Efficient Production Of Acetoin And 2,3-butanediol By Metabolically Engineered Corynebacterium Crenatum

As an important platform compound, acetoin is widely used in medicine and food industries; 2,3-butanediol as its reduced product, is also an important chemical material and liquid fuel, widely used in chemistry and aerospace fields. Lately, to produce acetoin and 2,3-butanediol by microbiological fermentation has received increasing attention. In this work, the strain C. crenatum SYPA5-5 which is Generally Recognized As Safe（GRAS）, was studied and engineered for the production of acetoin and 2,3-butanediol respectively.The alsS. alsD and bdh A genes（encoding α-acetolactate synthase（ALS）. α-acetolactate decarboxylase（ALDC）. 2,3-butanediol dehydrogenase（BDH） respectively） from B. subtilis 168 were cloned, and inserted into C. crenatum-E. coli shuttle plasmid pXMJ19, The three key enzymes named as ALS. ALDC and BDH were expressed successfully in C. crenatum. Shake flask fermentation was carried out with glucose as substrate, original C. crenatum as control. The recombinant strain C. crenatum/pXMJ19-alsSD not only produced 13.59 g·L^-1 acetoin, but also accumulated up to 10.61 g·L^-1 2,3-butanediol, indicating that there may existing some enzyme catalyzing the reaction between acetoin and 2,3-butanediol. The recombinant strain C. crenatum/pXMJ19-alsSD-bdhA produced 15.16 g·L^-1 2,3-butanediol, improved by 43%.Under aerobic fermentation, the pyruvate that generated from glycolytic pathway was metabolized through TCA cycle, the metabolism of acetoin and 2,3-butanediol with pyruvate as precursor was at a disadvantage. The recombinant strain can produce acetoin and 2,3-butanediol by whole-cell biocatalyst with glucose as substrate. The recombinant strain C. crenatum/pXMJ19-alsSD cells were used to convert glucose to acetoin. This strain produced 9.86 g·L^-1 acetoin and 12.76 g·L^-1 2,3-butanediol, 24.50 g·L^-1 lactate; The recombinant strain C. crenatum/pXMJ19-alsSD-bdhA cells were used to convert glucose to 2,3-butanediol. The strain generated 5.29 g·L^-1 acetoin and 17.08 g·L^-1 2,3-butanediol, 36.01 g·L^-1 lactate.This work characterized the 2,3-butanediol dehydrogenase（2,3-BDH） from C. crenatum. The optimum temperature for 2,3-BDH activity was 35 °C, the optimum pH was pH 4.0 and pH 10.0 respectively for reduction reaction and for oxidation reaction. Enzyme kinetics indicated that diacetyl was also an important substrate for this enzyme as acetoin.The yield of lactate with pyruvate as precursor was also high during whole-cell biocatalyst, so the ldh gene was deleted in C. crenatum. After deletion of ldh gene, the yield of 2,3-butanediol was increased to 25.93 g·L^-1, improved by 52%, the lactate was reduced by 87%. After inactivation of both butA and ldh genes, the recombinant strain C. crenatumΔbutAΔldh/pXMJ19-alsSD accumulated up to 23.56 g·L^-1 acetoin, improved by 1.39 folds, little 2,3-butanediol and lactate were detected. The productivity of acetoin was improved by 1.12 folds, the molar yield from glucose was increased by 1.52 folds. At the same time, the yield of acetate was also increased.