纪念王义翘教授：解脂耶氏酵母替代植物油脂的技术瓶颈及展望

doi:10.12211/2096-8280.2021-041

摘要/Abstract

摘要：

构建细胞农业经济是解决资源短缺、降低温室气体排放、延缓全球变暖和实现可持续性经济发展的重要手段之一。微生物细胞工厂具有易于遗传改造、便于工程放大和高效利用可再生资源的优势，成为了现代生物制造的重要组成部分。王义翘教授是现代生化工程技术的开创者和奠基人，本人及同事（乔康健博士、胡鹏博士、周康博士等人）在Stephanopoulos实验室所从事的构建油脂细胞工厂的工作，受益于王先生在MIT所创建的生物工程技术中心。植物油脂具有2000多亿美元的年均市场需求，本文从植物油脂需求激增所造成的负面环境效应出发，分析了目前植物油脂的市场供应现状。产油酵母细胞工厂具有替代植物源油脂的巨大潜能。本文围绕产油酵母的代谢工程遗传改造策略，总结了如何提高碳源转化率、油脂产量、油脂生产速率和菌体生长适用性；进一步归纳了构建高效解脂耶氏酵母细胞工厂的主要技术瓶颈，其中包括产油菌株的高通量筛选和表型鉴定技术、产油酵母的代谢调控机制和发酵动力学模型等。作者进一步探讨了以蔗糖作为原材料，生产植物源功能性油脂的经济可行性和技术可行性。作者预测解脂耶氏酵母具有极大的潜力，可以解决当前高附加值油脂（比如用于巧克力生产的可可脂，潜在市场为500亿美元）的市场需求。开发产油酵母微生物资源，提供健康的功能性油脂，将会解决一系列能源、健康食品和环境资源等问题，促进我们迈向低碳性和可持续性的经济运转模式。

关键词: 细胞工厂, 代谢工程, 产油酵母, 植物源可替代油脂, 细胞农业经济, 可持续性发展

Abstract:

Developing cellular agriculture economy is one of the solutions to mitigate resource limitation, reduce greenhouse gas emission, slow down global warming as well as achieve true sustainability. Microbial cell factory has become a critical component to advance biomanufacturing due to the availability of versatile genetic tools, ease scale-up and the high conversion efficiency of low-cost renewable feedstocks. Prof. Daniel I.C. Wang was one of the trailblazers and founders of biochemical engineering, who built and led the Biotechnology Process Engineering Center (BPEC) at MIT. When I and my colleagues worked as postdoc associates and research scientists in Prof. Stephanopoulos' lab, part of our work on engineering oleaginous yeast cell factory was a direct result of the analytical, imaging and cell culture facility at BPEC. Plant-based oil and fats have an overall market value about $200 billion. Recently, the world has seen a craving for plant oil products, which negatively impacted our environment due to massive-scale deforestation and loss of ecological diversity in tropical regions. We thought oleaginous yeast could be a solution to this problem. Centering around the important genetic targets of the oleaginous species Yarrowia lipolytica, we summarized the essential metabolic engineering strategies for improving the carbon conversion efficiency (yield), lipid titer, lipid production rate (productivity) and cell growth fitness. We further analyzed technical constraints that limit our ability to build high oil-yielding yeast cell factory, including high throughput strain screening or phenotyping techniques, the incomplete understanding of the lipogenesis and underlying regulatory mechanism, as well as the lack of well-defined biochemical models to guide bioprocess optimization and scale-up. Further we discussed the technical and economic feasibility of converting sugarcane feedstock to high value plant-based lipids with metabolically engineered Y. lipolytica. Our analysis indicates that Y. lipolytica has a great potential to address the current market gap of high value plant-based fats (i.e. cocoa butter equivalent to make chocolate with a potential market of $50 billions). Engineering oleaginous yeast to provide plant-based healthy fats will help us address energy, foods, environment and resource challenges, which will surely move us one step closer to a society of low-carbon footprint and sustainable economy.

Key words: microbial cell factory, metabolic engineering, oleaginous yeast, plant-based alternative fats, cellular agriculture economy, sustainable development

中图分类号:

徐鹏. 纪念王义翘教授：解脂耶氏酵母替代植物油脂的技术瓶颈及展望[J]. 合成生物学, 2021, 2(4): 509-527.

Peng XU. In memory of Prof. Daniel I.C. Wang: Engineering Yarrowia lipolytica for the production of plant-based lipids: technical constraints and perspectives for a sustainable cellular agriculture economy[J]. Synthetic Biology Journal, 2021, 2(4): 509-527.

图/表 8

图1 油脂的分子结构及重要脂肪酸分子

Fig. 1 The basic structure of triglyceride and the four important fatty acids: palmitic acid (C16∶0), oleic acid (C18∶1), linoleic acid (C18∶2), eicosapentaenoic acid (EPA, C20∶5) and α-linolenic acid (ALA, C18∶3).

图2 油脂决定了一些肉类食物的口感

Fig. 2 Lipids determine the mouthful feeling of major meat products: Iberico ham, Peking roasted duck, Kobe beef and Foie gras.

图3 脂肪酸碳链延伸过程需要还原性辅因子NADPH（n表示碳链长度）

Fig. 3 The enzymatic elongation cycle of fatty acids needs reducing equivalents NADPH(n is the chain-length of carbon backbones)

图4 不同还原性辅因子替代途径对脂肪酸合成效率的影响［56］（图示中包含了以下四种途径：①磷酸戊糖途径；②NADPH-特异性的3-磷酸甘油醛脱氢酶；③丙酮酸-草酰乙酸-苹果酸转氢反应；④非氧化性糖酵解途径）

Fig. 4 Carbon conversion efficiency from NADPH for fatty acids synthesis pathways [56](OxPP—oxidative pentose phosphate pathway; GAPHD—NADPH-specific glyceraldehyde-3-phosphate dehydrogenase; POM cycle—pyruvate-oxaloacetate-malate transhydrogenase cycle; NOG—non-oxidative glycolytic pathway)

图5 解脂耶氏酵母合成功能性油脂的生化过程

Fig. 5 The biochemical scheme of synthesizing triglycerides in Y. lipolytica

表1 产油酵母胞内积累油脂的分批发酵动力学描述

Tab. 1 Fermentation kinetics of the oil accumulation process in oleaginous yeast in batch culture

方程编号 Equation No.	方程 Equations	描述 Description
(1)	$μ = μ m a x S K S + S 1 - γ P S Y P / S$	细胞比生长速率 Specific growth rate
(2)	$d d t X t = μ X t - k d X t$	非油脂生物量积累 Oil-free cell growth
(3)	$d d t P t = α μ + β X t$	油脂积累 Lipid accumulation
(4)	$d d t S t = - μ X t Y X / S - α μ + β X t Y P / S$	底物消耗 Substrate consumption
(5)	$X t o t a l t = P t + X t$	总生物量 Total biomass
(6)	$θ t = P t P t + X t$	动态含油量 Oil content
(7)	$Y p r o c e s s t = P f t - P 0 S 0 - S f t$	动态过程得率 Process yield
(8)	$r t = P f t - P 0 t f - t 0$	总的生产强度 Overall productivity

表1 产油酵母胞内积累油脂的分批发酵动力学描述

Tab. 1 Fermentation kinetics of the oil accumulation process in oleaginous yeast in batch culture

方程编号 Equation No.	方程 Equations	描述 Description
(1)	$μ = μ m a x S K S + S 1 - γ P S Y P / S$	细胞比生长速率 Specific growth rate
(2)	$d d t X t = μ X t - k d X t$	非油脂生物量积累 Oil-free cell growth
(3)	$d d t P t = α μ + β X t$	油脂积累 Lipid accumulation
(4)	$d d t S t = - μ X t Y X / S - α μ + β X t Y P / S$	底物消耗 Substrate consumption
(5)	$X t o t a l t = P t + X t$	总生物量 Total biomass
(6)	$θ t = P t P t + X t$	动态含油量 Oil content
(7)	$Y p r o c e s s t = P f t - P 0 S 0 - S f t$	动态过程得率 Process yield
(8)	$r t = P f t - P 0 t f - t 0$	总的生产强度 Overall productivity

表2 世界主要油料作物的单位面积产量及产油得率

Tab. 2 The unit area output and maximal oil yield from major oil crops.

油料作物 Oil crop	产量/[t/(hm²·a)] Production	含油量/% Oil content	最大产油得率/[t/(hm²·a)] Maximal oil yield
黄豆 Soybean	3.1	20	0.62
葵花籽 Sunflower seed	1.7	50	0.85
油菜籽 Canola	2.0	45	0.90
可可果 Cocoa bean	0.8	50	0.40
棕榈果 Oil palm	9.1	41	3.69

表3 以糖料作物为原料经解脂耶氏酵母转化后的产油得率

Tab. 3 The maximal oil yield from Y. lipolytica converting sugar feedstock from corn, sweet potato or sugarcane.

糖料作物 Sugar crop	产量/[t/(hm²·a)] Production	含糖量/% Sugar content	最大产油得率^①/[t/(hm²·a)] Maximal oil yield
玉米 Corn	10.7 (dry, 干重)	74 (dry, 干重)	2.15
红薯 Sweet potato	25 (wet, 湿重)	20 (wet, 湿重)	1.36
甘蔗 Sugarcane	80.9 (wet, 湿重)	14 (wet, 湿重)	3.07

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