Cloning, over-expression, and characterization of a new carboxypeptidase A gene of Bacillus pumilus ML413 in Bacillus subtilis 168

Carboxypeptidase A (CPAs) are a well-studied group of zinc-containing exopeptidases that facilitate thebreakdown of proteins and peptides during metabolism. Carboxypeptidase A is typically produced in mammalian pancreatic, brain and other tissues. A new gene encoding carboxypeptidase A in the prokaryote Bacillus pumilus was amplified by polymerase chain reaction (PCR), ligated into the shuttle vector pMA5, and cloned in a GRAS bacteria-Bacillus subtilis 168 host. This gene sequence contained a 1621 bp open reading frame that encodes a protein of 540 amino acids. The optimum pH and temperature for enzyme activity were 7.5 and 50°C, respectively. The enzyme was quite stable at neutral pH and maintained about 65% activity following a 24 h incubation at 40°C. The Km of this CPA was 0.1 mM, much higher than in mammalian species. Glycerol, ammonium sulfate, and sodium citrate improved enzyme activity under optimal culture condition. The carboxypeptidase activity in recombinant B. subtilis 168 reached a maximum of 179 U ml-1 in a 5 L fermentator when cultured on improved medium. The over expression of  carboxypeptidase A in Bacillus subtilis has commercial applications.Key words: Bacillus pumilus, Bacillus subtilis 168, over-expression, orthogonal arrays, carboxypeptidase A,metallocarboxypeptidase

Over-expression of Mycobacterium neoaurum 3-ketosteroid-\u3941-dehydrogenase in Corynebacterium crenatum for efficient bioconversion of 4-androstene-3,17-dione to androst-1,4-diene-3,17-dione

Background: 3-Ketosteroid-\u3941-dehydrogenase (KSDD), a flavoprotein enzyme, catalyzes the bioconversion of 4-androstene-3,17-dione (AD) to androst-1,4-diene-3,17-dione (ADD). To date, there has been no report about characterization of KSDD from Mycobacterium neoaurum strains, which were usually employed to produce AD or ADD by fermentation. Results: In this work, Corynebacterium crenatum was chosen as a new host for heterologous expression of KSDD from M. neoaurum JC-12 after codon optimization of the KSDD gene. SDS-PAGE and western blotting results indicated that the recombinant C. crenatum harboring the optimized ksdd (ksddII) gene showed significantly improved ability to express KSDD. The expression level of KSDD was about 1.6-fold increased C. crenatum after codon optimization. After purification of the protein, we first characterized KSDD from M. neoaurum JC-12, and the results showed that the optimum temperature and pH for KSDD activity were 30\ub0C and pH 7.0, respectively. The Km and Vmax values of purified KSDD were 8.91 \u3bcM and 6.43 mM/min. In this work, C. crenatum as a novel whole-cell catalyst was also employed and validated for bioconversion of AD to ADD. The highest transformation rate of AD to ADD by recombinant C. crenatum was about 83.87% after 10 h reaction time, which was more efficient than M. neoaurum JC-12 (only 3.56% at 10 h). Conclusions: In this work, basing on the codon optimization, overexpression, purification and characterization of KSDD, we constructed a novel system, the recombinant C. crenatum SYPA 5-5 expressing KSDD, to accumulate ADD from AD efficiently. This work provided new insights into strengthening sterol catabolism by overexpressing the key enzyme KSDD, for efficient ADD production

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

Over-expression of Mycobacterium neoaurum 3-ketosteroid-Δ1-dehydrogenase in Corynebacterium crenatum for efficient bioconversion of 4-androstene-3,17-dione to androst-1,4-diene-3,17-dione

Background: 3-Ketosteroid-Δ1-dehydrogenase (KSDD), a flavoprotein enzyme, catalyzes the bioconversion of 4-androstene-3,17-dione (AD) to androst-1,4-diene-3,17-dione (ADD). To date, there has been no report about characterization of KSDD from Mycobacterium neoaurum strains, which were usually employed to produce AD or ADD by fermentation. Results: In this work, Corynebacterium crenatum was chosen as a new host for heterologous expression of KSDD from M. neoaurum JC-12 after codon optimization of the KSDD gene. SDS-PAGE and western blotting results indicated that the recombinant C. crenatum harboring the optimized ksdd (ksddII) gene showed significantly improved ability to express KSDD. The expression level of KSDD was about 1.6-fold increased C. crenatum after codon optimization. After purification of the protein, we first characterized KSDD from M. neoaurum JC-12, and the results showed that the optimum temperature and pH for KSDD activity were 30°C and pH 7.0, respectively. The Km and Vmax values of purified KSDD were 8.91 μM and 6.43 mM/min. In this work, C. crenatum as a novel whole-cell catalyst was also employed and validated for bioconversion of AD to ADD. The highest transformation rate of AD to ADD by recombinant C. crenatum was about 83.87% after 10 h reaction time, which was more efficient than M. neoaurum JC-12 (only 3.56% at 10 h). Conclusions: In this work, basing on the codon optimization, overexpression, purification and characterization of KSDD, we constructed a novel system, the recombinant C. crenatum SYPA 5-5 expressing KSDD, to accumulate ADD from AD efficiently. This work provided new insights into strengthening sterol catabolism by overexpressing the key enzyme KSDD, for efficient ADD production

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

Intracellular Environment Improvement of Mycobacterium neoaurum for Enhancing Androst-1,4-Diene-3,17-Dione Production by Manipulating NADH and Reactive Oxygen Species Levels

As one of the most significant steroid hormone precursors, androst-1,4-diene-3,17-dione (ADD) could be used to synthesize many valuable hormone drugs. The microbial transformation of sterols to ADD has received extensive attention in recent years. In a previous study, Mycobacterium neoaurum JC-12 was isolated and converted sterols to the major product, ADD. In this work, we enhanced ADD yield by improving the cell intracellular environment. First, we introduced a nicotinamide adenine dinucleotide (NADH) oxidase from Bacillus subtilis to balance the intracellular NAD+ availability in order to strengthen the ADD yield. Then, the catalase gene from M. neoaurum was also over-expressed to simultaneously scavenge the generated H2O2 and eliminate its toxic effects on cell growth and sterol transformation. Finally, using a 5 L fermentor, the recombinant strain JC-12yodC-katA produced 9.66 g/L ADD, which increased by 80% when compared with the parent strain. This work shows a promising way to increase the sterol transformation efficiency by regulating the intracellular environment

One-Pot Biocatalytic Preparation of Enantiopure Unusual α-Amino Acids from α-Hydroxy Acids via a Hydrogen-Borrowing Dual-Enzyme Cascade

Unusual α-amino acids (UAAs) are important fundamental building blocks and play a key role in medicinal chemistry. Here, we constructed a hydrogen-borrowing dual-enzyme cascade for efficient synthesis of UAAs from α-hydroxy acids (α-HAs). D-mandelate dehydrogenase from Lactobacillus brevis (LbMDH) was screened for the catalysis of α-HAs to α-keto acids but with low activity towards aliphatic α-HAs. Therefore, we rational engineered LbMDH to improve its activity towards aliphatic α-HAs. The substitution of residue Leu243 located in the substrate entrance channel with nonpolar amino acids like Met, Trp, and Ile significantly influenced the enzyme activity towards different α-HAs. Compared with wild type (WT), variant L243W showed 103 U/mg activity towards D-α-hydroxybutyric acid, 1.7 times of the WT’s 60.2 U/mg, while its activity towards D-mandelic acid decreased. Variant L243M showed 2.3 times activity towards D-mandelic acid compared to WT, and its half-life at 40 °C increased to 150.2 h comparing with 98.5 h of WT. By combining LbMDH with L-leucine dehydrogenase from Bacillus cereus, the synthesis of structurally diverse range of UAAs from α-HAs was constructed. We achieved 90.7% conversion for L-phenylglycine production and 66.7% conversion for L-α-aminobutyric acid production. This redox self-sufficient cascade provided high catalytic efficiency and generated pure products

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

<div><p>Background</p><p>Previously, a safe strain, <i>Bacillus amyloliquefaciens</i> B10-127 was identified as an excellent candidate for industrial-scale microbial fermentation of 2,3-butanediol (2,3-BD). However, <i>B. amyloliquefaciens</i> fermentation yields large quantities of acetoin, lactate and succinate as by-products, and the 2,3-BD yield remains prohibitively low for commercial production.</p> <p>Methodology/Principal Findings</p><p>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 <i>B. amyloliquefaciens</i>. 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 <i>B. amyloliquefaciens</i>, 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.</p> <p>Conclusions/Significance</p><p>Co-overexpression of <i>bdh</i> and <i>gapA</i> 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.</p> </div

A Novel Method to Screen Strong Constitutive Promoters in <i>Escherichia coli</i> and <i>Serratia marcescens</i> for Industrial Applications

Promoters serve as the switch of gene transcription, playing an important role in regulating gene expression and metabolites production. However, the approach to screening strong constitutive promoters in microorganisms is still limited. In this study, a novel method was designed to identify strong constitutive promoters in E. coli and S. marcescens based on random genomic interruption and fluorescence-activated cell sorting (FACS) technology. First, genomes of E. coli, Bacillus subtilis, and Corynebacterium glutamicum were randomly interrupted and inserted into the upstream of reporter gene gfp to construct three promoter libraries, and a potential strong constitutive promoter (PBS) suitable for E. coli was screened via FACS technology. Second, the core promoter sequence (PBS76) of the screened promoter was identified by sequence truncation. Third, a promoter library of PBS76 was constructed by installing degenerate bases via chemical synthesis for further improving its strength, and the intensity of the produced promoter PBS76-100 was 59.56 times higher than that of the promoter PBBa_J23118. Subsequently, promoters PBBa_J23118, PBS76, PBS76-50, PBS76-75, PBS76-85, and PBS76-100 with different strengths were applied to enhance the metabolic flux of L-valine synthesis, and the L-valine yield was significantly improved. Finally, a strong constitutive promoter suitable for S. marcescens was screened by a similar method and applied to enhance prodigiosin production by 34.81%. Taken together, the construction of a promoter library based on random genomic interruption was effective to screen the strong constitutive promoters for fine-tuning gene expression and reprogramming metabolic flux in various microorganisms