Amplification and disruption of the phenylacetyl-CoA ligase gene of Penicillium chrysogenum encoding an aryl-capping enzyme that supplies phenylacetic acid to the isopenicillin N-acyltransferase
Amplification and disruption of the phenylacetyl-CoA ligase gene of Penicillium chrysogenum encoding an aryl-capping enzyme that supplies phenylacetic acid to the isopenicillin N-acyltransferase
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DOI：
10.1042/bj20051599
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发表时间：
2006-04-01
影响因子：
4.1
通讯作者：
Martín, JF
中科院分区：
文献类型：
--
作者：
Lamas-Maceiras, M;Vaca, I;Martín, JF
从产黄青霉AS-P-78的噬菌体文库中克隆了编码苯乙酰辅酶A连接酶的基因phl。通过逆转录-PCR证实了phi基因中存在5个内含子。phi基因编码的芳基辅酶A连接酶与拟南芥4-香豆酰辅酶A连接酶密切相关。Phi蛋白含有定义芳基-CoA（4-香豆酰-CoA）连接酶底物特异性密码的大多数氨基酸，并且不同于乙酰-CoA连接酶和其他酰基-CoA连接酶。phl基因与青霉素基因簇不连锁。在自主复制质粒中扩增phi导致苯乙酰辅酶A连接酶活性增加8倍，青霉素产量增加35%。含有扩增的phl基因的转化体对高浓度的苯乙酸（超过2.5g/l）具有抗性。phi基因的破坏导致青霉素产量减少40%，苯乙酰辅酶A连接酶活性也有类似的降低。突变株对苯乙酸高度敏感。被破坏的突变体与phi基因的互补恢复了青霉素生产的正常水平和对苯乙酸的抗性。因此，由phi基因编码的plenylacetyl-CoA连接酶参与青霉素的产生，尽管第二个芳基-CoA连接酶似乎对苯乙酸活化有部分贡献。Phi蛋白缺乏肽-载体-蛋白结构域，并且表现为激活苯乙酸并将其转移到异青霉素N酰基转移酶的芳基加帽酶。Phi蛋白含有过氧化物酶体靶向序列，该序列也存在于异青霉素N酰基转移酶中。这两种蛋白质的过氧化物酶体共定位表明青霉素途径的最后两种酶形成过氧化物酶体功能复合物。
A gene, phl, encoding a phenylacetyl-CoA ligase was cloned from a phage library of Penicillium chrysogenum AS-P-78. The presence of five introns in the phi gene was confirmed by reverse transcriptase-PCR. The phi gene encoded an aryl-CoA ligase closely related to Arabidopsis thaliana 4-coumaroyl-CoA ligase. The Phi protein contained most of the amino acids defining the aryl-CoA (4-coumaroyl-CoA) ligase substrate-specificity code and differed from acetyl-CoA ligase and other acyl-CoA ligases. The phl gene was not linked to the penicillin gene cluster. Amplification of phi in an autonomous replicating plasmid led to an 8-fold increase in phenylacetyl-CoA ligase activity and a 35 % increase in penicillin production. Transformants containing the amplified phl gene were resistant to high concentrations of phenylacetic acid (more than 2.5 g/l). Disruption of the phi gene resulted in a 40 %, decrease in penicillin production and a similar reduction of phenylacetyl-CoA ligase activity. The disrupted mutants were highly susceptible to phenylacetic acid. Complementation of the disrupted mutants with the phi gene restored normal levels of penicillin production and resistance to phenylacetic acid. The plenylacetyl-CoA ligase encoded by the phi gene is therefore involved in penicillin production, although a second aryl-CoA ligase appears to contribute partially to phenylacetic acid activation. The Phi protein lacks a peptide-carrier-protein domain and behaves as an aryl-capping enzyme that activates phenylacetic acid and transfers it to the isopenicillin N acyltransferase. The Phi protein contains the peroxisome-targeting sequence that is also present in the isopenicillin N acyltransferase. The peroxisomal colocalization of these two proteins indicates that the last two enzymes of the penicillin pathway form a peroxisomal functional complex.