Rewiring The Glucose Transportation Pathway And Central Metabolic Pathway For Overproduction Of N-acetylglucosamine In Bacillus Subtilis

Glucosamine（GlcN）and its derivative N-acetylglucosamine（GlcNAc）are important functional monosaccharides,which have been widely used in medicine,food,health care and cosmetics.It is reported that the global glucosamine（including GlcN and GlcNAc）market is about$5 billion in 2019.The demand of GlcN and GlcNAc products will continue to increase due to the improvements of life quality and aggravation of the aging of the population.Currently,industrial production methods of GlcN and GlcNAc include chemical hydrolysis,enzyme conversion and microbial fermentation.Compared with chemical hydrolysis and enzyme conversion,microbial fermentation has some significantly superiorities,including sustainability,environmental protection and renewable of raw materials,thus,it is an important way to solve the problems of resources and environment.For this,this study started with a GlcNAc producing Bacillus subtilis strain BSGN6P_{xyl A}-glmS,and further,using metabolic engineering strategies,including transporter engineering,carbon metabolism engineering and cofacter engineering,improved the performances of engineering strains.The main results of this study include:（1）For optimization of the glucose transportation,genes yyzE,ypqE,and ptsG,encoding EIIA^Glc and EIIBC^Glc components,respectively,were firstly deleted to disrupt the PTS^Glclc transport system in strain BSGN6 P_xylA-glmS.Then,the promoters of glucose/H⁺symporter and glucokinase encoding genes glcP and glcK were replaced with the strong constitutive promoter P₄₃ to strengthen NPTS^Glc transport system.In line with this,several glucose/H⁺symporters were tested,and that from B.subtilis gave the best result,which increased GlcNAc titer to 10.1 g·L^-1.Next,initiation codon engineering strategy was performed to optimize the activities of competitive pathways,including pentose phosphate pathway,glycolysis and peptidoglycan synthesis pathway,which further increased GlcNAc titer,giving13.2 g·L^-1 in shake flasks and 42.1 g·L^-1 in 3-L batch fermentation;（2）Based on carbonic mass balance,a mathematical model of GlcNAc production was established,which indicated that GlcNAc yield depends on GlcNAc pathway rigidity and theoretical pathway yield.To improve GlcNAc pathway rigidity,carbon flux of glycolysis was firstly forced to GlcNAc pathway by blocking pyruvate synthesis,which increased yield to 0.263 g·g^-1 _Glucose.Subsquently,promoters of key genes pfkA and fba A in glycolysis were replaced with P₄₃ promoter,and gene pckA,encoding phosphoenolpyruvate carboxykinase,was knocked out in the engineering strain to avoid the feedback regulation caused by accumulated phosphoenolpyruvate,which gave the 10.1 g·L^-1 titer of GlcNAc.Next,for relieving the overflow metabolism and avoiding pyruvate replenishment,the promoter of gene pycA,encoding pyruvate carboxylase,was replaced with P₄₃ promoter,and malate dehydrogenase encoding genes melA,malS,ywkA and ytsJ were knocked out.Specifically,introduction of pyruvate carboxylase from B.cereus completely eliminated the synthesis of central metabolic byproducts,as a result,the enginerring strain gave 13.3 g·L^-1 titer and 0.325g·g^-1 _Glucose yield of GlcNAc in shake flask;（3）To further improve GlcNAc pathway rigidity,several glucosamine-phosphate N-acetyltransferases were tested,and that from Anthracocystis flocculosa was comfirmed as the best one.Then,the expression cassette of gene glmS,encoding glutamine-fructose-6-phosphate transaminase,under the control of P₄₃ promoter was integrated in the engineering strain to avoid the suppression of xylose-induced promoter P_xylAylA acitivity by glucose,and thus,circumvented the feedback inhibition of the expression of gene glmS by GlmS ribozyme,increasing GlcNAc titer to 15.9 g·L^-1.Furthermore,using P₄₃promoter regulated the expression of LysR family protein GltC,and genome integrataion of E.coli’s glutamate dehydrogenase coding gene EcglnA was performed in engineering strain.As a result,GlcNAc reached to 17.1 g·L^-1.Additionally,expression of genes pduA and RmmH from Salmonella enterica and Mycobacterium smegmatis could form artificial organelle structure in B.subtilis.However,the immobilization of GlcNAc key enzymes afGNA1 and GlmS in organelles via CC-Di-A/B showed a negative result;（4）The concentration of intracellular NADH in BP17-afGNA1 is 3.22μmol·g^-1 _DCW,which is 3.5-fold of that in BP17,indicating that the synthesis of GlcNAc would lead to the accumulation of NADH.However,the accumulation of NADH will reduce the theoretical yield of GlcNAc pathway.In addition,the titer and yield of GlcNAc significantly reduced when the synthesis of 2,3-butanediol was blocked by deleting gene bdhA,encoding butanediol dehydrogenase,which indicates that the synthesis of 2,3-butanediol is to balance the reducing equivalents during the process of GlcNAc production.To balance the reducing equivalents,NADH oxidase was expressed under the control of different domestic-strength promoter,however,that all resulted in reduced GlcNAc titer,indicating expression of NADH oxidase with non-metabolic coupling conditions caused intracellular energy disturbance,and thus,reduced the performance of engineering strains.Expecially,in order to balance the reducing equivalents,we introduced non-native NADH-independent reactions into the GlcNAc synthesis pathway,including reactions of glyceraldehyde-3-phosphate to 3-phospho-glycerate,pyruvate to acetyl-CoA,malic acid to oxaloacetate,and regenerating oxidized ferredoxin.As a result,the yield of GlcNAc in shake flask fermentation reached 24.5 g·L^-1,and yield reached 0.468 g·g^-11 _Glucose;（5）The transcriptome analysis of different engineering strains,including BSGN6-P_xylA-glmS,BP22,BSGN6-P_xylA-glmS-GNA1 and BP22-afGNA1,was performed based on RNA-seq technology by using Illumina HiSeq platform.Then,we detailly analyzed the differences of gene transcriptions in membrane transport and carbohydrate metabolism,and tested the effects of expressing the up-regualted genes on GlcNAc production.Specifically,expression of gene acoC（encoding dihydrolipoamide acetyltransferase）could increase the production of GlcNAc,which reached 26.8 g·L^-1 in shake flask.Moreover,the maximum titer of GlcNAc reached 72.1 g·L^-1 in the a 3-L fermenter,and the yield was 0.41g·g^-1 _Glucose with the productivity of 0.68 g·（L·h）^-1.