Enhancement and selective production of phoslactomycin B, a protein phosphatase IIa inhibitor, through identification and engineering of the corresponding Biosynthetic gene cluster
Enhancement and selective production of phoslactomycin B, a protein phosphatase IIa inhibitor, through identification and engineering of the corresponding Biosynthetic gene cluster
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DOI：
10.1074/jbc.m305082200
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发表时间：
2003-09-12
影响因子：
4.8
通讯作者：
Reynolds, KA
中科院分区：
文献类型：
--
作者：
Palaniaappan, N;Kim, BS;Reynolds, KA
磷内酰胺霉素（PLMs）是丝氨酸苏氨酸磷酸酶的有效选择性抑制剂，具有抗肿瘤和抗病毒活性。发酵过程中的多种类似物和低滴度阻碍了这类天然产物的开发。对链霉菌HK-803的75-kb PLM生物合成基因簇进行了克隆、测序和分析。PLM聚酮合酶的加载域和七个延伸模块从环己烷羧酸（CHC）引物产生含有E-和Z-双键的不寻常的线性不饱和聚酮链。通过PlmS（2）羟基化所得PLM-B的CHC衍生侧链，并随后酯化，产生剩余的PLM类似物。一种新的PCR靶向技术允许plmS的快速和容易的等位基因替换（2）。所得突变体选择性地产生PLM-B，滴度比野生型菌株高6倍。该突变体和生物合成基因簇将促进具有改善的性质的杂合PLM的工程微生物生产。
Phoslactomycins (PLMs), potent and selective inhibitors of serine threonine phosphatases, are of interest for their antitumor and antiviral activity. Multiple analogs and low titers in the fermentation process have hampered the development of this class of natural products. The entire 75-kb PLM biosynthetic gene cluster of Streptomyces sp. HK-803 was cloned, sequenced, and analyzed. The loading domain and seven extension modules of the PLM polyketide synthase generate an unusual linear unsaturated polyketide chain containing both E- and Z-double bonds from a cyclohexanecarboxylic acid (CHC) primer. Hydroxylation of the CHC-derived side chain of the resulting PLM-B by PlmS(2), and a subsequent esterification, produces the remaining PLM analogs. A new PCR targeting technology allowed rapid and facile allelic replacement of plmS(2). The resulting mutant selectively produced the PLM-B, at 6-fold higher titers than the wild type strain. This mutant and the biosynthetic gene cluster will facilitate engineered microbial production of hybrid PLMs with improved properties.