Nonsterile l-Lysine Fermentation Using Engineered Phosphite-Grown Corynebacterium glutamicum.

Fermentation using Corynebacterium glutamicum is an important method for the industrial production of amino acids. However, conventional fermentation processes using C. glutamicum are susceptible to microbial contamination and therefore require equipment sterilization or antibiotic dosing. To establish a more robust fermentation process, l-lysine-producing C. glutamicum was engineered to efficiently utilize xenobiotic phosphite (Pt) by optimizing the expression of Pt dehydrogenase in the exeR genome locus. This ability provided C. glutamicum with a competitive advantage over common contaminating microbes when grown on media containing Pt as a phosphorus source instead of phosphate. As a result, the engineered strain could produce 41.00 g/L l-lysine under nonsterile conditions during batch fermentation for 60 h, whereas the original strain required 72 h to produce 40.78 g/L l-lysine under sterile conditions. Therefore, the recombinant strain can efficiently produce l-lysine under nonsterilized conditions with unaffected production efficiency. Although this anticontamination strategy has been previously reported for other species, this is the first time it has been demonstrated in C. glutamicum; these findings should aid in the further development of cost-efficient amino acid fermentation processes.

Effects of Spo0A on Clostridium acetobutylicum with an emphasis on biofilm formation.

Clostridium acetobutylicum is a well-known strain for biofuel production. In previous work, it was found that this strain formed biofilm readily during fermentation processes. Biofilm formation could protect cells and enhance productivities under environmental stresses in our previous work. To explore the molecular mechanism of biofilm formation, Spo0A of C. acetobutylicum was selected to investigate its influences on biofilm formation and other physiological performances. When spo0A gene was disrupted, the spo0A mutant could hardly form biofilm. The aggregation and adhesion abilities of the spo0A mutant as well as its swarming motility were dramatically reduced compared to those of wild type strain. Sporulation was also negatively influenced by spo0A disruption, and solvent production was almost undetectable in the spo0A mutant fermentation. Furthermore, proteomic differences between wild type strain and the spo0A mutant were consistent with physiological performances. This is the first study confirming a genetic clue to C. acetobutylicum biofilm and will be valuable for biofilm optimization through genetic engineering in the future.

Efficient Xylitol Production from Cornstalk Hydrolysate Using Engineered Escherichia coli Whole Cells.

Economic transformation of lignocellulose hydrolysate into valued-added products is of particular importance for energy and environmental issues. In this study, xylose reductase and glucose dehydrogenase were cloned into plasmid pETDuet-1 and then simultaneously expressed in Escherichia coli BL21(DE3), which was used as whole-cell catalyst for the first time to convert xylose into xylitol coupled with gluconate production. When tested with reconstituted xylose and glucose solution, 0.1 g/mL cells could convert 1 M xylose and 1 M glucose completely and produced 145.81 g/L xylitol with a yield of 0.97 (g/g) and 184.85 g/L gluconic acid with a yield of 1.03 (g/g) in 24 h. Subsequently, the engineered cells were applied in real cornstalk hydrolysate, which generated 30.88 g/L xylitol and 50.89 g/L gluconic acid. The cells were used without penetration treatment, and CaCO3 was used to effectively regulate the pH during the production, which further saved costs.

Clostridium acetobutylicum grows vegetatively in a biofilm rich in heteropolysaccharides and cytoplasmic proteins.

BACKGROUND: Biofilms are cell communities wherein cells are embedded in a self-produced extracellular polymeric substances (EPS). The biofilm of Clostridium acetobutylicum confers the cells superior phenotypes and has been extensively exploited to produce a variety of liquid biofuels and bulk chemicals. However, little has been known about the physiology of C. acetobutylicum in biofilm as well as the composition and biosynthesis of the EPS. Thus, this study is focused on revealing the cell physiology and EPS composition of C. acetobutylicum biofilm. RESULTS: Here, we revealed a novel lifestyle of C. acetobutylicum in biofilm: elimination of sporulation and vegetative growth. Extracellular polymeric substances and wire-like structures were also observed in the biofilm. Furthermore, for the first time, the biofilm polysaccharides and proteins were isolated and characterized. The biofilm contained three heteropolysaccharides. The major fraction consisted of predominantly glucose, mannose and aminoglucose. Also, a great variety of proteins including many non-classically secreted proteins moonlighting as adhesins were found considerably present in the biofilm, with GroEL, a S-layer protein and rubrerythrin being the most abundant ones. CONCLUSIONS: This study evidenced that vegetative C. acetobutylicum cells rather than commonly assumed spore-forming cells were essentially the solvent-forming cells. The abundant non-classically secreted moonlighting proteins might be important for the biofilm formation. This study provides the first physiological and molecular insights into C. acetobutylicum biofilm which should be valuable for understanding and development of the biofilm-based processes.

Towards acetone-uncoupled biofuels production in solventogenic Clostridium through reducing power conservation.

Microbial production of butanol by solventogenic Clostridium has long been complicated with the formation of acetone as an unwanted product, which causes poor product yields and creates a most important problem concerning substrate transformation. Intensive attempts concentrate on carbon conversion pathways to eliminate acetone, but have actually achieved little so far. Here, we believe microbial product distribution can largely depend on how the cell plays its energetic cofactors in central metabolism, and demonstrate that by introducing a synthetic 2,3-butanediol synthesis pathway in Clostridium acetobutylicum as an NADH-compensating module to readjust the reducing power at a systems level, the production of acetone can be selectively and efficiently eliminated (< 0.3 g/L). H2 evolution was reduced by 78%, and the total alcohol yield was strikingly increased by 19% to 0.44 g/g glucose, much higher than those yet reported for butanol fermentation. These findings highlight that it is the loss of reducing power rather than typically manipulated solventogenesis genes that dominates acetone formation. Further study revealed that the NADH-module triggered apparent regulation of pathways involved in electron transfer and reducing power conservation. The study also suggested the key to conservation of intracellular reducing power might essentially lie in the intermediate processes in central metabolism that are related to redox partners, butyrate or C4 branches, and possibly NADH and NADPH specificity. This study represents the first effective redox-based configuration of C. acetobutylicum and provides valuable understandings for redox engineering of native Clostridium species towards advanced production of biofuels and alcohols.

Enhanced production of butanol and acetoin by heterologous expression of an acetolactate decarboxylase in Clostridium acetobutylicum.

Butanol is an important industrial chemical and an attractive transportation fuel. However, the deficiency of reducing equivalents NAD(P)H in butanol fermentation results in a large quantity of oxidation products, which is a major problem limiting the atom economy and economic viability of bio-butanol processes. Here, we integrated the butanol fermentation process with a NADH-generating, acetoin biosynthesis process to improve the butanol production. By overexpressing the α-acetolactate decarboxylase gene alsD from Bacillus subtilis in Clostridium acetobutylicum, acetoin yield was significantly increased at the cost of acetone. After optimization of fermentation conditions, butanol (12.9g/L), acetoin (6.5g/L), and ethanol (1.9g/L) were generated by the recombinant strain, with acetone no more than 1.8g/L. Thus, both mass yield and product value were greatly improved. This study demonstrates that reducing power compensation is effective to improve the atom economy of butanol fermentation, and provides a novel approach to improve the economic viability of bio-butanol production.