Preliminary Study On Metabolic Engineering Of Bacillus Subtilis For Acetoin Production

Acetoin is an important platform compound, widely used in food and otherindustries. Focusing on the acetoin biosynthetic reactions, Bacillus subtilis168wasengineered as the starting strain, aiming at enhancing the acetoin yield andproductivity with Bacillus subtilis by metabolic engineering. The bdhA and acoAgenes were disrupted, the alsSD operon was overexpressed, and the pta and ldh geneswere deleted, to increase the acetoin biosynthis and reduce the accumulation of itscompeting compounds.Applying the approaches of constructing the double-crossover knock-out plasmidand double-crossover overexpressing plasmid, the bdhA and acoA genes wereinactiviated in B. subtilis168to obtain the strains with single disruption of bdhA oracoA and the strain BSUWO3with disruption of both genes. Under the mirco-aerobicflask fermentations, for the bdhA-disrupted mutant,2,3-butanediol production wasalmost abolished in early stationary phase and was not detected until9h after glucosewas exhausted. And the maximum acetoin yield increased by10%compared to that ofB. subtilis168. Acetoin was almost not degraded in acoA-inactiviated strain, henceproduced more acetoin. The bdhA and acoA double-knockout strain exhibited anacetoin yield of0.72mol/mol, which increased by16.4%compared to B. subtilis168.Subsequently, overexpression of the alsSD operon enhanced the acetolactatesynthase activity by52%and66%in exponential and stationary phase, respectively.And0.78mol acetoin/mol glucose was obtained in alsSD-overexpressed strain.However, by constucting the pta-deletion plasmid through inserting the tetracyclineresistence gene tet, further deletion of pta gene caused little increase of acetoinproduction. But after comparing the acetoin production peformance of differentstrains, the pta-deleted strain BSUW06was demonstrated to be the best acetoinproducer.To elevate the acetoin productivity of the engineered strain, different medium andoxygen conditions were used for high concentration glucose flask fermentation. Foracetoin production by the engineered strain BSUW06, the acetoin volumetricproductivity was improved from0.087g/（L·h）^-1using M9medium plus30g/L glucose under micro-aerobic condition, to0.273g/（L·h）^-1using LB medium plus50gglucose/l under aerobic condition. Under this aerobic condition, the decrease ofacetoin yield was not obvious. In fermentor culture, BSUW06could achieve a39.4%increase of acetoin titer （up to19.8g/L） than B. subtilis168. These resultsdemonstrate that enhancement of acetoin production by B. subtilis could besuccessfully achieved by metabolic engineering, providing some valuable informationfor other studies.