title: Novel T7-like expression systems used for Halomonas.
abstract: 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 in vivo characterization in non-model strains Halomonas spp TD01 and Pseudomonas entomophila. 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 Ptac) in Halomonas sp. TD01, a chassis strain with a high industrial value. The expression under the corresponding T7-like promoter libraries persisted with striking correlations (R2>0.94) between Escherichia coli and Halomonas sp. TD01, implying suitability of broad-host range. Three Halomonas TD strains were then constructed based upon these expression systems that enabled interchangeable and controllable gene expression. One of the strains termed Halomonas TD-MmP1 was used to express the cell-elongation cassette (minCD 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.
authors.affiliation: Center for Synthetic and Systems Biology, School of Life Sciences, Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing 100084, China.
authors.forename: Han
authors.initials: H
authors.lastname: Zhao
authors.affiliation: Peking-Tsinghua Joint Centre for Life Sciences, Center for Quantitative Biology, Peking University, Beijing 100871, China; Bluepha Co., Ltd., Beijing 100084, China.
authors.forename: Haoqian M
authors.initials: HM
authors.lastname: Zhang
authors.affiliation: Center for Synthetic and Systems Biology, School of Life Sciences, Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing 100084, China.
authors.forename: Xiangbin
authors.initials: X
authors.lastname: Chen
authors.affiliation: Center for Synthetic and Systems Biology, School of Life Sciences, Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing 100084, China; Bluepha Co., Ltd., Beijing 100084, China.
authors.forename: Teng
authors.initials: T
authors.lastname: Li
authors.affiliation: Center for Synthetic and Systems Biology, School of Life Sciences, Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing 100084, China.
authors.forename: Qiong
authors.initials: Q
authors.lastname: Wu
authors.affiliation: Peking-Tsinghua Joint Centre for Life Sciences, Center for Quantitative Biology, Peking University, Beijing 100871, China. Electronic address: qi@pku.edu.cn.
authors.forename: Qi
authors.initials: Q
authors.lastname: Ouyang
authors.affiliation: Center for Synthetic and Systems Biology, School of Life Sciences, Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing 100084, China. Electronic address: chengq@mail.tsinghua.edu.cn.
authors.forename: Guo-Qiang
authors.initials: GQ
authors.lastname: Chen
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D017123:Bacteriophage T7 / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D020632:Halomonas / Q000378:metabolism / Q000502:physiology*; D006885:Hydroxybutyrates / Q000378:metabolism*; D060847:Metabolic Engineering / Q000379:methods*; D053858:Metabolic Networks and Pathways / Q000235:genetics; D014161:Transduction, Genetic / Q000379:methods*
chemical_list: D001426:Bacterial Proteins; D006885:Hydroxybutyrates
keywords: Expression system; Morphological engineering; Non-model microorganism; PHB; Part mining; Synthetic biology