•A completely chromosomally engineered efficient butanol producing E. coli strain was developed.•This E. coli strain could produce 20 g/L butanol, with a mass yield of 34%, without adding any antibiotics or inducers.•This is the first vector-free recombinant E. coli producing the highest titer of butanol without in situ removal of butanol.Biotechnological production of butanol in heterologous hosts has recently attracted many interests. Of the heterologous hosts investigated to date, engineered Escherichia coli has shown a superior butanol yield than the natural butanol-producing clostridial strains. However, all reported butanol-producing E. coli strains contain vectors and inducible promoters, which means antibiotics and inducers are required in the fermentation. The aim of this study was to develop a completely chromosomally engineered E. coli strain capable of producing butanol efficiently in the absence of vectors, antibiotics, and inducers. The challenges are the expression strength of chromosomally engineered genes under constitutive promoters is much weaker than the vector engineered genes under inducible promoters. To address these challenges, the butanol pathway was engineered into the chromosome in the first place, then the host and the butanol pathway was iteratively engineered through rational and non-rational strategies to develop an efficient butanol producer where the heterologous butanol pathway fits the host well. Finally, a systematically chromosomally engineered E. coli strain EB243, in which 33 native genes were deleted and 5 heterologous genes were introduced, was developed. Strain EB243 could produce 20 g/L butanol with a yield of 34% (w/w, 83% of theoretical yield) in batch fermentation without any antibiotics and inducers, thus showed great potential for industrial application. This work also demonstrated a procedure on how to integrate the existing knowledge to engineer a strain with industrial application potential.

Recycling of carbon dioxide (CO2) into fuels and chemicals is a potential approach to reduce CO2 emission and fossil-fuel consumption. Autotrophic microbes can utilize energy from light, hydrogen, or sulfur to assimilate atmospheric CO2 into organic compounds at ambient temperature and pressure. This provides a feasible way for biological production of fuels and chemicals from CO2 under normal conditions. Recently great progress has been made in this research area, and dozens of CO2-derived fuels and chemicals have been reported to be synthesized by autotrophic microbes. This is accompanied by investigations into natural CO2-fixation pathways and the rapid development of new technologies in synthetic biology. This review first summarizes the six natural CO2-fixation pathways reported to date, followed by an overview of recent progress in the design and engineering of CO2-fixation pathways as well as energy supply patterns using the concept and tools of synthetic biology. Finally, we will discuss future prospects in biological fixation of CO2.

Photosynthetic CO2 fixation is the ultimate source of organic carbon on earth and thus is essential for crop production and carbon sequestration. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the first step of photosynthetic CO2 fixation. However, the extreme low carboxylation efficiency of Rubisco makes it the most attractive target for improving photosynthetic efficiency. Extensive studies have focused on re-engineering a more efficient enzyme, but the effort has been impeded by the limited understanding of its structure-function relationships and the lack of an efficient selection system towards its activity. To address the unsuccessful molecular engineering of Rubisco, we developed an Escherichia coli-based activity-directed selection system which links the growth of host cell solely to the Rubisco activity therein. A Synechococcus sp. PCC7002 Rubisco mutant with E49V and D82G substitutions in the small subunit was selected from a total of 15,000 mutants by one round of evolution. This mutant showed an 85% increase in specific carboxylation activity and a 45% improvement in catalytic efficiency towards CO2. The small-subunit E49V mutation was speculated to influence holoenzyme catalysis through interaction with the large-subunit Q225. This interaction is conserved among various Rubisco from higher plants and Chlamydomonas reinhardtii. Knowledge of these might provide clues for engineering Rubisco from higher plants, with the potential of increasing the crop yield.

Development of controllable hypermutable cells can greatly benefit understanding and harnessing microbial evolution. However, there have not been any similar systems developed for Clostridium, an important bacterial genus. Here we report a novel two-step strategy for developing controllable hypermutable cells of Clostridium acetobutylicum, an important and representative industrial strain. Firstly, the mutS/L operon essential for methyldirected mismatch repair (MMR) activity was inactivated from the genome of C. acetobutylicum to generate hypermutable cells with over 250-fold increased mutation rates. Secondly, a proofreading control system carrying an inducibly expressed mutS/L operon was constructed. The hypermutable cells and the proofreading control system were integrated to form a controllable hypermutable system SMBMutC, of which the mutation rates can be regulated by the concentration of anhydrotetracycline (aTc). Duplication of the miniPthl-tetR module of the proofreading control system further significantly expanded the regulatory space of the mutation rates, demonstrating hypermutable Clostridium cells with controllable mutation rates are generated. The developed C. acetobutylicum strain SMBMutC2 showed higher survival capacities than the control strain facing butanol-stress, indicating greatly increased evolvability and adaptability of the controllable hypermutable cells under environmental challenges.

Fermentative production of solvents (acetone, butanol, and ethanol) by Clostridium acetobutylicum is generally a biphasic process consisting of acidogenesis and solventogenesis. We report that the biphasic metabolism of C. acetobutylicum could be changed by oxidoreduction potential (ORP) regulation. When using air to control the ORP of the fermentation broth at −290 mV, an earlier initiation of solventogenesis was achieved. Solvent production reached 25.6 g l−1 (2.8 g acetone l−1, 16.8 g butanol l−1, 6.0 g ethanol l−1), a 35% increase compared with the ORP uncontrolled process. Metabolic flux analysis revealed that there was a general increase of the central carbon flux in the first 24 h of fermentation when ORP was controlled at −290 mV, compared with the control. Specifically, the solvent ratio (acetone:butanol:ethanol) was changed from 25:64:11 to 11:66:23 at ORP level of −290 mV, which might have resulted from the rigidity at acetyl-CoA node and the flexibility at acetoacetyl-CoA and butyryl-CoA nodes in response to ORP regulation.

The solventogenic bacterium Clostridium acetobutylicum is the most important species of Clostridium used in the fermentation industry. However, the intolerance to butanol hampers the efficient production of solvents. Butanol toxicity has been attributed to the chaotropic effect on the cell membrane, but the knowledge on the effect of butanol on membrane associated proteins is quite limited. Using 2-DE combined with MALDI-TOF MS/MS and 1-DE integrated with LC-MS/MS, 341 proteins in the membrane fractions of cell lysate were identified, thus establishing the first comprehensive membrane proteome of C. acetobutylicum. The identified proteins are mainly involved in transport, cellular membrane/wall machinery, formation of surface coat and flagella, and energy metabolism. Comparative analysis on the membrane proteomes of the wild type strain DSM 1731 and its butanol-tolerant mutant Rh8 revealed 73 differentially expressed proteins. Hierarchical clustering analysis suggested that mutant Rh8 may have evolved a more stabilized membrane structure, and have developed a cost-efficient energy metabolism strategy, to cope with the butanol challenge. This comparative membrane proteomics study, together with our previous published work on comparative cytoplasmic proteomics, allows us to obtain a systemic understanding of the effect of butanol on cellular physiology of C. acetobutylicum.

The solventogenic bacterium Clostridium acetobutylicum is an important species of the Clostridium community. To develop a fundamental tool that is useful for biological studies of C. acetobutylicum, we established a high resolution proteome reference map for this species. We identified 1206 spots representing 564 different proteins by mass spectrometry, covering approximately 50% of major metabolic pathways. To better understand the relationship between butanol tolerance and butanol yield, we performed a comparative proteomic analysis between the wild type strain DSM 1731 and the mutant Rh8, which has higher butanol tolerance and higher butanol yield. Comparative proteomic analysis of two strains at acidogenic and solventogenic phases revealed 102 differentially expressed proteins that are mainly involved in protein folding, solvent formation, amino acid metabolism, protein synthesis, nucleotide metabolism, transport, and others. Hierarchical clustering analysis revealed that over 70% of the 102 differentially expressed proteins in mutant Rh8 were either upregulated (e.g., chaperones and solvent formation related) or downregulated (e.g., amino acid metabolism and protein synthesis related) in both acidogenic and solventogenic phase, which, respectively, are only upregulated or downregulated in solventogenic phase in the wild type strain. This suggests that Rh8 cells have evolved a mechanism to prepare themselves for butanol challenge before butanol is produced, leading to an increased butanol yield. This is the first report on the comparative proteome analysis of a mutant strain and a base strain of C. acetobutylicum. The fundamental proteomic data and analyses will be useful for further elucidating the biological mechanism of butanol tolerance and/or enhanced butanol production.

The world’s energy and global warming crises call for sustainable, renewable, carbon-neutral alternatives to replace fossil fuel resources. Currently, most biofuels are produced from agricultural crops and residues, which lead to concerns about food security and land shortage. Compared to the current biofuel production system, cyanobacteria, as autotrophic prokaryotes, do not require arable land and can grow to high densities by efficiently using solar energy, CO2, water, and inorganic nutrients. Moreover, powerful genetic techniques of cyanobacteria have been developed. For these reasons, cyanobacteria, which carry out oxygenic photosynthesis, are attractive hosts for production of fuels and chemicals. Recently, several chemicals including ethanol, isobutanol and isoprene have been produced by engineered cyanobacteria directly using solar energy, CO2, and water. Cyanobacterium is therefore a potential novel cell factory for fuels and chemicals production to address global energy security and climate change issues.

Lactic acid bacteria (LAB) are a heterogeneous group of bacteria contributing to various industrial applications, ranging from food and beverage fermentation, bulk and fine chemicals production to pharmaceuticals manufacturing. Genome sequencing is booming; hitherto, 25 genomes of LAB have been published and many more are in progress. Based on genomic content of LAB, this review highlights some findings related to applications revealed by genomics and functional genomics analyses. Finally, this review summarizes mathematical modeling strategies of LAB in the context of genomics, to further our understanding of industrial related features.

Enzyme® product is a popular macrobiotic food in Asia, which is naturally fermented using vegetables, fruits, plant leaves, and flour as raw materials. To study the bacterial diversity in a commercial available Enzyme® product, culture-dependent BOX-PCR analysis and culture-independent PCR-DGGE analysis were used simultaneously. The amount of the cultivable bacteria in the product is about 2.5×107 CFU/g. A total of 329 isolated strains were grouped into six fingerprint types according to BOX-PCR analysis. Four dominant species were identified among the six groups, which are Bacillus coagulans (60.2%), Lactobacillus plantarum (23.7%), Lactobacillus oris (8.2%), and Staphylococcus epidermidis (7.9%). Other five unculturable species, which were not cultivated by the condition used in this study, were identified according to PCR-DGGE analysis. Among them, Phyllobacterium myrsinacearum, Rhodococcus erythropolis, and Acidovorax konjaci were frequently found to be associated with plant materials. Therefore, combinatory application of culture-dependent and -independent techniques allowed us to unravel the microbial diversity in a naturally fermented health-promoting food product.

Microbial fermentations and bioconversions play a central role in the production of pharmaceuticals, enzymes and chemicals. To meet the demands of industrial production, it is desirable that microbes maintain a maximized carbon flux towards target metabolites regardless of fluctuations in intracellular or extracellular environments. This requires cellular systems that maintain functional stability and dynamic homeostasis in a given physiological state, or manipulate transitions between different physiological states. Stable maintenance or smooth transition can be achieved through engineering of dynamic controllability, modular and hierarchical organization, or functional redundancy, three key features of biological robustness in a cellular system. This review summarizes how synthetic biology can be used to improve the robustness of industrial microbes.

Lactic acid bacteria (LAB) that are widely used in the food fermentations often encounter various environmental stresses during manufacturing and application. Improving the antioxidative properties of LAB was found to be an effective approach for increasing their general robustness. Here we review the recent progress of engineering the antioxidative properties of LAB, with focus on engineering the thiol compounds production. Engineering of the enzymes involved in oxidative stress resistance are also discussed. To further improve the industrial relevant robustness, engineering LAB at a higher control level is expected.Graphical abstractDownload high-res image (152KB)Download full-size imageHighlights► Thiol compounds (glutathione) protect LAB from multiple environmental stresses. ► Engineering antioxidative properties of LAB can improve the overall robustness. ► Genomics approaches helped to reveal the molecular mechanism of stress response.

A process for efficient production of an alkaline β-mannanases from Bacillus sp. N16-5 was established by heterologous expression using Pichia pastoris. A high producing strain was generated by removing the native β-mannanases signal peptide and increasing the copy number of the mature β-mannanases gene. High cell density fermentation of this strain in 1-L bioreactor led to a production level of 4164 U/mL after 96 h of induction. Sorbitol co-feeding and temperature-lowering strategies both increased the β-mannanase production levels. Combined usage of these two strategies achieved the most effective result—the enzyme level reached 6336 U/mL within 84 h, which to our best knowledge is the highest production level reported for the expression of extreme β-mannanase thus far. The strategy described in this work can also be adapted to express other important industrial enzymes with extreme properties.Highlights► Removal of the native signal peptide is a prerequisite to achieve high yield. ► Increasing the gene dosage dramatically increased the mannanase production level. ► The combinatorial strategy clearly led to better performance than either alone. ► The enzyme activity of the expressed alkaline β-mannanase reached 6336 U/ml. ► This is the highest alkaline β-mannanases level produced to date.