Yuan Luke Z, Rouvière Pierre E, Larossa Robert A, Suh Wonchul
DuPont Central Research and Development, Experimental Station, Wilmington, DE 19880, USA.
Metab Eng. 2006 Jan;8(1):79-90. doi: 10.1016/j.ymben.2005.08.005. Epub 2005 Oct 28.
For metabolic engineering it is advantageous in terms of stability, genetic regulation, and metabolic burden to modulate expression of relevant genes on the chromosome rather than relying on over-expression of the genes on multi-copy vectors. Here we have increased the production of beta-carotene in Escherichia coli by replacing the native promoter of the chromosomal isoprenoid genes with the strong bacteriophage T5 promoter (P(T5)). We recombined PCR fragments with the lambda-Red recombinase to effect chromosomal promoter replacement, which allows direct integration of a promoter along with a selectable marker that can subsequently be excised by the Flp/FRT site-specific recombination system. The resulting promoter-engineered isoprenoid genes were combined by serial P1 transductions into a host strain harboring a reporter plasmid containing beta-carotene biosynthesis genes allowing a visual screen for yellow color indicative of beta-carotene accumulation. Construction of an E. coli P(T5)-dxs P(T5)-ispDispF P(T5)-idi P(T5)-ispB strain resulted in producing high titers (6mg/g dry cell weight) of beta-carotene. Surprisingly, over-expression of the ispB gene, which was expected to divert carbon flow from the isoprenoid pathway to quinone biosynthesis, resulted in increased beta-carotene production. We thus demonstrated that chromosomal promoter engineering of the endogenous isoprenoid pathway yielded high levels of beta-carotene in a non-carotenogenic E. coli. The high isoprenoid flux E. coli can be used as a starting strain to produce various carotenoids by introducing heterologous carotenoid genes.
对于代谢工程而言，从稳定性、基因调控和代谢负担方面考虑，调节染色体上相关基因的表达比依赖多拷贝载体上基因的过表达更具优势。在此，我们通过用强噬菌体T5启动子（P(T5)）替换染色体类异戊二烯基因的天然启动子，提高了大肠杆菌中β-胡萝卜素的产量。我们用λ-Red重组酶重组PCR片段以实现染色体启动子替换，这允许启动子与一个可选择标记直接整合，随后该可选择标记可被Flp/FRT位点特异性重组系统切除。通过连续P1转导将所得的启动子工程化类异戊二烯基因组合到一个携带含有β-胡萝卜素生物合成基因的报告质粒的宿主菌株中，从而可以通过视觉筛选黄色来指示β-胡萝卜素的积累。构建大肠杆菌P(T5)-dxs P(T5)-ispDispF P(T5)-idi P(T5)-ispB菌株导致产生高滴度（6mg/g干细胞重量）的β-胡萝卜素。令人惊讶的是，预期会使碳流从类异戊二烯途径转向醌生物合成的ispB基因的过表达却导致β-胡萝卜素产量增加。因此，我们证明了内源性类异戊二烯途径的染色体启动子工程在非产胡萝卜素的大肠杆菌中产生了高水平的β-胡萝卜素。这种具有高类异戊二烯通量的大肠杆菌可以用作起始菌株，通过引入异源类胡萝卜素基因来生产各种类胡萝卜素。
