초록
<P><B>Abstract</B></P> <P>To engineer non-model organisms, suitable genetic parts must be available. However, biological parts are often host strain sensitive. It is therefore necessary to develop genetic parts that are functional regardless of host strains. Here we report several novel phage-derived expression systems used for transcriptional control in non-model bacteria. Novel T7-like RNA polymerase-promoter pairs were obtained by mining phage genomes, followed by <I>in vivo</I> characterization in non-model strains <I>Halomonas</I> spp TD01 and <I>Pseudomonas entomophila</I>. Three expression systems, namely, MmP1, VP4, and K1F, were developed displaying orthogonality (crosstalk<0.7%), tight regulation (3085-fold induction), and high efficiency (2.5-fold of P<SUB>tac</SUB>) in <I>Halomonas</I> sp. TD01, a chassis strain with a high industrial value. The expression under the corresponding T7-like promoter libraries persisted with striking correlations (R<SUP>2</SUP> >0.94) between <I>Escherichia coli</I> and <I>Halomonas</I> sp. TD01, implying suitability of broad-host range. Three <I>Halomonas</I> TD strains were then constructed based upon these expression systems that enabled interchangeable and controllable gene expression. One of the strains termed <I>Halomonas</I> TD-MmP1 was used to express the cell-elongation cassette (<I>minCD</I> genes) and polyhydroxybutyrate (PHB) biosynthetic pathway, resulting in a 100-fold increase in cell lengths and high levels of PHB production (up to 92% of cell dry weight), respectively. We envision these T7-like expression systems to benefit metabolic engineering in other non-model organisms.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Novel T7-like expression systems were developed for non-model bacteria including <I>Halomonas</I> and <I>Pseudomonas entomophila.</I> </LI> <LI> The expression systems displayed low crosstalk, tight regulation and high efficiency. </LI> <LI> Cross-species transferring of pathway control was enabled by the T7-like systems. </LI> <LI> Ultrahigh PHB accumulation (92% of CDW) was achieved in <I>Halomonas</I> TD01 using T7-like MmP1. </LI> </UL> </P>