Feng G H, Leonard T J
Department of Genetics, University of Wisconsin, Madison 53706, USA.
J Bacteriol. 1995 Nov;177(21):6246-54. doi: 10.1128/jb.177.21.6246-6254.1995.
Aflatoxins are potent toxic and carcinogenic compounds, produced by Aspergillus parasiticus and A. flavus as secondary metabolites. In this research, a polyketide synthase gene (pksL1), the key gene for aflatoxin biosynthesis initiation in A. parasiticus, has been functionally identified and molecularly characterized. PCR-derived DNA probes were used to find the pksL1 gene from subtracted, aflatoxin-related clones. Gene knockout experiments generated four pksL1 disruptants which lost both the ability to produce aflatoxins B1, B2, and G1 and the ability to accumulate norsolorinic acid and all other intermediates of the aflatoxin biosynthetic pathway. A pksL1 DNA probe detected a 6.6-kb poly(A)+ RNA transcript in Northern (RNA) hybridizations. This transcript, associated with aflatoxin production, exhibited a regulated expression that was influenced by growth phase, medium composition, and culture temperature. DNA sequencing of pksL1 revealed an open reading frame for a polypeptide (PKSL1) of 2,109 amino acids. Sequence analysis further recognized four functional domains in PKSL1, acyl carrier protein, beta-ketoacyl-acyl carrier protein synthase, acyltransferase, and thioesterase, all of which are usually present in polyketide synthases and fatty acid synthases. On the basis of these results, we propose that pksL1 encodes the polyketide synthase which synthesizes the backbone polyketide and initiates aflatoxin biosynthesis. In addition, the transcript of pksL1 exhibited heterogeneity at the polyadenylation site similar to that of plant genes.
黄曲霉毒素是由寄生曲霉和黄曲霉作为次级代谢产物产生的强效毒性和致癌化合物。在本研究中，已对寄生曲霉中黄曲霉毒素生物合成起始的关键基因——聚酮合酶基因（pksL1）进行了功能鉴定和分子表征。利用聚合酶链反应（PCR）衍生的DNA探针，从消减的、与黄曲霉毒素相关的克隆中寻找pksL1基因。基因敲除实验产生了四个pksL1破坏株，它们丧失了产生黄曲霉毒素B1、B2和G1的能力，以及积累诺索林酸和黄曲霉毒素生物合成途径中所有其他中间体的能力。一个pksL1 DNA探针在Northern（RNA）杂交中检测到一个6.6 kb的聚腺苷酸化（poly(A)+）RNA转录本。这个与黄曲霉毒素产生相关的转录本表现出受生长阶段、培养基组成和培养温度影响的调控表达。pksL1的DNA测序揭示了一个编码2109个氨基酸多肽（PKSL1）的开放阅读框。序列分析进一步在PKSL1中识别出四个功能结构域，即酰基载体蛋白、β-酮酰基-酰基载体蛋白合酶、酰基转移酶和硫酯酶，所有这些结构域通常都存在于聚酮合酶和脂肪酸合酶中。基于这些结果，我们提出pksL1编码合成聚酮主链并启动黄曲霉毒素生物合成的聚酮合酶。此外，pksL1的转录本在聚腺苷酸化位点表现出与植物基因类似的异质性。
