Taowei Yang

The rebalanced pathway significantly enhances acetoin production by disruption of acetoin reductase gene and moderate-expression of a new water-forming NADH oxidase in Bacillus subtilis

Bacillus subtilis produces acetoin as a major extracellular product. However, the by-products of 2,3-butanediol, lactic acid and ethanol were accompanied in the NADH-dependent pathways. In this work, metabolic engineering strategies were proposed to redistribute the carbon flux to acetoin by manipulation the NADH levels. We first knocked out the acetoin reductase gene bdhA to block the main flux from acetoin to 2,3-butanediol. Then, among four putative candidates, we successfully screened an active water-forming NADH oxidase, YODC. Moderate-expression of YODC in the bdhA disrupted B. subtilis weakened the NADH-linked pathways to by-product pools of acetoin. Through these strategies, acetoin production was improved to 56.7g/l with an increase of 35.3%, while the production of 2,3-butanediol, lactic acid and ethanol were decreased by 92.3%, 70.1% and 75.0%, respectively, simultaneously the fermentation duration was decreased 1.7-fold. Acetoin productivity by B. subtilis was improved to 0.639g/(lh).

Production of 2,3-butanediol from glucose by GRAS microorganism Bacillus amyloliquefaciens

In the current study, a GRAS (Generally Recognized As Safe) strain of Bacillus amyloliquefaciens producing 2,3‐butanediol (2,3‐BD) designated as B10‐127 was isolated in our lab. The strain B10‐127 produced 2,3‐BD effectively under the condition of 20% glucose (quality concentration), showed a high‐glucose tolerance. The effects of initial glucose concentration, temperature, pH and agitation on 2,3‐BD production were investigated in this work and the proper parameters were identified. Accordingly, the fed‐batch culture of B10‐127 in larger scales (5 l) showed a remarkable 2,3‐BD producing potency. The maximum 2,3‐BD concentration reached 92.3 g/l at 96 h with a 2,3‐BD productivity of 0.96 g/l h. To our knowledge, the results were new records on 2,3‐BD fermentation by Bacillus, which shown an excellent candidate for the microbial fermentation of 2,3‐BD on an industrial scale. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Improved Production of 2,3-Butanediol in Bacillus amyloliquefaciens by Over-Expression of Glyceraldehyde-3-Phosphate Dehydrogenase and 2,3-butanediol Dehydrogenase

Background Previously, a safe strain, Bacillus amyloliquefaciens B10-127 was identified as an excellent candidate for industrial-scale microbial fermentation of 2,3-butanediol (2,3-BD). However, B. amyloliquefaciens fermentation yields large quantities of acetoin, lactate and succinate as by-products, and the 2,3-BD yield remains prohibitively low for commercial production. Methodology/Principal Findings In the 2,3-butanediol metabolic pathway, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the conversion of 3-phosphate glyceraldehyde to 1,3-bisphosphoglycerate, with concomitant reduction of NAD+ to NADH. In the same pathway, 2,3-BD dehydrogenase (BDH) catalyzes the conversion of acetoin to 2,3-BD with concomitant oxidation of NADH to NAD+. In this study, to improve 2,3-BD production, we first over-produced NAD+-dependent GAPDH and NADH-dependent BDH in B. amyloliquefaciens. Excess GAPDH reduced the fermentation time, increased the 2,3-BD yield by 12.7%, and decreased the acetoin titer by 44.3%. However, the process also enhanced lactate and succinate production. Excess BDH increased the 2,3-BD yield by 16.6% while decreasing acetoin, lactate and succinate production, but prolonged the fermentation time. When BDH and GAPDH were co-overproduced in B. amyloliquefaciens, the fermentation time was reduced. Furthermore, in the NADH-dependent pathways, the molar yield of 2,3-BD was increased by 22.7%, while those of acetoin, lactate and succinate were reduced by 80.8%, 33.3% and 39.5%, relative to the parent strain. In fed-batch fermentations, the 2,3-BD concentration was maximized at 132.9 g/l after 45 h, with a productivity of 2.95 g/l·h. Conclusions/Significance Co-overexpression of bdh and gapA genes proved an effective method for enhancing 2,3-BD production and inhibiting the accumulation of unwanted by-products (acetoin, lactate and succinate). To our knowledge, we have attained the highest 2,3-BD fermentation yield thus far reported for safe microorganisms.

Bioconversion of 4-androstene-3,17-dione to androst-1,4-diene-3,17-dione by recombinant Bacillus subtilis expressing ksdd gene encoding 3-ketosteroid-Δ1-dehydrogenase from Mycobacterium neoaurum JC-12

The enzyme 3-ketosteroid-Δ(1)-dehydrogenase (KSDD), involved in steroid metabolism, catalyzes the transformation of 4-androstene-3,17-dione (AD) to androst-1,4-diene-3,17-dione (ADD) specifically. Its coding gene was obtained from Mycobacterium neoaurum JC-12 and expressed on the plasmid pMA5 in Bacillus subtilis 168. The successfully expressed KSDD was analyzed by native-PAGE. The activities of the recombinant enzyme in B. subtilis were 1.75 U/mg, which was about 5-fold that of the wild type in M. neoaurum. When using the whole-cells as catalysts, the products were analyzed by tin-layer chromatography and high-performance liquid chromatography. The recombinant B. subtilis catalyzed the biotransformation of AD to ADD in a percent conversion of 65.7% and showed about 18 folds higher than M. neoaurum JC-12. The time required for transformation of AD to ADD was about 10h by the recombinant B. subtilis, much shorter than that of the wild-type strain and other reported strains. Thus, the efficiency of ADD production could be improved immensely. For industrial applications, the recombinant B. subtilis containing KSDD provides a new pathway of producing steroid medicines.

Efficient whole-cell biocatalyst for acetoin production with NAD+ regeneration system through homologous co-expression of 2,3-butanediol dehydrogenase and NADH oxidase in engineered Bacillus subtilis.

Acetoin (3-hydroxy-2-butanone), an extensively-used food spice and bio-based platform chemical, is usually produced by chemical synthesis methods. With increasingly requirement of food security and environmental protection, bio-fermentation of acetoin by microorganisms has a great promising market. However, through metabolic engineering strategies, the mixed acid-butanediol fermentation metabolizes a certain portion of substrate to the by-products of organic acids such as lactic acid and acetic acid, which causes energy cost and increases the difficulty of product purification in downstream processes. In this work, due to the high efficiency of enzymatic reaction and excellent selectivity, a strategy for efficiently converting 2,3-butandiol to acetoin using whole-cell biocatalyst by engineered Bacillus subtilis is proposed. In this process, NAD+ plays a significant role on 2,3-butanediol and acetoin distribution, so the NADH oxidase and 2,3-butanediol dehydrogenase both from B. subtilis are co-expressed in B. subtilis 168 to construct an NAD+ regeneration system, which forces dramatic decrease of the intracellular NADH concentration (1.6 fold) and NADH/NAD+ ratio (2.2 fold). By optimization of the enzymatic reaction and applying repeated batch conversion, the whole-cell biocatalyst efficiently produced 91.8 g/L acetoin with a productivity of 2.30 g/(L·h), which was the highest record ever reported by biocatalysis. This work indicated that manipulation of the intracellular cofactor levels was more effective than the strategy of enhancing enzyme activity, and the bioprocess for NAD+ regeneration may also be a useful way for improving the productivity of NAD+-dependent chemistry-based products.

Enhanced Production of Androst-1,4-Diene-3,17-Dione by Mycobacterium neoaurum JC-12 Using Three-Stage Fermentation Strategy.

To improve the androst-1,4-diene-3,17-dione (ADD) production from phytosterol by Mycobacterium neoaurum JC-12, fructose was firstly found favorable as the initial carbon source to increase the biomass and eliminate the lag phase of M. neoaurum JC-12 in the phytosterol transformation process. Based on this phenomenon, two-stage fermentation by using fructose as the initial carbon source and feeding glucose to maintain strain metabolism was designed. By applying this strategy, the fermentation duration was decreased from 168 h to 120 h with the ADD productivity increased from 0.071 g/(L·h) to 0.108 g/(L·h). Further, three-stage fermentation by adding phytosterol to improve ADD production at the end of the two-stage fermentation was carried out and the final ADD production reached 18.6 g/L, which is the highest reported ADD production using phytosterol as substrate. Thus, this strategy provides a possible way in enhancing the ADD production in pharmaceutical industry.

Two-Stage pH Control Strategy Based on the pH Preference of Acetoin Reductase Regulates Acetoin and 2,3-Butanediol Distribution in Bacillus subtilis

Acetoin reductase/2,3-butanediol dehydrogenase (AR/BDH), which catalyzes the interconversion between acetoin and 2,3-butanediol, plays an important role in distribution of the products pools. This work characterized the Bacillus subtilis AR/BDH for the first time. The enzyme showed very different pH preferences of pH 6.5 for reduction and pH 8.5 for oxidation. Based on these above results, a two-stage pH control strategy was optimized for acetoin production, in which the pH was controlled at 6.5 for quickly converting glucose to acetoin and 2,3-butanediol, and then 8.0 for reversely transforming 2,3-butanediol to acetoin. By over-expression of AR/BDH in the wild-type B. subtilis JNA 3-10 and applying fed-batch fermentation based on the two-stage pH control strategy, acetoin yield of B. subtilis was improved to a new record of 73.6 g/l, with the productivity of 0.77 g/(l·h). The molar yield of acetoin was improved from 57.5% to 83.5% and the ratio of acetoin/2,3-butanediol was switched from 2.7∶1 to 18.0∶1.

Regulation of the NADH pool and NADH/NADPH ratio redistributes acetoin and 2,3‐butanediol proportion in Bacillus subtilis

Bacillus subtilis produces acetoin as a major product along with several NADH-dependent byproducts, especially 2,3-butanediol. In this study, the down-regulation of the NADH pool and the redistribution of NADH/NADPH were targeted using external and genetic processes, as a means by which to redistribute the metabolic flux in favor of acetoin synthesis. First, it was found that the use of carbon sources of different oxidation states resulted in very different intracellular NADH/NAD(+) ratios that dictated the total process yield of acetoin. A mixture of glucose and gluconate as substrate produced a relatively low NADH/NAD(+) ratio, and resulted in an increase in acetoin production while byproducts significantly decreased. Metabolic engineering methods using glucose as a substrate could yield a similar effect. Acetoin production was significantly enhanced by overexpression of the oxidative pentose phosphate pathway: increased expression of glucose-6-phosphate dehydrogenase resulted in a decrease in the intracellular NADH/NADPH ratio (1.9-fold) and NADH/NAD(+) ratio (1.7-fold). In fed-batch culture the engineered strain yielded an acetoin concentration of 43.3 g L(-1) , while the production of 2,3-butanediol was only 1.7 g L(-1) . The concept of the manipulation of cofactor levels to redistribute carbon flux by external and genetic means as explored in this paper provides a novel strategy for improving industrial acetoin fermentation.

Systems pathway engineering of Corynebacterium crenatum for improved L-arginine production

L-arginine is an important amino acid in food and pharmaceutical industries. Until now, the main production method of L-arginine in China is the highly polluting keratin acid hydrolysis. The industrial level L-arginine production by microbial fermentation has become an important task. In previous work, we obtained a new L-arginine producing Corynebacterium crenatum (subspecies of Corynebacterium glutamicum) through screening and mutation breeding. In this work, we performed systems pathway engineering of C. crenatum for improved L-arginine production, involving amplification of L-arginine biosynthetic pathway flux by removal of feedback inhibition and overexpression of arginine operon; optimization of NADPH supply by modulation of metabolic flux distribution between glycolysis and pentose phosphate pathway; increasing glucose consumption by strengthening the preexisting glucose transporter and exploitation of new glucose uptake system; channeling excess carbon flux from glycolysis into tricarboxylic acid cycle to alleviate the glucose overflow metabolism; redistribution of carbon flux at α-ketoglutarate metabolic node to channel more flux into L-arginine biosynthetic pathway; minimization of carbon and cofactor loss by attenuation of byproducts formation. The final strain could produce 87.3 g L−1 L-arginine with yield up to 0.431 g L-arginine g−1 glucose in fed-batch fermentation.

High-level production of melanin by a novel isolate of Streptomyces kathirae

Forty-five bacterial strains that produced diffusive pigments were isolated from 40 soil samples. Maximum pigment production was from a Streptomyces kathirae strain designated SC-1. The diffused pigment was characterized by UV-visual and infrared spectroscopy, MS and (1) H nuclear magnetic resonance imaging, and was confirmed as melanin. This may be the first report of melanin production by S. kathirae. To enhance melanin production, the culture medium was optimized by conducting a series of batch fermentations in a defined medium, and the results were analysed statistically using a response surface method. The optimal culture medium comprised 3.3 g L(-1) amylodextrine, 37 g L(-1) yeast extract, 5 g L(-1) NaCl, 0.1 g L(-1) CaCl2 and 54.4 μM CuSO4 . The pH of this medium was 6.0. Under optimal conditions, the melanin concentration was maximized at 13.7 g L(-1) , c. 8.6-fold higher than obtained in suboptimal medium. To our knowledge, the results provide novel data on melanin fermentation, and identify an excellent candidate for industrial-scale microbial fermentation of melanin.