Directional Breeding Of 2-keto-L-gulonic Acid High-yield Strain

2-keto-L-gulonic acid（2-KLG）is a direct precursor of vitamin C produced by fermentation.Sorbose dehydrogenase（SDH）is one of the key nodes for one-step fermentation to produce vitamin C.In the preliminary study,we obtained a FAD-dependent sorbose dehydrogenase derived from the Gluconobacter oxydans WSH-004,which can achieve expression in different expression systems.Due to the low activity of sorbose dehydrogenase,if it needs to be used for one-step fermentation to produce 2-KLG,its enzyme activity needs to be greatly improved.In this paper,the sorbose dehydrogenase derived from G.oxydans WSH-004 was heterologously expressed in Escherichia coli,which it was found that the enzyme can directly ferment L-sorbose to 2-KLG.By optimizing the promoter,screening the expression host,optimizing the fermentation temperature,knocking out aldosterone reductase enzyme genes,knocking out relateing enzyme genes in phosphotransferase system and mutation screening of the sorbose dehydrogenase based on error-prone PCR to constructed a high-throughput screening platform suitable for sorbose dehydrogenase research.These results have important reference value for metabolic engineering modified microorganisms to realize2-KLG production by one-step fermentation.The main research results of the paper were as follows:（1）Based on metabolic engineering,this experiment mainly studied the factors that affected the activity of sorbose dehydrogenase and the production of 2-KLG in E.coli.First,selection of expression conditions suitable for sorbose dehydrogenase in E.coli to determine the better expressed host E.coli BL21（DE3）,and the expressed promoter P_cspA,and the fermentation temperature of 37^oC,to make 2-KLG yield reached 2.83 g·L^-1 with 10 g·L^-1 L-sorbose as substrate.Through gene knockout,it was found that the aldosterone reductase enzyme affected the yield of 2-KLG,and by optimizing the cultivation method,the maximum yield of 2-KLG reached 8.31 g·L^-1 with 20 g·L^-1 L-sorbose as substrate,and two key enzymes PtsI and PtsH were identified that affected the metabolic pathway from L-sorbose to 2-KLG in the phosphotransferase system.Based on the above experimental results,a screening platform suitable for expressing sorbose dehydrogenase was initially established in E.coli.（2）Applying microbial microdroplet culture system（MMC）,the problem was studied that the starting strain does not tolerate high concentration of L-sorbose.Based on the MMC culture,two strains 1-18 and 2-F6 were obtained that capable of tolerating high concentrations of L-sorbose.When the SDH was overexpressed in the evolved strain for 48h,the yield of 2-KLG in the medium of 1%L-sorbose,2%L-sorbose and 3%L-sorbose was 2.43 g·L^-1,4.11 g·L^-1,4.42 g·L^-1.When SDH/SNDH was co-expressed in the evolved strains for 48 h,the yield of 2-KLG in 1%L-sorbose,2%L-sorbose,and 3%L-sorbose medium was 5.10 g·L^-1,5.40 g·L^-1,6.05 g·L^-1.（3）Combining error-prone PCR and high-throughput screening technology to modified sorbose dehydrogenase that improved the production of 2-KLG from L-sorbose catalyzed by sorbose dehydrogenase expressed in E.coli.Based on the pre-established sorbose dehydrogenase screening platform for research.After two rounds of error-prone PCR and high-throughput screening,a positive mutant was selected from 1.3×10⁴ mutants,and 2-KLG production reached 5.92 g·L^-1,an increase of 14.1%compared to the control 2-KLG production.In addition,a mutant was found in the beneficial mutant.Amino acid 316 of this mutant was mutated to a stop codon,and sorbose dehydrogenase was divided into two modules for expression.On this basis,an in-depth study of the original sorbose dehydrogenase found that the sorbose dehydrogenase was divided into two parts,with the help of sorbosone dehydrogenase（SNDH）,which can also catalyze the production of 2-KLG from L-sorbose.