Yin Jin, Wang Huan, Fu Xiao-Zhi, Gao Xue, Wu Qiong, 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.
Appl Microbiol Biotechnol. 2015 Jul;99(13):5523-34. doi: 10.1007/s00253-015-6510-8. Epub 2015 Mar 12.
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.
由于具有遗传表达稳定性，异源基因的染色体整合和表达在工业生物技术中受到青睐。然而，染色体表达通常比质粒表达弱。使用由phaCAB操纵子编码的聚羟基丁酸酯（PHB）合成途径作为报告基因，研究了在大肠杆菌13个染色体位置对基因表达水平的影响。当将11份phaCAB分别随机整合到13个染色体位置中的11个时，产生了5.2 wt%的PHB。使用Cre-loxP重组方法，在活性asnB位点将50份phaCAB染色体插入重组大肠杆菌中，积累了PHB（34.1 wt%）。这个PHB积累水平等同于中拷贝数质粒表达系统，表明染色体基因拷贝数对大肠杆菌生产PHB的重要性。该结果被用于改造嗜盐单胞菌菌株。将编码甲基丙二酰辅酶A变位酶和甲基丙二酰辅酶A脱羧酶的scpAB基因的一份拷贝插入2-甲基柠檬酸合酶（prpC）缺失突变体嗜盐单胞菌TD08染色体中最强的表达位点孔蛋白，从而能够以葡萄糖作为唯一碳源合成聚（3-羟基丁酸酯-co-3-羟基戊酸酯）（PHBV）。与中拷贝数质粒系统中3HV含量不稳定地在7-25 mol%波动相比，染色体工程改造的菌株稳定地产生了由5-12 mol% 3-羟基戊酸酯（3HV）组成的PHBV。这些结果表明，与在大肠杆菌中相比，基于活性转录位点和基因拷贝数的染色体工程在嗜盐单胞菌TD08中用于聚羟基脂肪酸酯（PHA）合成更可行。
