Improved protein glycosylation enabled heterologous biosynthesis of monoterpenoid indole alkaloids and their unnatural derivatives in yeast

IF 3.7 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Metabolic Engineering Communications Pub Date : 2023-06-01 DOI:10.1016/j.mec.2022.e00215

Mohammadamin Shahsavarani , Joseph Christian Utomo , Rahul Kumar , Melina Paz-Galeano , Jorge Jonathan Oswaldo Garza-García , Zhan Mai , Dae-Kyun Ro , Yang Qu

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引用次数: 7

Abstract

With over 3000 reported structures, monoterpenoid indole alkaloids (MIAs) constitute one of the largest alkaloid groups in nature, including the clinically important anticancer drug vinblastine and its semi-synthetic derivatives from Catharanthus roseus (Madagascar’s periwinkle). With the elucidation of the complete 28-step biosynthesis for anhydrovinblastine, it is possible to investigate the heterologous production of vinblastine and other medicinal MIAs. In this study, we successfully expressed the flavoenzyme O-acetylstemmadenine oxidase in Saccharomyces cerevisiae (baker’s yeast) by signal peptide modification, which is a vinblastine biosynthetic gene that has not been functionally expressed in this system. We also reported the simultaneous integration of ∼18 kb MIA biosynthetic gene cassettes as single copies into four genomic loci of baker’s yeast by CRISPR-Cas9, which enabled the biosynthesis of vinblastine precursors catharanthine and tabersonine from the feedstocks secologanin and tryptamine. We further demonstrated the biosynthesis of fluorinated and hydroxylated catharanthine and tabersonine derivatives using our yeasts, which showed that the MIA biosynthesis accommodates unnatural substrates, and the system can be further explored to produce other complex MIAs.

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改进的蛋白质糖基化使单萜吲哚生物碱及其非天然衍生物在酵母中异源生物合成成为可能

单萜类吲哚生物碱（MIAs）具有3000多种已报道的结构，是自然界中最大的生物碱类之一，包括临床上重要的抗癌药物长春碱及其来自长春花（马达加斯加长春花）的半合成衍生物。随着脱水长春碱完整的28步生物合成的阐明，有可能研究长春碱和其他药用MIA的异源生产。在本研究中，我们通过信号肽修饰成功地在酿酒酵母（面包酵母）中表达了风味酶O-乙酰基茎胺氧化酶，这是一种尚未在该系统中功能表达的长春碱生物合成基因。我们还报道了通过CRISPR-Cas9将~18kb MIA生物合成基因盒以单拷贝形式同时整合到面包酵母的四个基因组位点中，这使得长春碱前体长春花碱和色胺能够从饲料中生物合成。我们使用我们的酵母进一步证明了氟化和羟基化的长春花碱和tabersonine衍生物的生物合成，这表明MIA生物合成适应非天然底物，该系统可以进一步探索产生其他复杂的MIA。

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来源期刊

Metabolic Engineering Communications Medicine-Endocrinology, Diabetes and Metabolism

CiteScore

13.30

自引率

1.90%

发文量

审稿时长

18 weeks

期刊介绍： Metabolic Engineering Communications, a companion title to Metabolic Engineering (MBE), is devoted to publishing original research in the areas of metabolic engineering, synthetic biology, computational biology and systems biology for problems related to metabolism and the engineering of metabolism for the production of fuels, chemicals, and pharmaceuticals. The journal will carry articles on the design, construction, and analysis of biological systems ranging from pathway components to biological complexes and genomes (including genomic, analytical and bioinformatics methods) in suitable host cells to allow them to produce novel compounds of industrial and medical interest. Demonstrations of regulatory designs and synthetic circuits that alter the performance of biochemical pathways and cellular processes will also be presented. Metabolic Engineering Communications complements MBE by publishing articles that are either shorter than those published in the full journal, or which describe key elements of larger metabolic engineering efforts.