초록
<P><B>Background</B></P><P>Promoters regulate the expression of metabolic pathway genes to control the flux of metabolism. Therefore, fine-tuning of metabolic pathway gene expression requires an applicable promoter system. In this study, a dissolved oxygen-dependent <I>nar</I> promoter was engineered for fine-tuning the expression levels of biosynthetic pathway enzymes in <I>Escherichia coli</I>. To demonstrate the feasibility of using the synthetic <I>nar</I> promoters in production of biochemicals in <I>E. coli</I>, the <SMALL>D</SMALL>-lactate pathway consisting of one enzyme and the 2,3-butanediol (BDO) pathway consisting of three enzymes were investigated.</P><P><B>Results</B></P><P>The spacer sequence of 15 bp between the − 35 and − 10 elements of the upstream region of the wild-type <I>nar</I> promoter was randomized, fused to the GFP gene, transduced into <I>E. coli</I>, and screened by flow cytometry. The sorted synthetic <I>nar</I> promoters were divided into three groups according to fluorescence intensity levels: strong, intermediate, and weak. The selected three representative <I>nar</I> promoters of strong, intermediate, and weak intensities were used to control the expression level of the <SMALL>D</SMALL>-lactate and 2,3-BDO biosynthetic pathway enzymes in <I>E. coli</I>. When the <I>ldhD</I> gene encoding <SMALL>D</SMALL>-lactate dehydrogenase was expressed under the control of the strong synthetic <I>nar</I> promoter in fed-batch cultures of <I>E. coli</I>, the <SMALL>D</SMALL>-lactate titers were 105.6 g/L, 34% higher than those using the wild-type promoter (79.0 g/L). When the three 2,3-BDO pathway genes (<I>ilvBN</I>, <I>aldB</I>, and <I>bdh1</I>) were expressed under the control of combinational synthetic <I>nar</I> promoters (strong–weak–strong) in fed-batch cultures of <I>E. coli</I>, the titers of 2,3-BDO were 88.0 g/L, 72% higher than those using the wild-type promoter (51.1 g/L).</P><P><B>Conclusions</B></P><P>The synthetic <I>nar</I> promoters, which were engineered to have strong, intermediate, and weak intensities, were successfully applied to metabolic engineering of <SMALL>D</SMALL>-lactate and 2,3-BDO pathways in <I>E. coli</I>. By controlling expression levels of <SMALL>D</SMALL>-lactate and 2,3-BDO pathway enzymes using the synthetic <I>nar</I> promoters, the production of <SMALL>D</SMALL>-lactate and 2,3-BDO was increased over that using the wild-type promoter by 34 and 72%, respectively. Thus, this synthetic promoter module system will support the improved production of biochemicals and biofuels through fine-tuning of gene expression levels.</P>