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.
['Huang|Qin-Geng|QG|', 'Zeng|Bang-Ding|BD|', 'Liang|Ling|L|', 'Wu|Song-Gang|SG|', 'Huang|Jian-Zhong|JZ|']
D001388:Aziridines D061249:Batch Cell Culture Techniques D018533:Biomass D019149:Bioreactors D040142:Brevibacterium flavum D032621:DNA Shuffling D005285:Fermentation D016680:Genome, Bacterial D042822:Genomic Instability D005947:Glucose D057166:High-Throughput Screening Assays D007218:Industrial Microbiology D008561:Membrane Fusion D016296:Mutagenesis D009154:Mutation D011523:Protoplasts D013997:Time Factors D014466:Ultraviolet Rays D014633:Valine
Genome shuffling High-throughput screening Brevibacterium flavum L-valine Fermentation