木糖生产柠檬烯的棉实代谢工程研究。

Gloria Muñoz-Fernández, Rubén Martínez-Buey, José Luis Revuelta, Alberto Jiménez
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引用次数: 2

摘要

背景:柠檬烯是一种环单萜,在食品、化妆品和制药工业中有着广泛的应用。通过植物提取工业生产柠檬烯及其衍生物存在季节性和气候问题、原料限制、效率低和环境问题等重要缺陷。因此,实施高效和生态友好的生产柠檬烯和其他萜烯的生物工艺构成了微生物生物技术的一个有吸引力的目标。在这种情况下,具有从替代碳源生产柠檬烯的能力的新型生物催化剂将有助于满足柠檬烯的工业需求。结果:已培育出以木糖为原料生产柠檬烯的工业木耳工程菌株。在利用木糖的棉皮a.s gossypii菌株中,首先将柠檬烯合成酶(LS)与天然HMG1基因(编码HMG-CoA还原酶)过表达,建立了产柠檬烯的平台。此外,还设计了几种提高柠檬烯产量的策略。因此,我们利用外源neryl二磷酸合酶,结合合成正交途径,对ERG20突变等位基因(erg20F95W和erg20F126W)的影响进行了评价。棉蚜双突变体erg20F95W-F126W的致死率凸显了法尼酯二磷酸对合成必需甾醇的不可或缺性。此外,利用正交途径,通过neryl二磷酸绕过Erg20活性,引发柠檬烯滴度大幅增加(33.6 mg/L),而没有严重改变工程菌株的适应度。最后,原生ERG12基因的过表达进一步提高了柠檬烯的产量,以木糖为碳源培养96 h后柠檬烯产量达到336.4 mg/L。结论:木糖基碳源棉蚜工程菌株可用于生产柠檬烯。利用正交合成途径结合过表达ERG12是棉蚜产生柠檬烯的一种非常有利的策略。本研究提出的菌株为从棉蚜中富含木糖的水解物等农业工业废物中生产柠檬烯和其他萜烯提供了原理证明。
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 Ashbya gossypii for limonene production from xylose.

Metabolic engineering of Ashbya gossypii for limonene production from xylose.

Metabolic engineering of Ashbya gossypii for limonene production from xylose.

Metabolic engineering of Ashbya gossypii for limonene production from xylose.

Background: Limonene is a cyclic monoterpene that has applications in the food, cosmetic, and pharmaceutical industries. The industrial production of limonene and its derivatives through plant extraction presents important drawbacks such as seasonal and climate issues, feedstock limitations, low efficiency and environmental concerns. Consequently, the implementation of efficient and eco-friendly bioprocesses for the production of limonene and other terpenes constitutes an attractive goal for microbial biotechnology. In this context, novel biocatalysts with the ability to produce limonene from alternative carbon sources will help to meet the industrial demands of limonene.

Results: Engineered strains of the industrial fungus Ashbya gossypii have been developed to produce limonene from xylose. The limonene synthase (LS) from Citrus limon was initially overexpressed together with the native HMG1 gene (coding for HMG-CoA reductase) to establish a limonene-producing platform from a xylose-utilizing A. gossypii strain. In addition, several strategies were designed to increase the production of limonene. Hence, the effect of mutant alleles of ERG20 (erg20F95W and erg20F126W) were evaluated together with a synthetic orthogonal pathway using a heterologous neryl diphosphate synthase. The lethality of the A. gossypii double mutant erg20F95W-F126W highlights the indispensability of farnesyl diphosphate for the synthesis of essential sterols. In addition, the utilization of the orthogonal pathway, bypassing the Erg20 activity through neryl diphosphate, triggered a substantial increase in limonene titer (33.6 mg/L), without critically altering the fitness of the engineered strain. Finally, the overexpression of the native ERG12 gene further enhanced limonene production, which reached 336.4 mg/L after 96 h in flask cultures using xylose as the carbon source.

Conclusions: The microbial production of limonene can be carried out using engineered strains of A. gossypii from xylose-based carbon sources. The utilization of a synthetic orthogonal pathway together with the overexpression of ERG12 is a highly beneficial strategy for the production of limonene in A. gossypii. The strains presented in this work constitute a proof of principle for the production of limonene and other terpenes from agro-industrial wastes such as xylose-rich hydrolysates in A. gossypii.

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