Effects of proteases on L-glutamic acid fermentation.

In the process of L-glutamic acid fermentation, there are proteins that cannot be decomposed and utilized by bacteria and that secreted by bacteria at the same time, which cause problems such as increased foam production in the fermentation broth that lowers the dissolved oxygen, which makes the total fermentation efficiency low. Therefore, these proteins can be decomposed by adding proteases in the fermentation broth, and it is found that the best results are obtained by adding 0.5 g/L of trypsin. Proteins can be used by bacteria after being decomposed as well. The final L-glutamic acid production in our research was 177.0 g/L, which is 14.9% more than the control fermentation (154.0 g/L). Similarly, the glucose conversion rate was 68.3%, which is an increase of 4.0% as compared to the control fermentation (65.6%).

Identification of Brevibacterium flavum genes related to receptors involved in bacteriophage BFK20 adsorption.

Phage infection of bacterial cells is a process requiring the interaction between phage receptor binding proteins and receptors on the bacterial cell surface. We prepared a Brevibacterium flavum CCM 251 EZ-Tn5 transposon insertional library and isolated phage-resistant mutants. Analysis of the DNA fragments produced by single-primer PCR was used to determine the EZ-Tn5 transposon insertion sites in the genomes of phage-resistant B. flavum mutants. Seven disrupted genes were identified in forty B. flavum mutants. The phage resistance of these mutants was demonstrated by cultivation analysis in the presence of BFK20, and the adsorption rate of BFK20 to these mutants was tested. B. flavum mutants displayed significantly reduced adsorption rates; the lowest rate was observed for mutants containing interrupted major facilitator superfamily (MFS) protein and glycosyltransferase genes. Uninterrupted forms of these genes were cloned into corynebacterial vector pJUP06 and used for in trans complementation of the corresponding B. flavum mutants. The growth of these complemented mutants when infected with BFK20 closely resembled that of wild-type B. flavum. These complemented mutants also exhibited similar BFK20 adsorption as the wild-type control. We infer that the disrupted MFS protein and glycosyltransferase genes are responsible for the phage-resistant phenotype of these B. flavum transposition mutants.

Genome shuffling and high-throughput screening of Brevibacterium flavum MDV1 for enhanced L-valine production.

L-valine is an essential branched-amino acid that is widely used in multiple areas such as pharmaceuticals and special dietary products and its use is increasing. As the world market for L-valine grows rapidly, there is an increasing interest to develop an efficient L-valine-producing strain. In this study, a simple, sensitive, efficient, and consistent screening procedure termed 96 well plate-PC-HPLC (96-PH) was developed for the rapid identification of high-yield L-valine strains to replace the traditional L-valine assay. L-valine production by Brevibacterium flavum MDV1 was increased by genome shuffling. The starting strains were obtained using ultraviolet (UV) irradiation and binary ethylenimine treatment followed by preparation of protoplasts, UV irradiation inactivation, multi-cell fusion, and fusion of the inactivated protoplasts to produce positive colonies. After two rounds of genome shuffling and the 96-PH method, six L-valine high-yielding mutants were selected. One genetically stable mutant (MDVR2-21) showed an L-valine yield of 30.1 g/L during shake flask fermentation, 6.8-fold higher than that of MDV1. Under fed-batch conditions in a 30 L automated fermentor, MDVR2-21 accumulated 70.1 g/L of L-valine (0.598 mol L-valine per mole of glucose; 38.9% glucose conversion rate). During large-scale fermentation using a 120 m3 fermentor, this strain produced > 66.8 g/L L-valine (36.5% glucose conversion rate), reflecting a very productive and stable industrial enrichment fermentation effect. Genome shuffling is an efficient technique to improve production of L-valine by B. flavum MDV1. Screening using 96-PH is very economical, rapid, efficient, and well-suited for high-throughput screening.

Report: Bioconversion of agriculture waste to lysine with UV mutated strain of brevibacterium flavum and its biological evaluation in broiler chicks.

Lysine executes imperative structural and functional roles in body and its supplementation in diet beneficial to prevent the escalating threat of protein deficiency. The physical mutagenesis offers new fascinating avenues of research for overproduction of lysine through surplus carbohydrate containing agriculture waste especially in developing countries. The current study was aimed to investigate the potential of UV mutated strain of Brevibacterium flavum at 254 nm for lysine production. The physical and nutritional parameters were optimized and maximum lysine production was observed with molasses (4% substrate water ratio). Moreover, supplementation of culture medium with metal cations (i.e. 0.4% CaSO4, 0.3% NaCl, 0.3% KH2PO4, 0.4% MgSO4, and 0.2% (NH4) 2SO4w/v) together with 0.75% v/v corn steep liquor significantly enhanced the lysine production up to 26.71 ± 0.31 g/L. Though, concentrations of urea, ammonium nitrate and yeast sludge did not exhibit any profound effect on lysine production. Biological evaluation of lysine enriched biomass in terms of weight gain and feed conversion ratio reflected non-significant difference for experimental and control (+ve) groups. Conclusively, lysine produced in the form of biomass was compatible to market lysine in its effectiveness and have potential to utilize at commercial scale.

(L)-Valine production with minimization of by-products' synthesis in Corynebacterium glutamicum and Brevibacterium flavum.

Corynebacterium glutamicum ATCC13032 and Brevibacterium flavum JV16 were engineered for L-valine production by over-expressing ilvEBN ( r ) C genes at 31 °C in 72 h fermentation. Different strategies were carried out to reduce the by-products' accumulation in L-valine fermentation and also to increase the availability of precursor for L-valine biosynthesis. The native promoter of ilvA of C. glutamicum was replaced with a weak promoter MPilvA (P-ilvAM1CG) to reduce the biosynthetic rate of L-isoleucine. Effect of different relative dissolved oxygen on L-valine production and by-products' formation was recorded, indicating that 15 % saturation may be the most appropriate relative dissolved oxygen for L-valine fermentation with almost no L-lactic acid and L-glutamate formed. To minimize L-alanine accumulation, alaT and/or avtA was inactivated in C. glutamicum and B. flavum, respectively. Compared to high concentration of L-alanine accumulated by alaT inactivated strains harboring ilvEBN ( r ) C genes, L-alanine concentration was reduced to 0.18 g/L by C. glutamicum ATCC13032MPilvA△avtA pDXW-8-ilvEBN ( r ) C, and 0.22 g/L by B. flavum JV16avtA::Cm pDXW-8-ilvEBN ( r ) C. Meanwhile, L-valine production and conversion efficiency were enhanced to 31.15 g/L and 0.173 g/g by C. glutamicum ATCC13032MPilvA△avtA pDXW-8-ilvEBN ( r ) C, 38.82 g/L and 0.252 g/g by B. flavum JV16avtA::Cm pDXW-8-ilvEBN ( r ) C. This study provides combined strategies to improve L-valine yield by minimization of by-products' production.

Improvement of L-valine production at high temperature in Brevibacterium flavum by overexpressing ilvEBNrC genes.

Brevibacterium flavum ATCC14067 was engineered for L: -valine production by overexpression of different ilv genes; the ilvEBN(r)C genes from B. flavum NV128 provided the best candidate for L: -valine production. In traditional fermentation, L: -valine production reached 30.08 ± 0.92 g/L at 31°C in 72 h with a low conversion efficiency of 0.129 g/g. To further improve the L: -valine production and conversion efficiency based on the optimum temperatures of L: -valine biosynthesis enzymes (above 35°C) and the thermotolerance of B. flavum, the fermentation temperature was increased to 34, 37, and 40°C. As a result, higher metabolic rate and L: -valine biosynthesis enzymes activity were obtained at high temperature, and the maximum L: -valine production, conversion efficiency, and specific L: -valine production rate reached 38.08 ± 1.32 g/L, 0.241 g/g, and 0.133 g g(-1) h(-1), respectively, at 37°C in 48 h fermentation. The strategy for enhancing L: -valine production by overexpression of key enzymes in thermotolerant strains may provide an alternative approach to enhance branched-chain amino acids production with other strains.

Construction of a novel shuttle vector for use in Brevibacterium flavum, an industrial amino acid producer.

Brevibacterium flavum has been developed to produce amino acids l-valine, l-lysine and l-threonine. However, there are not enough vectors available for the research on metabolic engineering in Brevibacterium flavum. Here we have constructed a shuttle expression vector pDXW-8 between Escherichia coli and B. flavum. The vector harbors an origin oriE for replicating in E. coli, a second origin oriC for replicating in B. flavum, a large multiple cloning site including 11 single restriction enzyme sites and suitable for cloning multiple genes or large DNA fragments, a tac promoter and a lacI(PF104) fragment which tightly controls the tac promoter. The applicability of pDXW-8 was confirmed by the expression of the vhb gene in B. flavum. The vector pDXW-8 will be very useful for research on metabolic engineering in corynebacteria.