caa a22 4500 605506000 CHVBK 20210128100624.0 cr unu---uuuuu 210128e20150701xx s 000 0 eng 10.1007/s00253-015-6510-8 doi (NATIONALLICENCE)springer-10.1007/s00253-015-6510-8 Effects of chromosomal gene copy number and locations on polyhydroxyalkanoate synthesis by Escherichia coli and Halomonas sp [Elektronische Daten] [Jin Yin, Huan Wang, Xiao-Zhi Fu, Xue Gao, Qiong Wu, Guo-Qiang Chen] 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, 5.2wt% of PHB was produced. PHB (34.1wt%) 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 5-12mol% 3-hydroxyvalerate (3HV) stably compared with unstable fluctuation of 7-25mol% 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. Springer-Verlag Berlin Heidelberg, 2015 PHB nationallicence Polyhydroxyalkanoates nationallicence PHA nationallicence Genomic expression nationallicence Chromosomal integration nationallicence Escherichia coli nationallicence Halomonas nationallicence Yin Jin 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 aut Wang Huan 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 aut Fu Xiao-Zhi 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 aut Gao Xue 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 aut Wu Qiong 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 aut Chen Guo-Qiang 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 aut Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/13(2015-07-01), 5523-5534 0175-7598 99:13<5523 2015 99 253 https://doi.org/10.1007/s00253-015-6510-8 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00253-015-6510-8 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Yin Jin 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 aut NATIONALLICENCE 700 1- Wang Huan 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 aut NATIONALLICENCE 700 1- Fu Xiao-Zhi 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 aut NATIONALLICENCE 700 1- Gao Xue 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 aut NATIONALLICENCE 700 1- Wu Qiong 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 aut NATIONALLICENCE 700 1- Chen Guo-Qiang 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 aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/13(2015-07-01), 5523-5534 0175-7598 99:13<5523 2015 99 253