Genome Shuffling Of Bacillus Subtilis For Improved Riboflavin Production

Genome shuffling has been used in this study to obtain new Bacillus subtilisstrains for high-yield riboflavin production, and optimization of the medium andfed-batch fermentation process were carried out. The main results obtained are asfollows:Zwf and gnd genes from Corynebacterium glutamicum were firstly cloned, andexpression of the site-directed mutant zwf243and gnd361in B.subtilis RH33led toapproximately29.7%increased riboflavin production. The new engineering strainnamed B.subtilis RH33-SGZ was conducted to be parent strain for the genomeshuffling. And then screening was conducted to obtain another parent strain B.subtilisX42, whose riboflavin production restored to7.08g/L.Genome shuffling was then conducted by two parent strains to get fusants, andone fusant named B.subtilis F311was screened out, which has significantenhancement of riboflavin productivity. Riboflavin production of B.subtilis F311byshake flask fermentation was about8g/L, which is14.2%higher than B.subtilis X42and66.7%higher than B.subtilis RH33-SGZ.Plackett-Burman (PB) design was implemented to screen medium componentsthat significantly influence riboflavin production. And three significant effect factorsamong the8variables were found out which were glucose, urea, CuCl2. One of thenegative impact factors was CuCl2whose bottom level was0, so CuCl2was not addedin the futher experiment. Glucose and urea were selected for the response surfaceanalysis for the purpose of getting the optimal medium of riboflavin production. Theproduction of riboflavin was8.58±0.32g/L by the optimized medium in shake flasks,and increased6.98%comparing with the control medium.In the end, fermentation was conducted in the5L-bioreactor, and mediumcomponents and fed-batch fermentation process were optimized, the production ofriboflavin by fed-batch fermentation was sbout12.8g/L.