Using CRISPR/Cas System To Enhance The Synthesis Of Oxidized Coenzyme Ⅰ

Nicotinamide Adenine Dinucleotide（referred to as NAD⁺）is an essential coenzyme involved in many physiological processes.In recent years,with the development of green biotechnology,NAD⁺has shown great application value in the field of biocatalysis and pharmaceutical industry.Although biotechnology for the construction of NAD⁺-producing strains has been developed,its low productivity still hinders its large-scale application.In this paper,the intracellular NAD⁺content of Escherichia coli BL21（DE3）was used to construct a high-yield NAD⁺strain by weakening the NAD（H）degradation pathway,strengthening the key enzymes in the synthesis pathway,increasing the supply of synthetic substrates and regulating energy metabolism.The intracellular NAD⁺concentration was increased from 3.23μmol·g^-1DCW to 41.6μmol·g^-1DCW at shake flask level and the main results are as follows:（1）Multi-strategy modified Escherichia coli to enhance intracellular NAD⁺synthesis.First of all,in order to weaken the degradation of NAD（H）,the genes nud E,nud C,ush A and maz G related to the degradation pathway were knocked out.The results of shake flask fermentation showed that in the constructed single gene knockout strain,the knockout of gene ush A resulted in a high level of NAD⁺in the cell.The polygene knockout strain constructed on the basis of it can increase the intracellular NAD⁺concentration by up to 39%.Secondly,the key enzyme genes nad E and pnc B in the Preiss-Handler synthesis pathway of NAD⁺were over-expressed in ush A site by replacing the integrated gene at the knockout locus.The results of shaking flask showed that the intracellular NAD⁺concentration increased by 221%.Third,the PRPP synthesis module is combined with the Preiss-Handler pathway to continue to integrate gnd,zwf and prs genes in the pentose phosphate pathway to increase the supply of coenzyme synthesis precursors.Compared with the original strain,the intracellular NAD⁺content increased by 520%.Fourth,the adenine salvage pathway is introduced to indirectly regulate the level of intracellular ATP by knocking out gene add/amn and integrating gene ado1.The increase of ATP concentration increased the intracellular concentration of NAD⁺by170%.Finally,based on all the metabolic modifications mentioned above,a highly efficient strain producing NAD⁺was constructed by using multi-strategy metabolic engineering.The results of shake flask fermentation showed that the intracellular NAD⁺concentration of the strain was 734%higher than that of the original strain.（2）Study the effect of induction conditions on the intracellular NAD⁺synthesis of the recombinant strain.The recombinant strains with high NAD⁺production were optimized from three aspects:induction temperature,inducer concentration and induction time.The optimum induction conditions were determined as follows:when the induction temperature was 17℃and OD₆₀₀ was 0.6,0.8 mmol.L^-1 IPTG was added.Compared with that before the optimization of culture conditions,the content of intracellular NAD⁺increased by 26%.（3）Analysis of the correlation between the transcriptional level of integrated overexpressed genes and NAD⁺synthesis.The effects of overexpressed genes on intracellular NAD⁺synthesis were analyzed by SPSS software.It has been found that the overexpression of genes nad E and pnc B can promote the synthesis of intracellular NAD⁺.（4）Increasing the copy number of positive regulatory genes nad E and pnc B in the constructed recombinant strain to solve the bottleneck of NAD⁺production in Escherichia coli.The results of shaking flask showed that the intracellular NAD⁺concentration increased again by 22% at the optimized level.The above results laid a foundation for further expanding the biosynthesis and industrial application of coenzyme natural products.