Effect Of GTP Biosynthesis On Toyocamycin Production In Streptomyces Diastatochromogenes 1628

Toyocamycin(TM)is a member of the nucleoside antibiotics family.TM is a potential fungicide that can control plant diseases,and it has become an attractive target for research.In the earlier study,a strain Streptomyces diastatochromogenes 1628 was isolated,which could produce TM.The metabolic pathway that guanosine triphosphate(GTP)as a precursor converted to TM through a multi-step reaction was revealed.A TM high-yield rifampicin-resistant mutant strain S.diastatochromogenes 1628-T15 was isolated by using ribosome engineering in the previous study.By using the comparative proteomics,the differential expression proteins between the wide-type strain and the mutant strain 1628-T15 were analyzed.In this study,we focused on the different proteins involved in the purine metabolism,and attempted to elucidate the between synthesis of precursor GTP and production of TM.The main contents of this paper are as follows:First,based on the data analysis of comparative proteomics and KEGG database comparison of mutant strain S.diastatochromogenes 1628-T15 and the wide-type strain S.diastatochromogenes 1628,five differential expression proteins involved in the purine metabolic pathway were screened.Among them,four differential expression proteins X0NBV6,X0P2N4,A0A059W736,and A0A059VSQ4,encoding ribose-phosphate pyrophosphokinase,GMP synthase,ribonucleoside diphosphate kinase and guanylate kinase respectively,exhibited a significantly increase in expression level,whereas the expression of 5'-nucleotidase that encoded by A0A059WBI4 was significantly reduced in the mutant strain1628-T15.Intracellular GTP concentration of the mutant strain 1628-T15 and the wide-type strain 1628 at different fermentation stage was determined.The results indicated that the concentration of the precursor GTP produced by mutant strain1628-T15 was higher than that produced by the wide-type strain 1628.In the initial-period of fermentation process(12-36 h),the rate of increase reached to90%-102.5%.It suggested that high production of TM of mutant 1628-T15 may be due to the increase of GTP.The encoding genes that correspond to those of five differential proteins in S.diastatochromogenes 1628 were named as rhp_sd,gua_sd,ndk_sd,gua K_sdand nuc_sdrespectively.According to the results of real-time quantitative PCR(q RT-PCR),the transcription level of genes rhp_sd,gua_sd,ndk_sdand gua K_sdin mutant strain 1628-T15 were 4.50-,2.23-,1.78-,and 1.48-fold higher compared with that of wide-type strain 1628,while the transcription level of nuc_sdwas only 37%as that of the wide-type strain 1628.Subsequently,in order to verify the correlation among these five differential expression proteins,GTP synthesis and TM production,genes of rhp_sd,gua_sd,ndk_sd,gua K_sdand nuc_sdencoding five differential expression proteins were amplified by PCR using S.diastatochromogenes 1628 genomic DNA as template.The DNA fragment was placed under the control of constitutive promoter perm E^*in plasmid p IB139 to generate the recombinant plasmids p IB139-rhp_sd,p IB139-gua_sd,p IB139-ndk_sd,p IB139-gua K_sdand p IB139-nuc_sd,respectively.Integration of the recombinant plasmid p IB139-rhp_sd,p IB139-gua_sd,p IB139-ndk_sd,p IB139-gua K_sdand p IB139-nuc_sd,into S.diastatochromogenes 1628 by conjugation resulted in the strain 1628-RP,1628-GA,1628-NK,1628-GK and 1628-NC,respectively.This work provided the basis for further exploring the function of five differential genes.Finally,the transcription level,TM production and intracellular GTP concentration of above constructed five recombinant strains were analyzed during different fermentation time.The results of q RT-PCR showed that the expression level of overexpressed genes in each corresponding recombinant strains were higher compared to those of wild-type strain during the whole fermentation process.These results confirmed that overexpression of this five genes was successful in recombinant strains.The intracellular GTP concentration and TM production in recombinant strains 1628-RP,1628-GA,1628-NK and 1628-GK were all increased in different degrees comparing with wild-type strain 1628.In the recombinant strains,the increase of GTP concentration was the most obvious in the early stage of fermentation,which was ranging from 10%to 122%.The increase of TM production was ranging from37%to 134%in five recombinant strains compared to that of wild-type strain.Among them,the recombinant strain 1628-RP harboring overexpression of rhp_sdgene exhibited the best performance on the TM production.The highest level of TM production achieved by recombinant strain 1628-RP reached to 340.2 mg/L,which was 133.3%higher than that achieved by wild-type strain 1628.It indicated that the overexpression of genes rhp_sd,gua_sd,ndk_sd,and gua K_sdcould enhance the biosynthesis of GTP and further promote the production of TM.Whereas intracellular GTP concentration(7.2 mmol/mg DCW)of recombinant strain 1628-NC decreased36.1%compared to that of wild-type strain 1628 in the early stage of fermentation and its highest TM production was only 67.1 mg/L,which was 53.9%lower than that of wild-type strain.Therefore,the overexpression of nuc_sdgene did not favor to the accumulation of GTP,and it thus affected the TM biosynthesis.The above results,were consistent with the results from proteome analysis data,and this resultalso showed that precursor GTP was essential and helpful for the production of TM.This work will provide the basis for further improving TM production by utilizing precursor engineering.