Construction An Efficient Biosynthesis Pathway For 2-KGA Production In Ketogulonicigenium Based On Genomic Analysis

2-Keto-L-gulonic acid(2-KGA),the precursor of vitamin C,is produced from L-sorbose by the symbiosis system composed of Ketogulonicigenium vulgare and Bacillus spp.in the industrial fermentation.Although the continual improvement on the 2-KGA-producing strains,the capacity to produce 2-KGA was improved unsignificantly,and the metabolic pathway remianed unclear.In this study,the whole genome of Ketogulonigenium vulgare SPU B805(accompanied by Bacillus megatherium SPU_B806 for fermentation)and Ketogulonigenium robustum SPU_B003(independent fermentation)were sequenced.The comparative genome analyses were conducted with the reported four K.vulgare strains(K.vulgare WSH-001,K.vulgare Y25,K.vulgare Hbe602 and K.vulgare SKV)to reveal the underlying mechanism of different growth characteristics and 2-KGA yield.K.vulgare SPU_B805 harbors one circular chromosome with plasmid free.K.vulgare SPU_B805 was absent of the genes of gluconate 2-dehydrogenase(2-KGA decomposition,ga2dh),sorbosone dehydrogenase(conversion L-sorbosone to 2-KGA,sndh),and 2-keto-3-deoxy-6-phosphogluconic acid aldolase(the key enzyme of Entner-Doudoroff pathway,eda)and phosphogluconate dehydratase(the key enzyme of Entner-Doudoroff pathway,eda).Strain SPU_B805 was absent of the genes of 6-phosphofructokinase(pfk,the key gene of EMP pathway),edd and eda(the key gene of ED pathway),so the carbohydrate metabolism mainly through pentose phosphate pathway(PPP)and citric acid cycle(TCA).Meanwhile,the amino acid biosynthetic pathway was more complete than other K.vulgare,which was absent of the four key enzymes for the biosynthesis of histidine,alanine and asparagine(histidinol-phosphatase,leucine-alanine transaminase,alanine transaminase and asparagine synthetase).These factors are conducive to the cell growth and 2-KGA accumulation of K.vulgare SPU_B805.Moreover,the number of tRNAs,rRNAs,NAD and NADP biosynthetic genes,regulation and cell signaling related genes,as well as intracellular trafficking,secretion,and vesicular transport related genes in K.robustum SPU B003 are more than that of in K.vulgare,which are presumably the main reason for its ability to growth independently and higher 2-KGA yield.Metabolic pathway analyses showed that K.robustum SPU_B003 was mainly through PPP,ED pathway and TCA cycle for carbohydrate metabolism.In order to conduct gene expression in K.robustum SPU B003,BPROM and phiSITE were used for promoter prediction.Four promoters,PtufB,P1,P2 and P3,were isolated and their abilities were detected by initiating egfp gene expression in different bacteria.In K.robustum SPU_B003 and K.vulgare SPU_B805,the promoter activity from strong to weak was P1>P2>PtufB>P3;the order in Pseudomonas putida KT2440 was P1>P3>P2>PtufB;in Paracoccus denitrificans PD 1 222 was P3>P2>P1>PtufB.In Escherichia coli BL21(DE3),Escherichiacoli C43(DE3)and Bacillus subtilis,the promoter P1 showed the strongest strength,while promoter P2 was the strongest one in Raoultella ornithinolytica.The promoter P1 showed the strongest activity in K.robustum SPU_B003,K.vulgare SPU_B805,P.putida KT2440,E.coli BL21(DE3),E.coli C43(DE3)and B.subtilis,which demonstrated it was robust and applicable in cross-species.The formation of acetyl-CoA from L-sorbose through the PPP pathway is accompanied by two steps of decarboxylation,namely the generation of ribulose-5-phosphate(R5P)from 6-phosphogluconate(6PG)by 6-phosphogluconate dehydrogenase(PGD)and acetyl-CoA from pyruvate by pyruvate dehydrogenase(PDH),so the released CO2 in the processes decrease the carbon utilization rate and 2-KGA conversion rate from L-sorbose.To reduce the carbon loss,an innovative XFP-PTA pathway was introduced into K.robustum SPU_B003 by expressing heterologous phosphoketolase(XFP)and phosphotransacetylase(PTA)and optimizing the codons of xfp and pta.In the recombinant strain,K.robustum/pBBR-P1_xfp2502-P2_pta2145,the acetyl-CoA enhanced approximately 2.4-fold,the biomass enhanced by 17.27%,and the 2-KGA production increased 6.90 g/L(21.09%)compared to the control strain K.robustum/pBBRlMCS-2.Accordingly,the transcriptional level of pgd and pdh decreased by 24.33±6.67%and 8.67±5.51%,respectively.The key genes responsible for 2-KGA biosynthesis,sorbose dehydrogenase gene(sdh)and sorbosone dehydrogenase gene(sndh),were up-regulated to different degrees.Therefore,the XFP-PTA pathway can increase the intracellular acetyl-CoA level,improve the biomass and 2-KGA yield,and reduce the release of CO2.