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.

[Overexpression of Penicillium expansum lipase gene in Pichia pastoris].

The alkaline lipase gene of Penicillium expansum (PEL) was coloned into the yeast integrative plasmid pPIC3.5K, which was then transformed into His4 mutant yeast GS115. Recombinant Pichia strains were obtained by minimal olive oil-methanol plates screening and confirmed by PCR. The expression producus of PEL gene was analysis by SDS-PAGE and olive oil plate, the result indicated that PEL gene was functionally overexpressed in Pichia pastoris and up to 95% of the secreted protein. Recombinant lipase had a molecular mass of 28kD, showing a range similar to that of PEL, could hydrolyze olive oil and formed clear halos in the olive oil plates. Four different strategies (different media, pH, glycerol and methanol concentration) were applied to optimize the cultivation conditions, the activity of lipase was up to 260 u/mL under the optimal cultivation conditions. It is pointed out that the absence of the expensive biotin and yeast nitrogen base in the medium increased the lipase production. The possible reason of this result is absence of yeast nitrogen base increased the medium pH during cultivation, and PEL shows a higher stability at this condition. The lipase activity of the supernatant from the culture grown at pH 7 was higher than the one from the culture in the same medium at pH 6.0 is due to the pH stability of PEL too. The results also showed that the methanol and glycerol concentration had a marked effect on the production of lipase.