Functional Exchangeability of Oxidase and Dehydrogenase Reactions in the Biosynthesis of Hydroxyphenylglycine, a Nonribosomal Peptide Building Block

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• 作者：Veronica Diez ; Mark Loznik ; Sandra Taylor ; Michael Winn ; Nicholas J. W. Rattray ; Helen Podmore ; Jason Micklefield ; Royston Goodacre ; Marnix H. Medema ; Ulrike M眉ller ; Roel Bovenberg ; Dick B. Janssen ; Eriko Takano
• 刊名：ACS Synthetic Biology
• 出版年：2015
• 出版时间：July 17, 2015
• 年：2015
• 卷：4
• 期：7
• 页码：796-807
• 全文大小：500K
• ISSN：2161-5063

文摘

A key problem in the engineering of pathways for the production of pharmaceutical compounds is the limited diversity of biosynthetic enzymes, which restricts the attainability of suitable traits such as less harmful byproducts, enhanced expression features, or different cofactor requirements. A promising synthetic biology approach is to redesign the biosynthetic pathway by replacing the native enzymes by heterologous proteins from unrelated pathways. In this study, we applied this method to effectively re-engineer the biosynthesis of hydroxyphenylglycine (HPG), a building block for the calcium-dependent antibiotic of Streptomyces coelicolor, a nonribosomal peptide. A key step in HPG biosynthesis is the conversion of 4-hydroxymandelate to 4-hydroxyphenylglyoxylate, catalyzed by hydroxymandelate oxidase (HmO), with concomitant generation of H₂O₂. The same reaction can also be catalyzed by O₂-independent mandelate dehydrogenase (MdlB), which is a catabolic enzyme involved in bacterial mandelate utilization. In this work, we engineered alternative HPG biosynthetic pathways by replacing the native HmO in S. coelicolor by both heterologous oxidases and MdlB dehydrogenases from various sources and confirmed the restoration of calcium-dependent antibiotic biosynthesis by biological and UHPLC-MS analysis. The alternative enzymes were isolated and kinetically characterized, confirming their divergent substrate specificities and catalytic mechanisms. These results demonstrate that heterologous enzymes with different physiological contexts can be used in a Streptomyces host to provide an expanded library of enzymatic reactions for a synthetic biology approach. This study thus broadens the options for the engineering of antibiotic production by using enzymes with different catalytic and structural features.