Paramasivan Kalaivani, Mutturi Sarma
a CSIR-Central Food Technological Research Institute , Mysore , India.
Crit Rev Biotechnol. 2017 Dec;37(8):974-989. doi: 10.1080/07388551.2017.1299679. Epub 2017 Apr 20.
Terpenes are natural products with a remarkable diversity in their chemical structures and they hold a significant market share commercially owing to their distinct applications. These potential molecules are usually derived from terrestrial plants, marine and microbial sources. In vitro production of terpenes using plant tissue culture and plant metabolic engineering, although receiving some success, the complexity in downstream processing because of the interference of phenolics and product commercialization due to regulations that are significant concerns. Industrial workhorses' viz., Escherichia coli and Saccharomyces cerevisiae have become microorganisms to produce non-native terpenes in order to address critical issues such as demand-supply imbalance, sustainability and commercial viability. S. cerevisiae enjoys several advantages for synthesizing non-native terpenes with the most significant being the compatibility for expressing cytochrome P450 enzymes from plant origin. Moreover, achievement of high titers such as 40 g/l of amorphadiene, a sesquiterpene, boosts commercial interest and encourages the researchers to envisage both molecular and process strategies for developing yeast cell factories to produce these compounds. This review contains a brief consideration of existing strategies to engineer S. cerevisiae toward the synthesis of terpene molecules. Some of the common targets for synthesis of terpenes in S. cerevisiae are as follows: overexpression of tHMG1, ERG20, upc2-1 in case of all classes of terpenes; repression of ERG9 by replacement of the native promoter with a repressive methionine promoter in case of mono-, di- and sesquiterpenes; overexpression of BTS1 in case of di- and tetraterpenes. Site-directed mutagenesis such as Upc2p (G888A) in case of all classes of terpenes, ERG20p (K197G) in case of monoterpenes, HMG2p (K6R) in case of mono-, di- and sesquiterpenes could be some generic targets. Efforts are made to consolidate various studies (including patents) on this subject to understand the similarities, to identify novel strategies and to contemplate potential possibilities to build a robust yeast cell factory for terpene or terpenoid production. Emphasis is not restricted to metabolic engineering strategies pertaining to sterol and mevalonate pathway, but also other holistic approaches for elsewhere exploitation in the S. cerevisiae genome are discussed. This review also focuses on process considerations and challenges during the mass production of these potential compounds from the engineered strain for commercial exploitation.
萜类化合物是一类化学结构具有显著多样性的天然产物，由于其独特的应用，在商业上占有重要的市场份额。这些潜在的分子通常来源于陆生植物、海洋生物和微生物。利用植物组织培养和植物代谢工程在体外生产萜类化合物，尽管取得了一些成功，但由于酚类物质的干扰导致下游加工复杂，以及法规对产品商业化的影响，这些都是重大问题。工业上常用的大肠杆菌和酿酒酵母已成为生产非天然萜类化合物的微生物，以解决供需不平衡、可持续性和商业可行性等关键问题。酿酒酵母在合成非天然萜类化合物方面具有诸多优势，其中最重要的是能够兼容表达来自植物的细胞色素P450酶。此外，实现高产量，如40g/L的倍半萜类化合物紫穗槐二烯，提升了商业价值，并鼓励研究人员设想开发酵母细胞工厂生产这些化合物的分子和工艺策略。本综述简要考虑了工程改造酿酒酵母以合成萜类分子的现有策略。酿酒酵母中萜类化合物合成的一些常见靶点如下：对于所有类别的萜类化合物，tHMG1、ERG20、upc2-1过表达；对于单萜、二萜和倍半萜类化合物，用抑制性甲硫氨酸启动子取代天然启动子来抑制ERG9；对于二萜和四萜类化合物，BTS1过表达。在所有类别的萜类化合物中，如Upc2p（G888A）的定点诱变；在单萜类化合物中，如ERG20p（K197G）；在单萜、二萜和倍半萜类化合物中，如HMG2p（K6R），可能是一些通用靶点。本文致力于整合关于该主题的各种研究（包括专利），以了解其相似性，确定新策略，并思考构建强大的酵母细胞工厂用于萜类或萜类化合物生产的潜在可能性。重点不仅限于与甾醇和甲羟戊酸途径相关的代谢工程策略，还讨论了在酿酒酵母基因组中其他地方进行开发的其他整体方法。本综述还关注从工程菌株大规模生产这些潜在化合物用于商业开发过程中的工艺考虑和挑战。
