利用代谢工程改造酿酒酵母,从葡萄糖中高水平生产 (+)-ambrein 蛋白。

IF 2 4区 生物学 Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
Biotechnology Letters Pub Date : 2024-08-01 Epub Date: 2024-06-17 DOI:10.1007/s10529-024-03502-2
Chumin Lin, Xiaopeng Zhang, Zhongju Ji, Baolian Fan, Yaman Chen, Yuhong Wu, Yuhong Gan, Zhengping Li, Yi Shang, Lixin Duan, Feng Wang
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引用次数: 0

摘要

(+)-Ambrein 是龙涎香的主要成分,龙涎香是抹香鲸(Physeter microcephalus)体内发现的一种稀有产品。利用可持续资源进行微生物生产是取代动物提取和化学合成的一种可行方法。我们构建了一种可从头生产 (+)- 角鲨烯的工程酵母菌株。角鲨烯是(+)-安布林生物合成的底物。首先,通过优化甲羟戊酸途径,获得了角鲨烯产量为 384.4 mg/L 的菌株 LQ2。然后,我们通过过表达经过密码子优化的四烯-β-莪术烯环化酶(BmeTC)及其双突变体酶(BmeTCY167A/D373C),评估不同的启动子,敲除 GAL80,并将该蛋白与 BmeTC 和角鲨烯合成酶(AtSQS2)融合,设计出了一种以葡萄糖为碳源从头生产(+)-丹皮酚的方法。然而,(+)-羊毛脂的合成仍然有限,导致 BmeTC 的催化活性较低。我们对 BmeTC 进行了蛋白质表面氨基酸修饰。我们获得了第一步的显性突变体 BmeTCK6A/Q9E/N454A,以提高其催化活性。在摇瓶中,(+)-丹魄素的产量从 35.2 毫克/升增加到 59.0 毫克/升,最后在 2 升发酵罐中达到 457.4 毫克/升,这是目前酵母菌的最高滴度。高效的工程菌株和廉价的发酵条件可用于 (+)-ambrein 的工业生产。代谢工程工具为优化其他高价值三萜的生物合成提供了方向。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

Metabolic engineering of Saccharomyces cerevisiae for high-level production of (+)-ambrein from glucose.

Metabolic engineering of Saccharomyces cerevisiae for high-level production of (+)-ambrein from glucose.

(+)-Ambrein is the primary component of ambergris, a rare product found in sperm whales (Physeter microcephalus). Microbial production using sustainable resources is a promising way to replace animal extraction and chemical synthesis. We constructed an engineered yeast strain to produce (+)-ambrein de novo. Squalene is a substrate for the biosynthesis of (+)-ambrein. Firstly, strain LQ2, with a squalene yield of 384.4 mg/L was obtained by optimizing the mevalonate pathway. Then we engineered a method for the de novo production of (+)-ambrein using glucose as a carbon source by overexpressing codon-optimized tetraprenyl-β-curcumene cyclase (BmeTC) and its double mutant enzyme (BmeTCY167A/D373C), evaluating different promoters, knocking out GAL80, and fusing the protein with BmeTC and squalene synthase (AtSQS2). Nevertheless, the synthesis of (+)-ambrein is still limited, causing low catalytic activity in BmeTC. We carried out a protein surface amino acid modification of BmeTC. The dominant mutant BmeTCK6A/Q9E/N454A for the first step was obtained to improve its catalytic activity. The yield of (+)-ambrein increased from 35.2 to 59.0 mg/L in the shake flask and finally reached 457.4 mg/L in the 2 L fermenter, the highest titer currently available for yeast. Efficiently engineered strains and inexpensive fermentation conditions for the industrial production of (+)-ambrein. The metabolic engineering tools provide directions for optimizing the biosynthesis of other high-value triterpenes.

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来源期刊
Biotechnology Letters
Biotechnology Letters 工程技术-生物工程与应用微生物
CiteScore
5.90
自引率
3.70%
发文量
108
审稿时长
1.2 months
期刊介绍: Biotechnology Letters is the world’s leading rapid-publication primary journal dedicated to biotechnology as a whole – that is to topics relating to actual or potential applications of biological reactions affected by microbial, plant or animal cells and biocatalysts derived from them. All relevant aspects of molecular biology, genetics and cell biochemistry, of process and reactor design, of pre- and post-treatment steps, and of manufacturing or service operations are therefore included. Contributions from industrial and academic laboratories are equally welcome. We also welcome contributions covering biotechnological aspects of regenerative medicine and biomaterials and also cancer biotechnology. Criteria for the acceptance of papers relate to our aim of publishing useful and informative results that will be of value to other workers in related fields. The emphasis is very much on novelty and immediacy in order to justify rapid publication of authors’ results. It should be noted, however, that we do not normally publish papers (but this is not absolute) that deal with unidentified consortia of microorganisms (e.g. as in activated sludge) as these results may not be easily reproducible in other laboratories. Papers describing the isolation and identification of microorganisms are not regarded as appropriate but such information can be appended as supporting information to a paper. Papers dealing with simple process development are usually considered to lack sufficient novelty or interest to warrant publication.
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