Genome And Transcriptome Based Improvement Of Geldanamycin Production

Geldanamycin is an important antitumor natural product.Through binding to the heat shock protein HSP90,it exhibits potent antitumor activities against various cancer cells.The derivatives of geldanamycin,17-AAG and 17-DMAG have entered different stages of clinical trials.Because of the structure complexity,it is difficult to synthesize geldanamycin totally by chemical methods.Nowadays,geldanamycin is mainly produced by fermentation of several Streptomyces strains.Therefore,it is necessary to engineer geldanamycin producing strain and to improve the yield of geldanamycin.In this study,the genetic manipulation system of geldanamycin producer S.hygroscopicus XM201 was firstly established.In order to understand its basic metabolic condition,the genome and transcriptome of XM201 were then sequenced.XM201 has a linear chromosome of 12 Mb.It has a 6 Mb core region in the center of the chromosome,with genes mainly responsible for cell survival and division.There is also a 6 Mb non-core region,including genes responsible for secondary metabolite biosynthesis.The genes with functions such as DNA duplication,transcription,carbohydrates metabolism,fatty acids metabolism and energy metabolism are evenly distributed in core and non-core region in XM201,while in other Streptomyces strains they were mainly distributed in the core region.However,as indicated by the transcriptome,most of the latter genes in the non core region are weakly expressed under geldanamycin fermentation condition.In order to study the impact of primary metabolism on cell growth and geldanamycin production,we systematically knocked out the genes involved in the central metabolism.It is observed that under geldanamycin production condition,the cell growth competes with geldanamycin production,and weaken the TCA cycle would increase the yield of geldanamycin.After the genes involved in EMP pathway and TCA cycle were located in XM201 genome,it is obvious to see that most genes have multiple corresponding isozyme genes.As indicated by the transcriptomic data,most genes located in the core region are highly expressed,while genes located in the non-core region are weakly expressed.The 18 selected genes were then individually deleted.As shown by the fermentation result,the genes in the core region or with higher expression levels had stronger impacts on geldanamycin yield.In addition,inactivation of the genes involved in EMP pathway increased the final biomass of the mutant strains,but decreased the yield of geldanamycin.On the contrary,inactivation of the genes in TCA cycle decreased the final biomass but increased the yield of geldanamycin.It is clear to see that under geldanamycin fermentation condition,the accumulation of biomass competes with the biosynthesis of secondary metabolite.Through weaken the TCA cycle by inactivation of gltA,sdhA and fumC,the redundant materials used for biomass synthesis can be driven to support the biosynthesis of geldanamycin.It is also observed that after weakening of TCA cycle,the concentration of one geldanamycin precursor malonyl-CoA increased,which might be another reason for increased geldanamycin yield.On the other hand,we overexpressed geldanamycin PKS genes with screened endogenous promoter and increased the yield of geldanamycin by 39%,overexpressing the AHBA biosynthetic genes further increased geldanamycin production by 35%.After analyzing the transcriptome data of XM201,it is indicated that the expression level of the PKS genes was lower than those of other biosynthetic genes and this step was deduced to be rate-limiting in geldanamycin biosynthesis.qRT-PCR data further showed the transcriptional level of the PKS genes were lowest during the late stage of fermentation.Therefore,we expect to find an endogenous strong promoter.Through the process of evaluation according to the RNA-seq,qRT-PCR results,kanamycin resistance test and XylE enzymatic activity,promoters such as 5063 p,6214p,7359 p and 7138 p were found to have high activities under geldanamycin fermentation condition.Among them,5063 p exhibited the highest activity at the late stage of fermentation.After replacing the native PKS promoter with the strong promoter 5063 p,the expression levels of the PKS genes were found to be increased by 4-to 141-folds.During the late stage of fermentation,the production ability of geldanamycin PKS was increased and finally a 39% increase of geldanamycin yield was achieved.Tandem overexpression of the AHBA biosynthetic genes in the PKS overexpressing strain further increased geldanamycin yield by 35%.These two steps increased the yield of geldanamycin from 773 mg/L to 1,450 mg/L.In conclusion,with the help of genomic and transcriptomic information,we systematically investigated on the two main pathways involved in geldanamycin biosynthesis,and found the biosynthesis of geldanamycin was affected by genes involved in both primary metabolism and secondary metabolism.Through weakening the TCA cycle and overexpression of the PKS genes,we efficiently improved the yield of geldanamycin,and give guidance on studies about improving antibiotic yield based on genomic level in Streptomyces.