In vitro biosynthesis of chemicals: pathway design, component assembly and applications-a review

doi:10.12211/2096-8280.2021-019

Abstract

Abstract:

Chemical industry is one of the pillar industries in the modern society. The demand for refined chemicals with high-quality and diversity is rapidly increasing with the improvement of society and human being's living standards. As a supplement to conventional chemical synthesis, environmental friendly biosynthesis is attracting widespread attention and will be an important step to achieve a sustainable development. In vitro biosynthesis (cell free biosynthesis) is a synthetic method to prepare desired chemicals, which was catalyzed by purified enzymes or cell extracts. With the development of synthetic biotechnology, in vitro biosynthesis has gradually become one of the most important ways for chemicals production, exhibiting the advantages of environmental friendliness, high catalytic efficiency, good atom economy and strong controllability. Pathway design is the key for the construction of in vitro biosynthesis system. In this review, two important principles for designing in vitro biosynthetic pathways have been summarized, including atom economy and energy optimization. As one of the important concepts of green chemistry, principle atom economy means that the synthesis method or process should be designed to convert raw materials into the final product as much as possible. Principle energy optimization means that the ATP-free or ATP minimized process should be designed in the synthesis method. Assembly enzymes into a multi-enzyme complex via biological macromolecules can increase reaction rate and also reduce the side reactions of the in vitro biosynthesis. Thus, three common-used biological macromolecules for enzyme assembly will be introduced here, including peptide linkers, protein scaffolds, DNA. Some recent examples of chemicals produced viain vitro biosynthesis have also been summarized, including glucosamine, glycerol glucoside, pyruvate, α-ketoglutarate, ethanol, 1,3-propanediol, islatravir, azomycin, and etc. Through the introduction of in vitro biosynthesis of bulk chemicals (carbohydrate chemicals, organic acid chemicals, alcohol chemicals, and etc.), this article demonstrates the potentials of in vitro biosynthesis in chemical synthesis. By reviewing the pathway design, enzyme component assembly and chemicals production examples, the future of in vitro biosynthesis of chemicals has been prospected here. With the design improvement of in vitro biosynthesis, the pathway designing will be becoming more and more intelligent and efficient. It is believed that the efficiency of in vitro biosynthesis of chemicals will be gradually increased and the in vitro biosynthesis hopefully can cover all chemicals' production, which is expected to be one of the predominant ways for chemicals production in the future.

Key words: in vitro biosynthesis, chemicals production, pathway design, protein engineering, enzyme component

摘要：

随着合成生物技术的发展，体外生物合成逐渐成为化学品合成的重要方式之一，具有环境友好、催化效率高、原子经济性好、可控性强等优点。途径设计是构建整个体外生物合成系统的关键所在，本文分析总结了体外生物合成中应遵循的两个重要原则，包括原子经济性原则和能量最优原则。利用生物大分子将酶元件组装构建成多酶复合体，可提高体外生物合成的反应速率、减少副反应的发生，本文介绍了用于酶元件组装的3类常见生物大分子，包括连接肽、蛋白支架、DNA等。通过近年来体外生物合成在大宗化学品（糖类化学品、有机酸类化学品以及醇类化学品等）生产中的应用案例的介绍，展示了体外生物合成在化学品合成中的应用前景。随着体外生物合成设计能力的不断提高，体外生物合成的途径设计将朝着智能化、高效化发展，化学品体外生物合成的效率也将逐步提高，有望涵盖所有化学品的生物合成，成为未来化学品合成的主要方式之一。

关键词: 体外生物合成, 化学品制造, 途径设计, 蛋白质工程, 酶元件

CLC Number:

Q591.1

Yichen WAN, Kongliang XU, Renchao ZHENG, Yuguo ZHENG. In vitro biosynthesis of chemicals: pathway design, component assembly and applications-a review[J]. Synthetic Biology Journal, 2021, 2(6): 886-901.

万逸尘, 许孔亮, 郑仁朝, 郑裕国. 化学品体外生物合成途径设计、元件组装和应用[J]. 合成生物学, 2021, 2(6): 886-901.

Figures/Tables 11

Fig. 1 Designing in vitro biosynthetic pathways via principle atom economy［In vitro reconstruction （left） of in vivo synthetic pathway from cell； Reverse construction （middle） in vitro pathway for production of didanosine via bioretrosynthesis； Redesigning a new pathway （right） for conversion of starch to fructose to achieve a higher conversion rate］

Fig. 2 Designing in vitro biosynthetic pathways via principle energy optimization

Fig. 3 Biomolecules for multi-enzyme assembly Peptides linkers (top left): fusing two or more enzymes together to fabricate a multi-enzyme system can be produced by gene fusion to introduce a peptide linker, which can bring heterogeneous enzymes into close proximity with peptide mediated conjugation. Scaffoldin (bottom left): for the construction of cohesin-dockerin based multi-enzymatic system, enzymes fused with dockerin and corresponding cohesins fused together to form a chimaeric scaffoldin via position-specific self-assembly of enzymes. DNA (top right): DNA self-assembling into one-, two- and three-dimensional nanostructures by complementary base-pairing makes DNA-bioconjugation a promising approach for self-organization of enzymes. Enzymes can be attached onto DNA strips by chemical conjugation such as click chemistry.

Fig. 4 In vitro biosynthesis pathway for conversion of starch and inorganic ammonia to glucosamine(Metabolites and product were starch, glucose 1-phosphate, glucose 6-phosphate, fructose 6-phosphate, glucosamine 6-phosphate, phosphate, and glucosamine. Enzymes involved were α-glucan phosphorylase, phosphoglucomutase, phosphoglucose isomerase, glucosamine 6-phosphate deaminase, and glucosamine 6-phosphate phosphatase)

Tab. 1 In vitro biosynthesis of carbohydrates and its derivatives, including glucosamine, N-acetyl glucosamine, glycerol glucoside, glucose-6-phosphate, fructose, fructose-6-phosphate, fructose-1,6-diphosphate, 2-deoxy-5-ribose, d-tagatose, mannose and xylulose-5-phosphate.

化学品	原料	所使用的关键酶	参考文献
氨基葡萄糖	淀粉	氨基葡萄糖-6-磷酸脱氨酶、氨基葡萄糖-6-磷酸磷酸酶等7种酶	[14]
氨基葡萄糖	甲壳素	甲壳素酶、N-乙酰葡萄糖胺去乙酰酶	[65]
N-乙酰氨基葡萄糖	丙酮酸	N-乙酰谷氨酸合酶等4种酶	[68]
甘油葡萄糖苷	蔗糖、麦芽糖	葡萄糖基甘油磷酸酶、蔗糖磷酸酶、麦芽糖磷酸酶	[17]
葡萄糖-6-磷酸	甲醇	甲醇脱氢酶、3-己糖-6-磷酸合酶、6-磷酸-3-己糖异构酶	[49]
果糖	淀粉	转醛缩酶、3-磷酸甘油醛磷酸酶等5种酶	[22]
果糖-6-磷酸	3-磷酸甘油醛	基于蛋白支架的多酶组装体（果糖-1, 6-双磷酸酶、磷酸丙糖异构酶、醛缩酶）	[54]
果糖-6-磷酸	磷酸二羟基丙酮、3-磷酸甘油醛	基于蛋白支架的多酶组装体（磷酸丙糖异构酶、醛缩酶、果糖-1, 6-二磷酸酶）	[69]
果糖-1, 6-二磷酸	麦芽糊精	α-葡聚糖磷酸化酶、焦磷酸磷酸果糖激酶等4种酶	[70]
2-脱氧-5-核糖	淀粉	脱氧-D-核糖-5-磷酸醛缩酶等11种酶	[36]
2-脱氧-5-核糖	果糖	脱氧核糖醛缩酶等5种酶	[71]
D-塔格糖	D-半乳糖	基于支架蛋白的多酶组装体（β-琼脂酶、脱水半乳糖苷酶、L-阿拉伯糖异构酶）	[55]
甘露聚糖	α-D-甘露糖-1-磷酸、甘露糖	热活性糖苷磷酸化酶	[72]
甘露糖	淀粉	甘露糖-6-磷酸磷酸酶等6种酶	[73]
木酮糖-5-磷酸	果糖-1,6-二磷酸	木酮糖激酶等3种酶	[74]

Fig. 5 In vitro biosynthesis pathway for conversion of D-glucuronic acid to α-ketoglutarate(Metabolites and product were D-glucuronate, D-glucarate, 5-keto-4-deoxy-glucarate, α-ketoglutaricsemialdehyde and α-ketoglutarate. Enzymes involved were uronate dehydrogenase, glucarate dehydratase, 5-keto-4-deoxy-glucarate dehydratase, α-ketoglutaric semialdehyde dehydrogenase and NADH oxidase)

Tab. 2 In vitro biosynthesis for organic acid chemicals, including α-ketoglutarate, pyruvate, lactic acid, D-2-aminobutyric acid, cysteine and glucaric acid

化学品	原料	所使用的关键酶	参考文献
α-酮戊二酸	D-葡萄糖醛酸	α-酮戊二酸半醛脱氢酶、5-酮-4-脱氧葡萄糖酸脱水酶	[15]
丙酮酸	甲壳素	烯醇化酶等12种酶	[18]
乳酸	葡萄糖	葡萄糖酸脱水酶、乳酸脱氢酶	[42]
D-2-氨基丁酸	L-苏氨酸	L-苏氨酸裂解酶、D-氨基酸脱氢酶	[78]
半胱氨酸	葡萄糖	邻磷酸丝氨酸巯基化酶等11种酶	[28]
葡糖二酸	蔗糖	肌醇加氧酶、尿酸脱氢酶	[79]

Fig. 6 In vitro biosynthesis pathway for conversion of glucose to ethanol(Metabolites and product were glucose, gluconate, 2-keto-3-desoxy-gluconate, pyruvate, glyceraldehyde, glycerate, acetaldehyde and ethanol. Enzymes involved were glucose dehydrogenase, dihydroxy acid dehydratase, 2-keto-3-desoxygluconate aldolase, glyceraldehyde dehydrogenase, pyruvate decarboxylase, alcohol dehydrogenase)

Tab. 3 In vitro biosynthesis for alcohol chemicals, including ethanol, isobutanol, 1,3-propanediol, butanol, diaminosorbitol and inositol

化学品	原料	所使用的关键酶	参考文献
乙醇	葡萄糖	丙酮酸脱羧酶等6种酶	[16]
异丁醇	葡萄糖	乙酰乳酸合酶等9种酶	[16]
1, 3-丙二醇	甘油	甘油脱水酶等3种酶	[19]
正丁醇	葡萄糖	3-羟酰基辅酶A脱氢酶等16种酶	[23]
二氨基山梨醇	异山梨醇	基于连接肽的多酶组装体（ω-氨基转移酶、醇脱氢酶）	[50]
肌醇	淀粉	肌醇单磷酸酶等4个酶	[83-84]

Tab. 4 In vitro biosynthesis for other chemicals, including isoprene, phloroglucinol, islatravir, naringenin, deoxyinosine, cytidine-5-monophosphate, azomycin, 3-hydroxybutyryl limonene, pinene, hinokene and L-glutathione

化学品	原料	所使用的关键酶	参考文献
异戊二烯	葡萄糖	异戊二烯合酶等12种酶	[12]
间苯三酚	甲酸	间苯三酚合酶	[85]
Islatravir	乙炔基甘油、核碱基	嘌呤核苷磷酸化酶等9种酶	[20]
柚皮素	对香豆酸	查尔酮异构酶等3种酶	[26]
脱氧肌苷	双脱氧核糖	嘌呤核苷磷酸化酶等3种酶	[31]
胞苷-5-单磷酸	胞苷	胞苷激酶	[86]
氮霉素	L-精氨酸	二氢二吡啶甲酸合酶	[21]
3-羟基丁酰基CoA	泛硫乙胺	甲羟戊酸激酶等7种酶	[87]
柠檬烯	葡萄糖	柠檬烯合酶等12种酶	[88]
蒎烯	葡萄糖	蒎烯合酶等12种酶	[88]
桧烯	葡萄糖	柠檬烯合酶等12种酶	[88]
L-谷胱甘肽	谷氨酸、半胱氨酸、甘氨酸	谷胱甘肽合酶	[85]

Fig. 7 In vitro biosynthesis pathway for conversion of ethynyl glycerol, acetaldehyde and guanine to islatravir[Metabolites and product were ethynyl glycerol, (R)-enantiomer of aldehyde, 2-ethynylglyceraldehyde 3-phosphate and islatravir. Enzymes involved were galactose oxidase, horseradish peroxidase, catalase, pantothenate kinase, acetate kinase, deoxyribose 5-phosphate aldolase, phosphopentomutase and purine nucleoside phosphorylase]

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