Department of Chemistry, Portland State University, Portland, Oregon 97201, United States.
Biochemistry. 2012 Nov 20;51(46):9333-41. doi: 10.1021/bi300830q. Epub 2012 Nov 9.
The DEBS1-TE fusion protein is comprised of the loading module, the first two extension modules, and the terminal TE domain of the Saccharopolyspora erythraea 6-deoxyerythronolide B synthase. DEBS1-TE produces triketide lactones that differ on the basis of the starter unit selected by the loading module. Typical fermentations with plasmid-based expression of DEBS1-TE produce a 6:1 ratio of propionate to isobutyrate-derived triketide lactones. Functional dissection of the loading module from the remainder of DEBS1-TE results in 50% lower titers of triketide lactone and a dramatic shift in the production to a 1:4 ratio of propionate to isobutyrate-derived products. A series of radiolabeling studies of the loading module has shown that transfer from the AT to the ACP occurs much faster for propionate than for isobutyrate. However, the equilibrium occupancy of the AT favors isobutyrate such that propionate is outcompeted for ACP occupancy. Thus, propionyl-ACP is the kinetic product, while isobutyryl-ACP is the thermodynamic product. A slowed transfer from the loading domain ACP to first-extension module KS due to functional dissection of DEBS1-TE allows this isobutyryl-ACP-favored equilibrium to be realized and likely accounts for the observed shift in triketide lactone products.
DEBS1-TE 融合蛋白由加载模块、前两个延伸模块和 Saccharopolyspora erythraea 6-脱氧红霉内酯 B 合酶的末端 TE 结构域组成。DEBS1-TE 产生的三酮内酯在加载模块选择的起始单元的基础上有所不同。基于质粒表达 DEBS1-TE 的典型发酵产生丙酸酯与异丁酸盐衍生的三酮内酯的 6:1 比例。从 DEBS1-TE 的其余部分对加载模块进行功能剖析，导致三酮内酯的产量降低 50%，并且产物的产生从丙酸酯与异丁酸盐衍生产物的 1:4 比例急剧转变。对加载模块的一系列放射性标记研究表明，从 AT 到 ACP 的转移对于丙酸酯比异丁酸酯快得多。然而，AT 的平衡占据有利于异丁酸酯，使得 ACP 占据被丙酸酯竞争。因此，丙酰基-ACP 是动力学产物，而异丁酰基-ACP 是热力学产物。由于 DEBS1-TE 的功能剖析，从加载域 ACP 到第一延伸模块 KS 的转移减慢，使得这种有利于异丁酰基-ACP 的平衡得以实现，这可能解释了观察到的三酮内酯产物的转变。
