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<p>One of the characteristics of actinomycetes, especially streptomycetes, is the high GC content in their genome, which often leads to the failure of heterologous expression in <i>E. coli</i>, and thus hinders in vitro enzyme activity experiments. Therefore, we have developed a precisely regulated and efficient <i>Streptomyces</i> expression system, pTZYp, that relied on the strong promoter <i>stnY</i> and the <i>cmt</i> operon. As tested in three model <i>Streptomyces</i> strains (<i>S. albus</i> J1074, <i>S. coelicolor</i> M1152 and <i>S. lividans</i> TK24), the reporter protein sfGFP was not detected without the addition of the inducer cumate, whereas sfGFP was significantly produced when a certain amount of inducer cumate was added to the medium, demonstrating that the pTZYp expression system can achieve the goal of precise regulation and efficient expression. After optimization of the expression conditions, the maximum sfGFP production was obtained when the inducer was added to the final concentration of 100&#xa0;μM and cultivated for about 24&#xa0;h. pTZYp has also been used to express other six non-model proteins in <i>Streptomyces</i>, and all of them have been successfully expressed. The pTZYp expression system demonstrated robustness, high efficiency (relying on the <i>stnY</i> promotor), precise regulation (relying on <i>cmt</i> operon and moderate production of regulatory protein CymR) and low experimental cost (relying on the lower cost of the inducer cumate), which may be an efficient and widely applicable heterologous expression tool for genes with high GC content in actinomycetes.</p>

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A precisely regulated Streptomyces expression system pTZYp facilitated by the strong promoter stnY and cmt operon

  • 期刊论文

  • 发表日期：

    2025年8月12日

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  • Kai Zhang,
  • Ao-yu Wang,
  • Qiang Zhou,
  • Yi-feng Chen,
  • Shuang-jun Lin,
  • Gong-li Tang

摘要

One of the characteristics of actinomycetes, especially streptomycetes, is the high GC content in their genome, which often leads to the failure of heterologous expression in E. coli, and thus hinders in vitro enzyme activity experiments. Therefore, we have developed a precisely regulated and efficient Streptomyces expression system, pTZYp, that relied on the strong promoter stnY and the cmt operon. As tested in three model Streptomyces strains (S. albus J1074, S. coelicolor M1152 and S. lividans TK24), the reporter protein sfGFP was not detected without the addition of the inducer cumate, whereas sfGFP was significantly produced when a certain amount of inducer cumate was added to the medium, demonstrating that the pTZYp expression system can achieve the goal of precise regulation and efficient expression. After optimization of the expression conditions, the maximum sfGFP production was obtained when the inducer was added to the final concentration of 100 μM and cultivated for about 24 h. pTZYp has also been used to express other six non-model proteins in Streptomyces, and all of them have been successfully expressed. The pTZYp expression system demonstrated robustness, high efficiency (relying on the stnY promotor), precise regulation (relying on cmt operon and moderate production of regulatory protein CymR) and low experimental cost (relying on the lower cost of the inducer cumate), which may be an efficient and widely applicable heterologous expression tool for genes with high GC content in actinomycetes.