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Development of an enhanced chromosomal expression system based on porin synthesis operon for halophile Halomonas sp.

  • Applied genetics and molecular biotechnology
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Abstract

Since halophile Halomonas spp. can grow contamination free in seawater under unsterile and continuous conditions, it holds great promise for industrial biotechnology to produce low-cost chemicals in an economic way. Yet, metabolic engineering methods are urgently needed for Halomonas spp. It is commonly known that chromosomal expression is more stable yet weaker than plasmid one is. To overcome this challenge, a novel chromosomal expression method was developed for halophile Halomonas TD01 and its derivatives based on a strongly expressed porin gene as a site for external gene integration. The gene of interest was inserted downstream the porin gene, forming an artificial operon porin-inserted gene. This chromosome expression system was proven functional by some examples: First, chromosomal expression of heterologous polyhydroxybutyrate (PHB) synthase gene phaC Re from Ralstonia eutropha completely restored the PHB accumulation level in endogenous phaC knockout mutant of Halomonas TD01. The integrated phaC Re was expressed at the highest level when inserted at the locus of porin compared with insertions in other chromosome locations. Second, an inducible expression system was constructed in phaC-deleted Halomonas TD01 by integrating the lac repressor gene (lacI) into the porin site in the host chromosome. The native porin promoter was inserted with the key 21 bp DNA of lac operator (lacO) sequence to become an inducible promoter encoded in a plasmid. This inducible system allowed on-off switch of gene expression in Halomonas TD strains. Thus, the stable and strong chromosomal expression method in Halomonas TD spp. was established.

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Acknowledgments

We are grateful to Professor Víctor de Lorenzo of Centro Nacional de Biotecnología (CNB-CSIC) in Spain for the generous donation of plasmid pSEVA341. This research was financially supported by National High Tech 863 Grants (Grant No. 2012BAD32B02 to JCC) and 973 Basic Research Fund (Grant No. 2012CB725201 to GQC and No. 2012CB725204 to QW), as well as a Grant from National Natural Science Foundation of China (Grant No. 31270146 to GQC).

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Authors and Affiliations

  1. MOE Key Lab of Bioinformatics, Department of Biological Science and Biotechnology, School of Life Science, Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing, 100084, China

    Jin Yin, Xiao-Zhi Fu, Qiong Wu, Jin-Chun Chen & Guo-Qiang Chen

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  1. Jin Yin
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  2. Xiao-Zhi Fu
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  4. Jin-Chun Chen
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Correspondence to Guo-Qiang Chen.

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Yin, J., Fu, XZ., Wu, Q. et al. Development of an enhanced chromosomal expression system based on porin synthesis operon for halophile Halomonas sp.. Appl Microbiol Biotechnol 98, 8987–8997 (2014). https://doi.org/10.1007/s00253-014-5959-1

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