Metabolic Engineering Of Corynebacterium Glutamicum For L-leucine Fermentation

As one of eight essential amino acids,L-leucine that cannot be synthesized by themselves must be supplied from an external soure for humans and animals.It has various physiology functions,which is currently widely used in industries ranging from food,cosmetics,and infusion soultions in pharmaceutical industries.With the increase of people's health care awareness and the fast-growing market demand for L-leucine,the production of L-leucine by microbial fermentation has received more and more attention.Additionaly,with the continuous development of molecular biology technology and in-depth research on the mechanism of L-leucine synthesis of Corynebacterium glutamicum,metabolic engineering methods are used to perform microorganisms to produce L-leucine through rational design.This study focuses on the research of a L-leucine producing strain C.glutamicum XQ-9through mutation breeding,and the production of L-leucine is improved with the employment of metabolic engineering strategies.Furthermore,the L-leucine production of targed-strain is further improved with the optimization of medium and fermentation parameters.The main conclusions are as follows:?1?Improving the activity of key enzymes for L-leucine synthesis and optimization of cofactor supply for altering redox balance.Sequencing analysis of genes encoding key enzymes in the L-leucine biosynthesis of C.glutamicum XQ-9,there were multiple amino acid mutations in the key genes.The accumulation of L-leucine in the C.glutamicum XQ-9revealed that the feedback inhibition and repression of key enzymes in the L-leucine synthesis had been relieved.The deletion of ltbR and overexpression of genes?leuA,ilvBNC,and ilvE?were performed to enhance L-leucine biosynthesis.Then,according to the predicted analysis of the coenzyme binding site,the site-directed mutagenesis of amino acids 34,48 and 49 of acetohydroxyacid isomeroreductase?AHAIR?were changed from the original S,L and R to G,E and F,so that AHAIR^M changed the use of cofactor from NADPH to NADH.The NAD-specific leucine dehydrogenase?Leu DH?encoded by leuDH from Lysinibacillus sphaericus was in turn expressed heterologously,and the strain XL-2 was constructed.The strain XL-2/ABNC^MLDH increased the use of NADH and reduced the consumption of NADPH.The intracellular NADPH concentration increased to 5.34?mol.g^-1,and NADPH/NADP⁺was 0.81.Shake-flask fermentation of the XL-2/ABNC^MLDH strain resulted in the production of 22.9 g·L^-11 L-leucine,and the L-valine production was only 3.7 g·L^-1,which was beneficial to separate and purify L-leucine.?2?Enhancing the availability of pyruvate to improve L-leucine production.The availability of pyruvate was enhanced by regulating the TCA cycle complementation pathway and weakening the L-alanine biosynthesis.First,the pyc gene encoding pyruvate carboxylase was deleted in XL-2 strain for reducing the metabolic flux of TCA cycle and supplying more pyruvate for the L-leucine biosynthesis.Next,the avtA gene encoding alanine aminotransferase AvtA was deleted for reducing the competitive consumption of pyruvate.The transcriptional level of the alaT gene enconding alanine aminotransferase AlaT was downregulated by inserting suitable strength terminator for increasing the metabolic flux of L-leucine biosynthesis from pyruvate,and reducing the accumulation of L-alanine.The strain WL-8 was constructed.Shake-flask fermentation of the WL-8 strain resulted in the production of 18.1 g·L^-11 L-leucine.?3?Strengthing the metabolic flux of L-leucine biosynthesis.First,the genes involved in L-leucine biosynthesis were overexpressed by replacing the native promoters P_leuAeuA and P_ilvBNClvBNC of leuA gene and ilvBNC operon with the promoter P_tuf of eftu,encoding elongation factor Tu,and the metabolic flux of L-leucine biosynthesis was enhanced.Then,the overexpression of leuA was performed to enhance the?-isopropylmalate biosynthesis from?-ketoisovalerate,which was the direct precursor of L-valine,and the metabolic flux of L-leucine was enhanced.To avoid carring plasmid and slower growth,the IPMS was overexpressed by implementation of an additional copy of leuA gene in genome,and the strain WL-13 was constructed.Shake-flask fermentation of the WL-13 strain resulted in the production of 28.7 g·L^-1L-leucine,which was significantly higher than WL-8 strain.Additionally,the accumulation of L-valine and L-alanine was significantly decreased compared to WL-8 strain.?4?Improvement of the supply of acetyl-Co A and glucose metabolism.First,the transcriptional level of the glt A gene enconding cirate synthase was downregulated by inserting terminator in front of gltA gene for decreasing the metabolic flux of TCA cycle,and supplying more acetyl-CoA into L-leucine biosynthesis.Next,the acetyl-CoA synthease gene acs and its activator protein gene cobB from E.coli were overexpressed for increasing the supply of acetyl-CoA,resuting in the decrease of glucose consumption.Then,the transcriptional regulator SugR was stepwise deleted for releasing the transcriptional inhibition of the ptsG gene for increasing the utilization of glucose,and strain JL-3 was constructed.Shake-flask fermentation of the JL-3/AcsCobB strain resulted in the production of 30.9 g·L^-1L-leucine,?5?The composition of L-leucine fermentation medium and fermentation conditions were optimized by single factor experiment and response surface experiment.The optimal composition of medium was(g·L^-1):glucose 140.0,?NH₄?₂SO₄ 23,NH₄Ac 15,corn steep liquor 22,KH₂PO₄ 0.8,MnSO₄·H₂O 0.01,MgSO₄·7H₂O 0.5,Na₃C₆H₅O₇·2H₂O 3,urea 3,biotin 5×10^-5,thiamine 2×10^-4,L-methimine 0.7,L-isoleucine 0.06,L-glutamate 0.5,CaCO₃30.0.The optimal fermen-tation condition was:liquid volume 50 mL/500 mL,inoculum size10%,initial pH7.0,culture temperature 30°C.Under this optimized condition,L-isoleucine reached 32.8 g·L^-1 after 72 h,which was 6.1%higher than that of before optimization.The fermentation process parameters were also optimized at 5 L fermenter,including dissolved oxygen level and feeding.Under the condition that the residual sugar was controlled at 20g·L^-1,and the dissolved oxygen was controlled at 15%in the fed-batch fermentation,the final L-leucine yield and the glucose conversion rate reached 40.1 g·L^-1 and 0.25 g·g^-1.