金黄拟诺卡氏菌CGMCC 4.5723中Pictet-Spenglerase基因的克隆与功能鉴定

Cloning and Functional Identification of Pictet-Spenglerase Gene from Nocardiopsis flavescens CGMCC 4.5723

催化β-咔啉生物碱生物合成途径中的必需酶Pictet-Spenglerase(P-S酶)能够催化色氨酸或色胺与相应的醛酮底物缩合形成关键的β-咔啉骨架结构。金黄拟诺卡氏菌CGMCC 4.5723是一株能够生产Marinacarboline A类β-咔啉生物碱的稀有放线菌,相应的生物合成途径中存在1个潜在的P-S酶。因此,本研究以已知的P-S酶McbB/NscbB的编码基因序列为分子标记,定位了菌株中P-S酶的编码基因,并将该酶命名为NfmcbB。通过后续的蛋白表达纯化和酶学分析发现该酶同样能够利用色氨酸和丙酮醛缩合生成关键的β-咔啉骨架前体1-酰基-3-羧基-β-咔啉和1-酰基-β-咔啉。上述NfmcbB的发掘与鉴定工作,不但丰富了β-咔啉生物碱的生物合成研究工作,同时也为新颖P-S酶类生物催化剂开发提供了可选酶分子。

The Pictet-Spenglerase(P-S enzyme)is essential for the biosynthesis of carboline alkaloids.The P-S enzyme catalyzes the formation of theβ-carboline skeleton by condensing the tryptophan or tryptamine with the corresponding aldehyde or ketone substrate.Nocardiopsis flavescens CGMCC 4.5723 produces aβ-carboline alkaloid called Marinacarboline A,suggesting that a new P-S enzyme exists in the Marinacarboline A biosynthetic pathway.In this study,the gene sequence encoding the known P-S enzyme of McbB/NscbB was used as molecular marker,the coding gene of P-S enzyme in the strain of N.flavescens CGMCC 4.5723 was located,and the enzyme was named NfmcbB.Further characterization of NfmcbB confirmed that NfmcbB can condense the L-tryptophan and methylglyoxal to biosynthesize 1-acetyl-3-carboxyl-β-carboline and 1-acetyl-β-carboline,two precursors of Marinecarboline withβ-carboline skeleton.Our study expands the biosynthesis research ofβ-carboline alkaloid,and further provides a new P-S enzyme candidate for developing biocatalyst in the future.