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

Isolation, Identification, Screening and Fermentation Process Optimization of Bacillus Producing High Antimicrobial Lipopeptide

In order to screen out Bacillus sp. with high production of antimicrobial lipopeptide and determine its optimal fermentation conditions, the study isolated and identified antimicrobial lipopeptide-producing Bacillus sp. strains from traditional fermented soybean paste, and adopted orthogonal test to investigate how lipopeptide production by Bacillus sp was affected by fermentation conditions, such as fermentation inoculum amount, fermentation filling volume, fermentation time and fermentation temperature. The results showed that 27 strains of Bacillus sp. were isolated and screened out from 9 portions traditional fermented soybean paste of the Northeast, among which 6 strains were identified by 16S rDNA as having the genes sfp, fenB and ituA for synthesizing lipopeptide. According to the determination of lipopeptide yield and inhibition effect, Bacillus subtilis SN-20 and Bacillus amyloliquefaciens SN-46 showed excellent performance with the yield of 106 and 72 mg of lipopeptide per unit, and strong inhibition effect on both gram-positive and negative indicator bacteria. The optimal fermentation process of Bacillus subtilis SN-20 was found to be 3% inoculum, 20% fermentation filler, 36 h fermentation time and 32 ℃. The optimal fermentation process of Bacillus subtilis SN-46 was 2% inoculum, 40% fermentation filler, 24 h fermentation time and 32 ℃. Under these conditions, the unit biomass yields of lipopeptides for the two strains of Bacillus before optimization were 106.11 and 76.23 mg/g, respectively. After optimization, they increased by 21.85% and 23.84%, respectively. The study results effectively increased the production of antimicrobial lipopeptides from Bacillus

Whole Genome Sequencing of Bacillus subtilis SNBS-3 and Prediction of Its Antimicrobial Substances

As an extension of the previous research, this study aimed to comprehensively characterize the genome of Bacillus subtilis SNBS-3. Illumina second-generation sequencing technology and the third-generation high-throughput Pacbio sequencing platform were used for whole-genome sequencing of B. subtilis SNBS-3 isolated from traditional bean paste to obtain the key information of genome characteristics, gene function annotation and classification, phylogenetic evolution, and secondary metabolites. The results showed that the genome of SNBS-3 was a closed circular DNA of 4 076 387 bp in length containing 4 000 protein-coding genes. A total of 3 209, 2 824, 2 560, 147, 12 and 4 functional genes were annotated in the Clusters of Orthologous Groups (COG), Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), Carbohydrate-Active Enzymes (CAZyme), Comprehensive Antibiotic Resistance Database (CARD) and Virulence Factor Database (VFDB), respectively. Using the online software AntiSMASH and Bagel4, we found that it contained genes related to the synthesis of surfactin, mycosubtilin, plipastatin, bacilysin and bacillaene, as well as a complete gene cluster for the synthesis of the bacteriocin subtilosin A. Based on the results of antimicrobial test and proteinase K test, it was hypothesized that B. subtilis SNBS-3 had the ability to synthesize subtilosin A. In conclusion, the whole genome sequencing results of B. subtilis SNBS-3 show that it can produce a variety of bacteriostatic substances and thus have biocontrol potential. The results from this study provide a theoretical basis for further development and application of various bacteriostatic substances including the bacteriocin subtilosin A

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