Genetic engineering of Bacillus sp. and fermentation process optimizing for diacetyl production

Diacetyl, an important flavor extensively used in the food industry, can be produced from the non-enzymatic oxidative decarboxylation of alpha-acetolactate in bacteria fermentation. In previous work, we obtained a strain of Bacillus sp. DL01-Delta alpha lsD with low diacetyl accumulation. The strain was engineered and optimized for improving the production of diacetyl in this study. First, deletion of the gene encoding phosphotransacetylase (pta), by homologous recombination with high temperature sensitive shuttle plasmid vector pKS1, led to a reduction of acetate and 130% increase of diacetyl production in B. sp. DL01-Delta alpha lsD-alpha pta. Then overexpression of alpha-acetolactate synthase (ALS) from B. subtilis 168 in B. sp. DL01-Delta alpha lsD-Delta pta resulted in efficient diacetyl production with a titer of 5.43 g/L. To further increase diacetyl production, single factor and orthogonal experimental data were used to predict the optimal fermentation conditions by Back Propagation neural network. Optimal value of K(L)a (Dissolved oxygen volume coefficient) was 12.4 h(-1) with fermentation parameters of aeration rate 0.66 vvm, agitation speed 179 rpm and temperature 35.7 degrees C. A titer of 11.18 g/L diacetyl, the highest reported diacetyl production, was achieved by fed-batch fermentation at the optimal condition using the metabolic engineered strain of B. sp. DL01-Delta alpha lsD-alpha pta-alpha ls(168). These results are of great importance as a new way for the efficient production of diacetyl by food-safety bacteria.