PDF(1419 KB)
萜类香味成分及潜香物质生物合成研究进展*
常晋, 李乾, 魏新铎, 刘德裕, 徐永明, 魏甲欣, 杨金初, 王光路
中国生物工程杂志 ›› 2024, Vol. 44 ›› Issue (12) : 124-140.
PDF(1419 KB)
PDF(1419 KB)
萜类香味成分及潜香物质生物合成研究进展*
Research Progress on the Biosynthesis of Terpenoid Flavor and Latent Fragrance Compounds
({{custom_author.role_en}}), {{javascript:window.custom_author_en_index++;}}萜类化合物已广泛应用于医药、食品及香精香料等领域。天然萜类化合物在植物中合成浓度低,来源受限且提取成本高昂,具有复杂化学结构的萜类化合物化学合成难度高,反应条件苛刻且收率普遍较低,严重限制了萜类化合物的广泛应用。生物合成法在萜类化合物高效合成方面具有巨大应用潜力,已成为近年来的研究热点。随着合成生物学的快速发展,通过微生物底盘细胞的改造、新颖目标产物的合成途径设计,结合微生物大规模发酵技术,构建微生物细胞工厂应用于萜类化合物高效生物合成,其合成产品在食品、药品、化妆品等领域展现了广阔的市场前景。萜类香味物质(具有特殊香韵)及潜香物质(经转化后可释放香气成分)是目前天然产物产业化领域研究最具经济价值的化合物。总结了萜类香味成分及潜香物质生物合成的共性合成机制、前沿进展以及构建微生物细胞工厂工程策略(基因编辑、转录调控、途径设计及模块组装、细胞膜工程、适应性进化、共培养系统),以此促进萜类香味成分及潜香物质合成技术发展及其在香精香料行业的转化应用。
Terpenoids are widely used in medicine, food, fragrances, and flavors. However, natural terpenoids are produced at low concentrations in plants, making extraction costly and sources limited. The chemical synthesis of terpenoids, especially those with complex chemical structures, is challenging due to harsh conditions and low yields, hindering their widespread application. Biosynthesis offers a promising alternative for efficient production of terpenoids and has become a focal point of research in recent years. With advances in synthetic biology, the construction of microbial cell factories for terpenoid biosynthesis through microbial host modification, novel pathway design, and integration of microbial mass fermentation shows great potential for the food, medical, and cosmetic industries. Terpenoid flavors, which have distinctive aromas, and latent fragrance compounds, which release aromatic components when transformed, are the most economically valuable substances in the industrialization of natural products. This article reviews the synthetic mechanisms, recent advances, and engineering strategies for the biosynthesis of terpenoid flavors and latent fragrances. Key strategies include gene editing, transcriptional regulation, modular pathway engineering, cell membrane engineering, adaptive laboratory evolution, and co-culture systems. The goal is to advance terpenoid synthesis technologies for industrial applications in the fragrance and flavor industry.
萜类化合物 / 香精香料 / 合成生物学 / 微生物细胞工厂 {{custom_keyword}} /
Terpenoids / Flavors and fragrances / Synthetic biology / Microbial cell factory {{custom_keyword}} /
图1 萜类化合物生物合成途径[18]Glucose:葡萄糖;PYR:丙酮酸;G3P:3-磷酸甘油醛;DXP:1-脱氧-D-木酮糖-5-磷酸;MEP:2-C-甲基-D-4-磷酸-赤藓醇;CDP-ME:4-二磷核糖酰-2-C-甲基-D-赤藓糖醇;CDP-MEP: 4-二磷核糖酰-2-C-甲基-D-赤藓糖醇焦磷酸;MEcPP:2-C-甲基-D-赤藓糖醇-2,4-环二磷酸;HMB-PP:2-羟基-3-甲基-2-戊烷酸;Acetyl-CoA:乙酰辅酶A;AcAc-CoA:乙酰乙酰辅酶A;HMG-CoA:3-羟基-3-甲基戊二酰辅酶A;MVA:甲羟戊酸;MVA-5-P:甲羟戊酸-5-磷酸;MVA-PP:甲羟戊酸-5-焦磷酸;MG-CoA:3-甲基戊二酰辅酶A;MB:3-甲基-2-丁烯醛;Prenol:3-甲基-2-丁烯-1-醇;Isoprenol:3-甲基-3-丁烯-1-醇;IP:异戊烯基磷酸;DMAP:二甲基烯丙基磷酸;IPP:异戊二烯焦磷酸;DMAPP:二甲烯丙基焦磷酸;GPP:香叶基焦磷酸;FPP:法尼基焦磷酸;GGPP:香叶基香叶基焦磷酸;DXS:1-脱氧-D-木酮糖-5-磷酸合成酶;DXR:1-脱氧-D-木酮糖-5-磷酸还原异构酶;MCT:MEP 胞苷酰转移酶;CMK:CDP-ME激酶;MDS:MEcPP合酶;HDS:HMB-PP 合酶;HDR:HMB-PP 还原酶;ACCT:乙酰辅酶A C-乙酰转移酶;HMGS:HMG-CoA合酶;HMGR:HMG-CoA还原酶;MVK:MVA激酶;PMVK:磷酸甲羟戊酸激酶;MVD:焦磷酸甲羟戊酸脱羧酶;LiuC:烯酰基辅酶A水合酶;AibAB:谷胱甘肽酰辅酶A脱羧酶;cbjALD:脂肪酰辅酶A还原酶;YahK:醇脱氢酶;ThiM:羟乙基噻唑激酶;IPK:异戊烯基磷酸激酶;CK:胆碱激酶;IDI: 异戊二烯焦磷酸异构酶;GPPS:香叶基焦磷酸合酶;FPPS:法尼基焦磷酸合酶;GGPPS:香叶基香叶基焦磷酸合酶 Fig.1 Synthetic pathway of terpenoids PYR:Pyruvic acid; G3P:Glyceraldehyde 3-phosphate; DXP:1-Deoxy-D-xylulose 5-phosphate; MEP:2-C-methyl-D-erythritol-4-phosphate; CDP-ME:4-Diphosphocytidyl-2-C-methyl-D-erythritol; CDP-MEP:4-Diphosphocytidyl-2-C-methyl-D-erythritol 2-phosphate; MEcPP:2-C-methyl-D-erythritol-2,4-cyclodiphosphate; HMB-PP:2-Hydroxy-3-methylbut-2-enyl-diphosphate; Acetyl-CoA:Acetyl coenzyme; AcAc-CoA:Acetoaceyl-CoA; HMG-CoA:3-Hydroxy-3-methylglutaryl-CoA; MVA:Mevalonate; MVA-5-P:MVA-5-phosphate; MVA-PP:MVA-5-diphosphate; MG-CoA:3-Methylglutaconyl-CoA; MB:3-Methyl-2-butenal; Prenol:3-Methyl-2-butene-1-ol; Isoprenol:3-Methyl-3-butene-1-ol; IP:Isopentenyl monophosphate; DMAP:Dimethylallyl monophosphate; IPP:Isopentenyl pyrophosphate; DMAPP:Dimethylallyl pyrophosphate; GPP:Geranyl diphosphate; FPP:Farnesyl diphosphate synthase; GGPP:Geranylgeranyl diphosphate; DXS:1-Deoxy-D-xylulose-5-phosphate synthase; DXR:1-Deoxy-D-xylulose 5-phosphate reductoisomeras; MCT:MEP cytidylyltransferase; CMK:CDP-ME kinase; MDS:MEcPP synthase; HDS:HMB-PP synthase; HDR:HMB-PP reductase; ACCT:Acetyl-CoA C-acetyl transferase; HMGS:HMG-CoA synthase; HMGR:HMG-CoA reductase; MVK:MVA kinase; PMVK:Phosphomevalonate kinase; MVD:Diphosphomevalonate decarboxylase; LiuC:Enoyl-CoA hydratase; AibAB:Glutaconyl-CoA decarboxylase; cbjALD:Acyl-CoA reductase; YahK:Alcohol dehydrogenase; ThiM:Hydroxyethylthiazole kinase; IPK:Isopentenyl phosphate kinase; CK:Choline kinase; IDI:Isopentenyl diphosphate isomerase; GPPS:Geranyl diphosphate synthase; FPPS:Farnesyl diphosphate synthase; GGPPS:Geranylgeranyl diphosphate synthase |
表1 天然及人工设计的类异戊二烯前体合成途径比较[19]Table 1 Comparison of native and synthetic pathways for the generation of isoprenoid precursors |
| 合成途径 | 天然途径 | 人工设计途径 | ||
|---|---|---|---|---|
| MVA | DXP/MEP | IPA | IUP | |
| 起始中心碳代谢物 | Acetyl-CoA | Pyruvate; Glyceraldehyde 3-phosphate | Acetyl-CoA | Prenol/isoprenol |
| 合成步数 | 6 | 7 | 8 | 2 |
| 异戊二烯合成前体 | IPP | IPP和DMAPP | DMAPP | IPP和DMAPP |
| 碳转化效率* | 0.83 | 0.83 | 0.83 | 1.00 |
| ATP需求 | 3 ATP | 3 ATP | 2 ATP | 2 ATP |
| NAD(P)H需求 | 2 | 3 | 2 | 0 |
| 辅因子 | CoA、ATP | TPP, CTP, ATP,[4Fe-4S] | CoA, ATP | ATP |
| *碳摩尔质量 | |
| *Carbon molar basis |
图2 构建微生物细胞工厂生物合成萜类化合物研究策略示意图[46,49]A:基因编辑 B:转录调控 C:途径设计及模块组装 D:细胞膜工程 E:适应性进化 F:共培养系统 Fig.2 Schematics of synthetic biology strategies to engineer host cells for the bioproduction of terpenoids A: Gene edit (CRIPSR/Cas9/Cpf1) B: Transcriptional regulation C: Modular pathway engineering D: Cell membrane engineering E:Adaptive laboratory evolution F: Co-culture system |
表2 萜类香味物质生物合成水平及香气描述Table 2 Microbial cell factories for terpenoid flavor and fragrance compounds biosynthesis and aroma description |
| 萜类香味成分 | 平台宿主 | 浓度 | 发酵规模 | 生产率 | 气味描述 | 文献 | |||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| 橙花醇 | Escherichia coli | 0.967 g/L | 摇瓶发酵 | 0.012 4 g/(L·h) | 新鲜清甜的橙花和玫瑰花香,带些果香,有似覆盆子的果香韵味,味甜而略带轻微的苦味 | [53] | |||||
| 香叶醇 | Saccharomyces cerevisiae | 5.52 g/L | 摇瓶发酵 | - | 有似玫瑰的香气,留香较长,稍苦 | [54] | |||||
| 柠檬烯 | Escherichia coli | 7.3 g/L | 3.1 L发酵罐 | 0.15 g/(L·h) | 具有青酸带甜的新鲜橘子-柠檬果香,香气轻飘 | [55] | |||||
| (S)/(R)- 芳樟醇 | Pantoea ananatis | 5.60 g/L/ 3.71 g/L | 1 L发酵罐 | 0.17 g/(L·h)/ 0.077 g/(L·h) | 具有典型的花香香气,清新飘逸的香气,有淡弱的柑橘类果香韵调 | [56] | |||||
| α-松油醇 | Saccharomyces cerevisiae | 21.88 mg/L | 5 L发酵罐 | 0.18 mg/(L·h) | 具有紫丁香香气,气势淡弱,不够留长 | [57] | |||||
| α-檀香烯 | Saccharomyces cerevisiae | 2.92 g/L | 1.3 L发酵罐 | 0.48 mg/(L·h) | 甜而温和木香,香气令人愉快且留香持久 | [58] | |||||
| 橙花叔醇 | Saccharomyces cerevisiae | 7.01 g/L | 5 L发酵罐 | 0.049 g/(L·h) | 近似于玫瑰和苹果的微弱花香,非常甜美的、清鲜的、持久的香气 | [59] | |||||
| (+)-圆柚酮 | Saccharomyces cerevisiae | 2.39 g/L | 3 L发酵罐 | 0.016 g/(L·h) | 非常强烈的、柑橘的、葡萄的、水果的芳香和味道,气味阈值极低 | [60] | |||||
| 瓦伦西亚烯 | Saccharomyces cerevisiae | 5.61 g/L | 3 L发酵罐 | 0.032 g/(L·h) | 具有柑橘香气,富有饱满的木香质感 | [61] | |||||
| 二氢-β- 紫罗兰酮 | E. coli-S. cerevisiae | 27 mg/L | 摇瓶发酵 | 0.56 mg/(L·h) | 具有木香、花香、果香香气 | [48] | |||||
表3 潜香类香味成分生物合成水平及香气描述Table 3 Biosynthesis level and aroma description of latent fragrant compounds |
| 潜香类香味 成分 | 平台宿主 | 浓度 | 发酵规模 | 生产率 | 气味描述 | 文献 |
|---|---|---|---|---|---|---|
| 冷杉醇 | Escherichia coli | 1.375 g/L | 1.3 L发酵罐 | 0.012 g/(L·h) | 发生氧化降解可转化为具有独特香味的类琥珀化合物 | [85] |
| 西柏三烯二醇 | Saccharomyces cerevisiae | 1.05 mg/L | 3 L发酵罐 | 0.015 mg/(L·h) | 具有烤香味,稍甜味草药香,可可香味 | [86] |
| 龙涎香醇 | Pichia pastoris | 0.105 g/L | 5 L发酵罐 | 0.001 4 mg/ (L·h) | 被氧化降解后形成降龙涎香醚等香味物质,具有花香和植物油脂香味 | [87] |
| β-胡萝卜素 | Yarrowia lipolytica | 39.5 g/L | 3 L发酵罐 | 0.165 g/(L·h) | 降解可转化为芳樟醇、异佛尔酮等香味物质 | [88] |
| 叶黄素 | S. cerevisiae | 595.3 μg/L | 摇瓶发酵 | 6.20 μg/(L·h) | 降解可转化为芳樟醇、异佛尔酮等香味物质 | [89] |
| [1] |
The repetitive and inadequate application of pediculicidal products frequently results in the development of resistance to these compounds. Essential oils are a promising alternative to synthetic insecticides, although their mode of action remains to be explored. It has been proposed that one possible target of the essential oils is the inhibition of acetylcholinesterase (AChE). The role of monoterpenoids as possible AChE inhibitors and their relationship with the toxicity was investigated both in vitro and in vivo. Inhibition of electric eel AChE activity showed that the most effective inhibitor was 1,8-cineole with IC(50) 6 x 10(-3) M. The inhibition of AChE activity of head louse homogenate by 1,8-cineole showed IC(50) 7.7 x 10(-2) M. The intoxication symptoms of head lice exposed to vapors of 1,8-cineole was recorded before the in vivo head louse AChE inhibition assay. No correlation was found between neurotoxic symptoms and inhibition of AChE activity.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [2] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [3] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [4] |
Taxol (paclitaxel) is a potent anticancer drug first isolated from the Taxus brevifolia Pacific yew tree. Currently, cost-efficient production of Taxol and its analogs remains limited. Here, we report a multivariate-modular approach to metabolic-pathway engineering that succeeded in increasing titers of taxadiene--the first committed Taxol intermediate--approximately 1 gram per liter (~15,000-fold) in an engineered Escherichia coli strain. Our approach partitioned the taxadiene metabolic pathway into two modules: a native upstream methylerythritol-phosphate (MEP) pathway forming isopentenyl pyrophosphate and a heterologous downstream terpenoid-forming pathway. Systematic multivariate search identified conditions that optimally balance the two pathway modules so as to maximize the taxadiene production with minimal accumulation of indole, which is an inhibitory compound found here. We also engineered the next step in Taxol biosynthesis, a P450-mediated 5α-oxidation of taxadiene to taxadien-5α-ol. More broadly, the modular pathway engineering approach helped to unlock the potential of the MEP pathway for the engineered production of terpenoid natural products.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [5] |
Identification of genes that affect the product accumulation phenotype of recombinant strains is an important problem in industrial strain construction and a central tenet of metabolic engineering. We have used systematic (model-based) and combinatorial (transposon-based) methods to identify gene knockout targets that increase lycopene biosynthesis in strains of Escherichia coli. We show that these two search strategies yield two distinct gene sets, which affect product synthesis either through an increase in precursor availability or through (largely unknown) kinetic or regulatory mechanisms, respectively. Exhaustive exploration of all possible combinations of the above gene sets yielded a unique set of 64 knockout strains spanning the metabolic landscape of systematic and combinatorial gene knockout targets. This included a global maximum strain exhibiting an 8.5-fold product increase over recombinant K12 wild type and a twofold increase over the engineered parental strain. These results were further validated in controlled culture conditions.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [6] |
Ambergris, a sperm whale metabolite, has long been used as a fragrance and traditional medication, but it is now rarely available. The odor components of ambergris result from the photooxidative degradation of the major component, ambrein. The pharmacological activities of ambergris have also been attributed to ambrein. However, efficient production of ambrein and odor compounds has not been achieved. Here, we constructed a system for the synthesis of ambrein and odor components. First, we created a new triterpene synthase, "ambrein synthase," for mass production of ambrein by redesigning a bacterial enzyme. The ambrein yields were approximately 20 times greater than those reported previously. Next, an efficient photooxidative conversion system from ambrein to a range of volatiles of ambergris was established. The yield of volatiles was 8-15%. Finally, two biological activities, promotion of osteoclast differentiation and prevention of amyloid β-induced apoptosis, were discovered using the synthesized ambrein.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [7] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [8] |
The chassis is the cellular host used as a recipient of engineered biological systems in synthetic biology. They are required to propagate the genetic information and to express the genes encoded in it. Despite being an essential element for the appropriate function of genetic circuits, the chassis is rarely considered in their design phase. Consequently, the circuits are transferred to model organisms commonly used in the laboratory, such as Escherichia coli, that may be suboptimal for a required function. In this review, we discuss some of the properties desirable in a versatile chassis and summarize some examples of alternative hosts for synthetic biology amenable for engineering. These properties include a suitable life style, a robust cell wall, good knowledge of its regulatory network as well as of the interplay of the host components with the exogenous circuits, and the possibility of developing whole-cell models and tuneable metabolic fluxes that could allow a better distribution of cellular resources (metabolites, ATP, nucleotides, amino acids, transcriptional and translational machinery). We highlight Pseudomonas putida, widely used in many different biotechnological applications as a prominent organism for synthetic biology due to its metabolic diversity, robustness and ease of manipulation.© 2016 The Author(s).
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [9] |
Terpene synthases are the primary enzymes in the formation of low-molecular-weight terpene metabolites. Rapid progress in the biochemical and molecular analysis of terpene synthases has allowed significant investigations of their evolution, structural and mechanistic properties, and regulation. The organization of terpene synthases in large gene families, their characteristic ability to form multiple products, and their spatial and temporal regulation during development and in response to biotic and abiotic factors contribute to the time-variable formation of a diverse group of terpene metabolites. The structural diversity and complexity of terpenes generates an enormous potential for mediating plant-environment interactions. Engineering the activities of terpene synthases provides opportunities for detailed functional evaluations of terpene metabolites in planta.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [10] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [11] |
Isoprenoids represent the oldest class of known low molecular-mass natural products synthesized by plants. Their biogenesis in plastids, mitochondria and the endoplasmic reticulum-cytosol proceed invariably from the C5 building blocks, isopentenyl diphosphate and/or dimethylallyl diphosphate according to complex and reiterated mechanisms. Compounds derived from the pathway exhibit a diverse spectrum of biological functions. This review centers on advances obtained in the field based on combined use of biochemical, molecular biology and genetic approaches. The function and evolutionary implications of this metabolism are discussed in relation with seminal informations gathered from distantly but related organisms.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [12] |
Isoprenoids are a large and highly diverse group of natural products with many functions in plant primary and secondary metabolism. Isoprenoids are synthesized from common prenyl diphosphate precursors through the action of terpene synthases and terpene-modifying enzymes such as cytochrome P450 monooxygenases. Many isoprenoids have important applications in areas such as human health and nutrition, and much effort has been directed toward their production in microbial hosts. However, many hurdles must be overcome in the elucidation and functional microbial expression of the genes responsible for biosynthesis of an isoprenoid of interest. Here, we review investigations into isoprenoid function and gene discovery in plants as well as the latest advances in isoprenoid pathway engineering in both plant and microbial hosts.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [13] |
Terpenoids are a diverse class of natural products that have many functions in the plant kingdom and in human health and nutrition. Their chemical diversity has led to the discovery of over 40,000 different structures, with several classes serving as important pharmaceutical agents, including the anticancer agents paclitaxel (Taxol) and terpenoid-derived indole alkaloids. Many terpenoid compounds are found in low yield from natural sources, so plant cell cultures have been investigated as an alternate production strategy. Metabolic engineering of whole plants and plant cell cultures is an effective tool to both increase terpenoid yield and alter terpenoid distribution for desired properties such as enhanced flavor, fragrance or color. Recent advances in defining terpenoid metabolic pathways, particularly in secondary metabolism, enhanced knowledge concerning regulation of terpenoid accumulation, and application of emerging plant systems biology approaches, have enabled metabolic engineering of terpenoid production. This paper reviews the current state of knowledge of terpenoid metabolism, with a special focus on production of important pharmaceutically active secondary metabolic terpenoids in plant cell cultures. Strategies for defining pathways and uncovering rate-influencing steps in global metabolism, and applying this information for successful terpenoid metabolic engineering, are emphasized.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [14] |
Industrial biotechnology promises to revolutionize conventional chemical manufacturing in the years ahead, largely owing to the excellent progress in our ability to re-engineer cellular metabolism. However, most successes of metabolic engineering have been confined to over-producing natively synthesized metabolites in E. coli and S. cerevisiae. A major reason for this development has been the descent of metabolic engineering, particularly secondary metabolic engineering, to a collection of demonstrations rather than a systematic practice with generalizable tools. Synthetic biology, a more recent development, faces similar criticisms. Herein, we attempt to lay down a framework around which bioreaction engineering can systematize itself just like chemical reaction engineering. Central to this undertaking is a new approach to engineering secondary metabolism known as 'multivariate modular metabolic engineering' (MMME), whose novelty lies in its assessment and elimination of regulatory and pathway bottlenecks by re-defining the metabolic network as a collection of distinct modules. After introducing the core principles of MMME, we shall then present a number of recent developments in secondary metabolic engineering that could potentially serve as its facilitators. It is hoped that the ever-declining costs of de novo gene synthesis; the improved use of bioinformatic tools to mine, sort and analyze biological data; and the increasing sensitivity and sophistication of investigational tools will make the maturation of microbial metabolic engineering an autocatalytic process. Encouraged by these advances, research groups across the world would take up the challenge of secondary metabolite production in simple hosts with renewed vigor, thereby adding to the range of products synthesized using metabolic engineering.Copyright © 2011 Elsevier Inc. All rights reserved.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [15] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [16] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [17] |
Throughout human history, natural products have been the foundation for the discovery and development of therapeutics used to treat diseases ranging from cardiovascular disease to cancer. Their chemical diversity and complexity have provided structural scaffolds for small-molecule drugs and have consistently served as inspiration for medicinal design. However, the chemical complexity of natural products also presents one of the main roadblocks for production of these pharmaceuticals on an industrial scale. Chemical synthesis of natural products is often difficult and expensive, and isolation from their natural sources is also typically low yielding. Synthetic biology and metabolic engineering offer an alternative approach that is becoming more accessible as the tools for engineering microbes are further developed. By reconstructing heterologous metabolic pathways in genetically tractable host organisms, complex natural products can be produced from inexpensive sugar starting materials through large-scale fermentation processes. In this Perspective, we discuss ongoing research aimed toward the production of terpenoid natural products in genetically engineered Escherichia coli and Saccharomyces cerevisiae.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [18] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [19] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [20] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [21] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [22] |
The mevalonate pathway for the biosynthesis of the universal terpenoid precursors, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), is known in considerable detail. Only recently, the existence of a second mevalonate-independent pathway for the biosynthesis of IPP and DMAPP was detected in plants and certain eubacteria. Experiments with 13C and/or 2H-labelled precursors were crucial in the elucidation of this novel route. The pathway is essential in plants, many eubacteria and apicomplexan parasites, but not in archaea and animals. The genes, enzymes and intermediates of this pathway were rapidly unravelled over the past few years. Detailed knowledge about the mechanisms of this novel route may benefit the development of novel antibiotics, antimalarials and herbicides.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [23] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [24] |
Isoprenoids are synthesized by consecutive condensations of their five-carbon precursor, isopentenyl diphosphate, to its isomer, dimethylallyl diphosphate. Two pathways for these precursors are known. One is the mevalonate pathway, which operates in eucaryotes, archaebacteria, and cytosols of higher plants. The other is a recently discovered pathway, the nonmevalonate pathway, which is used by many eubacteria, green algae, and chloroplasts of higher plants. To date, five reaction steps in this new pathway and their corresponding enzymes have been identified. EC numbers of these enzymes have been assigned by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB) and are available at http://www.chem.qmw.ac.uk/iubmb/enzyme/reaction/terp/nonMVA.html.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [25] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [26] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [27] |
Cell-free systems are growing in importance for the biosynthesis of complex molecules. These systems combine the precision of traditional chemistry with the versatility of biology in creating superior overall processes. Recently, a new synthetic pathway for the biosynthesis of isoprenoids using the substrate isopentenol, dubbed the isopentenol utilization pathway (IUP), was demonstrated to be a promising alternative to the native 2C-methyl-d-erythritol-4-phosphate (MEP) and mevalonate (MVA) pathways. This simplified pathway, which contains a minimum of four enzymes to produce basic monoterpenes and only depends on ATP and isopentenol as substrates, allows for a highly flexible approach to the commercial synthesis of isoprenoid products. In this work, we use metabolic reconstitution to characterize this new pathway in vitro and demonstrate its use for the cell-free synthesis of mono-, sesquit-, and diterpenoids. Kinetic modeling and sensitivity analysis were also used to identify the most significant parameters for taxadiene productivity, and metabolic control analysis was employed to elucidate protein-level interactions within this pathway, which demonstrated that the IUP enzymatic system is primarily controlled by the concentration and kinetics of choline kinase (CK) and not regulated by any pathway intermediates. This is a significant advantage over the natural MEP or MVA pathways as it greatly simplifies future metabolic engineering efforts, both in vitro and in vivo, aiming at improving the kinetics of CK. Finally, we used the insights gathered to demonstrate an in vitro IUP system that can produce 220 mg/L of the diterpene taxadiene, in 9 hr, almost 3-fold faster than any system reported thus far.© 2019 Wiley Periodicals, Inc.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [28] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [29] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [30] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [31] |
Isoprenoids are constructed in nature using hemiterpene building blocks that are biosynthesized from lengthy enzymatic pathways with little opportunity to deploy precursor-directed biosynthesis. Here, an artificial alcohol-dependent hemiterpene biosynthetic pathway was designed and coupled to several isoprenoid biosynthetic systems, affording lycopene and a prenylated tryptophan in robust yields. This approach affords a potential route to diverse non-natural hemiterpenes and by extension isoprenoids modified with non-natural chemical functionality. Accordingly, the prototype chemo-enzymatic pathway is a critical first step toward the construction of engineered microbial strains for bioconversion of simple scalable building blocks into complex isoprenoid scaffolds.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [32] |
Terpenes are industrially relevant natural compounds the biosynthesis of which relies on two well-established-mevalonic acid (MVA) and methyl erythritol phosphate (MEP)-pathways. Both pathways are widely distributed in all domains of life, the former is predominantly found in eukaryotes and archaea and the latter in eubacteria and chloroplasts. These two pathways supply isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), the universal building blocks of terpenes.The potential to establish a semisynthetic third pathway to access these precursors has been investigated in the present work. We have tested the ability of a collection of 93 isopentenyl phosphate kinases (IPK) from the biodiversity to catalyse the double phosphorylation of isopentenol and dimethylallyl alcohol to give, respectively IPP and DMAPP. Five IPKs selected from a preliminary in vitro screening were evaluated in vivo in an engineered chassis E. coli strain producing carotenoids. The recombinant pathway leading to the synthesis of neurosporene and lycopene, allows a simple colorimetric assay to test the potential of IPKs for the synthesis of IPP and DMAPP starting from the corresponding alcohols. The best candidate identified was the IPK from Methanococcoides burtonii (UniProt ID: Q12TH9) which improved carotenoid and neurosporene yields ~ 18-fold and > 45-fold, respectively. In our lab scale conditions, titres of neurosporene reached up to 702.1 ± 44.7 µg/g DCW and 966.2 ± 61.6 µg/L. A scale up to 4 L in-batch cultures reached to 604.8 ± 68.3 µg/g DCW and 430.5 ± 48.6 µg/L without any optimisation shown its potential for future applications. Neurosporene was almost the only carotenoid produced under these conditions, reaching ~ 90% of total carotenoids both at lab and batch scales thus offering an easy access to this sophisticated molecule.IPK biodiversity was screened in order to identify IPKs that optimize the final carotenoid content of engineered E. coli cells expressing the lycopene biosynthesis pathway. By simply changing the IPK and without any other metabolic engineering we improved the neurosporene content by more than 45 fold offering a new biosynthetic access to this molecule of upmost importance.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [33] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [34] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [35] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [36] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [37] |
Sesquiterpenes represent a large class of terpene compounds found in plants with broad applications such as pharmaceuticals and biofuels. The plastidial MEP pathway in ripening tomato fruit is naturally optimized to provide the 5-carbon isoprene building blocks of all terpenes for production of the tetraterpene pigment lycopene and other carotenoids, making it an excellent plant system to be engineered for production of high-value terpenoids. We reconstituted and enhanced the pool of sesquiterpene precursor farnesyl diphosphate (FPP) in plastids of tomato fruit by overexpressing the fusion gene DXS-FPPS encoding a fusion protein of 1-deoxy-D-xylulose 5-phosphate synthase (DXS) linked with farnesyl diphosphate synthase (originally called farnesyl pyrophosphate synthase, and abbreviated as FPPS) under the control of fruit-ripening specific polygalacturonase (PG) promoter concomitant with substantial reduction in lycopene content and large production of FPP-derived squalene. The supply of precursors achieved by the fusion gene expression can be harnessed by an engineered sesquiterpene synthase that is retargeted to plastid to engineer high-yield sesquiterpene production in tomato fruit, offering an effective production system for high-value sesquiterpene ingredients.Copyright © 2023 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [38] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [39] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [40] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [41] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [42] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [43] |
Recent advances in CRISPR/Cas9 based genome editing have considerably advanced genetic engineering of industrial yeast strains. In this study, we report the construction and characterization of a toolkit for CRISPR activation and interference (CRISPRa/i) for a polyploid industrial yeast strain. In the CRISPRa/i plasmids that are available in high and low copy variants, dCas9 is expressed alone, or as a fusion with an activation or repression domain; VP64, VPR or Mxi1. The sgRNA is introduced to the CRISPRa/i plasmids from a double stranded oligonucleotide by in vivo homology-directed repair, allowing rapid transcriptional modulation of new target genes without cloning. The CRISPRa/i toolkit was characterized by alteration of expression of fluorescent protein-encoding genes under two different promoters allowing expression alterations up to ~ 2.5-fold. Furthermore, we demonstrated the usability of the CRISPRa/i toolkit by improving the tolerance towards wheat straw hydrolysate of our industrial production strain. We anticipate that our CRISPRa/i toolkit can be widely used to assess novel targets for strain improvement and thus accelerate the design-build-test cycle for developing various industrial production strains.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [44] |
Background: Transcriptional reprogramming is a fundamental process of living cells in order to adapt to environmental and endogenous cues. In order to allow flexible and timely control over gene expression without the interference of native gene expression machinery, a large number of studies have focused on developing synthetic biology tools for orthogonal control of transcription. Most recently, the nuclease-deficient Cas9 (dCas9) has emerged as a flexible tool for controlling activation and repression of target genes, by the simple RNA-guided positioning of dCas9 in the vicinity of the target gene transcription start site.Results: In this study we compared two different systems of dCas9-mediated transcriptional reprogramming, and applied them to genes controlling two biosynthetic pathways for biobased production of isoprenoids and triacylglycerols (TAGs) in baker's yeast Saccharomyces cerevisiae. By testing 101 guide-RNA (gRNA) structures on a total of 14 different yeast promoters, we identified the best-performing combinations based on reporter assays. Though a larger number of gRNA-promoter combinations do not perturb gene expression, some gRNAs support expression perturbations up to similar to threefold. The best-performing gRNAs were used for single and multiplex reprogramming strategies for redirecting flux related to isoprenoid production and optimization of TAG profiles. From these studies, we identified both constitutive and inducible multiplex reprogramming strategies enabling significant changes in isoprenoid production and increases in TAG.Conclusion: Taken together, we show similar performance for a constitutive and an inducible dCas9 approach, and identify multiplex gRNA designs that can significantly perturb isoprenoid production and TAG profiles in yeast without editing the genomic context of the target genes. We also identify a large number of gRNA positions in 14 native yeast target pomoters that do not affect expression, suggesting the need for further optimization of gRNA design tools and dCas9 engineering.\
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [45] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [46] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [47] |
Adaptive laboratory evolution is a frequent method in biological studies to gain insights into the basic mechanisms of molecular evolution and adaptive changes that accumulate in microbial populations during long term selection under specified growth conditions. Although regularly performed for more than 25 years, the advent of transcript and cheap next-generation sequencing technologies has resulted in many recent studies, which successfully applied this technique in order to engineer microbial cells for biotechnological applications. Adaptive laboratory evolution has some major benefits as compared with classical genetic engineering but also some inherent limitations. However, recent studies show how some of the limitations may be overcome in order to successfully incorporate adaptive laboratory evolution in microbial cell factory design. Over the last two decades important insights into nutrient and stress metabolism of relevant model species were acquired, whereas some other aspects such as niche-specific differences of non-conventional cell factories are not completely understood. Altogether the current status and its future perspectives highlight the importance and potential of adaptive laboratory evolution as approach in biotechnological engineering.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [48] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [49] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [50] |
胡智慧, 李弘轩, 郭学武, 等. 酿酒酵母异源合成柠檬烯的研究进展. 食品科学, 2022, 43(9): 354-363.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [51] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [52] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [53] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [54] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [55] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [56] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [57] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [58] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [59] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [60] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [61] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [62] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [63] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [64] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [65] |
Geraniol is a rose-scented monoterpene with significant commercial and industrial value in medicine, condiments, cosmetics, and bioenergy. Here, we first targeted geraniol as a reporter metabolite and explored the suitability and potential of as a heterologous host for monoterpenoid production. Subsequently, dual-pathway engineering was employed to improve the production of geraniol with a geraniol titer of 858.4 mg/L. We then applied a synthetic hybrid promoter approach to develop a decane-responsive hybrid promoter based on the native promoter P derived from itself. The hybrid promoter was able to be induced by -decane with 3.6 times higher transcriptional intensity than the natural promoter P. In particular, the hybrid promoter effectively reduces the conflict between cell growth and product formation in the production of geraniol. Ultimately, 1194.6 mg/L geraniol was obtained at the shake flask level. The strong and tunable decane-responsive hybrid promoter developed in this study provides an important tool for fine regulation of toxic terpenoid production in cells.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [66] |
Geraniol is a class of natural products that are widely used in the aroma industry due to their unique aroma. Here, to achieve the synthesis of geraniol and alleviate the intense competition from the yeast ergosterol pathway, a transcription factor-mediated ergosterol feedback system was developed in this study to autonomously regulate ergosterol metabolism and redirect carbon flux to geraniol synthesis. In addition, the modification of ergosterol-responsive promoters, the optimization of transcription factor expression intensity, and stepwise metabolic engineering resulted in a geraniol titer of 531.7 mg L. For sustainable production of geraniol, we constructed a xylose assimilation pathway in (). Then, the xylose metabolic capacity was ameliorated and the growth of the engineered strain was rescued by activating the pentose phosphate (PP) pathway. Finally, we obtained 1091.6, 862.4, and 921.8 mg L of geraniol in a 5 L bioreactor by using pure glucose, simulated wheat straw hydrolysates, and simulated sugarcane bagasse hydrolysates, with yields of 47.5, 57.9, and 59.1 mg g DCW, respectively. Our study demonstrated that has the potential to produce geraniol from lignocellulosic biomass, providing a powerful tool for the sustainable synthesis of other valuable monoterpenes.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [67] |
Geraniol is a valuable monoterpene extensively used in the fragrance, food, and cosmetic industries. Increasing environmental concerns and supply gaps have motivated efforts to advance the microbial production of geraniol from renewable feedstocks. In this study, we first constructed a platform geraniol Escherichia coli strain by bioprospecting the key enzymes geranyl diphosphate synthase (GPPS) and geraniol synthase (GES) and selection of a host cell background. This strategy led to a 46.4-fold increase in geraniol titer to 964.3 mg/L. We propose that the expression level of eukaryotic GES can be further optimized through fusion tag evolution engineering. To this end, we manipulated GES to maximize flux towards the targeted product geraniol from precursor geranyl diphosphate (GPP) via the utilization of fusion tags. Additionally, we developed a high-throughput screening system to monitor fusion tag variants. This common plug-and-play toolbox proved to be a robust approach for systematic modulation of protein expression and can be used to tune biosynthetic metabolic pathways. Finally, by combining a modified E1* fusion tag, we achieved 2124.1 mg/L of geraniol in shake flask cultures, which reached 27.2% of the maximum theoretical yield and was the highest titer ever reported. We propose that this strategy has set a good reference for enhancing a broader range of terpenoid production in microbial cell factories, which might open new possibilities for the bio-production of other valuable chemicals.Copyright © 2021. Published by Elsevier Inc.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [68] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [69] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [70] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [71] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [72] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [73] |
Establishment of an efficient isoprene fermentation process by adopting inorganic phosphate limitation as the trigger to direct metabolic flux to the isoprene synthetic pathway.We constructed isoprene-producing strains of Pantoea ananatis (a member of the Enterobacteriaceae family) by integrating a heterologous mevalonate pathway and a metabolic switch that senses external inorganic phosphate (Pi) levels. This metabolic switch enabled dual-phase isoprene production, where the initial cell growth phase under Pi-saturating conditions was uncoupled from the subsequent isoprene production phase under Pi-limiting conditions. In fed-batch fermentation using our best strain (SWITCH-PphoC/pIspSM) in a 1-l bioreactor, isoprene concentration in the off-gas was maintained between 300 and 460 ppm during the production phase and at 20 ppm during the cell growth phase, respectively. The strain SWITCH-PphoC/pIspSM produced totally 2·5 g l of isoprene from glucose with a 1·8% volumetric yield in 48 h.This proof-of-concept study demonstrated that our Pi-dependent dual-phase production system using a P. ananatis strain as a producer has potential for industrial-scale isoprene fermentation.This Pi-dependent dual-phase fermentation process could be an attractive and economically viable option for the production of various commercially valuable isoprenoids.© 2019 The Society for Applied Microbiology.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [74] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [75] |
李言, 笪心怡, 张雨晨, 等. 酿酒酵母芳樟醇耐受性的工程改造. 微生物学通报, 2022, 49(8): 3062-3078.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [76] |
Production of fuels and chemicals by industrial biotechnology requires efficient, safe and flexible cell factory platforms that can be used for production of a wide range of compounds. Here we developed a platform yeast cell factory for efficient provision of acetyl-CoA that serves as precursor metabolite for a wide range of industrially interesting products. We demonstrate that the platform cell factory can be used to improve the production of α-santalene, a plant sesquiterpene that can be used as a perfume by four-fold. This strain would be a useful tool to produce a wide range of acetyl-CoA-derived products.Copyright © 2012 Elsevier Inc. All rights reserved.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [77] |
Microbial cells engineered for efficient production of plant sesquiterpenes may allow for sustainable and scalable production of these compounds that can be used as e.g. perfumes and pharmaceuticals. Here, for the first time a Saccharomyces cerevisiae strain capable of producing high levels of α-santalene, the precursor of a commercially interesting compound, was constructed through a rationally designed metabolic engineering approach. Optimal sesquiterpene production was obtained by modulating the expression of one of the key metabolic steps of the mevalonate (MVA) pathway, squalene synthase (Erg9). To couple ERG9 expression to glucose concentration its promoter was replaced by the HXT1 promoter. In a second approach, the HXT2 promoter was used to express an ERG9 antisense construct. Using the HXT1 promoter to control ERG9 expression, it was possible to divert the carbon flux from sterol synthesis towards α-santalene improving the productivity by 3.4 fold. Combining this approach together with the overexpression of a truncated form of 3-hydroxyl-3-methyl-glutaryl-CoA reductase (HMGR) and deletion of lipid phosphate phosphatase encoded by LPP1 led to a strain with a productivity of 0.18mg/gDCWh. The titer was further increased by deleting DPP1 encoding a second FPP consuming pyrophosphate phosphatase yielding a final productivity and titer, respectively, of 0.21mg/gDCWh and 92mg/l of α-santalene.Copyright © 2012 Elsevier Inc. All rights reserved.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [78] |
Sandalwood oil has been widely used in perfumery industries and aromatherapy. Santalols are its major components. Herein, we attempted to construct santalol-producing yeasts. To alter flux from predominant triterpenoid/steroid biosynthesis to sesquiterpenoid production, expression of (encoding yeast squalene synthase) was depressed by replacing its innate promotor with and fermenting the resulting strains in galactose-rich media. And the genes related to santalol biosynthesis were overexpressed under control of promotors, which linked santalol biosynthesis to GAL regulatory system. GAL4 (a transcriptional activator of promotors) and PGM2 (a yeast phosphoglucomutase) were overexpressed to overall promote this artificial santalol biosynthetic pathway and enhance galactose uptake. 1.3 g/L santalols and 1.2 g/L -α-santalol were achieved in the strain WL17 expressing SaSS (α-santalene synthase from ) and WL19 expressing SanSyn (α-santalene synthase from ) by fed-batch fermentation, respectively. This study constructed the microbial santalol-producing platform for the first time.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [79] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [80] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [81] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [82] |
(+)-Nootkatone (4) is a high added-value compound found in grapefruit juice. Allylic oxidation of the sesquiterpene (+)-valencene (1) provides an attractive route to this sought-after flavoring. So far, chemical methods to produce (+)-nootkatone (4) from (+)-valencene (1) involve unsafe toxic compounds, whereas several biotechnological approaches applied yield large amounts of undesirable byproducts. In the present work 125 cytochrome P450 enzymes from bacteria were tested for regioselective oxidation of (+)-valencene (1) at allylic C2-position to produce (+)-nootkatone (4) via cis- (2) or trans-nootkatol (3). The P450 activity was supported by the co-expression of putidaredoxin reductase (PdR) and putidaredoxin (Pdx) from Pseudomonas putida in Escherichia coli.Addressing the whole-cell system, the cytochrome CYP109B1 from Bacillus subtilis was found to catalyze the oxidation of (+)-valencene (1) yielding nootkatol (2 and 3) and (+)-nootkatone (4). However, when the in vivo biooxidation of (+)-valencene (1) with CYP109B1 was carried out in an aqueous milieu, a number of undesired multi-oxygenated products has also been observed accounting for approximately 35% of the total product. The formation of these byproducts was significantly reduced when aqueous-organic two-liquid-phase systems with four water immiscible organic solvents - isooctane, n-octane, dodecane or hexadecane - were set up, resulting in accumulation of nootkatol (2 and 3) and (+)-nootkatone (4) of up to 97% of the total product. The best productivity of 120 mg l-1 of desired products was achieved within 8 h in the system comprising 10% dodecane.This study demonstrates that the identification of new P450s capable of producing valuable compounds can basically be achieved by screening of recombinant P450 libraries. The biphasic reaction system described in this work presents an attractive way for the production of (+)-nootkatone (4), as it is safe and can easily be controlled and scaled up.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [83] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [84] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [85] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [86] |
Cembranoids are one kind of diterpenoids with multiple biological activities. The tobacco cembratriene-ol (CBT-ol) and cembratriene-diol (CBT-diol) have high anti-insect and anti-fungal activities, which is attracting great attentions for their potential usage in sustainable agriculture. Cembranoids were supposed to be formed through the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway, yet the involvement of mevalonate (MVA) pathway in their synthesis remains unclear. Exploring the roles of MVA pathway in cembranoid synthesis could contribute not only to the technical approach but also to the molecular mechanism for cembranoid biosynthesis.We constructed vectors to express cembratriene-ol synthase (CBTS1) and its fusion protein (AD-CBTS1) containing an N-terminal GAL4 AD domain as a translation leader in yeast. Eventually, the modified enzyme AD-CBTS1 was successfully expressed, which further resulted in the production of CBT-ol in the yeast strain BY-T20 with enhanced MVA pathway for geranylgeranyl diphosphate (GGPP) production but not in other yeast strains with low GGPP supply. Subsequently, CBT-diol was also synthesized by co-expression of the modified enzyme AD-CBTS1 and BD-CYP450 in the yeast strain BY-T20.We demonstrated that yeast is insensitive to the tobacco anti-fungal compound CBT-ol or CBT-diol and could be applied to their biosynthesis. This study further established a feasibility for cembranoid production via the MVA pathway and provided an alternative bio-approach for cembranoid biosynthesis in microbes.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [87] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [88] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [89] |
The market-expanding lutein is currently mainly supplied by plant extraction, with microbial fermentation using engineered cell factory emerging as a promising substitution. During construction of lutein-producing yeast, α-carotene formation through asymmetric ε- and β-cyclization of lycopene was found as the main limiting step, attributed to intra-pathway competition of the cyclases for lycopene, forming β-carotene instead. To solve this problem, temperature-responsive expression of β-cyclase was coupled to constitutive expression of ε-cyclase for flux redirection to α-carotene by allowing ε-cyclization to occur first. Meanwhile, the ε-cyclase was engineered and re-localized to the plasma membrane for further flux reinforcement towards α-carotene. Finally, pathway extension with proper combination of carotenoid hydroxylases enabled lutein (438 μg/g dry cells) biosynthesis in S. cerevisiae. The success of heterologous lutein biosynthesis in yeast suggested temporospatial pathway control as a potential strategy in solving intra-pathway competitions, and may also be applicable for promoting the biosynthesis of other natural products.Copyright © 2021. Published by Elsevier Inc.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [90] |
王国平, 刘旦, 李洋洋, 等. 烟草冷杉醇合成关键基因NtCPS2的SNP功能标记开发与应用. 分子植物育种, 2020, 18(24): 8178-8186.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [91] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [92] |
王冬, 张小全, 杨铁钊, 等. 类西柏烷二萜代谢机理及调控研究进展. 中国烟草学报, 2014, 20(3): 113-118.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [93] |
张思琦, 何佳, 周方, 等. 不同烤烟品种(系)二萜类物质合成关键基因的表达及代谢差异. 西北农林科技大学学报(自然科学版), 2019, 47(9): 25-32.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [94] |
Terpenes have numerous applications, ranging from pharmaceuticals to fragrances and biofuels. With increasing interest in producing terpenes sustainably and economically, there has been significant progress in recent years in developing methods for their production in microorganisms. In Saccharomyces cerevisiae, production of the 20-carbon diterpenes has so far proven to be significantly less efficient than production of their 15-carbon sesquiterpene counterparts. In this report, we identify the modular structure of geranylgeranyl diphosphate synthesis in yeast to be a major limitation in diterpene yields, and we engineer the yeast farnesyl diphosphate synthase Erg20p to produce geranylgeranyl diphosphate. Using a combination of protein and genetic engineering, we achieve significant improvements in the production of sclareol and several other isoprenoids, including cis-abienol, abietadiene and β-carotene. We also report the development of yeast strains carrying the engineered Erg20p, which support efficient isoprenoid production and can be used as a dedicated chassis for diterpene production or biosynthetic pathway elucidation. The design developed here can be applied to the production of any GGPP-derived isoprenoid and is compatible with other yeast terpene production platforms. Copyright © 2014 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [95] |
cis-Abienol, a natural diterpene-diol isolated from balsam fir (Abies balsamea), can be employed as precursors for the semi-synthesis of amber compounds, which are sustainable replacement for ambergris and widely used in the fragmented industry. This study combinatorially co-expressed geranyl diphosphate synthase, geranylgeranyl diphosphate synthase, Labda-13-en-8-ol diphosphate synthase and diterpene synthase, with the best combination achieving ~ 0.3 mg/L of cis-abienol. An additional enhancement of cis-abienol production (up to 8.6 mg/L) was achieved by introducing an exogenous mevalonate pathway which was divided into the upper pathway containing acetyl-CoA acetyltransferase/HMG-CoA reductase and HMG-CoA synthase and the lower pathway containing mevalonate kinase, phosphomevalonate kinase, pyrophosphate mevalonate decarboxylase and isopentenyl pyrophosphate isomerase. The genetically modified strain carrying chromosomal copy of low genes of the mevalonate with the trc promoter accumulated cis-abienol up to 9.2 mg/L in shake flask. Finally, cis-abienol titers of ~ 220 mg/L could be achieved directly from glucose using this de novo cis-abienol-producing E. coli in high-cell-density fermentation. This study demonstrates a microbial process to apply the E. coli cell factory in the biosynthesis of cis-abienol.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [96] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [97] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [98] |
The functions of two key, trichome-expressed genes were assessed using different posttranscriptional gene silencing strategies (PTGS). Efficient RNA interference (RNAi) revealed the function of a cembratriene-ol (CBT-ol) cyclase gene responsible for conversion of geranylgeranyl pyrophosphate to CBT-ols, and verified the function of a P450 gene responsible for conversion of CBT-ols to CBT-diols. CBT-diols are abundant diterpenes that comprise about 60% and 10% of trichome exudate weight and leaf dry weight, respectively, in Nicotiana tabacum, T.I. 1068. The relative efficiencies and levels of suppression using antisense (AS), sense co-suppression (S), and RNAi were compared for these two genes. With a partial cDNA of the P450 gene, the suppression efficiencies (percent of primary transformants with high CBT-ols/CBT-diols) were low, 3.3% for AS and 0% for S plants. In contrast, using RNAi with a partial gene sequence, a knockdown efficiency of about 45% was achieved. For the CBT-ol cyclase gene, no suppression was observed using partial cDNAs in AS or S orientations, while RNAi with a partial gene sequence yielded an efficiency of about 64%. The efficiencies of gene silencing using full-length coding regions of both genes in AS and S orientations were </=20%. Our results identify the function of a CBT-ol cyclase gene and demonstrate the efficacy and superiority of RNAi for assessing the functions of two trichome-specific genes that encode enzymes having widely different functions.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [99] |
Trichome glands on the surface of many higher plants produce and secrete exudates affecting insects, microbes, and herbivores. Metabolic engineering of gland exudation has potential for improving pest/disease resistance, and for facilitating molecular farming. We identified a cytochrome P450 hydroxylase gene specific to the trichome gland and used both antisense and sense co-suppression strategies to investigate its function. P450-suppressed transgenic tobacco plants showed a > or =41% decrease in the predominant exudate component, cembratriene-diol (CBT-diol), and a > or =19-fold increase in its precursor, cembratriene-ol (CBT-ol). Thus, the level of CBT-ol was raised from 0.2 to > or =4.3% of leaf dry weight. Exudate from antisense-expressing plants had higher aphidicidal activity, and transgenic plants with exudate containing high concentrations of CBT-ol showed greatly diminished aphid colonization responses. Our results demonstrate the feasibility of significantly modifying the natural-product chemical composition and aphid-interactive properties of gland exudates using metabolic engineering. The results also have implications for molecular farming.
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [100] |
While chemical steps involved in bioactive cembranoid biosynthesis have been examined, the corresponding enzymatic mechanisms leading to their formation remain elusive. In the tobacco plant, a putative cembratriene-ol synthase (CBTS) initiates the catalytic cascade that lead to the biosynthesis of cembratriene-4,6-diols, which displays antibacterial- and anti-proliferative activities. We report here on structural homology models, functional studies, and mechanistic explorations of this enzyme using a combination of biosynthetic and computational methods. This approach guided us to develop an efficient production of five bioactive non- and monohydroxylated cembranoids. Our homology models in combination with quantum and classical simulations suggested putative principles of the CBTS catalytic cycle, and provided a possible rationale for the formation of premature olefinic side products. Moreover, the functional reconstruction of a -derived class II P450 with a cognate CPR, obtained by transcriptome mining provided for production of bioactive cembratriene-4,6-diols. Our combined findings provide mechanistic insights into cembranoid biosynthesis, and a basis for the sustainable industrial production of highly valuable bioactive cembranoids.© 2020 The Author(s).
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [101] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [102] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| [103] |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
| {{custom_ref.label}} |
{{custom_citation.content}}
{{custom_citation.annotation}}
|
本文得到中国烟草总公司重点研发(110202102020、110202202006)项目的资助
PDF(1419 KB)
图1 萜类化合物生物合成途径[18]
表1 天然及人工设计的类异戊二烯前体合成途径比较[19]图2 构建微生物细胞工厂生物合成萜类化合物研究策略示意图[46,49]表2 萜类香味物质生物合成水平及香气描述表3 潜香类香味成分生物合成水平及香气描述/
| 〈 |
|
〉 |
