Wu K, Chung L, Revill W P, Katz L, Reeves C D
Kosan Biosciences Inc., Hayward, CA 94545, USA.
Gene. 2000 Jun 13;251(1):81-90. doi: 10.1016/s0378-1119(00)00171-2.
FK520 (ascomycin) is a macrolide produced by Streptomyces hygroscopicus var. ascomyceticus (ATCC 14891) that has immunosuppressive, neurotrophic and antifungal activities. To further elucidate the biosynthesis of this and related macrolides, we cloned and sequenced an 80kb region encompassing the FK520 gene cluster. Genes encoding the three polyketide synthase (PKS) subunits (fkbB, fkbC and fkbA), the peptide synthetase (fkbP), the 31-O-methyltransferase (fkbM), the C-9 hydroxylase (fkbD) and the 9-hydroxyl oxidase (fkbO) had the same organization as the genes reported in the FK506 gene cluster of Streptomyces sp. MA6548 (Motamedi, H., Shafiee, A., 1998. The biosynthetic gene cluster for the macrolactone ring of the immunosuppressant FK506. Eur. J. Biochem. 256, 528-534). Disruption of a PKS gene in the cluster using the φC31 phage vector, KC515, led to antibiotic non-producing strains, proving the identity of the cluster. Previous labeling data have indicated that FK520 biosynthesis uses novel polyketide extender units (Byrne, K.M., Shafiee, A., Nielson, J., Arison, B., Monaghan, R.L., Kaplan, L., 1993. The biosynthesis and enzymology of an immunosuppressant, immunomycin, produced by Streptomyces hygroscopicus var, ascomyceticus. Dev. Ind. Microbiol. 32, 29-45). Genes in the flanking regions of the FK520 cluster were identified that appear to be involved in synthesis of these extender units. All but two of these genes were homologous to genes with known function. In addition to a crotonyl-CoA reductase gene (fkbS), at least two other genes are proposed to be involved in biosynthesis of the atypical PKS extender unit ethylmalonyl-CoA, which accounts for the ethyl side chain on C-21 of FK520. A set of five contiguous genes (fkbGHIJK) is proposed to be involved in biosynthesis of an unusual PKS extender unit bearing an oxygen on the alpha-carbon, and leading to the 13- and 15-methoxy side chains. These putative precursor synthesis genes in the flanking regions of the FK520 cluster are not found in the flanking regions of the rapamycin cluster (Molnár, I., Aparicio, J.F., Haydock, S.F., Khaw, L.E., Schwecke, T., König, A., Staunton, J., Leadlay, P.F., 1996. Organisation of the biosynthetic gene cluster for rapamycin in Streptomyces hygroscopicus: analysis of genes flanking the polyketide synthase. Gene 169, 1-7), consistent with labeling data showing that rapamycin biosynthesis uses only malonyl and methylmalonyl extender units.
FK520（子囊霉素）是由吸水链霉菌子囊菌变种（ATCC 14891）产生的一种大环内酯类化合物，具有免疫抑制、神经营养和抗真菌活性。为了进一步阐明该化合物及相关大环内酯类化合物的生物合成过程，我们克隆并测序了一个包含FK520基因簇的80kb区域。编码三个聚酮合酶（PKS）亚基（fkbB、fkbC和fkbA）、肽合成酶（fkbP）、31-O-甲基转移酶（fkbM）、C-9羟化酶（fkbD）和9-羟基氧化酶（fkbO）的基因，其组织方式与链霉菌MA6548的FK506基因簇中报道的基因相同（Motamedi, H., Shafiee, A., 1998. 免疫抑制剂FK506大环内酯环的生物合成基因簇。欧洲生物化学杂志。256, 528 - 534）。使用φC31噬菌体载体KC515破坏该簇中的一个PKS基因，导致产生不产生抗生素菌株，证明了该簇的一致性。先前的标记数据表明，FK520生物合成使用新型聚酮延伸单元（Byrne, K.M., Shafiee, A., Nielson, J., Arison, B., Monaghan, R.L., Kaplan, L., 1993. 吸水链霉菌子囊菌变种产生的免疫抑制剂免疫霉素的生物合成和酶学。工业微生物学进展。32, 29 - 45）。在FK520簇侧翼区域鉴定出的基因似乎参与了这些延伸单元的合成。除了两个基因外，这些基因中的其他基因都与已知功能的基因同源。除了巴豆酰辅酶A还原酶基因（fkbS）外，至少还有另外两个基因被认为参与了非典型PKS延伸单元乙基丙二酰辅酶A的生物合成，该延伸单元构成了FK520 C-21位的乙基侧链。一组五个相邻基因（fkbGHIJK）被认为参与了一种不寻常的PKS延伸单元的生物合成，该延伸单元在α-碳上带有一个氧原子，并导致产生了13-和15-甲氧基侧链。在雷帕霉素簇的侧翼区域未发现FK520簇侧翼区域中的这些假定前体合成基因（Molnár, I., Aparicio, J.F., Haydock, S.F., Khaw, L.E., Schwecke, T., König, A., Staunton, J., Leadlay, P.F., 1996. 吸水链霉菌中雷帕霉素生物合成基因簇的组织：聚酮合酶侧翼基因的分析。基因。169, 1 - 7），这与标记数据一致，表明雷帕霉素生物合成仅使用丙二酰和甲基丙二酰延伸单元。
