Metabolic Properties Analysis Of K. Pneumoniae Of ALDC Gene Knockout And Overexpression

Diacetyl is a safe flavoring compound meeting the Prescribed Standards, which gives a special buttery flavor to the finished product. At present, diacetyl produced by extraction methods is very high-priced because of raw materials in short supply. Diacetyl produced by chemical methods is not accepted in food service industry, in consideration of safety reasons. Bioconversion is the most potential method to produce diacetyl, while the low yield is the main limiting factor to be industrialized. Microbial fermentation method has been attracted much attention in recent years. The major focus of my thesis is put on the Klebsiella Pneumoniae CICC10011as the initial strain. Recombinant strains can be selected through genetic engineering and were cultivated to get the change in product yield. The gene of ALDC as a key enzyme was modified, including gene overexpression and gene knock-out. The function of ALDC was analyzed after researching into phenotype of recombinant strains.In the experiment of gene overexpression, the gene of ALDC encoding a-acetolactate decarboxylase was amplified by PCR from the genomic DNA of Klebsiella Pneumoniae CICC10011. After ALDC sequenced, the recombinant vector PBR322-ALDC was constructed and transformed into Klebsiella Pneumoniae CICC10011compentent cells by electroporation. Recombinant strains were selected by tetracycline resistance gene. It showed that the change of2,3-butanediol was not obvious during the cultivation,37g/L2,3-butanediol obtained by Klebsiella Pneumoniae fermenting80g/L glucose. While the yield of acetoin was obviously higher than initial strain, running up to13.11g/L in recombinant strains. And the conversion ratio was16.4%,28percent more than initial strain. The enzyme activity of a-acetolactate decarboxylase reached0.173U/mg in the recombinant strain,1.6times than initial strain. The results demonstrated that a-acetolactate decarboxylase was a key point to the production of acetoin and did not lead to the production of diacetyl through non-enzymatic oxidation.In the experiment of gene knock-out, recombination gene was amplified by overlapping PCR. Recombination gene was transformed into Klebsiella Pneumoniae CICC10011compentent cells by electroporation after being sequenced. Recombinant strains were selected by ampicillin gene. The results of cultivation had revealed that the yield of diacetyl was0.14g/L in recombinant strains, while there did not produce diacetyl in initial strains. The yield of acetoin was78.4percent less than initial strain. There was not2,3-butanediol and enzymatic activity of ALDC in recombinant strains. As stated previously, recombinant strains without gene of ALDC has helped to increase production of diacetyl through non-enzymatic oxidation and caused the change of yield of byproducts.In short, there were not diacetyl both in initial strain and recombinant strain of overexpression, while the increase of acetoin was obvious in recombinant strain of overexpression. It showed that non-enzymatic oxidation was quite weak in initial strain and recombinant strain of overexpression. After the overexpression of ALDC, the yield of acetoin was increased clearly. The increase of acetoin did not result in production of diacetyl, because the activity of acetoin oxidase was low or absent. There was a small quantity of diacetyl in recombinant strains without gene of ALDC. Compared with initial strain, the production of acetoin clearly fell off and the outputs of2,3-butanediol were approaching zero. This emphasized that the knock-out of ALDC made the non-enzymatic oxidation grow, and a handful of diacetyl had been found. Due to the effect of diacetyl reductase, diacetyl converted into acetoin. While a small quantity of acetoin can not convert into2,3-butanediol, besides knock-out of ALDC might influence the oxidation-reduction system of metabolic pathway.