Rude M A, Khosla C
Department of Chemical Engineering, Stanford University, Stanford, California 94305, USA.
J Antibiot (Tokyo). 2006 Aug;59(8):464-70. doi: 10.1038/ja.2006.65.
For the heterologous production of ansamycin polyketides such as rifamycin and geldanamycin in Escherichia coli, a number of unusual but important tools must be engineered into the bacterium. Here we demonstrate efficient production of the starter unit 3-amino-5-hydroxybenzoic acid (AHBA) and the methoxymalonyl extender unit in E. coli. Previous work has demonstrated the production of the ansamycin starter unit AHBA in E. coli in low yield. It was shown that the low yield is primarily due to acetylation of AHBA into N-acetyl-AHBA. Three methods for minimizing this side reaction were evaluated. First, a putative N-arylamine-acetyltransferase (NAT) was deleted from the E. coli chromosome, although this did not alter N-acetyl-AHBA production. Next, E. coli grown in media devoid of glucose yielded a nearly equal mixture of AHBA and N-acetyl-AHBA. Lastly, the NAT inhibitor glycyrrhizic acid was shown to further inhibit the acetylation reaction. The entire set of genes for synthesizing the methoxymalonyl extender unit was transferred from the geldanamycin producer Streptomyces hygroscopicus into E. coli. The pathway specific ACP isolated from the resulting recombinant strain was found to predominantly occur as methyoxymalonyl-ACP. Together, these findings set the stage for engineered biosynthesis of ansamycin polyketides in E. coli.
为了在大肠杆菌中异源生产诸如利福霉素和格尔德霉素等安莎霉素聚酮化合物，必须将一些不同寻常但很重要的工具引入该细菌。在此，我们展示了在大肠杆菌中高效生产起始单元3-氨基-5-羟基苯甲酸（AHBA）和甲氧基丙二酰延伸单元。先前的工作已证明大肠杆菌中安莎霉素起始单元AHBA的产量很低。结果表明，产量低主要是由于AHBA乙酰化为N-乙酰-AHBA。评估了三种尽量减少这种副反应的方法。首先，从大肠杆菌染色体中删除了一种假定的N-芳基胺乙酰转移酶（NAT），尽管这并未改变N-乙酰-AHBA的产量。其次，在不含葡萄糖的培养基中生长的大肠杆菌产生了几乎等量的AHBA和N-乙酰-AHBA混合物。最后，NAT抑制剂甘草酸被证明可进一步抑制乙酰化反应。合成甲氧基丙二酰延伸单元的整套基因从格尔德霉素产生菌吸水链霉菌转移到了大肠杆菌中。从所得重组菌株中分离出的途径特异性酰基载体蛋白（ACP）主要以甲氧基丙二酰-ACP的形式存在。总之，这些发现为大肠杆菌中安莎霉素聚酮化合物的工程化生物合成奠定了基础。
