ARTP+UV Compound Mutagenesis Combined With Heterologous YbgE Gene Expression To Construct A High-producing Strain Of L-isoleucine

In this study,Escherichia coli K12(Met-)was used as the starting strain.Atmospheric and room temperature plasma(ARTP)and ultraviolet combined mutagenesis breeding methods and heterologous gene overexpression strategies were used.After multiple rounds of mutagenesis,Screening and gene overexpression,a high-yield L-isoleucine strain was obtained,and the fermentation conditions were studied.The main findings are as follows:1.ARTP mutagenesis and selection of Escherichia coli ?-aminobutyric acid(?-AB)resistant mutant strains.Using E.coli K12(Met-)as the starting strain,using ARTP mutagenesis,screening through the microbial high-throughput droplet culture system(MMC),using ?-AB resistance as the selection marker,a high-yield L-isoleucine strain was obtained Acid mutant strain Escherichia coli NXU12(Met-+?ABr),and its L-isoleucine fermentation yield and genetic stability were studied.The results showed that the starting strain E.coli K12(Met-)was subjected to ARTP mutagenesis for 180 s,and a high-yielding strain was selected through single-factor multi-level experiment,adaptive evolution experiment,fermentation culture re-screening,genetic stability verification,etc.-Isoleucine mutagenesis of E.coli NXU12(Met-+?-ABr).The ?-AB resistance concentration of this strain was 49.44 g/L,which was 2.4 times that of the original strain and increased by 140%.After 40 h of fermentation,the growth rate of this strain was consistent with that of the original strain.The L-isoleucine in the fermentation broth was determined by HPLC.The concentration of isoleucine was 17.46 g/L,1.61 times that of the original strain,an increase of 61.22%,and after 10 generations,the genetic traits were stable.2.ARTP and ultraviolet(UV)combined mutagenesis to select high-yield L-isoleucine mutant strains.E.coli NXU12(Met-+?-ABr)was subjected to compound mutagenesis by ARTP and UV.After multiple rounds of mutagenesis and plate screening,lysine auxotrophic strains were obtained,and the fermentation yield and genetic stability of L-isoleucine were researched and some results showed that the mutant strain NXU12(Met-+?-ABr)was subjected to multiple rounds of mutagenesis by ARTP(90 s)+UV(60 s),and a mutant strain of Escherichia coli NXU121(Met-+?-ABr+Lys-)was obtained after preliminary screening and re-screening on the plate.The growth rate of this strain was not affected after 40 hours of fermentation.The concentration of L-isoleucine determined by HPLC reached 18.64 g/L,which was 1.72 times that of the starting strain K12(Met-)and increased by 72.43%,which was 1.07 times that of the control strain NXU12(Met+?-ABr),An increase of 6.94%,and after 10 generations,genetic traits are stable.3.Construction of heterologous gene ybgE expression vector and its effect on L-isoleucine fermentation.The recombinant plasmid vector pET-28a(+)-ybgE was amplified and constructed,and it was transferred into E.coli NXU121(Met-+?-ABr+Lys-)to induce expression.As a result,it was found that the protein band at 33?35 kDa was significantly enhanced compared with the control group.After 40 hours of fermentation,the growth rate of this strain has no effect.The L-isoleucine concentration determined by HPLC reached 20.02 g/L,which was 1.85 times that of the original strain K12(Met-),increased by 84.85%,and was 1.15 times that of the control strain NXU 12(Met-+?-ABr),and increase of 14.66%,1.07 times that of the control strain NXU121(Met-+?-ABr+Lys-),and increase of 7.41%,and after 10 generations,the genetic traits are stable.4.Research on fermentation conditions of recombinant strain L-isoleucine.The fermentation conditions in the shake flask of E coli NXU121(Mer+?-ABr+Lys-)-ybgE introduced with recombinant plasmid were optimized by single factor and response surface method,and the best fermentation and culture conditions of the strain obtained by response surface were:glucose 127.41 g/L and ammonium sulfate 32.97 g/L,corn steep liquor 15.97 g/L,potassium dihydrogen phosphate 3.0 g/L,calcium carbonate 2.0 g/L,magnesium sulfate 2.5 g/L,ferrous sulfate 0.01 g/L.After the fermentation conditions were optimized,the mutant strain was fermented and cultured for 40 hours,and the L-isoleucine concentration determined by HPLC reached 23.22 g/L,which was 15.98%higher than that before optimization.