Whole-cell Catalytic Synthesis Of NMN Based On Xylose Pathway And Process Optimization

β-Nicotinamide mononucleotide（NMN）can be used as a direct precursor of the important coenzyme NAD⁺in the human body,and has broad application prospects in health care and daily chemicals.At present,chemical methods or chemical-enzyme catalytic methods are mainly used in industry for synthesis,but there are problems such as the use of high-risk reagents,high pollution,and harsh reaction conditions.The synthesis of NMN by biocatalysis has attracted widespread attention due to its mild reaction conditions and green process.In order to improve the potential of NMN catalytic synthesis by recombinant bacteria,reduce production costs and better apply it to industrial production,this paper systematically carried out systematic work on the construction of xylose as substrate for NMN synthesis,knockout of 5-phosphate ribose 1-pyrophosphate（PRPP）and carbon flow competition bypass pathway,shake flask and optimization of whole-cell catalytic synthesis of NMN on 5 L fermenter.The main findings of the paper are as follows:（1）Construction and screening of NMN synthesis pathway.Firstly,with Escherichia coli BL21（DE3）as the starting host,p ET28a and p RSFDuet-1 as expression plasmids,heterologous expression of nicotinamide phosphoribosyltransferase from Chitinophaga pinensis,recombinant strains Bp-1 and Bp-2 carrying key enzymes of NMN salvage synthesis pathway were constructed,and whole cell catalysis was performed by providing nicotinamide（NAM）,and the experimental results showed that the NMN yield was up to 17.2 mg·L^-1,21.4 mg·L^-1,Bp-2 recombinant strain was selected for follow-up study.Further expressed multiple pathway enzymes for de novo synthesis of NMN from substrates such as glucose,ribose and xylose,and combined nicotinamide phosphoribosyltransferase with xyloshikinase,ribose phosphopyrokinase and other multiple enzymes to construct recombinant strains Bp-3,Bp-4and Bp-5,with yields of 75.7,32.8 and 190.3 mg·L^-1,respectively,so the Bp-5 strain with xylose as substrate was selected for subsequent research.（2）Knockout of competitive bypass pathways.The CRISPR/Cas9 editing tool was used to knock out 11 genes in two aspects,including the PRPP competitive bypass pathway and the carbon flow competitive bypass pathway,in order to stabilize and increase NMN production.By knocking out the ATP transphosphoribosylase-coding gene his G,adenine phosphoribosyltransferase-coding gene apt,and hypoxanthine guanine phosphoribosyltransferase-coding gene hpt,NMN yield reached 275.1 mg·L^-1.The transketoalcoholase 2-encoding genes tkt B and transaldodolase encoding genes tal A and tal B were knocked out of the carbon flow bypass pathway,blocking their entry into the glycolytic pathway,and the target product yield reached 332.7 mg·L^-1.In order to promote NMN accumulation,the yield was increased to 373.6 mg·L^-1 by further knocking out NMN aminohydrolase pnc C and regulating NAD⁺derivative gene nad R.（3）Optimization of conditions for whole-cell catalytic synthesis of NMN.The effects of optimization of induction conditions and whole-cell catalytic conditions on NMN yield were explored,and univariate optimizations were carried out on inducer concentration,reaction catalytic system,substrate concentration Mg²⁺concentration,etc,and the optimal induction conditions were explored:LB medium,inducer concentration of 1.0 mmol·L^-1,induction temperature of 20°C,OD₆₀₀ of 0.8 plus inducer,and addition of xylose 5.0 g·L^-1,nicotinamide5.0 g·L^-1 and Mg²⁺to the optimized catalytic reaction mmol/L was catalyzed by whole cells with a yield of 497.5 mg·L^-1.The results of expanded culture and catalytic synthesis of NMN under the condition of 5 L fermenter showed that the biomass OD₆₀₀ of recombinant bacteria on 5 L tank reached a maximum of about 40,and the catalytic yield of whole cells was 760.2mg·L^-1.In this study,a variety of metabolic engineering strategies were used to increase the yield of NMN synthesis by xylose pathway,which laid a theoretical foundation for the industrial application of biosynthetic NMN.