Reseach On Xylose Efficient Utilization And Transport Mechanism Of Corynebacterium Glutamicum

Lignocellulose is a cheap and widely available renewable resource on the earth.It can not only obtain various renewable bio-based materials through biological and chemical methods,but also fertilize it to produce high value-added chemicals through microorganisms.Xylose is the second content of sugar in the lignocellulosic hydrolysate.Constructing industrial strains that can efficiently utilize and transport xylose,which is necessary for biorefinery of lignocellulosic hydrolysate.Corynebacterium glutamicum has some own unique advantages,and is the preferred strain to produce high value-added chemicals such as amino acids by fermentation using xylose.Corynebacterium glutamicum ATCC 13032 does not have the ability to metabolize xylose.In this study,xylose was utilized by constructing xylose isomerization pathway and plasmids expressing xylA gene from different sources,xylose isomerase from E.coli had the best effect.By expressing three copies of the xylose isomerase-encoding by xylA derived from E.coli MG 1655 at the genomic level,the strain overexpressing the xylA gene with pXMJ19 was subjected to shake flask verification in a medium containing xylose as the sole carbon source,and the results showed with the increase of the copy number of xylA gene,the final biomass of the bacteria gradually increased,and the sugar consumption rate gradually increased,thereby determining the optimal expression intensity of xylA gene.The three-copy expression of xylA gene at the genomic level,the biomass reached the plasmid overexpression level.The genomic level expression has higher growth rate and shorter lag period than the plasmid over-expressing strain,and the final biomass(OD6oo)reaches 25±0.5,the sugar consumption rate reaches 6.4 g/(L·h-1)at 15 h-24 h.The xylose metabolism ability of the genomic level expression strain is improved.The high expression of the enzyme is beneficial to the xylose metabolism and the growth of Corynebacterium glutamicum.In order to further strengthen the xylose metabolism pathway of Corynebacterium glutamicum,this study optimized and expressed the endogenous xylulose kinase gene xylB by different means.The results showed that after knocking out the repressor gene atlR of xylB,the growth of the strain was severely inhibited because the overexpression of xylB gene caused the accumulation of xylulose-5-phosphate to inhibit the growth of the cells;Substitution of xylB gene with gene xyl3 from Pichia pastoris,or overexpression gene xyl3,the growth of the cells is not improved,and the growth of the replaced strain is significantly inhibited.In order to promote the metabolism of xylulose-5-phosphate,overexpressed the genes xyl2 and xyo,and the bacterial biomass is slightly increased.In order to strengthen the linkage between the pentose phosphate pathway and the glycolytic pathway,the PgapA promoter was used to perform the second copy of the genome of the tktltal operon gene,and at the same time,in order to increase the supply of intracellular coenzyme,gene was expressed at genomic level to increase the ability of NADPH synthesis,and gene nadK was expressed to increase the ability of NADP synthesis.The shake flask results showed that by increasing the activity of transketolase and transaldolase and increasing the expression of coenzyme,the growth of the cells was slightly increased,and the OD600 values were increased by 1.0±0.4 and 2.0±0.4,respectively.A comparison of the xylose consumption and glucose consumption of the strain revealed that the rate of extracellular xylose consumption was higher than the rate of glucose consumption,but the co-utilization of glucose and xylose showed that the xylose consumption rate was inhibited in the presence of glucose.In order to achieve the simultaneous utilization of xylose and glucose,The transport system of glucose and xylose in Corynebacterium glutamicum was studied.The necessity of the components of PTS system for glucose and xylose transport was studied by gene knockout.The results showed that after ptsI and ptsH were knocked out,the cells could only grow in xylose medium,and the final biomass was 7.0±0.5 and 8.0±0.5,did not grow in glucose medium;after ptsG was knocked out,the strain did not grow in glucose medium,and grew slower in xylose medium;the full component of EIIBCA was necessary for transporting xylose.Mutation of two phosphorylation sites of the glucose transporter PtsG revealed that the two single mutations or double mutations had no effect on xylose growth,indicating that PtsG transports xylose without undergoing phosphorylation.This study is of great significance for understanding the xylose transport pathway of Corynebacterium glutamicum.It also lays a solid foundation for the comprehensive utilization of lignocellulosic hydrolysate as raw material and by fermentation to produce histidine,aromatic amino acids and nucleosides.