Synthesis Of α-ketoisovalerate By Metabolic Engineered Escherichia Coli

As an important pharmaceutical intermediate,α-ketoisovalerate is one of the main components ofα-ketoacid tablets.It is widely used in the field of medicine and plays an important role in the treatment of chronic uremia.The use of metabolic engineering strategies to modify microbial strains to efficiently synthesizeα-ketoisovalerate by fermentation has great economic value and social significance.In this study,the efficient fermentation synthesis ofα-ketoisovalerate was achieved in Escherichia coli by weakening the competitive metabolic pathways,dynamically regulating bacterial growth metabolic pathways,and balancing the coenzyme circulatory system.The main results are as follows:（1）Static metabolic engineering strategies to weaken competitive metabolic pathways.The starting strain 050T4/p CTSDT was fermented using organic nitrogen source in a 5 L fermentor.The titer ofα-ketoisovalerate was 46.4 g·L^-1,and the by-product isobutanol was 13g·L^-1.In order to reduce the accumulation of isobutanol,the key enzyme acetolactate synthetase（Als S）in the synthetic pathway ofα-ketoisovalerate was modified to reduce the enzyme activity of synthesizing isobutyraldehyde with the substrate ofα-ketoisovalerate,and the binding site sequence of the ribosome of the enzyme was optimized.Then,the strain050T4/p CTSDTQ487S-RBS55 was obtained.The yield ofα-ketoisovalerate in shake flask fermentation was comparable to that of the original strain,and the by-product isobutanol accumulation was 56.3%lower than that of the original strain.In order to further improve the production and yield ofα-ketoisovalerate,the C-terminal of pyruvate dehydrogenase（pdh）was fused to the degradation tag DAS+4,and the tricarboxylic acid cycle（TCA）was weakened to obtain strain 050TY.The oxygen supply intensity and induction time of strain050TY/p CTSDTQ487S-RBS55 were optimized by shake-flask experiment,and the titer ofα-ketoisovalerate was increased by 14.8%compared with the original strain.The accumulation of isobutanol was only 1.5 g·L^-1.（2）Combining CRISPRi technology with degradation tags to dynamically regulate strain growth and metabolic pathways.In order to synthesizeα-ketoisovalerate using inorganic mineral salt medium to reduce the fermentation cost,the pox B gene of the valine prototrophic strain B0016-050 was integrated with the T7 RNA polymerase-encoded gene to obtain strain 050Y/p CTSDT.This strain was used as the starting strain for dynamic regulation.Using CRISPRi technology combined with DAS+4 degradation tag,the expression of ilv E gene and residual enzyme activity Ilv E were inhibited to dynamically regulate valine synthesis,and promoter of the dcas9 gene and its operator gene were optimized to obtain recombinant strain050Y2/p CTSDT+p ACYC-trc-dcas9-444.The titer ofα-ketoisovalerate in this strain was 12.4g·L^-1,and the accumulation of valine was only 0.05 g·L^-1.Meanwhile,using CRISPRi technology and degradation tags,the key enzyme for TCA cycle（pyruvate dehydrogenase）was dynamically regulated to obtain recombinant strain 050Y3/p CTSDT+p ACYC-trc-dcas9-444-478.This strain generated 15.8 g·L^-1α-ketoisovalerate using inorganic salt medium,which was58%higher than that of strain 050Y/p CTSDT,and the accumulation of valine by-product was reduced by 98.7%.（3）Balancing the coenzyme circulatory system.In theα-ketoisovalerate synthetic pathway,two molecules of NADH are produced and one molecule of NADPH is consumed at the same time.In this study,the recombinant strain 050Y4/p CTSDT+p ACYC-trc-dcas9-444-478 was obtained by strengthening the promoter of pyridine nucleotide transhydrogenase（pnt AB）for the regeneration of NADPH.The titer of theα-ketoisovalerate of this strain was increased to 17.1 g·L^-1,which was 10%higher than that of recombinant strain050Y3/p CTSDT+p ACYC-trc-dcas9-444-478.Finally,combining the above optimal strategies,the strain 050Y4/p CTSDTQ487S-RBS55+p ACYC-trc-dcas9-444-478 was obtained.The titer ofα-ketoisovalerate fermented in inorganic salt medium was 16.5 g·L^-1,and the isobutanol by-product was only 0.52 g·L^-1.The fermentation titer of the 5 L fermenter reached 35.2 g·L^-1,and the accumulation of isobutanol was only 1.2 g·L^-1.