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Effects of chromosomal gene copy number and locations on polyhydroxyalkanoate synthesis by Escherichia coli and Halomonas sp.

  • Applied genetics and molecular biotechnology
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Abstract

Chromosomal integration and expression of heterologous gene(s) are favored in industrial biotechnology due to the inheriting expression stability. Yet, chromosomal expression is commonly weaker than plasmid one. The effect on gene expression level at 13 chromosomal locations in Escherichia coli was investigated using the polyhydroxybutyrate (PHB) synthesis pathway encoded by a phaCAB operon as a reporter. When 11 copies of phaCAB were randomly integrated into 11 of the 13 chromosomal locations, respectively, 5.2 wt% of PHB was produced. PHB (34.1 wt%) was accumulated by a recombinant E. coli inserted chromosomally with 50 copies of phaCAB in the active asnB site using a Cre-loxP recombination method. This PHB accumulation level was equivalent to a medium-copy-number plasmid expression system, suggesting the importance of chromosomal gene copy number for PHB production by E. coli. This result was used to manipulate a Halomonas strain. One copy of genes scpAB encoding methylmalonyl-CoA mutase and methylmalonyl-CoA decarboxylase was inserted into the strongest expression site porin in the chromosome of the 2-methylcitrate synthase (prpC) deleted mutant Halomonas TD08, leading to the synthesis of poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) from glucose as the sole carbon source. The chromosome-engineered strain produced PHBV consisting of 5–12 mol% 3-hydroxyvalerate (3HV) stably compared with unstable fluctuation of 7–25 mol% 3HV by a medium-copy-number plasmid system. These results demonstrated that chromosome engineering based on active transcriptional site and gene copy number is more feasible for polyhydroxyalkanoate (PHA) synthesis in Halomonas TD08 compared with in E. coli.

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Acknowledgments

We are grateful to Professor Alexander Steinbüchel for the generous donation of the plasmid pBHR68. This research was financially supported by National High Tech 973 Basic Research Fund (Grant No. 2012CB725201 to GQC and No. 2012CB725204 to QW) and partial National High Tech 863 Grant (No. 2012AA02A702 and No. 2013AA020301 to QW). A grant from the National Natural Science Foundation of China (Grant No. 31270146 to GQC) also contributed to this study. The 6-L fermentation studies were conducted with kind assistance from Mr. Zhao Kai in Qingdao KDN Biotech Co., Ltd, China.

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Authors and Affiliations

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

    Jin Yin, Huan Wang, Xiao-Zhi Fu, Xue Gao, Qiong Wu & Guo-Qiang Chen

  2. College of Eco-Environmental Engineering, Qinghai University, Xining, 810016, China

    Huan Wang

  3. Center for Nano and Micro Mechanics, Tsinghua University, 100084, Beijing, China

    Guo-Qiang Chen

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  1. Jin Yin
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  2. Huan Wang
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Correspondence to Guo-Qiang Chen.

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Yin, J., Wang, H., Fu, XZ. et al. Effects of chromosomal gene copy number and locations on polyhydroxyalkanoate synthesis by Escherichia coli and Halomonas sp.. Appl Microbiol Biotechnol 99, 5523–5534 (2015). https://doi.org/10.1007/s00253-015-6510-8

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