Promoter Screening Based Expression Fine-tuning Of Erythromycin Biosynthesis

Erythromycin is a very important medicinal antibiotic with wide application and huge production demand,which still can not be synthesized directly by chemical method because of its complex structure.In recent years,the fast-developing synthetic biology facilitates flourishing studies on erythromycin and its derivatives.However,the improvement of industrial production of erythromycin still relies heavily on fermentation process optimization and random mutagenesis,which are usually inefficient and labor-consuming.In this study,two promoter mutation libraries were created in the native erythromycin host S.erythraea NRRL 23338,and high-throughput screening of the desired target promoter variants was performed using the droplet microfluidic screening platform.Then,the transcriptional levels of all genes in erythromycin biosynthesis gene cluster（ery）were determined and compared between the low-producing strain S.erythraea NRRL 23338 and a high-producing industrial strain S0,and the potential rate-limiting enzymes in erythromycin biosynthesis were characterized.Finally,the expression of key ery genes was fine-tuned using two screened synthetic promoter variants to increase erythromycin yield.In this study,the two mutant libraries of S.erythraea NRRL 23338,which were created starting from the strongest native promoter p _SACE2101 and the heterologous strong promoter p_{erm E*},which contained about 50,000 mutants respectively,were screened by droplet microfluidic platform.Two groups of promoter variants with different strengths derived from p SACE＿2101 and perm E*were obtained:p SACE＿2101＿s32,p SACE＿2101＿s33,p SACE＿2101＿s43,p SACE＿2101＿s35,perm E*＿S23,perm E*＿S41,perm E*＿s42,and perm E*＿s43.The strongest promoter variant p SACE＿2101＿s32was 21.5 times of the weakest promoter variant p_{erm E*s42}.Through the comparative transcriptional analysis of ery gene cluster in low-yield strain S.erythraea NRRL 23338 and the high-yield strain S0,six potential key genes SACE＿0716,SACE＿0717,SACE＿0718,SACE＿0719,SACE＿0720,SACE＿0731 in erythromycin biosynthesis were identified.Then,12 overexpression strains driven by two different synthetic promoters were constructed.The fermentation results showed that the highest erythromycin titer of single-gene-overexpressing strain was 275.83 mg/L,which was 131 times compared to that of the wild-type strain.Considering that both SACE＿0718 and SACE＿0719 single-gene overexpression led to significant improvements of erythromycin production,we also constructed a double-gene-overexpressing strain S.erythraea/p SET152-hyg-p _SACE2101s32-SACE＿0718-p_{erm E*S23}-SACE＿0719 harboring both p _SACE2101s32-SACE＿0718 and p_{erm E*S23}-SACE＿0719 expression cassettes,and its erythromycin titer was 288.06 mg/L,which was 137 times of the titer of the wild type.And this was also the highest titer of all overexpression strains.In this study,we established a systematic method of promoter engineering in the native erythromycin host S.erythraea NRRL 23338,and successfully obtained a panel of promoters with different strengths.These promoters were further used for expression fine-tuning of erythromycin biosynthesis and production improvement.This work will accelerate the researches on promoter engineering and natural product biosynthesis in the similar filamentous microorganism hosts.