of adopting an appropriate yeast strain and a suitable promoter. The displayed ZZ domain had an ability to bind to rabbit IgG and the displayed Fc part to protein A. These were confirmed by flow cytometry and fluorescence microscopy. Furthermore, the cells displaying the ZZ domain or Fc part were isolated from the model libraries constructed by mixing the control yeast cells with the target yeast cells. The ratio of the target cells was increased from 0.0001% to more than 70% by two cycles of cell sorting. These results indicate that we can achieve a rapid and highly efficient isolation method for the target cells with FACSCalibur and that this method will further extend the application of flow cytometric sorting to library selections.

We engineered a Corynebacterium glutamicum strain displaying α-amylase from Streptococcus bovis 148 (AmyA) on its cell surface to produce amino acids directly from starch. We used PgsA from Bacillus subtilis as an anchor protein, and the N-terminus of α-amylase was fused to the PgsA. The genes of the fusion protein were integrated into the homoserine dehydrogenase gene locus on the chromosome by homologous recombination. l-Lysine fermentation was carried out using C. glutamicum displaying AmyA in the growth medium containing 50 g/l soluble starch as the sole carbon source. We performed l-lysine fermentation at various temperatures (30–40°C) and pHs (6.0–7.0), as the optimal temperatures and pHs of AmyA and C. glutamicum differ significantly. The highest l-lysine yield was recorded at 30°C and pH 7.0. The amount of soluble starch was reduced to 18.29 g/l, and 6.04 g/l l-lysine was produced in 24 h. The l-lysine yield obtained using soluble starch as the sole carbon source was higher than that using glucose as the sole carbon source after 24 h when the same amount of substrates was added. The results shown in the current study demonstrate that C. glutamicum displaying α-amylase has a potential to directly convert soluble starch to amino acids.

We isolated the lipase B from Candida antarctica CBS 6678 (CALB CBS6678) and successfully constructed CALB-displaying yeast whole-cell biocatalysts using the Flo1p short (FS) anchor system. For the display of CALB on a yeast cell surface, the newly isolated CALB CBS6678 exhibited higher hydrolytic and ester synthesis activities than the well-known CALB, which is registered in GenBank (Z30645). A protease accessibility assay using papain as a protease showed that a large part of CALB, approximately 75%, was localized on an easily accessible part of the yeast cell surface. A comparison of the lipase hydrolytic activities of yeast whole cells displaying only mature CALB (CALB) and those displaying mature CALB with a Pro region (ProCALB) revealed that mature CALB is preferable for yeast cell surface display using the Flo1p anchor system. Lyophilized yeast whole cells displaying CALB were applied to an ester synthesis reaction at 60°C using adipic acid and n-butanol as substrates. The amount of dibutyl adipate (DBA) produced increased with the reaction time until 144 h. This indicated that CALB displayed on the yeast cell surface retained activity under the reaction conditions.

To achieve direct and efficient lactic acid production from starch, a genetically modified Lactococcus lactis IL 1403 secreting α-amylase, which was obtained from Streptococcus bovis 148, was constructed. Using this strain, the fermentation of soluble starch was achieved, although its rate was far from efficient (0.09 g l−1 h−1 lactate). High-performance liquid chromatography revealed that maltose accumulated during fermentation, and this was thought to lead to inefficient fermentation. To accelerate maltose consumption, starch fermentation was examined using L. lactis cells adapted to maltose instead of glucose. This led to a decrease in the amount of maltose accumulation in the culture, and, as a result, a more rapid fermentation was accomplished (1.31 g l−1 h−1 lactate). Maximum volumetric lactate productivity was further increased (1.57 g l−1 h−1 lactate) using cells adapted to starch, and a high yield of lactate (0.89 g of lactate per gram of consumed sugar) of high optical purity (99.2% of l-lactate) was achieved. In this study, we propose a new approach to lactate production by α-amylase-secreting L. lactis that allows efficient fermentation from starch using cells adapted to maltose or starch before fermentation.

We demonstrated ethanolysis of rapeseed oil to produce biodiesel fuel using lipase-producing filamentous fungi immobilized on biomass support particles (BSPs) as a whole-cell biocatalyst. We prepared two types of whole-cell biocatalyst: wild-type Rhizopus oryzae producing triacylglycerol lipase (w-ROL) and recombinant Aspergillus oryzae expressing Fusarium heterosporum lipase (r-FHL). Both w-ROL and r-FHL successfully catalyzed the ethanolysis of rapeseed oil, and the fatty acid ethyl ester yield was as high as 79% (w-ROL) or 94% (r-FHL). In the case of r-FHL, the residual monoglycerides (MGs) and diglycerides (DGs) were no more than 0.73 and 0.18%, respectively. In addition, r-FHL could be recycled for the ethanolysis of rapeseed oil, retaining over 85% fatty acid ethyl ester yield by the fifth cycle. r-FHL was revealed to be a promising catalyst for biodiesel production using rapeseed oil and ethanol.

The yeast Saccharomyces cerevisiae GRI-117-UK was transformed to either display or secrete β-glucosidase 1 (BGL1) from the koji mold, Aspergillus oryzae. The β-glucosidase activity of BGL1-displaying yeast strains reached 405.9 U/g dry cell mass after 72 h of cultivation in YPD medium. The optimal pH and temperature for BGL1 displayed on the cell surfaces of the yeast were 5.0 and 55 °C, while the optima for BGL1 secreted by the yeast were 4.0 and 55 °C. The displayed BGL1 was stable at higher pH compared with the secreted BGL1. In addition, the thermostability of BGL1 was improved by displaying the enzyme on the yeast cell surfaces. In addition, the displayed and secreted forms of BGL1 had similar substrate specificity. β-Glucosidase hydrolyzes daidzin and genistin, which are the glycoside forms of soybean isoflavones, to the aglycones. Isoflavone aglycones were efficiently produced by BGL1-displaying yeast from an isoflavone mixture; at optimal temperature and pH the rate of aglycone production was at least 15.8 g/(l h). After 144 h of reaction, almost isoflavones were converted to its aglycone by BGL1-displaying yeast. The results of the present study demonstrate that BGL1-displaying yeast strains are effective whole cell biocatalysts of isoflavone aglycone production.

•P. carnosa and other basidiomycetes have multiple expansin-related proteins.•Most of the expansin-related proteins in P. carnosa are homologous to loosenin.•Loosenin-like proteins were classified into two subgroups by sequence homology.•P. carnosa also has a protein distantly related to other loosenin-like proteins.•Genes of expansin-related proteins in P. carnosa showed distinct expression patterns.Expansin and expansin-related proteins loosen plant cell wall architectures and are widely distributed in several types of organisms, including plants, fungi and bacteria. Here we describe sequence diversity and unique gene expression profiles of multiple expansin-related proteins identified in the basidiomycete, Phanerochaete carnosa. The protein sequences were homologous to loosenin, an expansin-related protein reported in the basidiomycete, Bjerkandera adusta. We identified homologous sequences of each of those P. carnosa proteins in many basidiomycete species. Twelve P. carnosa loosenin-like proteins (LOOLs) were classified into two subgroups according to sequence homology. Conservation of polysaccharide-binding amino acid residues was stricter in subgroup A. Subgroup A sequences included a conserved 8–9 amino acid insertion in a polysaccharide-binding groove whereas subgroup B contained a 12–18 amino acid insertion next to the binding groove. The P. carnosa genome also encodes the expansin-related protein, DREX1, which adopts a loosenin-like structure but has lower sequence homology to other LOOLs. The gene expression analysis of those proteins showed distinct patterns that were not significantly related to subgroupings. The variation in the protein sequences and gene expression patterns, and wide distribution among the basidiomycota, suggest that the diverse cell wall loosening proteins contribute to effective plant cell wall association and utilization by basidiomycetes.

A bio-nanocapsule derived from the hepatitis B virus (HBV) is expected to be useful as a drug delivery system carrier. Because various types of bio-nanocapsules have been developed, a simple and versatile purification method for bio-nanocapsules would be useful. Therefore, this study was focused on establishing a simple purification method using affinity chromatography by inserting a histidine tag (His-tag) into a bio-nanocapsule. The method achieved a simple, one-step purification with a yield that was 2.5-fold higher than conventional ultracentrifugation, and thus would be a desirable alternative method for recombinant virus-like particle purification.

Hepatitis B Virus core (HBc) particles have been studied for their potential as drug delivery vehicles for cancer therapy. HBc particles are hollow nano-particles of 30–34 nm diameter and 7 nm thick envelopes, consisting of 180–240 units of 21 kDa core monomers. They have the capacity to assemble/dis-assemble in a controlled manner allowing encapsulation of various drugs and other biomolecules. Moreover, other functional motifs, i.e. receptors, receptor binding sequences, peptides and proteins can be expressed. This study focuses on the development of genetically modified HBc particles to specifically recognise and target human epidermal growth factor receptor-2 (HER2)-expressing cancer cells, in vitro and in vivo, for future cancer therapy. The non-specific binding capacity of wild type HBc particles was reduced by genetic deletion of the sequence encoding arginine-rich domains. A specific HER2-targeting was achieved by expressing the ZHER2 affibodies on the HBc particles surface. In vitro studies showed specific uptake of ZHER2-ΔHBc particles in HER2 expressing cancer cells. In vivo studies confirmed positive uptake of ZHER2-ΔHBc particles in HER2-expressing tumours, compared to non-targeted ΔHBc particles in intraperitoneal tumour-bearing mice models. The present results highlight the potential of these nanocarriers in targeting HER2-positive metastatic abdominal cancer following intra-peritoneal administration.

•Ectoine productivity from glucose was improved by using b3657 overexpressing H. elongata.•Ectoine productivity from xylose was improved by using HEO_0208 overexpressing H. elongata.•The transport of sugar is important for efficient ectoine production.•We demonstrated that the HEO_0208 is the major xylose transporter in H. elongata.We successfully enhanced the productivity of ectoine with Halomonas elongata by improvement of the transport of sugar. First, we carried out screening for sugar transporters capable of improving glucose and xylose consumption. We found two transporters: b3657 from Escherichia coli, which is capable of improving glucose consumption, and HEO_0208 from H. elongata, which is capable of improving xylose consumption. Using transporter-overexpressing strains, the productivity of ectoine was improved. These results indicate that sugar consumption is important for efficient ectoine production. As result of phenotypic analysis of a HEO_0208 deletion strain, we discovered that HEO_0208 is the major xylose transporter in H. elongata. This is the first report demonstrating improvement of ectoine productivity by enhancing the transport of sugar.

Metabolic inhibitors were applied for chemical regulation of central carbon metabolism in Saccharomyces cerevisiae. S. cerevisiae was treated with 10 metabolic inhibitors with various modes of action, and their activities were evaluated using a growth inhibition assay. Among the 6 active inhibitors, the effects of pyrazole (alcohol dehydrogenase inhibitor) and TTA (2-thenoyltrifluoloacetone, succinate dehydrogenase inhibitor) were analyzed in detail. The flask-scale batch-fermentation test showed that ethanol yield was reduced to 0.10 ± 0.01 g g−1 and glycerol yield increased to 0.26 ± 0.01 g g−1 on treatment with pyrazole at 5.0 g L−1, indicating that multiple isozymes of alcohol dehydrogenase were simultaneously inhibited. The multi-targeted metabolic profiling analysis revealed that, although the TTA and pyrazole treatments affected the profiles of all central carbon metabolites in distinct manners, the level of fructose-1,6-bisphosphate commonly increased in the TTA- and pyrazole-treated S. cerevisiae by an unknown mechanism. These results demonstrate that chemical regulation of the central carbon metabolism could be used as an alternative tool to control microbial cell factories for bioproduction, or as a chemical probe to investigate the metabolic systems of useful microorganisms.

This study aimed to optimize extracellular glutathione production by a Saccharomyces cerevisiae engineered strain and to concentrate the extracellular glutathione by membrane separation processes, including ultrafiltration (UF) and nanofiltration (NF). Synthetic defined (SD) medium containing 20 g L−1 glucose was fermented for 48 h; the fermentation liquid was passed through an UF membrane to remove macromolecules. Glutathione in this permeate was concentrated for 48 h to 545.1 ± 33.6 mg L−1 using the NF membrane; this was a significantly higher concentration than that obtained with yeast extract peptone dextrose (YPD) medium following 96 h NF concentration (217.9 ± 57.4 mg L−1). This higher glutathione concentration results from lower cellular growth in SD medium (final OD600 = 6.9 ± 0.1) than in YPD medium (final OD600 = 11.0 ± 0.6) and thus higher production of extracellular glutathione (16.0 ± 1.3 compared to 9.2 ± 2.1 mg L−1 in YPD medium, respectively). Similar fermentation and membrane processing of sweet sorghum juice containing 20 g L−1 total sugars provided 240.3 ± 60.6 mg L−1 glutathione. Increased extracellular production of glutathione by this engineered strain in SD medium and subsequent UF permeation and NF concentration in shortend time may help realize industrial recovery of extracellular glutathione.

Bio-nanocapsules (BNCs) are hollow nanoparticles composed of the L protein of hepatitis B virus (HBV) surface antigen (HBsAg), which can specifically introduce genes and drugs into various kinds of target cells. Although the classic electroporation method has typically been used to introduce highly charged molecules such as DNA, it is rarely adopted for proteins due to its very low efficiency. In this study, a novel approach to the preparation of BNC was established whereby a target protein was pre-encapsulated during the course of nanoparticle formation. Briefly, because of the process of BNC formation in a budding manner on the endoplasmic reticulum (ER) membrane, the association of target proteins to the ER membrane with lipidation sequences (ER membrane localization sequences) could directly generate protein-encapsulating BNC in collaboration with co-expression of the L proteins. Since the membrane-localized proteins are automatically enveloped into BNCs during the budding event, this method can be protect the proteins and BNCs from damage caused by electroporation and obviate the need for laborious consideration to study the optimal conditions for protein encapsulation. This approach would be a useful method for encapsulating therapeutic candidate proteins into BNCs.Highlights► Bio-nanocapsules (BNCs) are virus-like follow nanoparticles for drug delivery. ► The classic electroporation method is commonly used to encapsulate the drugs. ► We established a simple method for preparing protein-encapsulated BNC. ► It can be achieved only by genetically fusing ER membrane localization sequences. ► It never requires the laborious consideration of electroporation conditions.

•Bio-based chemicals have been produced from lignocellulosic biomass by SHF, SSF, and CBP.•Lignocellulosic feedstocks are more recalcitrant to bioprocessing than starch.•Bio-based chemicals serve as biopolymeric building blocks for polyester and polyimide.•More complete utilization of lignocellulose is needed for efficient bioprocessing.The feedstocks used for the production of bio-based chemicals have recently expanded from edible sugars to inedible and more recalcitrant forms of lignocellulosic biomass. To produce bio-based chemicals from renewable polysaccharides, several bioprocessing approaches have been developed and include separate hydrolysis and fermentation (SHF), simultaneous saccharification and fermentation (SSF), and consolidated bioprocessing (CBP). In the last decade, SHF, SSF, and CBP have been used to generate macromolecules and aliphatic and aromatic compounds that are capable of serving as sustainable, drop-in substitutes for petroleum-based chemicals. The present review focuses on recent progress in the bioprocessing of microbially produced chemicals from renewable feedstocks, including starch and lignocellulosic biomass. In particular, the technological feasibility of bio-based chemical production is discussed in terms of the feedstocks and different bioprocessing approaches, including the consolidation of enzyme production, enzymatic hydrolysis of biomass, and fermentation.Graphical abstractDownload high-res image (113KB)Download full-size image

Although the potential for biofuel production from microalgae via photosynthesis has been intensively investigated, information on the selection of a suitable operation strategy for microalgae-based biofuel production is lacking. Many published reports describe competitive strains and optimal culture conditions for use in biofuel production; however, the major impediment to further improvements is the absence of effective engineering strategies for microalgae cultivation and biofuel production. This comprehensive review discusses recent advances in understanding the effects of major environmental stresses and the characteristics of various engineering operation strategies on the production of biofuels (mainly biodiesel and bioethanol) using microalgae. The performances of microalgae-based biofuel-producing systems under various environmental stresses (i.e., irradiance, temperature, pH, nitrogen depletion, and salinity) and cultivation strategies (i.e., fed-batch, semi-continuous, continuous, two-stage, and salinity-gradient) are compared. The reasons for variations in performance and the underlying theories of the various production strategies are also critically discussed. The aim of this review is to provide useful information to facilitate development of innovative and feasible operation technologies for effectively increasing the commercial viability of microalgae-based biofuel production.

β-Glucosidase (BGL1) from Aspergillus oryzae was efficiently produced in recombinant A. oryzae using sodM promoter-mediated expression system. The yield of BGL1 was 960 mg/l in liquid culture, which is 20-fold higher than the yield of BGL1 produced using the yeast Saccharomyces cerevisiae. Recombinant BGL1 converted isoflavone glycosides into isoflavone aglycones more efficiently than β-glucosidase from almond. In addition, BGL1 produced isoflavone aglycones even in the presence of the insoluble form of isoflavone glycosides.

Water activity (aw) is a crucial parameter affecting enzymatic synthetic reactions in organic media. In this paper, we report on the aw dependence of surface-displayed lipases, genetically immobilized on yeast cells via fusion with cell wall proteins. When Saccharomyces cerevisiae displaying Rhizopus oryzae lipase was used for esterification in n-hexane, equilibrating the dried cells with water prior to the reaction markedly increased the reaction rate. An equilibration of the cells with various saturated salt solutions showed that the reaction rate increased with increasing aw of the salt solution, to give the best performance at aw of 1.0. Interestingly, this trend was extremely different from those of lipases in powder or resin-immobilized form. To determine whether the cell surface is responsible for the unique aw profiles, an investigation was carried out similarly using other lipase sources and yeast strains, which indicated that, in all the cells examined, a higher aw resulted in a higher reaction rate. Moreover, increasing aw was found to increase the cell surface hydrophobicity determined by an aqueous-hydrocarbon biphasic partitioning assay. These results indicate that lipases displayed on yeast cells show a unique aw dependence probably because of the variation in cell surface characteristics.

Efficient ethanol producing yeast Saccharomyces cerevisiae cannot produce ethanol from raw starch directly. Thus the conventional ethanol production required expensive and complex process. In this study, we developed a direct and efficient ethanol production process from high-yielding rice harvested in Japan by using amylase expressing yeast without any pretreatment or addition of enzymes or nutrients. Ethanol productivity from high-yielding brown rice (1.1 g/L/h) was about 5-fold higher than that obtained from purified raw corn starch (0.2 g/L/h) when nutrients were added. Using an inoculum volume equivalent to 10% of the fermentation volume without any nutrient supplementation resulted in ethanol productivity and yield reaching 1.2 g/L/h and 101%, respectively, in a 24-h period. High-yielding rice was demonstrated to be a suitable feedstock for bioethanol production. In addition, our polyploid amylase-expressing yeast was sufficiently robust to produce ethanol efficiently from real biomass. This is first report of direct ethanol production on real biomass using an amylase-expressing yeast strain without any pretreatment or commercial enzyme addition.

The goal of this research was to construct a stable and efficient process for the production of ethanol from raw starch, using a recombinant Saccharomyces cerevisiae, which is productive even under conditions such as non-selection or long-term operation. Three recombinant yeast strains were used, two haploid strains (MT8-1SS and NBRC1440SS) and one diploid strain (MN8140SS). The recombinant strains were constructed by integrating the glucoamylase gene from Rhizopus oryzae fused with the 3′-half of the α-agglutinin gene as the anchor protein, and the α-amylase gene from Streptococcus bovis, respectively, into their chromosomal DNA by homologous recombination. The diploid strain MN8140SS was constructed by mating these opposite types of integrant haploid strains in order to enhance the expression of integrated amylase genes. The diploid strain had the highest ethanol productivity and reusability during fermentation from raw starch. Moreover, the ethanol production rate of the integrant diploid strain was maintained when batch fermentation was repeated three times (0.67, 0.60, and 0.67 g/l/h in each batch). These results clearly show that a diploid strain developed by mating two integrant haploid strains is useful for the establishment of an efficient ethanol production process.

Co-utilization of several sugars, especially xylose and glucose, is essential for economically feasible processes with high ethanol productivity. However, the major problem during xylose/glucose co-fermentation is that xylose is used very slowly until after glucose is completely consumed. Here, we demonstrated an effective co-fermentation process using xylose- and cellobiose-assimilating recombinant Saccharomyces cerevisiae. The recombinant yeast is able to consume xylose during xylose/cellobiose co-fermentation as rapidly as during glucose fermentation. After 72 h, 95.6% of xylose was consumed, despite the co-fermentation conditions, and the ethanol yield was 0.358 g-ethanol/g-total sugar.

We used cutinase from the filamentous fungi Aspergillus oryzae to produce dairy flavors. Secretory and displayed forms of cutinase were investigated using salt-free butter, which is composed mostly of triacylglycerides, as the substrate. The secretory form of cutinase, which was produced in recombinant A. oryzae, was suitable for producing butyric acids (16.8 mol%). Also, cutinase displayed on the cell surface of the yeast Saccharomyces cerevisiae as a fusion protein with α-agglutinin released butyric acid at a 2.7-fold rate (45.4 mol%) higher than that of the secreted form. Yeasts carrying two copies of cutinase genes into their chromosomes, which were constructed using the HELOH method, released free fatty acids rapidly and showed 2-fold higher lipase activity compared with yeasts carrying one copy of the cutinase gene.

We attempted to integrate lipase-catalyzed ethanolysis into fermentative bioethanol production. To produce bioethanol, ethanol fermentation from brown rice was conducted using a tetraploid Saccharomyces cerevisiae expressing α-amylase and glucoamylase. The resultant ethanol was distilled and separated into three fractions with different concentrations of water and fusel alcohols. In ethanolysis using the first fraction with 89.3% ethanol, a recombinant Aspergillus oryzae whole-cell biocatalyst expressing Fusarium heterosporum lipase (r-FHL) afforded the highest ethyl ester content of 94.0% after 96 h. Owing to a high concentration of water in the bioethanol solutions, r-FHL, which works best in the presence of water when processing ethanolysis, was found to be more suitable for the integrative process than a commercial immobilized Candida antarctica lipase. In addition, r-FHL was used for repeated-batch ethanolysis, resulting in an ethyl ester content of more than 80% even after the fifth batch. Fusel alcohols such as 1-butanol and isobutyl alcohol are thought to decrease the lipase activity of r-FHL. Using this process, a high ethyl ester content was obtained by simply mixing bioethanol, plant oil, and lipase with an appropriate adjustment of water concentration. The developed process model, therefore, would contribute to biodiesel production from only biomass-derived feedstocks.Graphical abstract.Download full-size imageHighlights► Enzymatic production of biodiesel from biomass-derived alcohol and oil. ► Bioethanol was obtained through brown rice fermentation and distillation. ► An immobilized fungus expressing FHL (r-FHL) was used for ethanolysis. ► Ethanolysis using bioethanol and r-FHL attained an ethyl ester content of 94%. ► An ethyl ester content of more than 80% was maintained during 5 batch cycles.

Enhancing the sugar uptake ability of the yeast Saccharomyces cerevisiae is a potentially important factor for efficient ethanol production during fermentation of lignocellulosic biomass. Here, we attempted to express a Pichia stipitis gene encoding a sugar transporter, SUT1, in a xylose-assimilating S. cerevisiae strain that expresses xylose reductase, xylosedehydrogenase and xylulokinase. We next investigated xylose fermentation, glucose fermentation and glucose and xylose co-fermentation using the Sut1-expressing S. cerevisiae strain. Expression of Sut1 in xylose-assimilating S. cerevisiae increased both xylose uptake ability and ethanol productivity during xylose fermentation. Moreover, glucose uptake ability and ethanol productivity during glucose fermentation also increased by expressing of Sut1. The yield of ethanol during xylose and glucose co-fermentation by the Sut1-expressing yeast strain (0.44 g/g-consumed sugar) was significantly higher than that of the parental strain (0.39 g/g-consumed sugar).

•Metabolomic analysis was applied on the Rre37 mutant of Synechocystis.•Role of Rre37 under nitrogen starvation was investigated.•2-Oxoglutarate accumulation was abolished in the Rre37 mutant.•Rre37 regulated glycogen accumulation through glycogen anabolism.•Rre37 regulated accumulation of TCA cycle metabolites.Rre37 (sll1330) in a cyanobacterium Synechocystis sp. PCC 6803 acts as a regulatory protein for sugar catabolic genes during nitrogen starvation. Low glycogen accumulation in Δrre37 was due to low expression of glycogen anabolic genes. In addition to low 2-oxoglutarate accumulation, normal upregulated expression of genes encoding glutamate synthases (gltD and gltB) as well as accumulation of metabolites in glycolysis (fructose-6-phosphate, fructose-1,6-bisphosphate, and glyceraldehyde-3-phosphate) and tricarboxylic acid (TCA) cycle (oxaloacetate, fumarate, succinate, and aconitate) were abolished by rre37 knockout. Rre37 regulates 2-oxoglutarate accumulation, glycogen accumulation through expression of glycogen anabolic genes, and TCA cycle metabolites accumulation.

For efficient alkyl glucoside production from cellooligosaccharides, we constructed a yeast strain for alkyl glucoside synthesis by genetically inducing the display of β-glucosidase 1 (BGL1) from the filamentous fungus Aspergillus aculeatus No. F-50 on the cell surface. The localization of BGL1 on the cell surface was confirmed by immunofluorescence microscopy. The yeast strain displaying BGL1 catalyzed alkyl glucoside synthesis from p-nitrophenyl β-d-glucoside and primary alcohols. The highest yield of alkyl glucoside was 27.3% of the total sugar. The substrate specificities of the BGL1-displaying yeast strain and almond β-glucosidase were compared using different-chain-length cellooligosaccharides. The BGL1-displaying yeast showed efficient alkyl glucoside production from not only glucose but also cellohexaose. This yeast is applicable as a whole-cell biocatalyst for alkyl glucoside production from cellulose hydrolysates.

In order to decrease carbon emissions and negative environmental impacts of various pollutants, biofuel/biochemical production should be promoted for replacing fossil-based industrial processes. Utilization of abundant lignocellulosic biomass as a feedstock has recently become an attractive option. In this review, we focus on recent efforts of cell surface display using industrial microorganisms such as Escherichia coli and yeast. Cell surface display is used primarily for endowing cellulolytic activity on the host cells, and enables direct fermentation to generate useful fuels and chemicals from lignocellulosic biomass. Cell surface display systems are systematically summarized, and the drawbacks/perspectives as well as successful application of surface display for industrial biotechnology are discussed.

The expression of epidermal growth factor receptor (EGFR) across a wide range of tumor cells has attracted attention for use as a tumor marker in drug delivery systems. Therefore, binding molecules with the ability to target EGFR have been developed. Among them, we focused on affibodies that are binding proteins derived from staphylococcal protein A. By displaying affibody (ZEGFR) binding to EGFR on the surface of a bio-nanocapsule (BNC) derived from a hepatitis B virus (HBV), we developed an altered BNC (ZEGFR-BNC) with a high specificity to EGFR-expressing cells. We considered two different types of ZEGFR (Z955 and Z1907), and found that the Z1907 dimer-displaying BNC ([Z1907]2-BNC) could effectively bind to EGFR-expressing cells and deliver drugs to the cytosol. Since this study showed that [Z1907]2-BNC could target EGFR-expressing cells, we would use this particle as a drug delivery carrier for various cancer cells expressing EGFR.Figure optionsDownload full-size imageDownload high-quality image (69 K)Download as PowerPoint slide

•Bioconversion of high phospholipid-containing oil.•The catalyst used was immobilized whole-cell A. oryzae-expressing FHL.•Effect of reverse micelle formation on bioconversion.•The reduction of reverse micelle formation was crucial for bioconversion.•Conversion was improved by approximately 3-fold to more than 90%.The presence of phospholipids in oil has been a major hurdle in the production of biodiesel using immobilized Aspergillus oryzae whole-cell biocatalysts. A density of phospholipids within the range of 10–30% could reduce both the rate of production and the final yield of biodiesel. Phospholipids in the oil leads to the formation of water-in-oil phospholipid-based reverse micelles. The water that activates the enzymatic process is observed to be trapped inside these reverse micelles. This has resulted in the inactivation of the reaction systems and has subsequently led to the deactivation of the immobilized lipase by the extended residence time of the added methanol. A reaction system involving gentle agitation and higher amount of water was found to reduce the reverse micelles formation. This simple technique improved the conversion efficiency by approximately 3-folds, producing a final biodiesel of more than 90%, using immobilized A. oryzae whole cells expressing Fusarium heterosporum lipase. This demonstrates that, the above technique could be successfully applied to the enzymatic biodiesel conversion of oils containing high amounts of phospholipids such as that from microalgae.Download high-res image (140KB)Download full-size image

•A marked preference for enzyme secretion using immobilized Aspergillus oryzae.•Immobilized cells show high growth rates even with a limited nutrient supply.•Immobilization can maintain morphology of A. oryzae regardless of nutrients.•Repeated-batch enzyme production can be achieved by immobilized cell culture.Given the complex fungal morphology, we provide an alternative to enhancing secretory protein production by Aspergillus oryzae. Immobilized A. oryzae, constructed to overexpress phospholipase A1 (PLA1) and cultivated in the presence of reticulated polyurethane foams, attained the extracellular PLA1 activities of 51.2–62.0 U/ml after 96–120 h, higher than those of suspension cells (36.9–53.5 U/ml). Moreover, the extracellular PLA1 activity of the immobilized cells at 0.5% polypeptone concentration was 34.8 U/ml, which is more than one-half of the maximum activity attained using 2% polypeptone concentration. Further investigations suggested the contribution of high growth rates of immobilized cells toward the enhanced PLA1 production. The macroscopic morphology, which affects the supply of oxygen and nutrients to the interior of cell pellets, is likely the reason for the high growth rates. This is based on the findings that, at 0.5% polypeptone concentration, the suspension cells formed mycelial clumps growing to a diameter of 10 mm, whereas the immobilized cells maintained a dense film with a thickness of 0.4 mm at the surface of the reticulated matrix. Together with the potential utility of repeated-batch cultivation, immobilized cell culture can be a powerful tool for targeted control of fungal morphology in bioprocesses using A. oryzae.

•Aspergillus oryzae (r-BTL) harboring lipase gene (BTL2) was constructed.•r-BTL showed higher thermo- and solvent-tolerance lipase activity.•Enzymatic production of biodiesel from palm oil was performed.•r-BTL efficiently catalyzed methanolysis at elevated temperature (40–50 °C).•r-BTL efficiently catalyzed methanolysis at a high methanol concentrationTo develop a robust whole-cell biocatalyst that works well at moderately high temperature (40–50 °C) with organic solvents, a thermostable lipase from Geobacillus thermocatenulatus (BTL2) was introduced into an Aspergillus oryzae whole-cell biocatalyst. The lipase-hydrolytic activity of the immobilized A. oryzae (r-BTL) was highest at 50 °C and was maintained even after an incubation of 24-h at 60 °C. In addition, r-BTL was highly tolerant to 30% (v/v) organic solvents (dimethyl carbonate, ethanol, methanol, 2-propanol or acetone). The attractive characteristics of r-BTL also worked efficiently on palm oil methanolysis, resulting in a nearly 100% conversion at elevated temperature from 40 to 50 °C. Moreover, r-BTL catalyzed methanolysis at a high methanol concentration without a significant loss of lipase activity. In particular, when 2 molar equivalents of methanol were added 2 times, a methyl ester content of more than 90% was achieved; the yield was higher than those of conventional whole-cell biocatalyst and commercial Candida antarctica lipase (Novozym 435). On the basis of the results regarding the excellent lipase characteristics and efficient biodiesel production, the developed whole-cell biocatalyst would be a promising biocatalyst in a broad range of applications including biodiesel production.

A diploid yeast strain displaying both α-amylase and glucoamylase was developed for repeated fermentation from raw starch. First, the construct of α-amylase was optimized for cell surface display, as there have been no reports of α-amylase-displaying yeast. The modified yeast displaying both glucoamylase and α-amylase produced 46.5 g/l of ethanol from 200 g/l of raw corn starch after 120 h of fermentation, and this was 1.5-fold higher when compared to native α-amylase-displaying yeast. Using the glucoamylase and modified α-amylase co-displaying diploid strain, we repeated fermentation from 100 g/l of raw starch for 23 cycles without the loss of α-amylase or glucoamylase activity. The average ethanol productivity and yield during repeated fermentation were 1.61 g/l/h and 76.6% of the theoretical yield, respectively. This novel yeast may be useful for reducing the cost of bio-ethanol production and may be suitable for industrial-scale bio-ethanol production.Highlights► We developed optimized α-amylase for yeast cell surface display. ► We developed glucoamylase and optimized α-amylase displaying diploid yeast. ► Repeated fermentation from raw starch was carried out using the diploid yeast. ► The ethanol productivity and yield were 1.61 g/l/h and 76.6%. ► Amylase activities and ethanol productivity were maintained for 23 cycles.

Gamma-amino butyric acid (GABA) is a component of pharmaceuticals, functional foods, and the biodegradable plastic polyamide 4. Here, we report a simple and robust system to produce GABA from glucose using the recombinant Corynebacterium glutamicum strain GAD, which expresses GadB, a glutamate decarboxylase encoded by the gadB gene of Escherichia coli W3110. As confirmed by HPLC analysis, GABA fermentation by C. glutamicum GAD cultured at 30 °C in GABA Production 1 (GP1) medium containing 50 g/L glucose without the addition of glutamate yielded 8.07 ± 1.53 g/L extracellular GABA after 96 h. Addition of 0.1 mM pyridoxal 5′-phosphate (PLP) was found to enhance the production of GABA, whereas Tween 40 was unnecessary for GABA fermentation. Using the optimized GABA Production 2 (GP2) medium, which contained 50 g/L glucose and 0.1 mM PLP, fermentation was performed in a flask at 30 °C with 10% (v/v) seed culture of C. glutamicum GAD. GABA was produced in the culture supernatant with a yield of 12.37 ± 0.88 g/L after 72 h with a space–time yield of 0.172 g/L/h, which is the highest yield obtained to date for GABA from fermentation with glucose as a main carbon source.Highlights► GABA was produced directly from glucose by C. glutamicum expressing E. coli GadB. ► Without added glutamate, GABA productivity reached 12.3 g/L in culture supernatant. ► Addition of PLP to the medium was found to enhance the production of GABA. ► By using C. glutamicum GAD, GABA was produced in a maximum yield of 0.17 g/L/h.

With the exhaustion of fossil fuels and with the environmental issues they pose, utilization of abundant lignocellulosic biomass as a feedstock for biofuels and bio-based chemicals has recently become an attractive option. Lignocellulosic biomass is primarily composed of cellulose, hemicellulose, and lignin and has a very rigid and complex structure. It is accordingly much more expensive to process than starchy grains because of the need for extensive pretreatment and relatively large amounts of cellulases for efficient hydrolysis. Efficient and cost-effective methods for the production of biofuels and chemicals from lignocellulose are required. A consolidated bioprocess (CBP), which integrates all biological steps consisting of enzyme production, saccharification, and fermentation, is considered a promising strategy for reducing production costs.Establishing an efficient CBP using lignocellulosic biomass requires both lignocellulose degradation into glucose and efficient production of biofuels or chemicals from glucose. With this aim, many researchers are attempting to endow selected microorganisms with lignocellulose-assimilating ability. In this review, we focus on studies aimed at conferring lignocellulose-assimilating ability not only to yeast strains but also to bacterial strains by recombinant technology. Recent developments in improvement of enzyme productivity by microorganisms and in improvement of the specific activity of cellulase are emphasized.

Here we expand the yeast cell surface display system to display non-natural, functional molecules. The short biotin acceptor peptide (BAP) sequence of biotin ligase from E. coli (BirA) was genetically introduced to the N-terminus of the anchor protein, Flo428. Through co-expression of BAP-fused Flo428 with BirA, biotinylated BAP could be displayed on the yeast cell surface. Subsequent addition of streptavidin–FITC resulted in the display of streptavidin–FITC, and, the display of biotin–FITC was successful using streptavidin as a linker. Our strategy provides a powerful tool for displaying functional molecules on yeast cell surfaces.