Cox R J, Hitchman T S, Byrom K J, Findlow I S, Tanner J A, Crosby J, Simpson T J
School of Chemistry, University of Bristol, UK.
FEBS Lett. 1997 Apr 1;405(3):267-72. doi: 10.1016/s0014-5793(97)00202-0.
Expression in Escherichia coli of Streptomyces acyl carrier proteins (ACPs) associated with polyketide biosynthesis using the pT7-7 expression system of Tabor and Richardson led to the production predominantly of inactive apo-proteins lacking the 4'-phosphopantetheinyl prosthetic group essential for polyketide synthase activity. Modification of growth conditions led to an increase of production of active holo-protein for the actinorhodin (act) ACP, but this technique was ineffective for oxytetracycline (otc) and griseusin (gris) ACPs. Labelling experiments revealed that a low level of otc ACP expressed prior to induction was produced mainly as active holo-protein, while post-induction 15N-labelled protein was almost exclusively in the apo-ACP form. Limiting endogenous holo-acyl carrier protein synthase (ACPS) concentration was implicated as responsible for low apo-ACP to holo-ACP conversion, rather than limiting substrate (coenzyme A) and cofactor (Mg2+) concentrations. Co-expression of act and gris ACPs with ACPS in E. coli led to high levels of production of active holo-ACPs and ACPS. We have also made the significant observation that ACPS is able to transfer acylated CoA moieties to act apo-ACP.
利用Tabor和Richardson的pT7-7表达系统在大肠杆菌中表达与聚酮生物合成相关的链霉菌酰基载体蛋白（ACP），主要产生的是缺乏聚酮合酶活性所必需的4'-磷酸泛酰巯基乙胺辅基的无活性脱辅基蛋白。生长条件的改变使放线紫红素（act）ACP的活性全蛋白产量增加，但该技术对土霉素（otc）和灰黄霉素（gris）ACP无效。标记实验表明，诱导前表达的低水平otc ACP主要以活性全蛋白形式产生，而诱导后15N标记的蛋白几乎完全是脱辅基ACP形式。内源性全酰基载体蛋白合酶（ACPS）浓度受限被认为是脱辅基ACP向全蛋白ACP转化率低的原因，而不是底物（辅酶A）和辅因子（Mg2+）浓度受限。在大肠杆菌中act和gris ACP与ACPS共表达导致活性全蛋白ACP和ACPS的高水平产生。我们还进行了重要观察，即ACPS能够将酰化的辅酶A部分转移到act脱辅基ACP上。
