人工代谢途径合成有机醇有机酸的研究进展

doi:10.12211/2096-8280.2021-049

摘要/Abstract

摘要：

有机酸有机醇不仅是食品、化工、医药领域的原料，更是一类重要的生物基燃料，相比于传统的石油基燃料，具有原料可再生，生产过程清洁的特点，是应对能源危机、环境污染问题的有效对策。传统的代谢工程方法是通过在微生物中过表达产物相关的特异性酶实现的，应用时常有发酵原料单一，代谢效率不高，产物种类受限等问题。筛选并重新组合不同来源的酶来构建人工代谢途径是一个有效的解决方案，同时也是代谢工程方法发展的一个重要趋势。本文综述了近年来构建人工代谢途径生产有机酸有机醇取得的突破与进展，具体阐述了5种创新生物合成途径（一碳化合物同化途径、非磷酸化途径、酮酸途径、β-氧化逆循环途径和聚酮化合物途径）的相关机理，为未来构建低成本、高效、产物多样化的有机酸有机醇生物合成平台提供了可能。

关键词: 有机酸, 有机醇, 生物基燃料, 代谢工程, 人工代谢途径

Abstract:

Organic acids and alcohols are usually known as commercial feedstock in chemical engineering and pharmaceutical field. It is also known as potential significant biofuel. Compared with traditional fossil fuels, the characteristics of industrial application of organic acids and alcohols can provide great advantages such as renewable feedstock and green production process. They are effective countermeasures to deal with energy crisis and environmental pollution. Traditional metabolic engineering is realized by overexpression specific genes to elevate the corresponding enzyme level. With certain artificial genetic transformation and modification, a microbial cell factory can be constructed for efficient production of certain organic acids and alcohols. However, problems such as limited fermentation raw materials, low metabolic efficiency, and limited product types arise during practical implementation. Constructing artificial metabolic pathways through screening and recombining nonhomologous enzymes can become an effective solution, which is also a cutting-edge general trend in this field. In this paper, recent breakthroughs and progress in constructing artificial metabolic pathway to produce organic acids and alcohols are reviewed. Five novel biosynthetic pathways are expounded with details including C₁ compounds assimilation pathway, nonphosphorylation pathway, ketoacid/ammino acid pathway, RBO pathway (reversal of β-oxidation pathway), and PKS pathway (polyketide pathway). Furthermore, the advantages and limitations compared with traditional chemical-produced technique are also discussed. Potential problems such as the tolerance of one carbon compounds, the inefficient conversion of xylose, the inefficient catalysis of key enzyme reactions in ketoacid amino acid pathway, and the lack of diversity of RBO pathway and PKS pathway products are reported in respected to practical application of the technology. In conclusion, the feasibility of establishing new metabolic pathways for production of specific products is analyzed, which has the potential of providing the possibility for the construction of high cost-efficient and diversified organic acid and organic alcohol biosynthesis platform in the future.

Key words: organic acid, organic alcohol, biofuel, metabolic engineering, artificial metabolic pathway

中图分类号:

Q815

曹晨凯, 李佳隆, 张科春. 人工代谢途径合成有机醇有机酸的研究进展[J]. 合成生物学, 2021, 2(6): 902-919.

Chenkai CAO, Jialong LI, Kechun ZHANG. Progress in artificial metabolic pathways for biosynthesis of organic alcohols & acids[J]. Synthetic Biology Journal, 2021, 2(6): 902-919.

图/表 7

图1 RuMP途径、ED途径、EMP途径（a）和丝氨酸循环（b）［19］H6P—Hexulose-6-phosphate; Ru5P—ribulose-5-phosphate; X5P—xylulose 5-phosphate; F6P—fructose-6-phosphate; R5P—ribose-5-phosphate; G3P—glyceraldehyde-3-phosphate; S7P—sedoheptulose-7-phosphate; E4P—erythrose 4-phosphate; G6P—glucose-6-phosphate; 6PGL—6-phospho-D-glucono-1,5-lactone; 6PG—6-phospho-D-glonate; KDPG—2-dehydro-3-deoxy-D-gluconate-6-phosphate; FBP—fructose-1,6-bisphosphate; TCA—tricarboxylic acid

Fig. 1 C1 compound assimilation through ribulose-5-phosphate (RuMP), ED (Entner-Doudoroff), EMP (Embden-Meyerhof-Parnas) pathway (a) and serine cycle (b)[19]

表1 由甲基营养性菌发酵生产特定有机醇与有机酸实例

Tab. 1 Summary of methylotrophic production data

菌株	方法	培养基	产物	产量/(g/L)	时间	文献
甲醇芽孢杆菌	补料分批发酵	含200 mmol/L甲醇的MVcM培养基	γ-氨基丁酸	9.0	2016	[25]
α-变形杆菌甲基杆菌AM1	摇瓶发酵	含20 mmol/L乙胺的基础培养基	L-丁醇	25.5×10^-3	2016	[26]
扭脱甲基杆菌AM1	补料分批发酵	含有3 g/L甲醇的矿物盐培养基	2-羟基异丁酸	2.1	2016	[27]
扭脱甲基杆菌AM1	摇瓶发酵	含125 mmol/L甲醇的基础培养基	3-羟基丙酸	69.8×10^-3	2017	[28]
α-变形杆菌甲基杆菌AM1	补料分批发酵	含5%（体积分数）甲醇的基础培养基	甲羟戊酸	2.3	2018	[29]
毕赤酵母	试管发酵	酵母/蛋白胨/葡萄糖培养基	D-乳酸	3.5	2019	[30]

图2 不同微生物的木糖代谢途径X5P—Xylulose 5-phosphate; G3P—glyceraldehyde-3-phosphate; F6P—fructose-6-phosphate; Ru5P—ribulose-5-phosphate; Acetyl-P—acetylphosphate

Fig. 2 Metabolic pathways of xylose in different microorganisms

图3 酮酸/氨基酸途径合成醇、二醇

Fig. 3 Ketoacid/amino acid pathway for production of alcohol and diol

图4 β-氧化逆循环［110］

Fig. 4 Reversal of β-oxidation[110]

图5 Ⅰ型PKS装配线

Fig. 5 Assembly line of type Ⅰ polyketide synthase

图6 以合成己二酸为例的PKS途径［其中一共有3个模块，底物通过硫酯交换反应从前一个模块转移到下一个模块，最后通过硫酯酶（TEp）结构域释放］

Fig. 6 PKS pathway for production of adipic acid[There are 3 respective modules. Substrate is transferred from previous module to next one via thioester exchange reaction, and finally released by thioesterase (TEp) domain]

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