Jiayang Qin

A novel whole-cell biocatalyst with NAD+ regeneration for production of chiral chemicals.

Background The high costs of pyridine nucleotide cofactors have limited the applications of NAD(P)-dependent oxidoreductases on an industrial scale. Although NAD(P)H regeneration systems have been widely studied, NAD(P)+ regeneration, which is required in reactions where the oxidized form of the cofactor is used, has been less well explored, particularly in whole-cell biocatalytic processes. Methodology/Principal Findings Simultaneous overexpression of an NAD+ dependent enzyme and an NAD+ regenerating enzyme (H2O producing NADH oxidase from Lactobacillus brevis) in a whole-cell biocatalyst was studied for application in the NAD+-dependent oxidation system. The whole-cell biocatalyst with (2R,3R)-2,3-butanediol dehydrogenase as the catalyzing enzyme was used to produce (3R)-acetoin, (3S)-acetoin and (2S,3S)-2,3-butanediol. Conclusions/Significance A recombinant strain, in which an NAD+ regeneration enzyme was coexpressed, displayed significantly higher biocatalytic efficiency in terms of the production of chiral acetoin and (2S,3S)-2,3-butanediol. The application of this coexpression system to the production of other chiral chemicals could be extended by using different NAD(P)-dependent dehydrogenases that require NAD(P)+ for catalysis.

Non-sterilized fermentative production of polymer-grade L-lactic acid by a newly isolated thermophilic strain Bacillus sp. 2-6.

Background The demand for lactic acid has been increasing considerably because of its use as a monomer for the synthesis of polylactic acid (PLA), which is a promising and environment-friendly alternative to plastics derived from petrochemicals. Optically pure l-lactic acid is essential for polymerization of PLA. The high fermentation cost of l-lactic acid is another limitation for PLA polymers to compete with conventional plastics. Methodology/Principal Findings A Bacillus sp. strain 2–6 for production of l-lactic acid was isolated at 55°C from soil samples. Its thermophilic characteristic made it a good lactic acid producer because optically pure l-lactic acid could be produced by this strain under open condition without sterilization. In 5-liter batch fermentation of Bacillus sp. 2–6, 118.0 g/liter of l-lactic acid with an optical purity of 99.4% was obtained from 121.3 g/liter of glucose. The yield was 97.3% and the average productivity was 4.37 g/liter/h. The maximum l-lactic acid concentration of 182.0 g/liter was obtained from 30-liter fed-batch fermentation with an average productivity of 3.03 g/liter/h and product optical purity of 99.4%. Conclusions/Significance With the newly isolated Bacillus sp. strain 2–6, high concentration of optically pure l-lactic acid could be produced efficiently in open fermentation without sterilization, which would lead to a new cost-effective method for polymer-grade l-lactic acid production from renewable resources.

Production of 2,3-Butanediol by Klebsiella Pneumoniae Using Glucose and Ammonium Phosphate

The production of 2,3-butanediol by Klebsiella pneumoniae from glucose supplemented with different salts was studied. A suitable medium composition was defined by response surface experiments. In a medium containing glu- cose and (NH4)2HPO4, the strain could convert 137.0g of glucose into 52.4g of 2,3-butanediol and 8.4g of acetoin in shaking flasks. The diol yield amounted to 90% of its theoretical value and the productivity was 1—1.5g·L -1 ·h -1 . In fed-batch fermentation, the yield and productivity of diol were further enhanced by maintaining the pH at 6.0. Up to 92.4g of 2,3-butanediol and 13.1g of acetoin per liter were obtained from 215.0g of glucose per liter. The diol yield reached 98% of its theoretical value and the productivity was up to 2.1g·L -1 ·h -1 . In the production of 2,3-butanediol via a mixed acid pathway, acetoin is an intermediate prior to the formation of 2,3-butanediol during fermentation. The metabolic conversion of acetoin to 2,3-butandiol is reversible. The net reactions forming 2,3-butanediol and acetoin from glucose are Glucose butanediol+2CO2+NADH2+2ATP Glucose acetoin+2CO2+2NADH2+2ATP Therefore, on the mass basis, the theoretical yield of 2,3-butanediol and acetoin from glucose are 0.5g·g -1

Efficient Conversion of Phenylpyruvic Acid to Phenyllactic Acid by Using Whole Cells of Bacillus coagulans SDM

Background Phenyllactic acid (PLA), a novel antimicrobial compound with broad and effective antimicrobial activity against both bacteria and fungi, can be produced by many microorganisms, especially lactic acid bacteria. However, the concentration and productivity of PLA have been low in previous studies. The enzymes responsible for conversion of phenylpyruvic acid (PPA) into PLA are equivocal. Methodology/Principal Findings A novel thermophilic strain, Bacillus coagulans SDM, was isolated for production of PLA. When the solubility and dissolution rate of PPA were enhanced at a high temperature, whole cells of B. coagulans SDM could effectively convert PPA into PLA at a high concentration (37.3 g l−1) and high productivity (2.3 g l−1 h−1) under optimal conditions. Enzyme activity staining and kinetic studies identified NAD-dependent lactate dehydrogenases as the key enzymes that reduced PPA to PLA. Conclusions/Significance Taking advantage of the thermophilic character of B. coagulans SDM, a high yield and productivity of PLA were obtained. The enzymes involved in PLA production were identified and characterized, which makes possible the rational design and construction of microorganisms suitable for PLA production with metabolic engineering.

Production of (2S,3S)-2,3-butanediol and (3S)-acetoin from glucose using resting cells of Klebsiella pneumonia and Bacillus subtilis

Production of highly pure (2S,3S)-2,3-butanediol ((2S,3S)-2,3-BD) and (3S)-acetoin ((3S)-AC) in high concentrations is desirable but difficult to achieve. In the present study, glucose was first transformed to a mixture of (2S,3S)-2,3-BD and meso-2,3-BD by resting cells of Klebsiella pneumoniae CICC 10011, followed by biocatalytic resolution of the mixture by resting cells of Bacillus subtilis 168. meso-2,3-BD was transformed to (3S)-AC, leaving (2S,3S)-2,3-BD in the reaction medium. Using this approach, 12.5 g l(-1) (2S,3S)-2,3-BD and 56.7 g l(-1) (3S)-AC were produced. Stereoisomeric purity of (2S,3S)-2,3-BD and enantiomeric excess of (3S)-AC was 96.9 and 96.2%, respectively.

Genome Sequence of the Thermophilic Strain Bacillus coagulans2-6, an Efficient Producer of High-Optical-Purity l-Lactic Acid

Bacillus coagulans 2-6 is an efficient producer of lactic acid. The genome of B. coagulans 2-6 has the smallest genome among the members of the genus Bacillus known to date. The frameshift mutation at the start of the d-lactate dehydrogenase sequence might be responsible for the production of high-optical-purity l-lactic acid.

Genome Sequence of Lactobacillus rhamnosus Strain CASL, an Efficient l-Lactic Acid Producer from Cheap Substrate Cassava

Lactobacillus rhamnosus is a type of probiotic bacteria with industrial potential for L-lactic acid production. We announce the draft genome sequence of L. rhamnosus CASL (2,855,156 bp with a G+C content of 46.6%), which is an efficient producer of L-lactic acid from cheap, nonfood substrate cassava with a high production titer.