Co-reporter:Bin Jia, Hao Qi, Bing-Zhi Li, Shuo Pan, Duo Liu, Hong Liu, Yizhi Cai, and Ying-Jin Yuan
ACS Synthetic Biology November 17, 2017 Volume 6(Issue 11) pp:2108-2108
Publication Date(Web):August 7, 2017
DOI:10.1021/acssynbio.7b00148
Biocontainment systems are crucial for preventing genetically modified organisms from escaping into natural ecosystems. Here, we describe the orthogonal ribosome biofirewall, which consists of an activation circuit and a degradation circuit. The activation circuit is a genetic AND gate based on activation of the encrypted pathway by the orthogonal ribosome in response to specific environmental signals. The degradation circuit is a genetic NOT gate with an output of I-SceI homing endonuclease, which conditionally degrades the orthogonal ribosome genes. We demonstrate that the activation circuit can be flexibly incorporated into genetic circuits and metabolic pathways for encryption. The plasmid-based encryption of the deoxychromoviridans pathway and the genome-based encryption of lacZ are tightly regulated and can decrease the expression to 7.3% and 7.8%, respectively. We validated the ability of the degradation circuit to decrease the expression levels of the target plasmids and the orthogonal rRNA (O-rRNA) plasmids to 0.8% in lab medium and 0.76% in nonsterile soil medium, respectively. Our orthogonal ribosome biofirewall is a versatile platform that can be useful in biosafety research and in the biotechnology industry.Keywords: biocontainment; biofirewall; biosafety; genetically modified organisms; orthogonal ribosome; synthetic biology;
Chemical Society Reviews 2017 vol. 46(Issue 23) pp:7191-7207
Publication Date(Web):2017/11/27
DOI:10.1039/C7CS00208D
Following the discovery of the DNA double helix structure and the advancement of genome sequencing, we have entered a promising stage with regard to genome writing. Recently, a milestone breakthrough was achieved in the chemical synthesis of designer yeast chromosomes. Here, we review the systematic approaches to the de novo synthesis of designer eukaryotic chromosomes, with an emphasis on technologies and methodologies that enable design, building, testing and debugging. The achievement of chemically synthesized genomes with customized genetic features offers an opportunity to rebuild genome organization, remold biological functions and promote life evolution, which will be of great benefit for application in medicine and industrial manufacturing.
Energy & Environmental Science (2008-Present) 2017 vol. 10(Issue 7) pp:1600-1609
Publication Date(Web):2017/07/12
DOI:10.1039/C6EE03705D
Design and construction of synthetic microbial consortia is a promising strategy to enhance the performance of bioelectrochemical systems (BESs) and to facilitate practical applications in bioenergy production. According to the design principle of “division-of-labor”, we synthesized a three-species microbial consortium for power generation, consisting of engineered Escherichia coli, Bacillus subtilis and Shewanella oneidensis. In this consortium, E. coli digested glucose to produce lactate as a carbon source and an electron donor; B. subtilis produced riboflavin as an electron shuttle; and S. oneidensis served as the exoelectrogen to generate electricity. In return, S. oneidensis oxidized lactate to acetate, which fed E. coli and B. subtilis as the carbon source. Thus, the three species formed a cross-feeding microbial consortium, which performed “better together” for power generation. As a result, glucose (11 mM, total 0.28 g) was converted to electricity for more than 15 days with high energy conversion efficiency (up to 55.7%). The microbial composition and electricity output were stable throughout the operation cycle. Furthermore, the consortium exhibited highly functional robustness to fluctuations in the initial inoculation ratio of the three strains. This system provided new insight into the rational design of more efficient, stable, and robust synthetic microbial consortia applicable in bioenergy and environmental bioremediation.
Design and construction of an extensively modified yeast genome is a direct means to interrogate the integrity, comprehensiveness, and accuracy of the knowledge amassed by the yeast community to date. The international synthetic yeast genome project (Sc2.0) aims to build an entirely designer, synthetic Saccharomyces cerevisiae genome. The synthetic genome is designed to increase genome stability and genetic flexibility while maintaining cell fitness near that of the wild type. A major challenge for a genome synthesis lies in identifying and eliminating fitness-reducing sequence variants referred to as “bugs.”
RATIONALE
Debugging is imperative for successfully building a fit strain encoding a synthetic genome. However, it is time-consuming and laborious to replace wild-type genes and measure strain fitness systematically. The Sc2.0 PCRTag system, which specifies recoded sequences within open reading frames (ORFs), is designed to distinguish synthetic from wild-type DNA in a simple polymerase chain reaction (PCR) assay. This system provides an opportunity to efficiently map bugs to the related genes by using a pooling strategy and subsequently correct them. Further, as we identify bugs in designer sequences, we will identify gaps in our knowledge and gain a deeper understanding of genome biology, allowing refinement of future design strategies.
RESULTS
We chemically synthesized yeast chromosome X, synX, designed to be 707,459 base pairs. A high-throughput mapping strategy called pooled PCRTag mapping (PoPM) was developed to identify unexpected bugs during chromosome assembly. With this method, the genotypes of pools of colonies with normal or defective fitness are assessed by PCRTag analysis. The PoPM method exploits the patchwork structure of synthetic and wild-type sequences observed in the majority of putative synthetic DNA integrants or meiotic progeny derived from synthetic/wild-type strain backcross. PCRTag analysis with both synthetic and wild-type specific primers, carried out with genomic DNA extracted from the two pools of clones (normal fitness versus a specific growth defect), can be used to identify regions of synthetic DNA missing from the normal fitness pool and, analogously, sections of wild-type DNA absent from the specific growth-defect pool. In this way, the defect can be efficiently mapped to a very small overlapping region, and subsequent systematic analysis of designed changes in that region can be used to identify the bug. Several bugs were identified and corrected, including a growth defect mapping to a specific synonymously recoded PCRTag sequence in the essential FIP1 ORF and the effect of introducing a loxPsym site that unexpectedly altered the the promoter function of a nearby gene, ATP2. In addition, meiotic crossover was employed to repair the massive duplications and rearrangements in the synthetic chromosome. The debugged synX strain exhibited high fitness under a variety of conditions tested and in competitive growth with the wild-type strain.
CONCLUSION
Synthetic yeast chromosome X was chemically synthesized from scratch, a rigorous, incremental step toward complete synthesis of the whole yeast genome. Thousands of designer modifications in synX revealed extensive flexibility of the yeast genome. We developed an efficient mapping method, PoPM, to identify bugs during genome synthesis, generalizable to any watermarked synthetic chromosome, and several details of yeast biology were uncovered by debugging. Considering the numerous gene-associated PCRTags available in the synthetic chromosomes, PoPM may represent a powerful tool to map interesting phenotypes of mutated synthetic strains or even mutated wild-type strains to the relevant genes. It may also be useful to study yeast genetic interactions when an unexpected phenotype is generated by alterations in two or more genes, substantially expanding understanding of yeast genomic and cellular functions. The PoPM method is also likely to be useful for mapping phenotype(s) resulting from the genome SCRaMbLE system.
The Saccharomyces cerevisiae 2.0 project (Sc2.0) aims to modify the yeast genome with a series of densely spaced designer changes. Both a synthetic yeast chromosome arm (synIXR) and the entirely synthetic chromosome (synIII) function with high fitness in yeast. For designer genome synthesis projects, precise engineering of the physical sequence to match the specified design is important for the systematic evaluation of underlying design principles. Yeast can maintain nuclear chromosomes as rings, occurring by chance at repeated sequences, although the cyclized format is unfavorable in meiosis given the possibility of dicentric chromosome formation from meiotic recombination. Here, we describe the de novo synthesis of synthetic yeast chromosome V (synV) in the “Build-A-Genome China” course, perfectly matching the designer sequence and bearing loxPsym sites, distinguishable watermarks, and all the other features of the synthetic genome. We generated a ring synV derivative with user-specified cyclization coordinates and characterized its performance in mitosis and meiosis.
RATIONALE
Systematic evaluation of underlying Sc2.0 design principles requires that the final assembled synthetic genome perfectly match the designed sequence. Given the size of yeast chromosomes, synthetic chromosome construction is performed iteratively, and new mutations and unpredictable events may occur during synthesis; even a very small number of unintentional nucleotide changes across the genome could have substantial effects on phenotype. Therefore, precisely matching the physical sequence to the designed sequence is crucial for verification of the design principles in genome synthesis. Ring chromosomes can extend those design principles to provide a model for genomic rearrangement, ring chromosome evolution, and human ring chromosome disorders.
RESULTS
We chemically synthesized, assembled, and incorporated designer chromosome synV (536,024 base pairs) of S. cerevisiae according to Sc2.0 principles, based on the complete nucleotide sequence of native yeast chromosome V (576,874 base pairs). This work was performed as part of the “Build-A-Genome China” course in Tianjin University.
We corrected all mutations found—including duplications, substitutions, and indels—in the initial synV strain by using integrative cotransformation of the precise desired changes and by means of a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)–based method. Altogether, 3331 corrected base pairs were required to match to the designed sequence. We generated a strain that exactly matches all designer sequence changes that displays high fitness under a variety of culture conditions. All corrections were verified with whole-genome sequencing; RNA sequencing revealed only minor changes in gene expression—most notably, decreases in expression of genes relocated near synthetic telomeres as a result of design.
We constructed a functional circular synV (ring_synV) derivative in yeast by precisely joining both chromosome ends (telomeres) at specified coordinates. The ring chromosome showed restoration of subtelomeric gene expression levels. The ring_synV strain exhibited fitness comparable with that of the linear synV strain, revealed no change in sporulation frequency, but notably reduced spore viability. In meiosis, heterozygous or homozygous diploid ring_wtV and ring_synV chromosomes behaved similarly, exhibiting substantially higher frequency of the formation of zero-spore tetrads, a type that was not seen in the rod chromosome diploids. Rod synV chromosomes went through meiosis with high spore viability, despite no effort having been made to preserve meiotic competency in the design of synV.
CONCLUSION
The perfect designer-matched synthetic chromosome V provides strategies to edit sequence variants and correct unpredictable events, such as off-target integration of extra copies of synthetic DNA elsewhere in the genome. We also constructed a ring synthetic chromosome derivative and evaluated its fitness and stability in yeast. Both synV and synVI can be circularized and can power yeast cell growth without affecting fitness when gene content is maintained. These fitness and stability phenotypes of the ring synthetic chromosome in yeast provide a model system with which to probe the mechanism of human ring chromosome disorders.
Synthesis, cyclization, and characterization of synV.
(A) Synthetic chromosome V (synV, 536,024 base pairs) was designed in silico from native chromosome V (wtV, 576,874 base pairs), with extensive genotype modification designed to be phenotypically neutral. (B) CRISPR/Cas9 strategy for multiplex repair. (C) Colonies of wtV, synV, and ring_synV strains.
Biotechnology for Biofuels 2017 Volume 10( Issue 1) pp:189
Publication Date(Web):18 July 2017
DOI:10.1186/s13068-017-0872-3
Integration of heterogeneous genes is widely applied in synthetic biology and metabolic engineering. However, knowledge about the effect of integrative position on gene expression remains limited.We established a genome-wide landscape of position effect on gene expression in Saccharomyces cerevisiae. The expression cassette of red fluorescence protein (RFP) gene was constructed and inserted at 1044 loci, which were scattered uniformly in the yeast genome. Due to the different integrative loci on the genome, the maximum relative intensity of RFP is more than 13-fold over the minimum. Plots of the number of strains to RFP relative intensity showed normal distribution, indicating significant position effect on gene expression in yeast. Furthermore, changing the promoters or reporter genes, as well as carbon sources, revealed little consequences on reporter gene expression, indicating chromosomal location is the major determinant of reporter gene expression.We have examined the position effects to integration genes expression in large number loci around whole genome in S. cerevisiae. The results could guide the design of integration loci for exogenous genes and pathways to maximize their expression in metabolic engineering.
Microbial production of monoterpenes is often limited by their cytotoxicity and in vivo conversion. Therefore, alleviating cytotoxicity and reducing conversion by chassis engineering are highly desirable. On the other hand, engineering key enzymes is also critical for improving monoterpenes production through facilitating the biosynthesis process. Here we critically review recent advances in cytotoxicity alleviation, reducing in vivo conversion, selecting geranyl diphosphate synthase and engineering monoterpene synthases. These achievements would lead to the development of superior chassis with improved tolerance to cytotoxicity and rationally tailored metabolites profiles to improve titer, yield and productivity for the production of monoterpenes by microbial cells.
Co-reporter:Jing-Sheng Cheng;Yan Zhao;Bin Qiao;Hua Lu
Applied Biochemistry and Biotechnology 2016 Volume 179( Issue 5) pp:788-804
Publication Date(Web):2016 July
DOI:10.1007/s12010-016-2031-x
The intracellular proteomes of the Penicillium chrysogenum throughout pilot and industrial processes were investigated by using 2-DE combined with MALDI-TOF-TOF MS, respectively. We detected a total of 223 spots corresponding to 154 proteins and 231 spots corresponding to 157 proteins throughout pilot and industrial processes, respectively. The levels of glyceraldehyde-3-phosphate dehydrogenase increased (5.1- and 2.5-fold) under the pilot process, while its levels were no significant changes under the industrial process at 140 and 170 h when compared with that at 2 h. The levels of isocitrate lyase and fumarate hydratase were increased significantly under the industrial process, while their levels had no obvious changes after 20 h of fermentation throughout the pilot process. These results indicate that there were remarkable differences in carbohydrate metabolism (including glycolysis, gluconeogenesis, pentose phosphate pathway, and tricarboxylic acid cycle) of P. chrysogenum during the pilot and industrial fermentations, which likely result in alterations of the primary metabolism and penicillin biosynthesis. Moreover, the differences in the levels of proteins involved in amino acid metabolisms (including valine, cysteine, and α-aminoadipic acid biosynthesis) indicated that the pilot and industrial processes influenced the supplies of penicillin precursors. Compared with that at 2 h, the maximum levels of superoxide (6.9-fold, at 32 h) and catalase (9-fold, at 80 h) during the industrial process and the maximum levels of superoxide (1.2-fold, at 20 h) and catalase (7.7-fold at 128 h) during the pilot process revealed the significant difference in cell redox homeostasis and stress responses during scale-up fermentation. Particularly, 10 spots corresponding to isopenicillin N synthetase and 4 spots corresponding to isopenicillin N (IPN) acyltransferase in pilot and industrial processes were identified, respectively. The levels of IPN acyltransferase (spots 197 and 198) and CoA ligase at 80 h during the industrial process were around 2-fold of that during the pilot process, indicating that the industrial process with a higher penicillin production per cell might provide available environments to induce over-expression of IPN acyltransferase and accelerate penicillin formation. These results provide new insights into the globally potential responses of P. chrysogenum to variations of environments in different fermentation scales so as to consequently regulate the penicillin production.
Co-reporter:Duo Liu, Hong Liu, Bing-Zhi Li, Hao Qi, Bin Jia, Xiao Zhou, Hao-Xing Du, Wei Zhang, and Ying-Jin Yuan
ACS Synthetic Biology 2016 Volume 5(Issue 12) pp:
Publication Date(Web):July 7, 2016
DOI:10.1021/acssynbio.6b00123
Multigene pathway engineering usually needs amounts of part libraries on transcriptional and translational regulation as well as mutant enzymes to achieve the optimal part combinations of the target pathways. We report a new strategy for multigene pathway engineering with regulatory linkers (M-PERL) focusing on tuning the transcriptional start site (TSS) of yeast promoters. The regulatory linkers are composed of two homologous ends of two adjacent gene parts for assembly and a central regulatory region between them. We investigated the effect of the homologous end’s length on multigene assembly, analyzed the influences of truncated, replaced, and elongated TSS and the adjacent region on promoters, and introduced 5 to 40 random bases of N (A/T/C/G) in the central regulatory region of the linkers which effectively varied the promoter’s strengths. The distinct libraries of five regulatory linkers were used simultaneously to assemble and tune all five genes in the violacein synthesis pathway. The gene expressions affected the product profiles significantly, and the recombinants for enhanced single component synthesis and varied composition synthesis were obtained. This study offers an efficient tool to assemble and regulate multigene pathways.Keywords: DNA assembly; multigene pathway engineering; regulatory linker; regulatory region; yeast promoter;
Co-reporter:Yunzi Luo, Bing-Zhi Li, Duo Liu, Lu Zhang, Yan Chen, Bin Jia, Bo-Xuan Zeng, Huimin Zhao and Ying-Jin Yuan
Chemical Society Reviews 2015 vol. 44(Issue 15) pp:5265-5290
Publication Date(Web):11 May 2015
DOI:10.1039/C5CS00025D
Natural products produced by microorganisms and plants are a major resource of antibacterial and anticancer drugs as well as industrially useful compounds. However, the native producers often suffer from low productivity and titers. Here we summarize the recent applications of heterologous biosynthesis for the production of several important classes of natural products such as terpenoids, flavonoids, alkaloids, and polyketides. In addition, we will discuss the new tools and strategies at multi-scale levels including gene, pathway, genome and community levels for highly efficient heterologous biosynthesis of natural products.
Co-reporter:Qiuhui Lin, Bin Jia, Leslie A. Mitchell, Jingchuan Luo, Kun Yang, Karen I. Zeller, Wenqian Zhang, Zhuwei Xu, Giovanni Stracquadanio, Joel S. Bader, Jef D. Boeke, and Ying-Jin Yuan
We describe rapid assembly of DNA overlapping multifragments (RADOM), an improved assembly method via homologous recombination in Saccharomyces cerevisiae, which combines assembly in yeasto with blue/white screening in Escherichia coli. We show that RADOM can successfully assemble ∼3 and ∼10 kb DNA fragments that are highly similar to the yeast genome rapidly and accurately. This method was tested in the Build-A-Genome course by undergraduate students, where 125 ∼3 kb “minichunks” from the synthetic yeast genome project Sc2.0 were assembled. Here, 122 out of 125 minichunks achieved insertions with correct sizes, and 102 minichunks were sequenced verified. As this method reduces the time-consuming and labor-intensive efforts of yeast assembly by improving the screening efficiency for correct assemblies, it may find routine applications in the construction of DNA fragments, especially in hierarchical assembly projects.Keywords: Build-A-Genome; in vivo assembly; Sc2.0; synthetic biology; synthetic yeast genome
Co-reporter:Hao Song, Ming-Zhu Ding, Xiao-Qiang Jia, Qian Ma and Ying-Jin Yuan
Chemical Society Reviews 2014 vol. 43(Issue 20) pp:6954-6981
Publication Date(Web):14 Jul 2014
DOI:10.1039/C4CS00114A
Synthetic biology is an emerging research field that focuses on using rational engineering strategies to program biological systems, conferring on them new functions and behaviours. By developing genetic parts and devices based on transcriptional, translational, post-translational modules, many genetic circuits and metabolic pathways had been programmed in single cells. Extending engineering capabilities from single-cell behaviours to multicellular microbial consortia represents a new frontier of synthetic biology. Herein, we first reviewed binary interaction modes of microorganisms in microbial consortia and their underlying molecular mechanisms, which lay the foundation of programming cell–cell interactions in synthetic microbial consortia. Systems biology studies on cellular systems enable systematic understanding of diverse physiological processes of cells and their interactions, which in turn offer insights into the optimal design of synthetic consortia. Based on such fundamental understanding, a comprehensive array of synthetic microbial consortia constructed in the last decade were reviewed, including isogenic microbial communities programmed by quorum sensing-based cell–cell communications, sender–receiver microbial communities with one-way communications, and microbial ecosystems wired by two-way (bi-directional) communications. Furthermore, many applications including using synthetic microbial consortia for distributed bio-computations, chemicals and bioenergy production, medicine and human health, and environments were reviewed. Synergistic development of systems and synthetic biology will provide both a thorough understanding of naturally occurring microbial consortia and rational engineering of these complicated consortia for novel applications.
Fermentation of xylose in lignocellulosic hydrolysates by Saccharomyces cerevisiae has been achieved through heterologous expression of the xylose reductase (XR)–xylitol dehydrogenase (XDH) pathway. However, the fermentation efficiency is far from the requirement for industrial application due to high yield of the byproduct xylitol, low ethanol yield, and low xylose consumption rate. Through evolutionary engineering, an improved xylose-utilizing strain SyBE005 was obtained with 78.3 % lower xylitol production and a 2.6-fold higher specific ethanol production rate than those of the parent strain SyBE004, which expressed an engineered NADP+-preferring XDH. The transcriptional differences between SyBE005 and SyBE004 were investigated by quantitative RT-PCR. Genes including XYL1, XYL2, and XKS1 in the initial xylose metabolic pathway showed the highest up-regulation in SyBE005. The increased expression of XYL1 and XYL2 correlated with enhanced enzymatic activities of XR and XDH. In addition, the expression level of ZWF1 in the oxidative pentose phosphate pathway increased significantly in SyBE005, indicating an elevated demand for NADPH from XR. Genes involved in the TCA cycle (LAT1, CIT1, CIT2, KGD1, KGD, SDH2) and gluconeogenesis (ICL1, PYC1) were also up-regulated in SyBE005. Genomic analysis revealed that point mutations in transcriptional regulators CYC8 and PHD1 might be responsible for the altered expression. In addition, a mutation (Y89S) in ZWF1 was identified which might improve NADPH production in SyBE005. Our results suggest that increasing the expression of XYL1, XYL2, XKS1, and enhancing NADPH supply are promising strategies to improve xylose fermentation in recombinant S. cerevisiae.
The addition of precursors was one strategy to improve antibiotic production. The exogenous proline and glutamate, as precursors of streptolydigin, could significantly improve the streptolydigin production, but their underlying molecular mechanisms remain unknown. Herein, metabolomic analysis was carried out to explore the metabolic responses of Streptomyces lydicus to the additions of proline and glutamine. The significant differences in the quantified 53 metabolites after adding the exogenous proline and glutamate were enunciated by gas chromatography coupled to time-of-flight mass spectrometry. Among them, the levels of some fatty acids (e.g., dodecanoic acid, octadecanoic acid, hexadecanoic acid) were significantly decreased after adding glutamate and proline, indicating that the inhibition of fatty acid synthesis might be benefit for the accumulation of streptolydigin. Particularly, the dramatic changes of the identified metabolites, which are involved in glycolysis, the tricarboxylic acid cycle, and the amino acid and fatty acid metabolism, revealed that the additions of glutamate and proline possibly caused the metabolic cross-talk in S. lydicus. Additionally, the level of intracellular glutamate dramatically enhanced at 12 h after adding proline, showing that exogenous proline may be firstly convert into glutamate and consequently result in crease of the streptolydigin production. The high levels of streptolydigin at 12 and 24 h after adding glutamate unveiled that part glutamate were rapidly used to synthesize the streptolydigin. Furthermore, there is the significant difference in metabolomic characteristics of S. lydicus after adding glutamate and proline, uncovering that multiple regulatory pathways are involved in responses to the additions of exogenous glutamate and proline. Taken together, exogenous glutamate and proline not only directly provided the precursors of streptolydigin biosynthesis, but also might alter the metabolic homeostasis of S. lydicus E9 during improving the production of streptolydigin.
Cephalosporin C (CPC) is the precursor of a class of antibiotics that were more effective than traditional penicillins. CPC production is performed mainly through fermentation by Acremonium chrysogenum, whose secondary metabolism was sensitive to the environmental changes. In the present work, secondary metabolites were measured by ion-pair reversed-phase liquid chromatography tandemed with hybrid quadrupole time-of-flight mass spectrometry, and the disparity of them from two scales of CPC fermentations (pilot and industrial) and also two different post-treatment processes (oxalic acid and formaldehyde added and control) were investigated. When fermentation size was enlarged from pilot scale (50 l) to industrial scale (156,000 l), the remarkable disparities of concentrations and changing trends of the secondary metabolites in A. chrysogenum were observed, which indicated that the productivity of CPC biosynthesis was higher in the large scale of fermentation. Three environmental factors were measured, and the potential reasons that might cause the differences were analyzed. In the post-treatment process after industrial fermentation, the changes of these secondary metabolites in the tank where oxalic acid and formaldehyde were added were much less than the control tank where none was added. This indicated that the quality of the final product was more stable after the oxalic acid and formaldehyde were added in the post-treatment process. These findings provided new insight into industrial CPC production.
Tetramethylpyrazine (TMP) and butylidenephthalide (BP) are two bioactive components isolated from Ligusticum chuanxiong Hort and Angelica sinensis, respectively. These two traditional Chinese medicines have been widely used in clinical treatments for vascular disease. The mechanism by which TMP and BP protect endothelial cells (ECs) against oxidative stress remains unknown, as does their effects on the steady state of the lipidome of ECs. Here, we demonstrate that both compounds protect EA.hy926 cells against H2O2 induced injury in a dose-dependent manner. We then apply an integrated analysis of the lipidome and signal transduction pathways to explore the underlying mechanism of their protective effects. We found that TMP elevates the content of several phosphatidylcholine (PC) species, reduces the release of arachidonic acid (AA) and inhibits the phosphorylation of cytosolic phospholipase A2 (cPLA2). Compared to eicosatetraynoic acid (ETYA), a cPLA2 inhibitor, TMP preferentially increases the content of arachidonoyl PCs. We also show that BP mainly elevates the pool of phosphatidylinositol (PI) species and inhibits the phosphorylation of both phospholipase Cγ (PLCγ) and extracellular signal-regulated kinase 1/2 (ERK1/2). In contrast, specific inhibition of ERK1/2 by PD98059 decreases the cell viability and increases pool of phosphatidylserine (PS). Taken together, these results demonstrate that TMP protects oxidatively stressed ECs through inhibition of cPLA2 and preferential increase of arachidonoyl PC levels. Conversely, the effects of BP are tied to inhibition of PLCγ and an increase in PI levels. The current work suggests that the interaction of the lipidome and phospholipases can serve as a promising therapeutic target in oxidatively stressed ECs.
Many microbial consortia are established upon metabolic interactions. Elucidating such interactions is a priority in understanding the population dynamics of these microbial consortia. In this study, we investigated the interaction dynamics of the vitamin C biosynthesis consortium comprising of Ketogulonicigenium vulgare and Bacillus megaterium. We systematically quantified the dynamic evolution of the ecosystem’s population and metabolism in response to a wide range of seeding concentrations and compositions of the two microorganisms. The consortium population dynamics was determined by quantitative PCR. The metabolomic profile of the community was systematically investigated by gas chromatography coupled with time-of-flight mass spectrometry. Our results showed that B. megaterium was responsible for initiating the reproduction of K. vulgare, meanwhile, K. vulgare could promote the growth of B. megaterium. Principal component analysis of the metabolomic profiling elucidated variations of intermediates in central carbon metabolism, nucleotide and amino acids metabolism in this microbial consortium. These findings provided new insights into the characterization of the community dynamics and the optimization of co-culture fermentation for vitamin C biosynthesis.
Frontiers of Chemical Science and Engineering 2012 Volume 6( Issue 4) pp:461-469
Publication Date(Web):2012 December
DOI:10.1007/s11705-012-1223-3
Variations in the composition and level of phospholipids (PLs) in yeast cells during industrial ethanol fermentation processes were analyzed. A comparative lipidomic method was used to investigate the changes in total cellular PLs during continuous and fed-batch/batch processes. The phospholipid metabolism in yeast changed during both processes, mainly due to the presence of longchain poly unsaturated fatty acids (PUFA) that contained phosphatidyglycerol (PG), phosphatidylethanolamine (PE) and phosphatidylserine (PS). The complexity of the media affected the growth of the yeast and the membrane composition. Yeast incorporated lots of exogenous saturated and PUFAs from the feedstock during the fermentations. During the continuous fermentation, there was an increase in PLs with shorter chains as the fermentation progressed and early in process there were more longchains. During the fed-batch/batch process, the PG species increased as the fermentation progressed. This is probably due to an inositol deficiency in the earlier part of the fermentation.
Biotechnology and Bioprocess Engineering 2012 Volume 17( Issue 5) pp:997-1007
Publication Date(Web):2012 October
DOI:10.1007/s12257-012-0173-4
Streptomyces lydicus has been reported to produce antibiotic streptolydigin. Pitching ratios play crucial roles in primary and secondary metabolism of Streptomyces bacteria. The higher pitching ratio (30%, v/v) significantly enhanced the levels of streptolydigin products in S. lydicus. Proteome analysis revealed that betaglucosidase and UTP-glucose-1-phosphate uridylyltransferase were up-regulated to accelerate the starch hydrolyzation at the high pitching ratios. Enhancement in the levels of UDPN-acetylmuramoylalanyl-D-glutamate-2, 6-diaminopimelate ligase and glycine cleavage system aminomethyltransferase were involved in the conversion of amino acids into secondary metabolites. Additionally, the expression levels of PfkA2, PfkA3, Zwf2, SucD, GalE1, GatB, TktA1 and ThcA, associated with glycolysis, pentose phosphate pathway, TCA cycle and amino acid metabolism, were dramatically elevated at high pitching ratios, which play important roles in the enhanced streptolydigin production in S. lydicus E9. Interestingly, the levels of proteins (glutamine synthetase I, glutamate synthase subunit beta and glutamine synthetase) were down-regulated with the increases of pitching ratios and fermentation progress, revealing that pitching ratio altered the glutamine synthetase levels and consequently regulated the streptolydigin production of S. lydicus E9. The up-regulation of proteins (eg, aldehyde dehydrogenase and alkyl hydroperoxide reductase) was involved in the redox-based regulation network triggered by an imbalance of the intracellular cell redox homeostasis and by crosstalk with secondary metabolism at the higher pitching ratio. These results settle new insights into physiological facts of S. lydicus E9 in responses to pitching ratios and will eventually improve the antibiotic production schemes in industry.
Co-reporter:Rui-Juan Xu;Bin Qiao;Bing-Zhi Li;Hua Lu
Biotechnology and Bioprocess Engineering 2012 Volume 17( Issue 2) pp:259-269
Publication Date(Web):2012 April
DOI:10.1007/s12257-011-0494-8
Cephalosporium acremonium has been widely applied in industrial cephalosporin C fermentation. However, little is known about the molecular basis of fermentation behavior of this strain. In this study, comparative lipidomic analysis using LC/ESI/MSn technology was employed to investigate responses of Cephalosporium acremonium to multiple environment variations in realistic industrial cephalosporin C fermentation process and provide molecular basis for the discrepancies between industrial and pilot fermentations. Totally 77 phospholipids species were detected and 65 species were further quantified. Score plot revealed that phospholipids metabolism differed in industrial and pilot process. Loading pilot indicated that the main variables responsible for the discrimination of industrial and pilot process were phosphatidylinositols (PIs), phosphatidylserines (PSs) and phosphatic acids (PAs). Higher PIs content in industrial process indicated that cells were more vigorous in industrial process than those in pilot process. Larger increases of PSs, PAs and ratio of oleic acid to linoleic acid coincided well with the earlier and more thorough cellular morphological differentiation in industrial process. The synergetic reaction between cellular behavior and cells living environment led to titer discrepancies between industrial and pilot process. These findings provided lipidomic insights into industrial cephalosporin C production.
The metabolic responses of parental and inhibitors-tolerant yeasts in presence of the combination of three inhibitors (furfural, phenol and acetic acid) during ethanol fermentation were investigated by comparative metabolic profiling. Samples of parental and tolerant yeasts with/without three inhibitors in fermentation medium represented significantly different metabolic states. Further investigation on the specific responses of two strains revealed that the levels of most amino acids, inositol, and phenethylamine were dramatically increased in presence of inhibitors in parental yeast, while they kept relatively stable in tolerant yeast. It suggested that the protein degradation was increased and oxygen stress was induced by combined inhibitors in parental yeast. In addition, carbon metabolism (glycolysis and TCA) and pyrimidine ribonucleotides pathway (uracil and cytosine) were reduced in both strains in presence of combined inhibitors, which was considered as the general stress response. Higher levels of pyridimines in tolerant yeast suggested that they were responsible for counteracting the stress of combined inhibitors. These findings provided new insights into underlying mechanisms of yeast in resistance to the synergistic effects of inhibitors in lignocellulose hydrolysates.
Phospholipids in human endothelial cells (ECs), cell line EA.hy926, were profiled by a novel lipidomics approach, combining liquid chromatography (LC)–ion trap mass spectrometry (MS) and LC–tandem quadrupole MS. More than 200 species of phospholipids were quantified. Twenty-eight were identified as the most discriminant species in response to different levels of oxidative stress induced by hydrogen peroxide (H2O2). H2O2 treatment induced phosphorylation of cytosolic phospholipase A2 (cPLA2) via the activation of extracellular-regulated kinase 1/2 (ERK1/2), increasing the production of lysophosphatidylethanolamine (LPE) and lysophosphatidylcholine (LPC). The release of arachidonic acid (AA, 20:4) increased from no H2O2 exposure to 1 h exposure, decreased from 1 h to 2 h, and increased again from 2 h to 4 h exposure time. The particular increase seen of phosphatidylcholine (PC) species that include AA chains from 1 h to 2 h indicates that the released AA is reincorporating into PC molecules to reduce the extension of the AA cascade. The change in free AA levels seen suggests possible defense mechanisms to oxidative injury in ECs. We further verified nine species as potential biomarkers by adding inhibitor and demonstrated direct correlation to the activity of the cPLA2–AA pathway. The oxidative injury to cell line EA.hy926 provided a novel application for a combined lipidomics and signal transduction approach. This combined approach has enabled future investigations for possible therapeutic interventions in phospholipids and cPLA2 activity for defense against oxidative cellular stress.
Journal of Agricultural and Food Chemistry 2011 Volume 59(Issue 18) pp:9845-9853
Publication Date(Web):July 27, 2011
DOI:10.1021/jf201792u
Corn steep liquor (CSL) is one of the main raw materials in 2-keto-l-gulonic acid (2-KLG) fermentation by Ketogulonicigenium vulgare and Bacillus megaterium. Due to its natural origin and variations in the manufacturing process, unpredicted and uncontrolled variability of CSL has a great influence on 2-KLG production; however, conventional quality specifications are not enough to ensure stability of fermentation behaviors. A process analytical technology (PAT) could be considered to explore the relationship between CSL quality and 2-KLG production comprehensively. The compositions of CSL from six manufacturers were profiled by gas chromatography with time-of-flight mass spectrometry (GC-TOFMS) and inductively coupled plasma-atomic emission spectroscopy (ICP-AES), combined with orthogonal partial least-squares discriminant analysis (OPLS-DA). Seventeen components were identified as the most discriminant marker compounds related to 2-KLG production. Results revealed that they were responsible for providing nutrients and protecting osmotic pressure. Furthermore, nine amino acids were verified as potential group markers by addition to the medium and demonstration of the correlation to 2-KLG production. The comprehensive approach provided an important platform to explore CSL marker compounds for quality evaluation in 2-KLG fermentation.
Frontiers of Chemical Science and Engineering 2011 Volume 5( Issue 3) pp:318-324
Publication Date(Web):2011 September
DOI:10.1007/s11705-010-1026-3
Due to its merits of drought tolerance and high yield, sweet potatoes are widely considered as a potential alterative feedstock for bioethanol production. Very high gravity (VHG) technology is an effective strategy for improving the efficiency of ethanol fermentation from starch materials. However, this technology has rarely been applied to sweet potatoes because of the high viscosity of their liquid mash. To overcome this problem, cellulase was added to reduce the high viscosity, and the optimal dosage and treatment time were 8 U/g (sweet potato powder) and 1 h, respectively. After pretreatment by cellulase, the viscosity of the VHG sweet potato mash (containing 284.2 g/L of carbohydrates) was reduced by 81%. After liquefaction and simultaneous saccharification and fermentation (SSF), the final ethanol concentration reached 15.5% (v/v), and the total sugar conversion and ethanol yields were 96.5% and 87.8%, respectively.
Saccharomyces cerevisiae is widely applied in large-scale industrial bioethanol fermentation; however, little is known about the molecular responses of industrial yeast during large-scale fermentation processes. We investigated the global transcriptional responses of an industrial strain of S. cerevisiae during industrial continuous and fed-batch fermentation by oligonucleotide-based microarrays. About 28 and 62% of all genes detected showed differential gene expression during continuous and fed-batch fermentation, respectively. The overrepresented functional categories of differentially expressed genes in continuous fermentation overlapped with those in fed-batch fermentation. Downregulation of glycosylation as well as upregulation of the unfolded protein stress response was observed in both fermentation processes, suggesting dramatic changes of environment in endoplasmic reticulum during industrial fermentation. Genes related to ergosterol synthesis and genes involved in glycogen and trehalose metabolism were downregulated in both fermentation processes. Additionally, changes in the transcription of genes involved in carbohydrate metabolism coincided with the responses to glucose limitation during the early main fermentation stage in both processes. We also found that during the late main fermentation stage, yeast cells exhibited similar but stronger transcriptional changes during the fed-batch process than during the continuous process. Furthermore, repression of glycosylation has been suggested to be a secondary stress in the model proposed to explain the transcriptional responses of yeast during industrial fermentation. Together, these findings provide insights into yeast performance during industrial fermentation processes for bioethanol production.
The adaptive evolution of Saccharomyces cerevisiae to repeated vacuum fermentations was investigated by metabolomic analysis using gas chromatography coupled to time-of-flight mass spectrometry. The first round (VFI, 30 cycles) and second round (VFII, 10 cycles) of repeated fermentations could be clearly distinguished by principal components analysis on intracellular metabolites, indicating that significant difference of metabolic states occurred between them. Further investigation revealed that higher levels of glycerol, trehalose, myo-inositol and glutamate might be involved in response to vacuum stress during initial cycles, while the decreases in their levels indicated that yeast cells adapted to vacuum condition as the fermentation progressed. Furthermore, lower levels of glycerol, myo-inositol, trehalose and glutamate during VFII indicated that the adapted yeast represented better vacuum tolerance. Additionally, glycolysis and TCA cycle intermediates were enhanced whereas glycerol biosynthesis was depressed by vacuum. The decreases of most amino acids might be related to increases in intermediates of glycolysis and TCA cycle as VFI progressed. These findings provided new insights into underlying mechanisms in adaptive evolution of yeast under vacuum condition.
Chemistry and Physics of Lipids 2009 Volume 159(Issue 1) pp:13-20
Publication Date(Web):May 2009
DOI:10.1016/j.chemphyslip.2009.02.004
Degradation of membrane phospholipids is associated with apoptotic responses, but the signaling development of this degradation is not well understood. Cerium (Ce4+), an important rare earth element, induces cellular apoptosis and taxol biosynthesis in Taxus cuspidata suspension cultures. Here, using mass spectrometry and biochemical technique, we demonstrated that the phospholipase D (PLD) was rapidly activated by Ce4+ and hydrolyzed structural phospholipids to generate lipid signal molecule, phosphatidic acid (PA). 1-Butanol, an antagonist of PLD-dependent PA production, blocked the biphasic burst of superoxide anions (O2−) and thus mitigated cellular apoptosis. The time-course analysis of PA accumulation and ERK-like mitogen-activated protein kinase (MAPK) regulation indicated PA generation preceded MAPK activation, suggesting that the rapid accumulation of PA might be required for the initial MAPK activity. After 2 h of Ce4+ elicitation, however, PA-induced O2− burst, forming a negative regulation to MAPK activity, which in turn led to apoptotic signaling development.
Journal of Agricultural and Food Chemistry 2009 Volume 57(Issue 1) pp:99-108
Publication Date(Web):December 2, 2008
DOI:10.1021/jf802720t
To reveal differences between inhibitor-resistant Saccharomyces cerevisiae strains and their parental strain and to investigate the response of S. cerevisiae to furfural, phenol, and acetic acid, comparative lipidomics strategy was employed using an LC-ESI/MSn technique on four S. cerevisiae strains, which include an industrial strain (SC) and three tolerant strains screened by this laboratory by step adaptation—a furfural-tolerant strain (SCF), a phenol-tolerant strain (SCP), and an acetic acid-tolerant strain (SCA). Lipidome data were then analyzed using wavelet transform-principal component analysis (WT-PCA). Results revealed that phosphatidylcholines (PCs), phosphatidylinositols (PIs), and phosphatidic acids (PAs) were biomarkers for discriminating SC from SCF, SCP, and SCA, respectively. PIs were believed to be extraordinarily important in all inhibitor-tolerant processes because they were the biomarkers responsible for the discrimination of all four different strains. Further analysis of the distribution of different hydrocarbon chains revealed that both the saturation and the length of the chains helped in maintaining proper fluidity of membranes.
The intracellular metabolic profile characterization of Saccharomyces cerevisiae throughout industrial ethanol fermentation was investigated using gas chromatography coupled to time-of-flight mass spectrometry. A total of 143 and 128 intracellular metabolites in S. cerevisiae were detected and quantified in continuous and batch fermentations, respectively. The two fermentation processes were both clearly distinguished into three main phases by principal components analysis. Furthermore, the levels of some metabolites involved in central carbon metabolism varied significantly throughout both processes. Glycerol and phosphoric acid were principally responsible for discriminating seed, main and final phases of continuous fermentation, while lactic acid and glycerol contributed mostly to telling different phases of batch fermentation. In addition, the levels of some amino acids such as glycine varied significantly during both processes. These findings provide new insights into the metabolomic characteristics during industrial ethanol fermentation processes.
Applied Microbiology and Biotechnology 2009 Volume 83( Issue 5) pp:
Publication Date(Web):2009 July
DOI:10.1007/s00253-009-2037-1
The responses and adaptation mechanisms of the industrial Saccharomyces cerevisiae to vacuum fermentation were explored using proteomic approach. After qualitative and quantitative analyses, a total of 106 spots corresponding to 68 different proteins were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The differentially expressed proteins were involved in amino acid and carbohydrate metabolisms, various signal pathways (Ras/MAPK, Ras–cyclic adenosine monophosphate, and HOG pathway), and heat shock and oxidative responses. Among them, alternations in levels of 17 proteins associated with carbohydrate metabolisms, in particular, the upregulations of proteins involved in glycolysis, trehalose biosynthesis, and the pentose phosphate pathway, suggested vacuum-induced redistribution of the metabolic fluxes. The upregulation of 17 heat stress and oxidative response proteins indicated that multifactors contributed to oxidative stresses by affecting cell redox homeostasis. Taken together with upregulation in 14-3-3 proteins levels, 22 proteins were detected in multispots, respectively, indicating that vacuum might have promoted posttranslational modifications of some proteins in S. cerevisiae. Further investigation revealed that the elevations of the differentially expressed proteins were mainly derived from vacuum stress rather than the absence of oxygen. These findings provide new molecular mechanisms for understanding of adaptation and tolerance of yeast to vacuum fermentation.
Applied Microbiology and Biotechnology 2008 Volume 81( Issue 2) pp:327-338
Publication Date(Web):2008 November
DOI:10.1007/s00253-008-1733-6
A robust Saccharomyces cerevisiae strain has been widely applied in continuous and batch/fed-batch industrial fermentation. However, little is known about the molecular basis of fermentative behavior of this strain in the two realistic fermentation processes. In this paper, we presented comparative proteomic profiling of the industrial yeast in the industrial fermentation processes. The expression levels of most identified protein were closely interrelated with the different stages of fermentation processes. Our results indicate that, among the 47 identified protein spots, 17 of them belonging to 12 enzymes were involved in pentose phosphate, glycolysis, and gluconeogenesis pathways and glycerol biosynthetic process, indicating that a number of pathways will need to be inactivated to improve ethanol production. The differential expressions of eight oxidative response and heat-shock proteins were also identified, suggesting that it is necessary to keep the correct cellular redox or osmotic state in the two industrial fermentation processes. Moreover, there are significant differences in changes of protein levels between the two industrial fermentation processes, especially these proteins associated with the glycolysis and gluconeogenesis pathways. These findings provide a molecular understanding of physiological adaptation of industrial strain for optimizing the performance of industrial bioethanol fermentation.
PCA (principal components analysis) and ANN (artificial neural network) are two broadly used pattern recognition methods in metabolomics data-mining. Yet their limitations sometimes are great obstacles for researchers. In this paper the wavelet transform (WT) method was used to integrate with PCA and ANN to improve their performance in manipulating metabolomics data. A dataset was decomposed by wavelets and then reconstructed. The "hard thresholding" algorithm was used, through which the detail information was discarded, and the entire "metabolomics image" reconstructed on the significant information. It was supposed that the most relevant information was captured after this process. It was found that, thanks to its ability in denoising data, the WT method could significantly improve the performance of the non-linear essence-extracting method ANN in classifying samples; further integration of WT with PCA showed that WT could greatly enhance the ability of PCA in distinguishing one group of samples from another and also its ability in identifying potential biomarkers. The results highlighted WT as a promising resolution in bridging the gap between huge bytes of data and the instructive biological information.
Co-reporter:Guang-Rong Zhao, Zhi-Jun Xiang, Ting-Xiang Ye, Ying-Jin Yuan, Zhi-Xin Guo
Food Chemistry 2006 Volume 99(Issue 4) pp:767-774
Publication Date(Web):2006
DOI:10.1016/j.foodchem.2005.09.002
Traditional Chinese medicinal herb Salvia miltiorrhiza (SM) and Panax notoginseng (PN) have been widely used for the prevention and treatment of vascular diseases in the clinics. To better understand their mechanisms of pharmacological actions, the in vitro antioxidant activities of extract of Salvia miltiorrhiza (ESM) and extract of Panax notoginseng (EPN) were evaluated with different antioxidant testing systems. Their activities of scavenging superoxide anion radicals, DPPH radicals, hydroxyl radicals, and hydrogen peroxide, chelating Ferrous ion, and ferric ion reducing power were assessed. The results showed that the mechanisms of their antioxidant effects were distinct and diverse. ESM possessed strong reducing power and high scavenging activities against free radicals including superoxide anion, hydroxyl and DPPH radicals, but a weaker scavenging activity for hydrogen peroxide. ferrous ion chelating activity of ESM was undetectable at the tested concentrations. In contrary, EPN exhibited strong ferrous ion chelating activity and high scavenging activities against hydrogen peroxide, hydroxyl radicals, and a weak activity against superoxide anion and DPPH free radicals. EPN did not show any ferric ion reducing power. Since their antioxidant mechanisms are complementary, the combined use of ESM and EPN might be even more beneficial. These antioxidant properties of SM and PN are likely part of the reasons that they are effective in the prevention and treatment of vascular diseases.
Co-reporter:Xiaoqiang Jia, Chang Liu, Hao Song, Mingzhu Ding, Jin Du, Qian Ma, Yingjin Yuan
Synthetic and Systems Biotechnology (June 2016) Volume 1(Issue 2) pp:109-117
Publication Date(Web):1 June 2016
DOI:10.1016/j.synbio.2016.02.001
The rapid development of synthetic biology has conferred almost perfect modification on single cells, and provided methodological support for synthesizing microbial consortia, which have a much wider application potential than synthetic single cells. Co-cultivating multiple cell populations with rational strategies based on interacting relationships within natural microbial consortia provides theoretical as well as experimental support for the successful obtaining of synthetic microbial consortia, promoting it into extensive research on both industrial applications in plenty of areas and also better understanding of natural microbial consortia. According to their composition complexity, synthetic microbial consortia are summarized in three aspects in this review and are discussed in principles of design and construction, insights and methods for analysis, and applications in energy, healthcare, etc.
•Effect of different additions of soluble materials in biomass on SSCF was studied.•Soluble materials inhibit enzymatic hydrolysis but stimulate fermentation.•Soluble lignins at low concentrations stimulates xylose consumption in yeast.•YP addition enhances ethanol yield in SSCF containing high inhibitor concentrations.In this study, wash liquors isolated from ethylenediamine and dry dilute acid pretreated corn stover were used to evaluate the effect of soluble materials in pretreated biomass on simultaneous saccharification and co-fermentation (SSCF) for ethanol production, respectively. Both of the wash liquors had different impacts on enzymatic hydrolysis and fermentation. Enzymatic conversions of glucan and xylan monotonically decreased as wash liquor concentration increased. Whereas, with low wash liquor concentrations, xylose consumption rate, cell viability and ethanol yield were maximally stimulated in fermentation without nutrient supplementary. Soluble lignins were found as the key composition which promoted sugars utilization and cell viability without nutrient supplementary. The dual effects of soluble materials on enzymatic hydrolysis and fermentation resulted in the reduction of ethanol yield as soluble materials increased in SSCF.
Synthetic and Systems Biotechnology (December 2016) Volume 1(Issue 4) pp:230-235
Publication Date(Web):1 December 2016
DOI:10.1016/j.synbio.2016.08.004
The rapid development of synthetic biology enables the design, construction and optimization of synthetic microbial consortia to achieve specific functions. In China, the “973” project-“Design and Construction of Microbial Consortia” was funded by the National Basic Research Program of China in January 2014. It was proposed to address the fundamental challenges in engineering natural microbial consortia and reconstructing microbial consortia to meet industrial demands. In this review, we will introduce this “973” project, including the significance of microbial consortia, the fundamental scientific issues, the recent research progresses, and some case studies about synthetic microbial consortia in the past two and a half years.
Industrial Crops and Products (August 2017) Volume 102() pp:51-57
Publication Date(Web):1 August 2017
DOI:10.1016/j.indcrop.2017.03.026
•Key pretreatment factors on enzymatic digestion of EDA pretreated CS were discovered.•EDA pretreatment increased glucose yield to above 90% at the optimized conditions.•Temperature was found the most important factor on EDA removal in pretreated CS.•Two-stage pretreatment was designed to maximize both sugar yields and EDA removal.•Enzyme loadings for hydrolysis of EDA pretreated CS were optimized.Ethylenediamine (EDA) pretreatment is an effective pretreatment technology to improve enzymatic digestibility of corn stover for the production of fermentable sugars. In this study, key pretreatment parameters were identified and optimized to improve enzymatic digestibility of corn stover. We found that agitation and biomass stack height during pretreatment had significant impacts on enzymatic digestibility. Response surface experiment showed that optimal condition to achieve maximum total sugar enzymatic yield was 150 °C and 80 mL EDA/100 g corn stover. Under this condition, glucose yield was greater than 90% in enzymatic hydrolysis at 1% glucan loading. Optimized temperature to minimize residual EDA in pretreated corn stover was 200 °C. Two-stage pretreatment was carried out to maximize both sugar yields and EDA removal, in which glucose and xylose enzymatic yields reached 92% and 70% respectively at 1% glucan loading, and EDA residue reduced to 27 g/kg corn stover. With the optimal enzyme loadings (both enzymes of Ctec2 and Htec2 were loaded at 30 mg protein/g glucan), glucose and xylose yields at 6% glucan loading reached 81% and 58%, respectively.
Co-reporter:Yunzi Luo, Bing-Zhi Li, Duo Liu, Lu Zhang, Yan Chen, Bin Jia, Bo-Xuan Zeng, Huimin Zhao and Ying-Jin Yuan
Chemical Society Reviews 2015 - vol. 44(Issue 15) pp:NaN5290-5290
Publication Date(Web):2015/05/11
DOI:10.1039/C5CS00025D
Natural products produced by microorganisms and plants are a major resource of antibacterial and anticancer drugs as well as industrially useful compounds. However, the native producers often suffer from low productivity and titers. Here we summarize the recent applications of heterologous biosynthesis for the production of several important classes of natural products such as terpenoids, flavonoids, alkaloids, and polyketides. In addition, we will discuss the new tools and strategies at multi-scale levels including gene, pathway, genome and community levels for highly efficient heterologous biosynthesis of natural products.
![Diphosphoric acid,P-[(2E,6E,10E)-3,7,11,15-tetramethyl-2,6,10,14-hexadecatetraen-1-yl] ester](http://img.cochemist.com/ccimg/6700/6699-20-3.png)
![Diphosphoric acid,P-[(2E,6E,10E)-3,7,11,15-tetramethyl-2,6,10,14-hexadecatetraen-1-yl] ester](http://img.cochemist.com/ccimg/6700/6699-20-3_b.png)
