Research On The Metabolic Engineering Of L-malic Acid Biosynthesis From Xylose

L-malic acid is a naturally occurring organic acid with significant physiological function and promising marketing prospect.Xylose is a widespread biomass feedstock,which is the second most monosaccharide after glucose in lignocellulose.The development of microorganisms which could use xylose as substrate for industrial fermentation has a significant effect on improving the utilization of lignocellulose.Therefore,it is of great significance to develop strains capable of synthesizing malic acid from xylose.Firstly,with the help of ?-Red recombination technique,the endogenous tricarboxylic acid pathway of Escherichia coli was modified to remove the malate synthase gene maeAB,the malate dehydrogenase gene mdh and the fumacate gene cluster fumACB.The,the glycolic acid producing pathway from xylose was introduced to form the malic acid synthesis pathway.We found that with the increasement of inactivated genes,cell growth and xylose utilization rate gradually decreased.The MA-05 strain,which harboring all of the above-mentioned gene deletions,accumulated 1.99 g/L of L-malic acid while consuming 4.24 g/L D-xylose in the 72 h shake flask cultivation.The yield was 0.47 g malic acid/g xylose,reaching 52%of the theoretical yield.Secondly,the glycolic acid oxidase glcDEF gene cluster and the malic acid synthase gene glcB and aceB were overexpressed to strengthen the malate synthesis pathway.The results showed that overexpression of glcDEF and glcB promoted the synthesis of malic acid,while the overexpression of aceB strongly inhibited cell growth.The MA-10 strain overexpressing glcDEF and glcB simultaneously consumed 5.22 g/L xylose in 72 h shake flask fermentation and produced 4.53 g/L L-malic acid,with the yield of 0.87 g malic acid/g xylose,which is currently known as the highest yield of malic acid production using xylose as substrate.Finally,the intracellular redox levels was regulated by the expression of NADH oxidase gene noxE and catalase gene katE.The results showed that the expression of noxE gene could promote the cell growth,but had no effect on the synthesis of malic acid.The overexpression of the katE gene can significantly promote cell growth and accelerate the synthesis of L-malic acid.The MA-11 strain,which overexpressed the katE gene,can accumulate 5.9 g/L of L-malic acid,which is 30%higher than that of the control strain.Thus,the overexpression of katE probably reduce H2O2 accumulation,releasing its toxic effects on cell growth,thereby increasing the biomass and malic acid production.