| In recent years,synthetic biology has developed rapidly and has been widely used in the synthesis of drugs,chemicals,fragrances and other high value-added products.Precise control of the target genes transcription level is the basis for the construction of recombinant strains with high production of target products.Many studies found that when the target gene was expressed by high-intensity transcription elements,the production of the target product was decreased.This abnormal phenomenon found in Saccharomyces cerevisiae,Pichia pastoris and other chassis microorganisms,which is a potential obstacle to the transformation of chassis microorganisms by synthetic biology.In this study,we established a strong promoter-mediated transcriptional repression response regulation model,mined key genes,transcription elements and related pathways,and comprehensively analyzed the transcriptional repression response regulation mechanism.By regulating key pathways,the dynamic change trend of target gene transcription level was reconstructed to improve the production of target products,which provided a new strategy for the construction of recombinant strains in synthetic biology.(1)Red fluorescent protein(RFP)and enhanced green fluorescent protein(EGFP)as reporter genes demonstrated that the strength of the glyceraldehyde 3-phosphate dehydrogenase gene promoter TDH3 p in industrial strain IS45 was significantly higher than that of the phosphoglycerate kinase gene promoter PGK1 p.TDH3p and PGK1 p were used to construct recombinant strains ISH(::TDH3p-ATF1-PGK1t)and ISP(::PGK1p-ATF1-PGK1t)that overexpressed ATF1 encoding gene.The ethyl acetate production of the recombinant strain was evaluated by corn synthetic medium-standing fermentation,YPD-20medium-standing fermentation and YPD-20 medium-shaking fermentation.It was found that the ethyl acetate production of strain ISP was 53.34%,62.68%,8.81% higher than that of strain ISH.This proved that high intensity expression of target gene was not conducive to the improvement of target product production.(2)Yeast cells of IS45,ISP and ISH at 12,24 and 36 h during fermentation as samples,the effect of high expression of ATF1 gene on global gene transcription level was analyzed by RNA-seq.The results showed that the number of differential genes of strain ISH was greater than that of strain ISP at the three time points,indicating that the strong promotordriven target genes caused more obvious fluctuations in the transcription level of global genes.The KEGG enrichment analysis showed that metabolic pathways,secondary metabolites biosynthesis,antibiotic biosynthesis,carbon metabolism,amino acid biosynthesis and other pathways were closely related to the decrease of ethyl acetate production caused by strong promoter.Realtime-PCR was used to verify the dynamic change trend of ATF1 gene transcription levels of strain IS45,ISP and ISH at 2,4,8,12,24,36 and 60 h.The results showed that ATF1 transcription levels of strain ISH were significantly repressed at the early stage of fermentation(2 and 4 h).It is speculated that the abnormal reduction of ethyl acetate production may be related to this transcriptional repression reaction.(3)The transcription levels of MET14,HOM3,HAP4,ADR1,BTN2,HSP30,INO1 and DIP5 genes in ISH were significantly down-regulated compared with that of parental strain IS45.Using ISH as the parental strain,gene knockout strategy was used to verify the regulation effect of eight differentially expressed genes on the production of ethyl acetate.Ethyl acetate production of strains ISH?HSP30,ISH?MET14,ISH?HOM3,ISH?HAP4,ISH?ADR1,ISH?INO1,ISH?BTN2 and ISH?DIP5 decreased by 55.26%,24.17%,13.75%,12.18%,15.61%,11.51%,18.76% and 14.44% compared with strain IS45,respectively.Moreover,the overexpression of HSP30 gene and BTN2 gene increased the production of ethyl acetate by 13.12% and 19.81%,respectively,indicating that the up-regulation of the transcription level of HSP30 and BTN2 could alleviate the transcriptional inhibition caused by the overexpression of the target gene.The unfolded protein response(UPR)levels of ISP and ISH were analyzed,the transcription levels of HAC1 and IRE1 genes of ISH were 4.48 and 1.9 times higher than those of ISP,respectively.This implied that there was a correlation between transcriptional inhibition and the UPR pathway.(4)To investigate the transcriptional changes of heat shock protein family genes in strains IS45,ISP and ISH.It was found that the transcription levels of HSP26,HSP42,HSP78,HSP82,HSC82,HSP104,SSA1,SSA2 and SSA4 genes in ISH-36 samples were significantly down-regulated.The deletion of HSP26,HSP42,HSP78 and HSP82 genes reduced the production of ethyl acetate by 10.49%,12.14%,18.81% and 13.23%,respectively.RT-PCR results showed that Hsf1 regulated the transcription level of HSP30 gene,and the ethyl acetate yield of HSF1 overexpressed strain was increased by 49.81% and 57.03%,respectively,after fermentation in corn synthetic medium and YPD-20 medium.The combined overexpression of HSF1 and HSP30 genes further increased the ethyl acetate production,suggesting that the transcriptional inhibition of strain ISH was significantly mitigated.(5)The transcriptional levels of ATF1 gene in strains IS45,ISP,ISH,ISH::HSP30,ISH::HSP30::HSF1 were analyzed by RT-PCR.It was proved that the combined overexpression of HSP30 and HSF1 genes increased the transcriptional abundance of ATF1 gene in early fermentation.The change trend of ATF1 transcription level was the fundamental reason for the significant increase of ISH::HSP30::HSF1 strain ethyl acetate production.On the other hand,the overexpression of HSF1 gene down-regulated the UPR level of the strain,alleviating the endoplasmic reticulum stress caused by the high expression of the target gene.By overexpressing HSF1,HSP30,HSP26 and HSP82 genes,the ethyl acetate yield of recombinant strain ISH::HSP30:: HSP26 and ISH::HSP30::HSF1::HSP82 was increased by16.75% and 16.6% compared with that of strain ISP.This indicated that the transcriptional inhibition response of strain ISH was successfully reconstructed.Finally,the regulation of Hsf1 on transcriptional inhibition induced by different promoters is proved to be universal,which provides a new idea and application strategy for the construction of recombinant strains in synthetic biology. |
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