电催化还原CO2耦合代谢工程改造解脂耶氏酵母实现高效异戊二烯生物合成

doi:10.12211/2096-8280.2025-046

摘要/Abstract

摘要：

生物合成技术因其绿色可持续特性，成为替代传统石化工艺的重要方向。本研究通过电催化还原CO₂与代谢工程改造解脂耶氏酵母相结合的策略，实现了高效异戊二烯的生物合成。首先，在解脂耶氏酵母中异源表达不同来源的异戊二烯合酶，筛选出葛根来源的异戊二烯合酶，异戊二烯产量为560 μg/L。随后，通过强化甲羟戊酸途径关键基因erg10、erg12、erg13、erg8、erg19、thmgr、idi及下调竞争基因erg20，构建工程菌株Misps13，摇瓶发酵异戊二烯滴度提高至12.23 mg/L。进一步整合电催化-微生物耦合系统，利用自组装纳米材料L-Bi-sh-H₂O和L-Cu-sh-3将CO₂高效转化为甲酸和乙酸，其法拉第效率分别为88.67%和50.14%。在Misps13中引入ACS与FDH模块，使工程菌Misps1315能够利用电催化合成的甲酸和乙酸，增强胞内乙酰辅酶A和NAD（P）H供应。通过响应曲面法优化外源甲酸和乙酸添加量，其中甲酸2.8 g/L、乙酸6.5 g/L，最终实现异戊二烯滴度32.14 mg/L，较基础菌株提升2.6倍。本研究为生物-电化学协同制造高值化合物提供了新范式。

关键词: 异戊二烯, 解脂耶氏酵母, 甲羟戊酸途径, 弱化竞争途径, CO₂电还原反应

Abstract:

Isoprene is a valuable platform chemical essential for manufacturing synthetic rubber, elastomers, and specialty materials, yet its conventional production depends heavily on petroleum resources with significant carbon emissions. Here, we present an innovative bio-electrocatalytic process that synergistically combines CO₂ electroreduction with metabolically engineered Yarrowia lipolytica for sustainable isoprene production. We began by screening several plant-derived isoprene synthases and identified the enzyme from Pueraria lobata (Isps-Pu) as the most effective in this yeast host, yielding initial production of 560 μg/L. Subsequent rational metabolic engineering involved systematic overexpression of the mevalonate pathway genes, including erg10, erg12, erg13, erg8, erg19, thmgr, and idi, via iterative genomic integration. Crucially, competitive metabolic flux was reduced by replacing the native promoter of erg20 with the weak promoter P_KI1, resulting in a significantly improved isoprene titer of 12.23 mg/L.To establish an efficient electrocatalytic module, we systematically developed and characterized multiple nanomaterials for CO₂ reduction. For formate production, bismuth-based electrodes (L-Bi-sh-C and L-Bi-sh-H₂O) were synthesized through distinct self-assembly and thermal treatment pathways. Comparative analysis revealed that L-Bi-sh-H₂O, prepared via hydrothermal treatment, demonstrated superior performance with a remarkable Faradaic efficiency of 88.67% for formate at -1.8 V versus Ag/AgCl. For acetate production, we engineered a series of copper-based catalysts (L-Cu-sh-3, L-Cu-sh-3-H₂O, L-Cu-sh-EtOH, and L-Cu-sh-EtOH-H₂O) using different solvent systems and processing methods. Among these, L-Cu-sh-3 electrode synthesized in acetonitrile/methanol/tetrahydrofuran solvent mixture exhibited optimal performance, achieving 50.14% Faradaic efficiency for acetate at -0.8 V.The integration of these electrocatalytic components with biological conversion was achieved by introducing Saccharomyces cerevisiae-derived acetyl-CoA synthase and formate dehydrogenase into the engineered yeast, enabling efficient assimilation of electro-generated formate and acetate to enhance intracellular acetyl-CoA and NADPH pools. Through response surface methodology optimization, we determined optimal concentrations of formate (2.8 g/L) and acetate (6.5 g/L), leading to a final isoprene titer of 32.14 mg/L—representing a 2.6-fold enhancement over the baseline strain. This integrated approach not only demonstrates a carbon-negative strategy for isoprene biosynthesis but also establishes a versatile platform for producing acetyl-CoA-derived chemicals from CO₂.

Key words: Isoprene, Yarrowia lipolytica, MVA pathway, approaches to reducing competitive intensity, CO? electroreduction

中图分类号:

Q939.9

兰云龙, 李嵩, 张伟, 常允蕴, 刘艳辉. 电催化还原CO₂耦合代谢工程改造解脂耶氏酵母实现高效异戊二烯生物合成[J]. 合成生物学, DOI: 10.12211/2096-8280.2025-046.

LAN Yunlong, LI Song, ZHANG Wei, CHANG Yunyun, LIU Yanhui. Electrocatalytic CO₂ Reduction Coupled with Metabolic Engineering of Yarrowia lipolytica for Efficient Isoprene Biosynthesis[J]. Synthetic Biology Journal, DOI: 10.12211/2096-8280.2025-046.

图/表 16

图1 MVA与MEP途径示意图

Fig. 1 Schematic Diagram of MVA and MEP Pathways

图2 代谢改造示意图

Fig. 2 Schematic diagram of metabolic engineering modification

表1-1 原始菌株

Table 1-1 Original strains

Name	Description	Source
E·coli-transT1	Resistance-deficient Escherichia coli strains used for cloning shuttle plasmids	Laboratory Preservation
E·coli-trans10	Resistance-deficient Escherichia coli strains used for cloning shuttle plasmids	Laboratory Preservation
SC-CENPK-113-5D	△leu、△ura、△his,Non-model strains of Saccharomyces cerevisiae	Laboratory Preservation
Po1f	△leu、△ura,Non-model strains of Yarrowia lipolytica	Laboratory Preservation
Misps	Po1f:Isps-Pu+idi	This study
Misps2	Misps:erg10+erg13	This study
Misps4	Misps2:tHMGR+erg12	This study
Misps6	Misps4:erg8+erg19	This study
Misps8	Misps6:tHMGR+Isps-Pu	This study
Misps10	Misps8:Isps-Pu +tHMGR	This study

表1-2 通用质粒表

Table 1-2 General Plasmids

Name	Description	Source
Puc57-amp⁺	An Escherichia coli cloning plasmid containing an ampicillin resistance selection gene	Synthesis
Puc57-kan⁺	An Escherichia coli cloning plasmid containing a kanamycin resistance selection gene	Synthesis
Pmo-ura3⁺	A high-copy plasmid for Yarrowia lipolytica containing an ampicillin resistance selection gene and a URA3 expression cassette	Synthesis
pcas-leu⁺	A low-copy plasmid for Yarrowia lipolytica containing an ampicillin resistance selection gene and a LEU2 expression cassette	Synthesis

图3 Bi电极扫描电镜图（a）L-Bi-sh-C；（b）L-Bi-sh-H2O（a）L-Bi-sh-C，（b）L-Bi-sh-H2O

Fig.3 Bi electrode scanning electron microscope

图4 扫描电镜图（a）L-Cu-sh-3；（b）L-Cu-sh-3-H2O；（c）L-Cu-sh-EtOH；（d）L-Cu-sh-EtOH- H2O（a）L-Cu-sh-3；（b）L-Cu-sh-3-H2O；（c）L-Cu-sh-EtOH；（d）L-Cu-sh-EtOH-H2O

Fig. 4 Sem figure:

表1-3 本研究材料电极的FE

Table 1-3 FE of the this study electrode

voltage（V）	S	Q	FE（%）
L-Bi-sh-C
-1.5	0.00	-0.15841	0
-1.6	0.01	-0.18058	75.23
-1.7	0.00	-0.55684	0
-1.8	0.05	-0.77842	87.26
-1.9	0.01	-0.21081	64.44
L-Bi-sh-H₂O
-1.5	0.08	-1.80135	60.31
-1.6	0.03	-1.00196	40.66
-1.7	0.10	-1.68801	80.48
-1.8	0.13	-1.99172	88.67
-1.9	0.05	-1.00719	67.44
L-Cu-sh-EtOH- H₂O
-0.8	0.13	0.6228	22.54
-1.0	0.11	0.3057	38.85
-1.2	0.13	0.3789	37.05
-1.5	0.09	0.68471	14.29
-1.7	0.15	1.3296	12.26
L-Cu-sh-EtOH
-0.8	0.02	0.1479	14.6
-1.0	0.03	0.1714	18.91
-1.2	0.02	0.0827	26.27
-1.5	0.02	0.1135	19.14
-1.7	0.02	0.1561	13.83
L-Cu-sh-3
-0.8	0.06	0.1300	50.14
-1.0	0.10	0.2546	42.69
-1.2	0.10	0.3013	36.06
-1.5	0.13	0.5301	26.65
-1.7	0.16	0.7862	22.12
L-Cu-sh-3-H₂O
-0.8	0.10	0.2431	44.43
-1.0	0.10	0.4598	23.64
-1.2	0.11	0.5533	21.61
-1.5	0.04	1.2853	3.38
-1.7	0.28	20.95	1.45

图5 Po1f-P-Pu菌株的发酵合成异戊二烯结果

Fig.5 isoprene synthesis by fermentation of Po1f-P-Pu strain

图6 四种携带Isps菌株的生长曲线及葡萄糖消耗图（a）发酵菌群浓度曲线；（b）发酵葡萄糖消耗图

Fig. 6 Growth curves and glucose consumption graphs of four strains carrying Isps(a) Concentration curves of four strains; (b) Figure of glucose consumption in fermentation of strains

图7 经过纯化后的SDS-PAGE胶图

Fig. 7 SDS-PAGE after Purification

图8 MVA途径强化菌株72 h摇瓶发酵生长曲线及异戊二烯产量（a）发酵72 h的OD监测曲线；（b）72 h摇瓶发酵后异戊二烯检测结果

Fig. 8 Growth curve and isoprene yield of MVA pathway-enhanced strain in 72-hour shake-flask fermentation(a)OD monitoring curve of fermentation for 72 h;(b)Detection results of isoprene after 72 h shaker fermentation

图9 竞争途径弱化菌株的生长曲线及异戊二烯产量（a）发酵72 h菌落生长曲线；（b）72 h摇瓶发酵异戊二烯产量图

Fig. 9 Growth curve and isoprene yield of strains with weakened competitive pathways(a)Colony growth curve for 72 h of fermentation;(b)Production of isoprene during 72 h shaker fermentation

图10 CO2RR高效合成CO的气质检测图

Fig. 10 Temperament detection of CO2RR efficiently synthesized CO

图11 Flow Cell阴极液核磁共振波谱图（a）乙酸溶液出峰图；（b）甲酸溶液出峰图

Fig.11 Flow Cell nuclear magnetic resonance spectroscopy of liquid cathode(a)Peak appearance chart of acetic acid solution;(b)Peak appearance chart of formic acid solution

图12 Misps1313菌株不同pH下发酵结果

Fig. 12 Fermentation results of Misps1313 at different pH

图13 Misps1313和Misps1315响应曲面设计结果：a）Misps1313的第一轮响应曲面设计结果；b）Misps1315的第一轮响应曲面设计结果；c）Misps1315的第二轮响应曲面设计结果；d）Misps1315的第三轮响应曲面设计结果

Fig. 13 Results of the response surface designs for Misps1313 and Misps1315:a) Results of the first round response surface design of Misps1313-3;b) First round response surface design results of Misps1315;c) Results of the second round response surface design of Misps1315;d) Third round response surface design results of Misps1315

参考文献 19

[1]	CHATZIVASILEIOU A O, WARD V, EDGAR S M, et al. Two-step pathway for isoprenoid synthesis [J]. Proceedings of the National Academy of Sciences, 2019, 116(2): 506-11.
[2]	CUI L, XIAO Y, HU W, et al. Enhanced dataset of global marine isoprene emissions from biogenic and photochemical processes for the period 2001–2020 [J]. Earth System Science Data, 2023, 15(12): 5403-25.
[3]	SANADZE G. Biogenic isoprene (a review) [J]. Russian Journal of Plant Physiology, 2004, 51: 729-41.
[4]	KUMAR V, JOHNSON B P, DIMAS D A, et al. Novel Homologs of Isopentenyl Phosphate Kinase Reveal Class-Wide Substrate Flexibility [J]. ChemCatChem, 2021, 13(17): 3781-8.
[5]	WANG C-H, HOU J, DENG H-K, et al. Microbial production of mevalonate [J]. Journal of Biotechnology, 2023, 370: 1-11.
[6]	GASTALDO C, LIPKO A, MOTSCH E, et al. Biosynthesis of isoprene units in Euphorbia lathyris Laticifers vs. other tissues: MVA and MEP pathways, compartmentation and putative endophytic fungi contribution [J]. Molecules, 2019, 24(23): 4322.
[7]	KIM J, BAIDOO E E, AMER B, et al. Engineering Saccharomyces cerevisiae for isoprenol production [J]. Metabolic Engineering, 2021, 64: 154-66.
[8]	ZHENG Y, LIU Q, LI L, et al. Metabolic engineering of Escherichia coli for high-specificity production of isoprenol and prenol as next generation of biofuels [J]. Biotechnology for biofuels, 2013, 6: 1-13.
[9]	YANG J, NIE Q, LIU H, et al. A novel MVA-mediated pathway for isoprene production in engineered E. coli [J]. BMC biotechnology, 2016, 16: 1-9.
[10]	YANG C, GAO X, JIANG Y, et al. Synergy between methylerythritol phosphate pathway and mevalonate pathway for isoprene production in Escherichia coli [J]. Metabolic Engineering, 2016, 37: 79-91.
[11]	ZHANG X-S, LI S, CAI H-X, et al. A DFT study of carbon dioxide reduction catalyzed by group 3 metal complexes of silylamides [J]. Chemical Physics Letters, 2022, 788: 139291.
[12]	PENG Z, DENG N, LI X, et al. Controllable Preparation, Working Mechanisms, and Actual Application of Various One-Dimensional Nanomaterials as Catalysts for CO2RR: A Review [J]. Industrial & Engineering Chemistry Research, 2023, 62(50): 21511-35.
[13]	YANG S, JIANG M, ZHANG W, et al. In situ structure refactoring of bismuth nanoflowers for highly selective electrochemical reduction of CO2 to formate [J]. Advanced Functional Materials, 2023, 33(37): 2301984.
[14]	QIU X F, HUANG J R, YU C, et al. A stable and conductive covalent organic framework with isolated active sites for highly selective electroreduction of carbon dioxide to acetate [J]. Angewandte Chemie, 2022, 134(36): e202206470.
[15]	LI H, OPGENORTH P H, WERNICK D G, et al. Integrated electromicrobial conversion of CO2 to higher alcohols [J]. Science, 2012, 335(6076): 1596-.
[16]	BI H, WANG K, XU C, et al. Biofuel synthesis from carbon dioxide via a bio-electrocatalysis system [J]. Chem Catalysis, 2023, 3(3).
[17]	LIU Q, BI H, WANG K, et al. Revealing the Mechanisms of Enhanced β-Farnesene Production in Yarrowia lipolytica through Metabolomics Analysis [J]. International Journal of Molecular Sciences, 2023, 24(24): 17366.
[18]	KAMINENI A, CHEN S, CHIFAMBA G, et al. Promoters for lipogenesis-specific downregulation in Yarrowia lipolytica [J]. FEMS Yeast Research, 2020, 20(5): foaa035.
[19]	DUAN G Y, LI X Q, DU Y R, et al. Efficient electrocatalytic reduction of CO₂ to CO on highly dispersed Ag nanoparticles confined by Poly(ionic liquid) [J]. Chemical Engineering Journal, 2023, 455.

电催化还原CO₂耦合代谢工程改造解脂耶氏酵母实现高效异戊二烯生物合成

Electrocatalytic CO₂ Reduction Coupled with Metabolic Engineering of Yarrowia lipolytica for Efficient Isoprene Biosynthesis

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