重组大肠杆菌全细胞催化合成β-烟酰胺单核苷酸

Whole-cell Catalytic Synthesis ofβ-nicotinamide Mononucleotide by the Recombinant Escherichia Coli

β-烟酰胺单核苷酸(NMN)是一种天然存在的维生素B族活性衍生物,具有改善哺乳动物生育能力和肠道功能的作用。以大肠杆菌BW25113为表达宿主,根据NCBI数据库中烟酰胺核糖激酶(NRK)的蛋白序列,同源比对分析合成不同来源的Nrk基因,筛选获得1株高效转化烟酰胺核糖(NR)合成NMN的重组菌株NRK-Gs/BW,在含有50 mmol·L^(-1)NR和10 mmol·L^(-1)ATP体系中能够合成43 mmol·L^(-1)的NMN。进一步引入多聚磷酸激酶(PPK2)构建ATP循环系统,ATP用量降低了80%,反应体系中AMP生成量≤3 mmol·L^(-1)。最后,通过析因设计对反应体系进行优化,在50 mmol·L^(-1)NR和10 mmol·L^(-1)ATP的反应体系中,NMN产量为49.96 mmol·L^(-1),摩尔转化率达到99.96%。通过构建了含有ATP循环系统的NR合成NMN工程菌,大幅度降低了ATP的使用摩尔比,为后续的NMN生产化生产奠定了基础。

β-nicotinamide mononucleotide(NMN)was a naturally occurring active derivative of B vitamins,which had the function of improving the mammalian fertility ability and intestinal function.Escherichia coli BW25113 was used as the expression host,and according to the protein sequence of nicotinamide ribokinase(NRK)in the NCBI database,Nrk genes from different sources were synthesized by homologous alignment analysis.Then,a recombinant strain NRK-Gs/BW with high efficiency in converting nicotinamide ribose(NR)to NMN was screened.43 mmol·L^(-1)NMN could be synthesized in the reaction mixture containing 50 mmol·L^(-1)NR and 10 mmol·L^(-1)ATP.Polyphosphate kinase(PPK2)was further introduced to construct the ATP circulation system.The amount of ATP was reduced by 80%,and the AMP production in the reaction mixture was less than or equal to 3 mmol·L^(-1).Finally,the reaction mixture was optimized by factorial design.In the reaction mixture of 50 mmol·L^(-1)NR and 10 mmol·L^(-1)ATP,the yield of NMN was 49.96 mmol·L^(-1),with a molar yield of 99.96%.By constructing the NMN engineering bacteria synthesized by NR containing the ATP circulation system,the molar ratio of ATP was greatly reduced,which laid a foundation for the subsequent production of NMN.