| As a branched-chain keto-acid,?-ketoisovalerate is an important pharmaceutical intermediate.It is widely used in clinical medicine,such as the tablet of?-ketoacid which treats uremia.Using metabolic engineering strategies to construct a recombinant strain for efficient?-ketoisovalerate synthesis is bound to promote commercial application of?-ketoisovalerate.In this study,efficient fermentation of?-ketoisovalerate was achieved by coordinating the expression of three key genes in the?-ketoisovalerate synthesis pathway,deleting its competitive metabolic pathways,and enhancing the supply of coenzyme.Thereafter,oxygen switches were constructed to realize the synthesis of?-ketoisovalerate with high titer and yield under a simple fermentation process.The main results are as follows:?1?Coordinating the expression of key enzymes.Three key genes for?-ketoisovalerate synthesis which were acetolactate synthase encoding gene alsS derived from B.subtilis 168,acetolactate isomerase reductase encoding gene ilvC and dihydroxyacid dehydratase encoding gene ilvD derived from E.coli MG1655 were cloned and overexpressed on plasmid.The effects of different arrangement orders of the three genes and terminator addition on the plasmid were investigated.After comparing the?-ketoisovalerate synthetic efficiency from pyruvate substrate in vitro,an optimal plasmid p CTSDT was selected.Recombinant strain E.coli BL21?DE3?/pCTSDT can produce 1.5 g·L-1?-ketoisovalerate during 16 h cultivation,which was 3.4-fold higher than the original strain.?2?Deleting the competitive metabolic pathways.Change a recombinant strain B0016-050 with deletions of synthetic routes for acetic acid,formic acid,ethanol,succinic acid and lactic acid used as the host strain.Competing routes for?-ketoisovalerate production such as the branched-chain amino acid transaminase B?ilvE?,isopropylmalate synthase?leuA?and threonine dehydratase?ilvA?were knoked out in strain B0016-050.Meanwhile,T7 RNA polymerase encoding gene was integrated on the chromosome.As a result,deletions of ilvE and leuA could improve?-ketoisovalerate production.The recombinant strain 050T3/pCTSDT can produced 8.5 g·L-1?-ketoisovaleric acid after 26 h fermentation which was increased by4.7 times compared with recombinant strain E.coli BL21?DE3?/pCTSDT.?3?Coordinating the coenzyme circulation and fermentation optimization.In order to balance the redox circulation,the NADPH regeneration system was intensified by overexpressing the transgenic enzyme encoding gene pntAB from E.coli.The expression of the gene pntAB on the chromosome was finely regulated and obtained the recombinant strain050T4/pCTSDT.Then we optimized the fermentation conditions.The optimal fermentation conditions were:cultivating the strain with shaking at 200 r·min-1,and adding 0.4 mmol·L-1IPTG to the broth when the cell density reached OD600 of 2.5.Under these conditions,the titer of?-ketoisovalerate reached 19.2 g·L-1 after 36 h fermentation.The yield of?-ketoisovalerate achieved 0.80 mol·mol-1 glucose.?4?Improving?-ketoisovalerate production through oxygen switch.Oxygen switches were constructed to regulate the?-ketoisovalerate synthetic pathway and pyruvate consumption way.The repressor-encoding gene lacI was knocked out,and the microaerobic promoter Pst was inserted before the T7 RNA polymerase on the chromosome.At the same time,the microaerobic promoter Psc was used to regulate the expression of pyruvate dehydrogenase complex encoding gene lpd through artificial synthetic s RNA.Finally,we obtained strain 050T8/pCTSDT-1.Under microaerobic fermentation with shaking at 100r·min-1,the yield of?-ketoisovalerate reached 0.85 mol·mol-1 glucose,under the micro-oxygen stage,it reached 0.98 mol·mol-1,which closed to theoretical conversion rate,and the specific productivity was 33.3%higher than strain 050T4/pCTSDT. |
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