Advances in microbial production of liquid biofuels

doi:10.12211/2096-8280.2023-040

Synthetic Biology Journal

Advances in microbial production of liquid biofuels

Shuyuan GUO¹^,², Qiannan ZHANG¹^,², Gulikezi·MAIMAITIREXIATI¹^,², Yiqun YANG¹^,², Tao YU¹^,²

^1.Center for Synthetic Biochemistry，Shenzhen Institute of Synthetic Biology，Shenzhen Institutes of Advanced Technology，Chinese Academy of Sciences （CAS），Shenzhen 518055，Guangdong，China
^2.CAS key laboratory of Quantitative Engineering Biology，Shenzhen Institute of Synthetic Biology，Shenzhen Institutes of Advanced Technology，Chinese Academy of Sciences，Shenzhen 518055，Guangdong，China

Received:2023-06-13 Revised:2024-01-30 Published:2024-02-28
Contact: Shuyuan GUO, Tao YU

液体生物燃料合成与炼制的研究进展

郭姝媛¹^,², 张倩楠¹^,², 姑丽克孜·买买提热夏提null¹^,², 杨一群¹^,², 于涛¹^,²

^1.中国科学院深圳先进技术研究院，深圳合成生物学创新研究院，合成生物化学研究中心，广东深圳 518055
^2.中国科学院深圳先进技术研究院，深圳合成生物学创新研究院，中国科学院定量工程生物学重点实验室，广东深圳 518055

通讯作者: 郭姝媛,于涛
作者简介:郭姝媛（1991—），女，博士，助理研究员。研究方向为甲醇生物转化及产物合成。E-mail：sy.guo@siat.ac.cn
于涛（1986—），男，博士，研究员。研究方向为酿酒酵母的合成生物学。E-mail：tao.yu@siat.ac.cn
基金资助:
国家重点研发计划(2021YFA0911000);广东省重点区域研究与发展计划项目(2022B1111080005);国家自然科学基金(NSFC32071416);深圳合成生物学创新研究院科研基金(JCHZ20200003);深圳市科技计划(ZDSYS20210623091810032);中国科学院战略重点研究项目(XDB0480000);招商局集团先进技术研究院有限公司（基于电催化CO2转化与生物炼制的绿色制造项目），二氧化碳绿色转化与生物制造联合实验室(E2Z152);深圳先进院跨所联合攻关青年团队项目（电驱动CO2转化与生物炼制规模化示范）

Abstract

Abstract:

With the development of the economy and technology, the dependence of human beings on fossil fuels has led to the aggravation of energy shortage and climate change. This has created an urgent need for the development of advanced biofuels as alternatives to fossil fuels. Nowadays, widely recognized biofuels like bioethanol and biodiesel face challenges in terms of the insufficient production scale and high costs. Hence, the integration of metabolic engineering and synthetic biology has opened avenues for utilizing diverse substrates from renewable sources, such as solar energy, light energy, electric energy and biomass energy. Microbial cell factories, including microalgae, bacteria, and yeast, play a crucial role in synthesizing biofuels from these substrates. The review traces the evolution of four generations of biofuels, encompassing bioethanol, biodiesel, bio-gasoline, jet fuel and aviation fuel. In this review, we discuss how microorganisms can be explored for the production of third- and fourth-generation biofuels, including a variety of substrates (biomass, carbon dioxide, methanol, and methane), multi-energy coupling to synthesize biofuels (the utilization of lignocellulose by bacterial or yeast, CO₂ conversion by microalgae or electrochemical-biological system, the conversion of methanol and methane by methyltrophic microbes) and the application of synthetic biology. Furthermore, we overview biosynthetic pathways and the engineering strategies for optimize biofuels production. These strategies can convert raw materials to various fuel compounds, including fatty acids and esters, advanced alcohols and esters, isoprenoids and polyketides. Finally, we also highlight some challenges in biofuel production, including raw material supply and cost issues, low production yields, and limited product variety. Meanwhile, to address the difficulties, we propose corresponding solutions. For example, by optimizing carbon fixation pathways, and converting carbon dioxide into low-carbon substrates like methanol, autotrophic microorganisms, methylotrophic microorganisms, and other cell factories can utilize carbon dioxide as the main raw material to synthesize various biofuels and expand the production capacity, which can broaden the application fields of biofuels and further promote the industrialization of biofuels.

Key words: synthetic biofuels, new bioenergy, one-carbon substrates, renewable energy, microbial metabolic engineering

摘要：

人类社会发展对化石燃料的依赖导致了能源枯竭的加剧及强烈的气候变化，迫切需要开发能够代替化石资源的新型生物燃料。虽然已有生物乙醇、生物柴油等生物能源，但其生产规模和价格成本仍然是大规模应用的主要问题。近年来，随着可再生能源技术的发展，结合代谢工程及新兴的合成生物学技术，开发基于 CO₂ 的新兴生物燃料，逐渐成为未来绿色能源的重要研究方向。本文综述了生物燃料的种类及四代生物燃料的发展情况，并着重介绍了第三代和第四代生物燃料丰富的底物原材料、多能源偶联合成生物燃料的研究现状、合成生物学技术在其中的应用及现阶段的研究进展。文章最后概括了合成生物燃料所面临的困境，主要包括原料的供应及成本，新型液体生物燃料产量低、产品种类少等问题，并提出相应的解决办法，以二氧化碳作为主要原材料，结合自养型微生物、甲基营养型微生物等细胞工厂，通过优选固碳途径、转化二氧化碳为甲醇等低碳底物、多能源耦合等方式，利用广泛存在的二氧化碳实现多种生物燃料的合成，以期扩大生物燃料的产能及应用范围，进一步推动新型生物燃料的产业化进程。

关键词: 合成生物燃料, 新型生物能源, 一碳底物, 可再生能源, 微生物代谢工程

CLC Number:

Q815

Shuyuan GUO, Qiannan ZHANG, Gulikezi·MAIMAITIREXIATI, Yiqun YANG, Tao YU. Advances in microbial production of liquid biofuels[J]. Synthetic Biology Journal, DOI: 10.12211/2096-8280.2023-040.

郭姝媛, 张倩楠, 姑丽克孜·买买提热夏提null, 杨一群, 于涛. 液体生物燃料合成与炼制的研究进展[J]. 合成生物学, DOI: 10.12211/2096-8280.2023-040.

Figures/Tables 5

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生物燃料	宿主	主要底物	发酵培养基	产物和产量	主要途径	改造策略及相关基因	备注	参考文献
生物高级醇、酮,短链酸类物质	大肠杆菌	葡萄糖	Luria-Bertani(LB)	高级醇混合物 (1.8 g/L)	逆β氧化途径	1. 表达酰基CoA还原酶(TER),硫解酶(FadA),羟基酰基CoA还原酶(FADB)	1. 1L生物反应器; 2. 产物:丁醇、己醇、辛醇、癸醇、十二醇、十四醇、十六醇;	[63]
生物高级醇、酮,短链酸类物质	大肠杆菌	葡萄糖	无机盐基础培养基,多种氨基酸	丁醇(14 g/L); 3-酮丁酸(500 mg/L); 脂肪酸(7 g/L)	逆β氧化途径	1. 丁醇合成:ΔyqhD,ΔeutE,表达酰基转移酶(YQEF),丙二醇氧化还原酶(FUCO); 2.羧酸(C>4)合成:ΔfadB,ΔydiO,表达硫酯酶(TESA,TESB),脂肪酰转移酶(YAQF); 3. 脂肪酸合成:ΔyqhD,ΔfucO,ΔfadD,表达硫酯酶(TESA,TESB,FADM,YCIA); 4. 长链醇(C>4):表达酰基CoA还原酶,醇脱氢酶(YIAY,BETA,EUTG)	1. 生产高级醇(>C4)和脂肪酸(>C10)具有更高的效率; 2. 丁醇产率:0.33 g/g 葡萄糖; 3. 脂肪酸产率: 0.28 g/g葡萄糖; 4. 不同硫酯酶的使用可以产生不同碳链的脂肪酸; 5. 利用生物反应器生产丁醇和脂肪酸;	[47]
生物高级醇、酮,短链酸类物质	大肠杆菌	葡萄糖	Terrifc broth(TB)	3-羟基丁酸 (29.8 g/L)	逆β氧化途径	1.多元重组酶调控系统; 2.表达3-羟基丁酸酰基CoA脱氢酶(HBD),3-羟基丁酸酰基脱水酶(CRT),烯酰CoA还原酶(TER),酰基CoA酯化酶(TESB); 3. 表达RPOS,σ-38	1. 建立二元或多元重组酶依赖的开关调控系统用于延长菌株的复制周期; 2. 通过提高菌株的复制周期提高物质产量; 3. 5 L生物反应器;	[64]
生物高级醇、酮,短链酸类物质	大肠杆菌	葡萄糖	TB	1-丁醇 (30 g/L)	逆β氧化途径	1.表达烯酰CoA还原酶(TER); 2.打断NADH竞争利用途径:ΔldhA,ΔadhE,ΔfrdBC; 3.Δpta; 4.表达甲酸脱氢酶(FDH)	1.厌氧发酵; 2.产率:70%～88%;	[48]
	大肠杆菌	葡萄糖	无机盐基础培养基,多种氨基酸	异丁醇(23 mmol/L), 1-丁醇(0.6 mmol/L)	酮酸途径	1. 表达酮酸脱羧酶(KDC),醇脱氢酶(ADH); 2. 表达缬氨酸或亮氨酸合成途径	1. 以苏氨酸、缬氨酸、异亮氨酸、亮氨酸等氨基酸生物合成途径为基础; 2. 供应不同底物可以产生不同化合物,如2-甲基-1-丁醇,3-甲基-1-丁醇或2-苯乙醇。	[65]
	酿酒酵母	葡萄糖	酵母合成培养基(SC), 硫酸铜	异丁醇 (263.2 mg/L)	酮酸途径	1.表达α-乙酰乳酸合酶(ALSS,Bacillus subtilis),酮醇酸还原异构酶(ILV5),二羟基酸脱氢酶(ILV3); 2.表达酮酸脱羧酶(KDC),醇脱氢酶(ADH)	1.通过引入铜诱导启动子CUP1缓解中间产物乙酰乳酸毒性; 2.Delta位点多拷贝整合; 3.以缬氨酸合成途径为基础;	[49]
	酿酒酵母	葡萄糖	无机盐基础培养基、酵母合成培养基	异丁醇(635±23 mg/L);异戊醇(95±12 mg/L);2-甲基-1-丁醇 (118±28 mg/L)	酮酸途径	1.表达α-乙酰乳酸合酶(ALSS),酮醇酸还原异构酶(ILV5),二羟基酸脱氢酶(ILV3); 2.表达酮酸脱羧酶(KDC),醇脱氢酶(ADH)	1.线粒体靶向表达; 2.以缬氨酸、亮氨酸、异亮氨酸合成途径为基础;	[50]
生物高级醇、酮,短链酸类物质	酿酒酵母	葡萄糖	YP	异丁醇 (2.09 g/L)	酮酸途径	1. 乙酰乳酸合成酶(ALSS),乙酰羟基酸还原异构酶(ILV5),二醇酸脱水酶(ILV3); 2. ΔILV2; 3. 敲除合成副产物的基因	1.以缬氨酸合成途径为基础; 2.转化率:59.55 mg/g葡萄糖;	[66]
	谷氨酸棒状杆菌	葡萄糖	CGXII 培养基	2-甲基-1-丁醇 (0.37 g/L); 3-甲基-1-丁醇 (2.76 g/L)	酮酸途径	1. 表达酮酸脱羧酶(KDC), 醇脱氢酶(ADH)	1. 以缬氨酸和异亮氨酸合成途径为基础;	[67]
	毕赤酵母	甘油	无机盐基础培养基	异戊醇; 3-甲基-1-丁醇 (191.0±9.6 mg/L)	酮酸途径	1. 表达乙酰乳酸合成酶(ILV2),乙酰羟基酸还原异构酶(ILV5),二醇酸脱水酶(ILV3),酮酸脱羧酶(KDC),乙醇脱氢酶(ADH); 2. 下调丙酮酸脱羧酶(PDC)	1.通过表达缬氨酸和亮氨酸合成途径增加中间产物2-酮异戊酸的产量;	[68]
	毕赤酵母	葡萄糖/甘油	无机盐基础培养基	异丁醇(2.22 g/L); 乙酸异丁酯(24 mg/L)	酮酸途径	1. 表达缬氨酸合成途径(ILV2,ILV5,ILV3),酮酸脱羧酶(KDC),醇脱氢酶(ADH); 2. 表达醇氧酰基转移酶用于乙酸异丁酯合成(ATF)	1.以缬氨酸合成途径为基础;	[51]
生物高级醇、酮,短链酸类物质	黄色短杆菌(Brevibacterium flavum)	葡萄糖	无机盐培养基,酵母提取物	异丁醇(5362 mg/L); 2-甲基-1-丁醇 (1945 mg/L); 3-甲基-1-丁醇(785.34 mg/L)	酮酸途径	1. 表达酮酸脱羧酶(KDC),酮基异戊酸脱羧酶(KIVD),醇脱氢酶(ADH); 2. 苯丙酮酸脱羧酶(ARO10)	1. 诱变结合高通量筛选; 2. 以亮氨酸、异亮氨酸、缬氨酸为基础合成;	[69]
	枯草芽孢杆菌	葡萄糖	LB和无机盐混合培养基;	异丁醇(2.62 g/L); 乙醇(1.2 g/L); 苯乙醇(1.06 g/L)	酮酸途径	1. 乙酰乳酸合酶(ALSS),酮酸还原异构酶(ILVC),二羟酸脱水酶(ILVD),酮酸脱羧酶(KDC),醇脱氢酶(ADH)	1. 以缬氨酸合成途径为基础; 2. 丙酮酸和磷酸烯醇式丙酮酸为乙醇和苯乙醇的前体物质; 3. 1 L摇瓶发酵;	[70]
	解脂耶氏酵母(Yarrowia lipolytica)	甘油	YP	异丙醇(1.94 g/L)		1. 表达丙酮酰CoA合成酶(nphT7),表达异丙醇合成酶	1. 利用该酵母生长异丙醇的最高滴度; 2. 纯甘油作为碳源可产生1.94 g/L异丙醇;利用原油作为碳源可产生1.6 g/L异丙醇; 3. 5 L生物反应器;	[71]
生物高级醇、酮,短链酸类物质	链霉菌(Streptomyces albus)	葡萄糖, 木糖		短链酮 (C5～C7)	聚酮合成途径	1. 表达聚酮合成酶(PKS)	1. 利用多结构域融合酶合成燃料; 2. C6-C7乙基酮: >1 g/L;C5-C6甲基酮:250 mg/L; 3. 原料为玉米秸秆; 4. 2 L生物反应器;	[72]
生物高级醇、酮,短链酸类物质	富养罗尔斯通氏菌Re21 33(Cupriavidus necator)	果糖	无机盐基础培养基	异丙醇 (3.44g/L)		1. 表达酮硫解酶(THL),CoA转移酶(CTF),乙酰乙酸脱羧酶 (ADC),醇脱氢酶(ADH); 2. ΔphaB, ΔphaC		[73]
萜类物质	紫色非硫光合细菌 (Rhodobactercapsulatus)	葡萄糖	无机盐基础培养基,酵母提取物	红没药烯 (1 g/L)	类异戊二烯途径	1.筛选红没药烯合成酶表达启动子; 2.Δzwf1; 3.增加NADPH:ΔgltBD, ΔphbC; 4.敲除FBB竞争途径: 5.表达异源MVA途径;乙酰CoA酰基转移酶(ATOB),HMG-CoA合成酶(HMGCS),HMG-CoA还原酶(HMGCR),甲羟戊酸激酶(MK),磷酸甲羟戊酸激酶(PMK),甲羟戊酸二磷酸脱羧酶(PMD),异戊二烯二磷酸异构酶(IDI),法尼基二磷酸合酶(ISPA)	1. 摇瓶产量1 g/L; 2. 5 L生物反应器中,产量:9.8 g/L,产率>0.196 g/g葡萄糖	[60]
萜类物质	大肠杆菌	葡萄糖	EZ-Rich,YP	红没药烯 (900 mg/L)	类异戊二烯途径	1. 表达没药烯合成酶(TPS, Abies grandis); 2. 表达MVA途径:乙酰CoA酰基转移酶(ATOB),HMG-CoA合成酶(HMGCS),HMG-CoA 还原酶(HMGCR),甲羟戊酸激酶(MK),磷酸甲羟戊酸激酶(PMK),甲羟戊酸二磷酸脱羧酶(PMD),异戊二烯二磷酸异构酶(IDI),法尼基二磷酸合酶(ISPA)		[58]
萜类物质	大肠杆菌	葡萄糖	无机盐基础培养基	异戊二烯 (587±47 mg/L)	类异戊二烯途径	1. 表达MVA途径:乙酰乙酰辅酶A硫代酶(MVAE),合成酶(MVAS),激酶(MVK),磷酸甲羟戊酸激酶(PMK),二磷酸甲羟戊酸脱羧酶(MVAD),异戊烯基二磷酸异构酶(IDI),异戊二烯合酶(ISPS)		[59]
	大肠杆菌	葡萄糖	EZ-Rich	柠烯 (435 mg/L)	类异戊二烯途径	1.表达柠烯合成酶(LS),细胞色素P450; 2.表达MVA途径:乙酰CoA酰基转移酶(ATOB),HMG-CoA合成酶(HMGCS),HMG-CoA还原酶(HMGCR),甲羟戊酸激酶(MK),磷酸甲羟戊酸激酶(PMK),甲羟戊酸二磷酸脱羧酶(PMD); 3.香叶基焦磷酸合成酶(GPPS)	1. MVA途径为基础;	[74]
	酿酒酵母	葡萄糖,蔗糖	无机盐培养基	法呢烯 (130 g/L)	类异戊二烯途径	1.表达磷酸转酮酶(XPK),磷脂酰转移酶(PTA),乙醛脱氢酶(ADA),HMG-CoA还原酶(HMGCR),法呢烯合成酶(FS); 2.Δacs2,Δacs1,Δacs6,Δhr2;	1. 首次在酿酒酵母中高效合成法呢烯; 2. 产率:17.3% g/g 葡萄糖;	[61]
萜类物质	解脂耶氏酵母 (Yarrowia lipolytica)	葡萄糖	YP	β-法呢烯 (22.8 g/L)	类异戊二烯途径	1. 表达MVA途径:HMG-CoA还原酶(HMGCR),法呢基二磷酸合成酶(ERG20),法呢烯合成酶(FS); 2. ∆DGA1,∆DGA2;	1. 以MVA途径为基础; 2. 2 L生物反应器;	[75]
萜类物质	解脂耶氏酵母 (Yarrowia lipolytica)	葡萄糖	YP	α-法呢烯 (25.55 g/L)	类异戊二烯途径	1.表达MVA途径:乙酰CoA酰基转移酶(ATOB),HMG-CoA还原酶(HMGCR); 2.法呢基二磷酸合成酶(ERG20),法呢烯合成酶(FS)	1.以MVA途径为基础; 2.1 L生物反应器;	[76]
脂肪酸及其衍生物	大肠杆菌	甘油	无机盐基础培养基,酵母提取物	游离脂肪酸 (30 g/L)		ihfAL--aidB+-ryfAM--gadAH-	1. 利用CRISPRi高通量筛选结合组学分析探究提高脂肪酸产量的靶基因; 2. 5 L生物反应器;	[77]
脂肪酸及其衍生物	大肠杆菌	葡萄糖	无机盐基础培养基,多种氨基酸	脂肪酸异丙酯 (203.4 mg/L)	脂肪酸合成途径,逆β氧化途径	1. 表达酰基CoA-酰基转移酶(ATOB),乙酰乙酰CoA转移酶(ATOAD),乙酰乙酸脱羧酶(ADC),乙醇脱氢酶(ADH); 2. 硫酯酶(TESA),脂酰辅酶A合成酶(FADD),酰基转移酶(DGAT)	1. 逆β氧化途径和脂肪酸合成途径共同作用;	[78]
脂肪酸及其衍生物	大肠杆菌	甘油	无机盐基础培养基,酵母提取物	脂肪酸短链酯 (1 g/L)	酮酸途径,脂肪酸合成途径	1. 表达酮酸脱羧酶(ARO10),乙醇脱氢酶(ADH),酰基转酯酶(DGAT); 2. 表达硫酯酶(TESA),脂酰辅酶A合成酶(FADD); 3. ΔfadE	1. 酮酸途径合成短链醇,脂肪酸合成途径提供乙酰CoA,随后酯化形成终产物; 2. 6 L生物反应器;	[79]
	大肠杆菌	甘油	无机盐基础培养基,酵母提取物,胰蛋白胨	脂肪酸乙酯 (813 mg/L)	脂肪酸合成途径	1. 表达酰基辅酶A:二酰基甘油酰基转移酶(ATFA)	1. 5 L生物反应器;	[80]
	酿酒酵母	葡萄糖	YP	C12～C18脂肪醇 (6 g/L)	脂肪酸合成途径	1. Δhfd1,Δadh6,Δgdh1,Δdga1; 2. 表达脂肪酸还原酶(FAR,Mus musculus),乙酰CoA羧化酶(ACC1),脂肪酸合成酶(FAS),脂肪酸去饱和酶(OLE1)	1. 木质纤维素作为原材料; 2. 产率:葡萄糖最大理论转化率的20%; 3. 2 L生物反应器;	[81]
	酿酒酵母	葡萄糖,半乳糖	无机盐基础培养基	超长链脂肪酸 (83.5 mg/L)	脂肪酸合成途径	1.表达脂肪酸合成酶(FAS); 2.ΔElo3,Δgal1; 3.表达脂肪酸还原酶(FAR); 4.表达乙酰CoA羧化酶(ACC1),延长酶(ELO1,ELO2)	1.C22脂肪酸及脂肪醇为主; 2.不同链长脂肪酸表达不同的延长酶;	[82]
脂肪酸及其衍生物	酿酒酵母	葡萄糖	无机盐基础培养基	脂肪酸 (33.4 g/L)	脂肪酸合成途径	1. 增强乙酰CoA供应:表达丙酮酸羧化酶(PYC1),乙酰CoA 羧化酶(ACC1),线粒体丙酮酸载体(MPC),柠檬酸合成酶(CIT1),柠檬酸裂解酶(ACL),胞质异柠檬酸脱氢酶(IDP2),柠檬酸穿梭蛋白(YHM2); 2. 加强PPP途径,降低葡萄糖磷酸异构酶(PGI1); 3. 降低异柠檬酸脱氢酶 1(IDH1); 4. Δpdc(丙酮酸脱羧酶); 5. pyk突变	1. 葡萄糖生产脂肪酸的最高产量; 2. 1 L生物反应器; 3. 挖掘进化的关键基因并通过反向工程验证,阐明高产油机制;	[56]
脂肪酸及其衍生物	酿酒酵母	葡萄糖	无机盐基础培养基	中链脂肪酸 (C6～C12: 1.39±0.05 g/L)	脂肪酸合成途径	1. 工程化改造脂肪酸合成酶(FAS); 2. Δhfd1; 3. 膜转运蛋白(TOP1)易错PCR结合进化筛选; 4. 菌株进化结合代谢重塑	1. 摇瓶发酵; 2. 产率:18.9±0.6;	[83]
脂肪酸及其衍生物	酿酒酵母	葡萄糖	无机盐基础培养基	脂肪酸 (20 g/L)	脂肪酸合成途径	1. Δpgi,Δpdc1,Δpdc5,Δpdc6; 2. 增加胞质NADH:表达谷氨酸脱氢酶(GDH1, GDH2); 3. 表达琥珀酸生成途径:延胡索酸酶(FUM1),苹果酸酶(tMDH3),丙酮酸羧化酶(PYC2),富马酸还原酶(FDR1); 4. 下调PFK1,Δpfk2; 5. 捕获胞质NADH进入呼吸链:表达NADH脱氢酶(NDE1,NDE2); 6. 表达脂肪酸合成途径:脂肪酸合成酶(FAS),硫酯酶(TESA),乙酰CoA羧化酶(ACC1); 7. Δfaa1,Δfaa4,Δpox1; 8. 过表达 PPP 途径:6-磷酸葡萄糖脱氢酶(ZWF1),磷酸葡糖酸脱氢酶 (GND1),转酮酶(TKL1),转醛酶(TAL1); 9.利用不同启动子表达不同来源的果糖1,6-二磷酸酶(FBP); 9. 表达NOG途径:磷酸转酮酶(XFPK),磷脂酰转移酶(PTA)	1. 利用合成的能量系统代替TCA 进行能量供应,用于脂肪酸生成; 2.产率:0.134 g/g 葡萄糖,40%的产率为已知报道的最高;	[57]
脂肪酸及其衍生物	斯达油脂酵母 (Lipomyces starkeyi), 解脂耶氏酵母 (Yarrowia lipolytica)	葡萄糖,木糖	无机盐基础培养基	脂肪醇	脂肪酸合成途径	1. 表达脂肪酰辅酶A还原酶 (FAR,Marinobactor aquaeolei VT8)	1. 十六烷醇(C16:0)和十八醇(C18:0)占主导地位; 2. 不同底物所得的脂肪醇产量不同;	[54]

生物燃料	宿主	主要底物	发酵培养基	产物和产量	主要途径	改造策略及相关基因	备注	参考文献
生物高级醇、酮,短链酸类物质	大肠杆菌	葡萄糖	Luria-Bertani(LB)	高级醇混合物 (1.8 g/L)	逆β氧化途径	1. 表达酰基CoA还原酶(TER),硫解酶(FadA),羟基酰基CoA还原酶(FADB)	1. 1L生物反应器; 2. 产物:丁醇、己醇、辛醇、癸醇、十二醇、十四醇、十六醇;	[63]
生物高级醇、酮,短链酸类物质	大肠杆菌	葡萄糖	无机盐基础培养基,多种氨基酸	丁醇(14 g/L); 3-酮丁酸(500 mg/L); 脂肪酸(7 g/L)	逆β氧化途径	1. 丁醇合成:ΔyqhD,ΔeutE,表达酰基转移酶(YQEF),丙二醇氧化还原酶(FUCO); 2.羧酸(C>4)合成:ΔfadB,ΔydiO,表达硫酯酶(TESA,TESB),脂肪酰转移酶(YAQF); 3. 脂肪酸合成:ΔyqhD,ΔfucO,ΔfadD,表达硫酯酶(TESA,TESB,FADM,YCIA); 4. 长链醇(C>4):表达酰基CoA还原酶,醇脱氢酶(YIAY,BETA,EUTG)	1. 生产高级醇(>C4)和脂肪酸(>C10)具有更高的效率; 2. 丁醇产率:0.33 g/g 葡萄糖; 3. 脂肪酸产率: 0.28 g/g葡萄糖; 4. 不同硫酯酶的使用可以产生不同碳链的脂肪酸; 5. 利用生物反应器生产丁醇和脂肪酸;	[47]
生物高级醇、酮,短链酸类物质	大肠杆菌	葡萄糖	Terrifc broth(TB)	3-羟基丁酸 (29.8 g/L)	逆β氧化途径	1.多元重组酶调控系统; 2.表达3-羟基丁酸酰基CoA脱氢酶(HBD),3-羟基丁酸酰基脱水酶(CRT),烯酰CoA还原酶(TER),酰基CoA酯化酶(TESB); 3. 表达RPOS,σ-38	1. 建立二元或多元重组酶依赖的开关调控系统用于延长菌株的复制周期; 2. 通过提高菌株的复制周期提高物质产量; 3. 5 L生物反应器;	[64]
生物高级醇、酮,短链酸类物质	大肠杆菌	葡萄糖	TB	1-丁醇 (30 g/L)	逆β氧化途径	1.表达烯酰CoA还原酶(TER); 2.打断NADH竞争利用途径:ΔldhA,ΔadhE,ΔfrdBC; 3.Δpta; 4.表达甲酸脱氢酶(FDH)	1.厌氧发酵; 2.产率:70%～88%;	[48]
	大肠杆菌	葡萄糖	无机盐基础培养基,多种氨基酸	异丁醇(23 mmol/L), 1-丁醇(0.6 mmol/L)	酮酸途径	1. 表达酮酸脱羧酶(KDC),醇脱氢酶(ADH); 2. 表达缬氨酸或亮氨酸合成途径	1. 以苏氨酸、缬氨酸、异亮氨酸、亮氨酸等氨基酸生物合成途径为基础; 2. 供应不同底物可以产生不同化合物,如2-甲基-1-丁醇,3-甲基-1-丁醇或2-苯乙醇。	[65]
	酿酒酵母	葡萄糖	酵母合成培养基(SC), 硫酸铜	异丁醇 (263.2 mg/L)	酮酸途径	1.表达α-乙酰乳酸合酶(ALSS,Bacillus subtilis),酮醇酸还原异构酶(ILV5),二羟基酸脱氢酶(ILV3); 2.表达酮酸脱羧酶(KDC),醇脱氢酶(ADH)	1.通过引入铜诱导启动子CUP1缓解中间产物乙酰乳酸毒性; 2.Delta位点多拷贝整合; 3.以缬氨酸合成途径为基础;	[49]
	酿酒酵母	葡萄糖	无机盐基础培养基、酵母合成培养基	异丁醇(635±23 mg/L);异戊醇(95±12 mg/L);2-甲基-1-丁醇 (118±28 mg/L)	酮酸途径	1.表达α-乙酰乳酸合酶(ALSS),酮醇酸还原异构酶(ILV5),二羟基酸脱氢酶(ILV3); 2.表达酮酸脱羧酶(KDC),醇脱氢酶(ADH)	1.线粒体靶向表达; 2.以缬氨酸、亮氨酸、异亮氨酸合成途径为基础;	[50]
生物高级醇、酮,短链酸类物质	酿酒酵母	葡萄糖	YP	异丁醇 (2.09 g/L)	酮酸途径	1. 乙酰乳酸合成酶(ALSS),乙酰羟基酸还原异构酶(ILV5),二醇酸脱水酶(ILV3); 2. ΔILV2; 3. 敲除合成副产物的基因	1.以缬氨酸合成途径为基础; 2.转化率:59.55 mg/g葡萄糖;	[66]
	谷氨酸棒状杆菌	葡萄糖	CGXII 培养基	2-甲基-1-丁醇 (0.37 g/L); 3-甲基-1-丁醇 (2.76 g/L)	酮酸途径	1. 表达酮酸脱羧酶(KDC), 醇脱氢酶(ADH)	1. 以缬氨酸和异亮氨酸合成途径为基础;	[67]
	毕赤酵母	甘油	无机盐基础培养基	异戊醇; 3-甲基-1-丁醇 (191.0±9.6 mg/L)	酮酸途径	1. 表达乙酰乳酸合成酶(ILV2),乙酰羟基酸还原异构酶(ILV5),二醇酸脱水酶(ILV3),酮酸脱羧酶(KDC),乙醇脱氢酶(ADH); 2. 下调丙酮酸脱羧酶(PDC)	1.通过表达缬氨酸和亮氨酸合成途径增加中间产物2-酮异戊酸的产量;	[68]
	毕赤酵母	葡萄糖/甘油	无机盐基础培养基	异丁醇(2.22 g/L); 乙酸异丁酯(24 mg/L)	酮酸途径	1. 表达缬氨酸合成途径(ILV2,ILV5,ILV3),酮酸脱羧酶(KDC),醇脱氢酶(ADH); 2. 表达醇氧酰基转移酶用于乙酸异丁酯合成(ATF)	1.以缬氨酸合成途径为基础;	[51]
生物高级醇、酮,短链酸类物质	黄色短杆菌(Brevibacterium flavum)	葡萄糖	无机盐培养基,酵母提取物	异丁醇(5362 mg/L); 2-甲基-1-丁醇 (1945 mg/L); 3-甲基-1-丁醇(785.34 mg/L)	酮酸途径	1. 表达酮酸脱羧酶(KDC),酮基异戊酸脱羧酶(KIVD),醇脱氢酶(ADH); 2. 苯丙酮酸脱羧酶(ARO10)	1. 诱变结合高通量筛选; 2. 以亮氨酸、异亮氨酸、缬氨酸为基础合成;	[69]
	枯草芽孢杆菌	葡萄糖	LB和无机盐混合培养基;	异丁醇(2.62 g/L); 乙醇(1.2 g/L); 苯乙醇(1.06 g/L)	酮酸途径	1. 乙酰乳酸合酶(ALSS),酮酸还原异构酶(ILVC),二羟酸脱水酶(ILVD),酮酸脱羧酶(KDC),醇脱氢酶(ADH)	1. 以缬氨酸合成途径为基础; 2. 丙酮酸和磷酸烯醇式丙酮酸为乙醇和苯乙醇的前体物质; 3. 1 L摇瓶发酵;	[70]
	解脂耶氏酵母(Yarrowia lipolytica)	甘油	YP	异丙醇(1.94 g/L)		1. 表达丙酮酰CoA合成酶(nphT7),表达异丙醇合成酶	1. 利用该酵母生长异丙醇的最高滴度; 2. 纯甘油作为碳源可产生1.94 g/L异丙醇;利用原油作为碳源可产生1.6 g/L异丙醇; 3. 5 L生物反应器;	[71]
生物高级醇、酮,短链酸类物质	链霉菌(Streptomyces albus)	葡萄糖, 木糖		短链酮 (C5～C7)	聚酮合成途径	1. 表达聚酮合成酶(PKS)	1. 利用多结构域融合酶合成燃料; 2. C6-C7乙基酮: >1 g/L;C5-C6甲基酮:250 mg/L; 3. 原料为玉米秸秆; 4. 2 L生物反应器;	[72]
生物高级醇、酮,短链酸类物质	富养罗尔斯通氏菌Re21 33(Cupriavidus necator)	果糖	无机盐基础培养基	异丙醇 (3.44g/L)		1. 表达酮硫解酶(THL),CoA转移酶(CTF),乙酰乙酸脱羧酶 (ADC),醇脱氢酶(ADH); 2. ΔphaB, ΔphaC		[73]
萜类物质	紫色非硫光合细菌 (Rhodobactercapsulatus)	葡萄糖	无机盐基础培养基,酵母提取物	红没药烯 (1 g/L)	类异戊二烯途径	1.筛选红没药烯合成酶表达启动子; 2.Δzwf1; 3.增加NADPH:ΔgltBD, ΔphbC; 4.敲除FBB竞争途径: 5.表达异源MVA途径;乙酰CoA酰基转移酶(ATOB),HMG-CoA合成酶(HMGCS),HMG-CoA还原酶(HMGCR),甲羟戊酸激酶(MK),磷酸甲羟戊酸激酶(PMK),甲羟戊酸二磷酸脱羧酶(PMD),异戊二烯二磷酸异构酶(IDI),法尼基二磷酸合酶(ISPA)	1. 摇瓶产量1 g/L; 2. 5 L生物反应器中,产量:9.8 g/L,产率>0.196 g/g葡萄糖	[60]
萜类物质	大肠杆菌	葡萄糖	EZ-Rich,YP	红没药烯 (900 mg/L)	类异戊二烯途径	1. 表达没药烯合成酶(TPS, Abies grandis); 2. 表达MVA途径:乙酰CoA酰基转移酶(ATOB),HMG-CoA合成酶(HMGCS),HMG-CoA 还原酶(HMGCR),甲羟戊酸激酶(MK),磷酸甲羟戊酸激酶(PMK),甲羟戊酸二磷酸脱羧酶(PMD),异戊二烯二磷酸异构酶(IDI),法尼基二磷酸合酶(ISPA)		[58]
萜类物质	大肠杆菌	葡萄糖	无机盐基础培养基	异戊二烯 (587±47 mg/L)	类异戊二烯途径	1. 表达MVA途径:乙酰乙酰辅酶A硫代酶(MVAE),合成酶(MVAS),激酶(MVK),磷酸甲羟戊酸激酶(PMK),二磷酸甲羟戊酸脱羧酶(MVAD),异戊烯基二磷酸异构酶(IDI),异戊二烯合酶(ISPS)		[59]
	大肠杆菌	葡萄糖	EZ-Rich	柠烯 (435 mg/L)	类异戊二烯途径	1.表达柠烯合成酶(LS),细胞色素P450; 2.表达MVA途径:乙酰CoA酰基转移酶(ATOB),HMG-CoA合成酶(HMGCS),HMG-CoA还原酶(HMGCR),甲羟戊酸激酶(MK),磷酸甲羟戊酸激酶(PMK),甲羟戊酸二磷酸脱羧酶(PMD); 3.香叶基焦磷酸合成酶(GPPS)	1. MVA途径为基础;	[74]
	酿酒酵母	葡萄糖,蔗糖	无机盐培养基	法呢烯 (130 g/L)	类异戊二烯途径	1.表达磷酸转酮酶(XPK),磷脂酰转移酶(PTA),乙醛脱氢酶(ADA),HMG-CoA还原酶(HMGCR),法呢烯合成酶(FS); 2.Δacs2,Δacs1,Δacs6,Δhr2;	1. 首次在酿酒酵母中高效合成法呢烯; 2. 产率:17.3% g/g 葡萄糖;	[61]
萜类物质	解脂耶氏酵母 (Yarrowia lipolytica)	葡萄糖	YP	β-法呢烯 (22.8 g/L)	类异戊二烯途径	1. 表达MVA途径:HMG-CoA还原酶(HMGCR),法呢基二磷酸合成酶(ERG20),法呢烯合成酶(FS); 2. ∆DGA1,∆DGA2;	1. 以MVA途径为基础; 2. 2 L生物反应器;	[75]
萜类物质	解脂耶氏酵母 (Yarrowia lipolytica)	葡萄糖	YP	α-法呢烯 (25.55 g/L)	类异戊二烯途径	1.表达MVA途径:乙酰CoA酰基转移酶(ATOB),HMG-CoA还原酶(HMGCR); 2.法呢基二磷酸合成酶(ERG20),法呢烯合成酶(FS)	1.以MVA途径为基础; 2.1 L生物反应器;	[76]
脂肪酸及其衍生物	大肠杆菌	甘油	无机盐基础培养基,酵母提取物	游离脂肪酸 (30 g/L)		ihfAL--aidB+-ryfAM--gadAH-	1. 利用CRISPRi高通量筛选结合组学分析探究提高脂肪酸产量的靶基因; 2. 5 L生物反应器;	[77]
脂肪酸及其衍生物	大肠杆菌	葡萄糖	无机盐基础培养基,多种氨基酸	脂肪酸异丙酯 (203.4 mg/L)	脂肪酸合成途径,逆β氧化途径	1. 表达酰基CoA-酰基转移酶(ATOB),乙酰乙酰CoA转移酶(ATOAD),乙酰乙酸脱羧酶(ADC),乙醇脱氢酶(ADH); 2. 硫酯酶(TESA),脂酰辅酶A合成酶(FADD),酰基转移酶(DGAT)	1. 逆β氧化途径和脂肪酸合成途径共同作用;	[78]
脂肪酸及其衍生物	大肠杆菌	甘油	无机盐基础培养基,酵母提取物	脂肪酸短链酯 (1 g/L)	酮酸途径,脂肪酸合成途径	1. 表达酮酸脱羧酶(ARO10),乙醇脱氢酶(ADH),酰基转酯酶(DGAT); 2. 表达硫酯酶(TESA),脂酰辅酶A合成酶(FADD); 3. ΔfadE	1. 酮酸途径合成短链醇,脂肪酸合成途径提供乙酰CoA,随后酯化形成终产物; 2. 6 L生物反应器;	[79]
	大肠杆菌	甘油	无机盐基础培养基,酵母提取物,胰蛋白胨	脂肪酸乙酯 (813 mg/L)	脂肪酸合成途径	1. 表达酰基辅酶A:二酰基甘油酰基转移酶(ATFA)	1. 5 L生物反应器;	[80]
	酿酒酵母	葡萄糖	YP	C12～C18脂肪醇 (6 g/L)	脂肪酸合成途径	1. Δhfd1,Δadh6,Δgdh1,Δdga1; 2. 表达脂肪酸还原酶(FAR,Mus musculus),乙酰CoA羧化酶(ACC1),脂肪酸合成酶(FAS),脂肪酸去饱和酶(OLE1)	1. 木质纤维素作为原材料; 2. 产率:葡萄糖最大理论转化率的20%; 3. 2 L生物反应器;	[81]
	酿酒酵母	葡萄糖,半乳糖	无机盐基础培养基	超长链脂肪酸 (83.5 mg/L)	脂肪酸合成途径	1.表达脂肪酸合成酶(FAS); 2.ΔElo3,Δgal1; 3.表达脂肪酸还原酶(FAR); 4.表达乙酰CoA羧化酶(ACC1),延长酶(ELO1,ELO2)	1.C22脂肪酸及脂肪醇为主; 2.不同链长脂肪酸表达不同的延长酶;	[82]
脂肪酸及其衍生物	酿酒酵母	葡萄糖	无机盐基础培养基	脂肪酸 (33.4 g/L)	脂肪酸合成途径	1. 增强乙酰CoA供应:表达丙酮酸羧化酶(PYC1),乙酰CoA 羧化酶(ACC1),线粒体丙酮酸载体(MPC),柠檬酸合成酶(CIT1),柠檬酸裂解酶(ACL),胞质异柠檬酸脱氢酶(IDP2),柠檬酸穿梭蛋白(YHM2); 2. 加强PPP途径,降低葡萄糖磷酸异构酶(PGI1); 3. 降低异柠檬酸脱氢酶 1(IDH1); 4. Δpdc(丙酮酸脱羧酶); 5. pyk突变	1. 葡萄糖生产脂肪酸的最高产量; 2. 1 L生物反应器; 3. 挖掘进化的关键基因并通过反向工程验证,阐明高产油机制;	[56]
脂肪酸及其衍生物	酿酒酵母	葡萄糖	无机盐基础培养基	中链脂肪酸 (C6～C12: 1.39±0.05 g/L)	脂肪酸合成途径	1. 工程化改造脂肪酸合成酶(FAS); 2. Δhfd1; 3. 膜转运蛋白(TOP1)易错PCR结合进化筛选; 4. 菌株进化结合代谢重塑	1. 摇瓶发酵; 2. 产率:18.9±0.6;	[83]
脂肪酸及其衍生物	酿酒酵母	葡萄糖	无机盐基础培养基	脂肪酸 (20 g/L)	脂肪酸合成途径	1. Δpgi,Δpdc1,Δpdc5,Δpdc6; 2. 增加胞质NADH:表达谷氨酸脱氢酶(GDH1, GDH2); 3. 表达琥珀酸生成途径:延胡索酸酶(FUM1),苹果酸酶(tMDH3),丙酮酸羧化酶(PYC2),富马酸还原酶(FDR1); 4. 下调PFK1,Δpfk2; 5. 捕获胞质NADH进入呼吸链:表达NADH脱氢酶(NDE1,NDE2); 6. 表达脂肪酸合成途径:脂肪酸合成酶(FAS),硫酯酶(TESA),乙酰CoA羧化酶(ACC1); 7. Δfaa1,Δfaa4,Δpox1; 8. 过表达 PPP 途径:6-磷酸葡萄糖脱氢酶(ZWF1),磷酸葡糖酸脱氢酶 (GND1),转酮酶(TKL1),转醛酶(TAL1); 9.利用不同启动子表达不同来源的果糖1,6-二磷酸酶(FBP); 9. 表达NOG途径:磷酸转酮酶(XFPK),磷脂酰转移酶(PTA)	1. 利用合成的能量系统代替TCA 进行能量供应,用于脂肪酸生成; 2.产率:0.134 g/g 葡萄糖,40%的产率为已知报道的最高;	[57]
脂肪酸及其衍生物	斯达油脂酵母 (Lipomyces starkeyi), 解脂耶氏酵母 (Yarrowia lipolytica)	葡萄糖,木糖	无机盐基础培养基	脂肪醇	脂肪酸合成途径	1. 表达脂肪酰辅酶A还原酶 (FAR,Marinobactor aquaeolei VT8)	1. 十六烷醇(C16:0)和十八醇(C18:0)占主导地位; 2. 不同底物所得的脂肪醇产量不同;	[54]

生物燃料	宿主	主要底物	发酵培养基	产物和产量	主要途径	改造策略及相关基因	备注	参考文献
生物高级醇、酮,短链酸类物质	大肠杆菌	甲醇	木糖, 核糖	乙醇(4.6 g/L); 1-丁醇(2 g/L)	RuMP, 逆β氧化途径	1.表达RuMP相关酶; 2.ΔAdhE(甲醛脱氢酶),Δald(乙醛脱氢酶),ΔrpiAB(核糖磷酸异构酶); 3.表达腺苷酸环化酶; 4.表达丙酮酸脱羧酶(PDC),乙醛脱氢酶(ADH); 5.表达丁醇合成途径	1.构建甲醇依赖型木糖菌株; 2.甲醇与木糖利用率为1:1; 3.RuMP和逆β氧化途径共同作用;	[131]
	大肠杆菌	甲醇,甲醛	葡萄糖, 硫酸素焦磷酸盐	1,3-丙二醇(508.3± 9.1 mg/L)	一磷酸核酮糖途径(RuMP),酮酸途径	1.表达甲醇脱氢酶; 2.ΔfrmA(甲醛脱氢酶); 3.表达羟丁酸醛缩酶,酮酸脱羧酶,1,3-丁二酸氧化还原酶	1.首次实现利用甲醇和丙酮酸合成1,3-丙二醇; 2.缩短途径,并有效提高1,3-丙二醇产量;	[128]
	大肠杆菌	甲醇	葡萄糖, 酵母提取物	丙酮 (13 mmol/L)	RuMP, 酮酸途径	1.Δpgi(6-磷酸葡萄糖异构酶),Δedd(磷酸葡萄糖酸脱氢酶),ΔrpiAB(核糖磷酸异构酶),ΔfrmA(甲醛脱氢酶); 2.表达RuMP相关酶; 3.表达丙酮生成途径(Clostridium acetobutylicum)	1.显著提升甲醇向丙酮的转化; 2.构建了甲醇依赖的菌株底盘;	[132]
生物高级醇、酮,短链酸类物质	大肠杆菌	甲醇	葡萄糖, 酵母提取物	丙酮(45.0±8.7 mM)	RuMP, 酮酸途径	1.Δpgi(6-磷酸葡萄糖异构酶)、ΔfrmA(甲醛脱氢酶); 2.表达RuMP途径相关酶; 3.表达磷酸核糖异构酶(RPE),转酮酶(TKT); 4.表达二磷酸果糖醛缩酶(FBA),景天庚糖双磷酸酶(GLPX),磷酸果糖激酶(PFK); 5.表达丙酮生成途径(C.acetobutylicum):硫解酶(THL),辅酶A转移酶,(CTFAB),乙酰乙酸脱羧酶(ADC)	1.两种策略共同提高甲醇利用率; 2.大肠杆菌利用甲醇合成丙酮;	[133]
生物高级醇、酮,短链酸类物质	扭脱甲基杆菌AM1(Methylobacterium extorquens)	甲醇	无机盐基础培养基	异丁醇 (19 mg/L)	丝氨酸循环,EMC途径,酮酸途径	1. ΔldhA; 2. 表达2-酮异戊酸脱羧酶(Lactococcus lactis),醇脱氢酶(Lactococcus lactis),乙酰乳酸合酶(Bacillus subtilis)	1.摇瓶培养;	[119]
酮,短链酸类物质	扭脱甲基杆菌AM1(Methylobacterium extorquens)	甲醇	无机盐基础培养基	1-丁醇 (25.5 mg/L)	丝氨酸循环,EMC途径,酮酸途径	1.表达烯酰辅酶A还原酶(Treponema denticola),乙醇脱氢酶(Clostridium acetobutylicum),巴豆酸酶(Methylobacterium extorquens AM1)	1.适应性进化筛选突变株耐受丁醇达到0.5%; 2.摇瓶培养;	[120]
	扭脱甲基杆菌AM1(Methylobacterium extorquens)	甲醇	无机盐基础培养基	3-羟基丙酸 (0.857 g/L)	RuMP途径,EMC途径	1.ΔhprA; 2.表达己糖磷酸合成酶(Bacillus methanolicus),磷酸己糖异构酶(Bacillus methanolicus),磷酸果糖激酶(Bacillus methanolicus),6-磷酸葡萄糖脱氢酶(Bacillus methanolicus); 3.丙二酰辅酶A还原酶(Chlorofexus aurantiacus)	1. 5L-生物反应器;	[124]
	富养罗尔斯通氏菌H16	CO₂	果糖,无机盐基础培养基	高级醇混合物(140 mg/L)	酮酸途径	1.表达α-乙酰乳酸合酶(ALSS,Bacillus subtilis),酮醇酸还原异构酶(ILVC),二羟基酸脱氢酶(ILVD); 2.敲除PHB合成基因:ΔphaB,ΔphaC	1.电催化产生甲酸,甲酸经由微生物转化为异丁醇或3-甲基-1-丁醇; 2.以缬氨酸和亮氨酸合成途径为基础;	[84]
生物高级醇、酮,短链酸类物质	富养罗尔斯通氏菌Re2133(Cupriavidus necator)	H₂,O₂,CO₂,N₂	果糖,无机盐基础培养基	异丙醇 (3.5 g/L)	酮酸途径	1.异丙醇产生菌株^[106]	1.多气体供给的加压生物反应器; 2.首次报道工程化自养菌利用CO₂产生克级别的化合物; 3.70%～80%的CO₂被回收;	[134]
	杨氏梭菌(Clostridium ljungdahlii)	CO₂,H₂		丁醇 (109 mg/L); 己醇 (393 mg/L)	还原型乙酰CoA途径,酮酸途径	1.表达硫解酶(THLA),羟基丁基CoA脱氢酶(HBD),巴豆酸酶(CRT),丁基CoA脱氢酶(BCD), 2.表达电子转移蛋白(ETF),醛醇脱氢酶(ADHE)	1.微生物可以利用CO和CO₂作为碳源; 2.CO₂和H₂作为碳源; 3. 2 L生物反应器;	[135]
	富养罗尔斯通氏菌(Ralstonia eutropha)	CO₂, H₂O	无机盐基础培养基	异丙醇 (600 mg/L);异丁醇+ 3-甲基-1-丁醇(220 mg/L)	酮酸途径	1.异丙醇产生菌株	1.CO₂在水电混合反应装置中转化为H₂,微生物利用H₂产生异丁醇等生物燃料; 2.CO₂还原效率达到10%,超过自然光合效率;	[85]
脂肪酸及其衍生物	酿酒酵母	CO₂	无机盐基础培养基,乙酸,	脂肪酸 (500 mg/L)	脂肪酸合成途径	1.Δfaa1,Δfaa4,Δpox1; 2.表达硫酯酶(TESA),酰基CoA羧化酶(ACC1),脂肪酸合成酶(FAS)	1.电催化和生物系统结合:CO₂经电催化合成乙酸,酿酒酵母利用乙酸合成长链化合物;	[118]
	毕赤酵母	甲醇	无机盐培养基	脂肪酸(23.4g/L);脂肪醇(2.0 g/L)	脂肪酸合成途径	1.Δfaa1,Δfaa4,Δpox1; 2.加强甲醇利用途径:过表达二酰丙酮磷酸合酶(DAS):3.增加乙酰CoA前体供应:过表达酰基磷酸转移酶(PTA),磷酸转酮酶(XFPK); 4.加强NADPH再生	1.成功利用甲醇作为唯一碳源合成脂肪酸; 2. 1 L生物反应器;	[122]
	富养罗尔斯通氏菌 (Ralstonia eutroph)	H₂,CO₂,O₂	果糖, 无机盐基础培养基	脂肪酸(124.48 mg/g果糖) (60.64 mg/g CO₂)	脂肪酸合成途径	1.ΔphaC; 2.表达脂肪酸合成酶(FAS),硫酯酶(TESA),乙酰CoA羧化酶(ACC1); 3.ACP合成酶(ACPS)	1.结合多气体生物反应器,自养菌利用CO₂生成脂肪酸;	[136]
脂肪酸及其衍生物	汉逊酵母(Ogataeapolym orpha)	甲醇	无机盐培养基	脂肪酸 (15.9 g/L)	脂肪酸合成途径	1.Δfaa1; 2.加强前体供应及辅因子供应:过表达果糖-1,6-二磷酸酶(FBP),磷酸核糖异构酶(RPE),柠檬酸裂解酶(ACL),异柠檬酸脱氢酶(ICL1),果糖6-磷酸脱氢酶(ZWF1);	1.适应性进化使得敲除细胞生长恢复,并解析机制是由于LPL1和IZH3缺失引起; 2. 1 L生物反应器	[137]
	蓝藻 (Synechocystis sp. PCC 6803)	CO₂		脂肪酸甲酯 (120 mg/L)		1.Δaas; 2.过表达硫酯酶(UcFatB1),O-甲基转移酶(DmJHAMT); 3.引入S-腺苷甲硫氨酸(SAM)循环供应甲基;	1.不利用甲醇作为甲基供体,利用SAM合成酶供应甲基; 2.可能产生的脂肪酸甲酯类物质:C12:0,C14:0,C16:0	[138]
	解脂耶氏酵母(Yarrowia lipolytica)	CO₂	酵母合成培养基,酵母提取物	脂肪酸 (10.7 g/L)		1.增强脂肪酸合成:过表达生物素羧化酶(BC); 2.引入CO₂利用途径:过表达碳酸酐酶(CA);	1.循环利用CO₂生产脂肪酸; 2. 250 ml摇瓶发酵	[139]
萜类物质	扭脱甲基杆菌AM1(Methylobacteriumextorquens)	甲醇	无机盐基础培养基	甲羟戊酸 (2.22 g/L)	丝氨酸循环,EMC途径,类异戊二烯途径	1.表达HMG-CoA合成酶(Enterococcus faecalistiters),HMG-CoA还原酶(Enterococcus faecalistiters),乙酰乙酰CoA硫解酶(Ralstonia eutropha)	1.产率:28.4 mg/g甲醇; 2. 5 L-生物反应器	[121]
	类黄色噬氢菌DSM1 084 (Hydrogenophaga pseudoflava)	CO₂, 合成气	醋酸盐, 果糖, 蔗糖等, 无机盐基础培养基	α-红没药烯 (59.0±7.9 μg)	卡尔文循环,Wood-Ljungdahl途径(WL),类异戊二烯途径	1. 表达没药烯合成酶( TPS, Abies grandis)	1.自养和异养条件皆可生长:自养条件下利用合成气作为碳源,异养条件下可以利用果糖、蔗糖等作为碳源; 2.自养条件利用卡尔文循环和WL途径,异养条件利用MEP途径;	[140]
	富养罗尔斯通氏菌 (Cupriavidus necator)	CO₂,H₂,O₂	果糖,无机盐基础培养基	α-蛇麻烯(10.8±2.5 mg/g DCW or 17 mg/g DCW)	类异戊二烯途径	1.表达MVA途径:焦磷酸法尼合成酶(ERG20),IPP异构酶,α-蛇麻烯合酶(ZSSI)	1.化学自养和电自养均可:化能自养主要利用CO₂等,电自养需要电极和水辅助;	[141]
萜类物质	嗜甲烷菌20Z(Methylomicrobium alcaliphilum)	50%甲烷	硝酸矿物盐培养基(NMS)	α-蛇麻烯 (0.75 mg/g DCW, 835 μg/L)	类异戊二烯途径	1.表达α-蛇麻烯合成酶(ZSS1); 2.表达1-脱氧木酮糖-5-磷酸合酶(DXS),HMBPP合成酶(ISPG),FPP合成酶(ISPA); 3.Δpgi; 4.提高NADPH:表达转氢酶(PNTAB),葡萄糖6-磷酸脱氢酶(ZWF),磷酸葡糖酸脱氢酶(PGD)	1.优化MEP途径;	[125]

生物燃料	宿主	主要底物	发酵培养基	产物和产量	主要途径	改造策略及相关基因	备注	参考文献
生物高级醇、酮,短链酸类物质	大肠杆菌	甲醇	木糖, 核糖	乙醇(4.6 g/L); 1-丁醇(2 g/L)	RuMP, 逆β氧化途径	1.表达RuMP相关酶; 2.ΔAdhE(甲醛脱氢酶),Δald(乙醛脱氢酶),ΔrpiAB(核糖磷酸异构酶); 3.表达腺苷酸环化酶; 4.表达丙酮酸脱羧酶(PDC),乙醛脱氢酶(ADH); 5.表达丁醇合成途径	1.构建甲醇依赖型木糖菌株; 2.甲醇与木糖利用率为1:1; 3.RuMP和逆β氧化途径共同作用;	[131]
	大肠杆菌	甲醇,甲醛	葡萄糖, 硫酸素焦磷酸盐	1,3-丙二醇(508.3± 9.1 mg/L)	一磷酸核酮糖途径(RuMP),酮酸途径	1.表达甲醇脱氢酶; 2.ΔfrmA(甲醛脱氢酶); 3.表达羟丁酸醛缩酶,酮酸脱羧酶,1,3-丁二酸氧化还原酶	1.首次实现利用甲醇和丙酮酸合成1,3-丙二醇; 2.缩短途径,并有效提高1,3-丙二醇产量;	[128]
	大肠杆菌	甲醇	葡萄糖, 酵母提取物	丙酮 (13 mmol/L)	RuMP, 酮酸途径	1.Δpgi(6-磷酸葡萄糖异构酶),Δedd(磷酸葡萄糖酸脱氢酶),ΔrpiAB(核糖磷酸异构酶),ΔfrmA(甲醛脱氢酶); 2.表达RuMP相关酶; 3.表达丙酮生成途径(Clostridium acetobutylicum)	1.显著提升甲醇向丙酮的转化; 2.构建了甲醇依赖的菌株底盘;	[132]
生物高级醇、酮,短链酸类物质	大肠杆菌	甲醇	葡萄糖, 酵母提取物	丙酮(45.0±8.7 mM)	RuMP, 酮酸途径	1.Δpgi(6-磷酸葡萄糖异构酶)、ΔfrmA(甲醛脱氢酶); 2.表达RuMP途径相关酶; 3.表达磷酸核糖异构酶(RPE),转酮酶(TKT); 4.表达二磷酸果糖醛缩酶(FBA),景天庚糖双磷酸酶(GLPX),磷酸果糖激酶(PFK); 5.表达丙酮生成途径(C.acetobutylicum):硫解酶(THL),辅酶A转移酶,(CTFAB),乙酰乙酸脱羧酶(ADC)	1.两种策略共同提高甲醇利用率; 2.大肠杆菌利用甲醇合成丙酮;	[133]
生物高级醇、酮,短链酸类物质	扭脱甲基杆菌AM1(Methylobacterium extorquens)	甲醇	无机盐基础培养基	异丁醇 (19 mg/L)	丝氨酸循环,EMC途径,酮酸途径	1. ΔldhA; 2. 表达2-酮异戊酸脱羧酶(Lactococcus lactis),醇脱氢酶(Lactococcus lactis),乙酰乳酸合酶(Bacillus subtilis)	1.摇瓶培养;	[119]
酮,短链酸类物质	扭脱甲基杆菌AM1(Methylobacterium extorquens)	甲醇	无机盐基础培养基	1-丁醇 (25.5 mg/L)	丝氨酸循环,EMC途径,酮酸途径	1.表达烯酰辅酶A还原酶(Treponema denticola),乙醇脱氢酶(Clostridium acetobutylicum),巴豆酸酶(Methylobacterium extorquens AM1)	1.适应性进化筛选突变株耐受丁醇达到0.5%; 2.摇瓶培养;	[120]
	扭脱甲基杆菌AM1(Methylobacterium extorquens)	甲醇	无机盐基础培养基	3-羟基丙酸 (0.857 g/L)	RuMP途径,EMC途径	1.ΔhprA; 2.表达己糖磷酸合成酶(Bacillus methanolicus),磷酸己糖异构酶(Bacillus methanolicus),磷酸果糖激酶(Bacillus methanolicus),6-磷酸葡萄糖脱氢酶(Bacillus methanolicus); 3.丙二酰辅酶A还原酶(Chlorofexus aurantiacus)	1. 5L-生物反应器;	[124]
	富养罗尔斯通氏菌H16	CO₂	果糖,无机盐基础培养基	高级醇混合物(140 mg/L)	酮酸途径	1.表达α-乙酰乳酸合酶(ALSS,Bacillus subtilis),酮醇酸还原异构酶(ILVC),二羟基酸脱氢酶(ILVD); 2.敲除PHB合成基因:ΔphaB,ΔphaC	1.电催化产生甲酸,甲酸经由微生物转化为异丁醇或3-甲基-1-丁醇; 2.以缬氨酸和亮氨酸合成途径为基础;	[84]
生物高级醇、酮,短链酸类物质	富养罗尔斯通氏菌Re2133(Cupriavidus necator)	H₂,O₂,CO₂,N₂	果糖,无机盐基础培养基	异丙醇 (3.5 g/L)	酮酸途径	1.异丙醇产生菌株^[106]	1.多气体供给的加压生物反应器; 2.首次报道工程化自养菌利用CO₂产生克级别的化合物; 3.70%～80%的CO₂被回收;	[134]
	杨氏梭菌(Clostridium ljungdahlii)	CO₂,H₂		丁醇 (109 mg/L); 己醇 (393 mg/L)	还原型乙酰CoA途径,酮酸途径	1.表达硫解酶(THLA),羟基丁基CoA脱氢酶(HBD),巴豆酸酶(CRT),丁基CoA脱氢酶(BCD), 2.表达电子转移蛋白(ETF),醛醇脱氢酶(ADHE)	1.微生物可以利用CO和CO₂作为碳源; 2.CO₂和H₂作为碳源; 3. 2 L生物反应器;	[135]
	富养罗尔斯通氏菌(Ralstonia eutropha)	CO₂, H₂O	无机盐基础培养基	异丙醇 (600 mg/L);异丁醇+ 3-甲基-1-丁醇(220 mg/L)	酮酸途径	1.异丙醇产生菌株	1.CO₂在水电混合反应装置中转化为H₂,微生物利用H₂产生异丁醇等生物燃料; 2.CO₂还原效率达到10%,超过自然光合效率;	[85]
脂肪酸及其衍生物	酿酒酵母	CO₂	无机盐基础培养基,乙酸,	脂肪酸 (500 mg/L)	脂肪酸合成途径	1.Δfaa1,Δfaa4,Δpox1; 2.表达硫酯酶(TESA),酰基CoA羧化酶(ACC1),脂肪酸合成酶(FAS)	1.电催化和生物系统结合:CO₂经电催化合成乙酸,酿酒酵母利用乙酸合成长链化合物;	[118]
	毕赤酵母	甲醇	无机盐培养基	脂肪酸(23.4g/L);脂肪醇(2.0 g/L)	脂肪酸合成途径	1.Δfaa1,Δfaa4,Δpox1; 2.加强甲醇利用途径:过表达二酰丙酮磷酸合酶(DAS):3.增加乙酰CoA前体供应:过表达酰基磷酸转移酶(PTA),磷酸转酮酶(XFPK); 4.加强NADPH再生	1.成功利用甲醇作为唯一碳源合成脂肪酸; 2. 1 L生物反应器;	[122]
	富养罗尔斯通氏菌 (Ralstonia eutroph)	H₂,CO₂,O₂	果糖, 无机盐基础培养基	脂肪酸(124.48 mg/g果糖) (60.64 mg/g CO₂)	脂肪酸合成途径	1.ΔphaC; 2.表达脂肪酸合成酶(FAS),硫酯酶(TESA),乙酰CoA羧化酶(ACC1); 3.ACP合成酶(ACPS)	1.结合多气体生物反应器,自养菌利用CO₂生成脂肪酸;	[136]
脂肪酸及其衍生物	汉逊酵母(Ogataeapolym orpha)	甲醇	无机盐培养基	脂肪酸 (15.9 g/L)	脂肪酸合成途径	1.Δfaa1; 2.加强前体供应及辅因子供应:过表达果糖-1,6-二磷酸酶(FBP),磷酸核糖异构酶(RPE),柠檬酸裂解酶(ACL),异柠檬酸脱氢酶(ICL1),果糖6-磷酸脱氢酶(ZWF1);	1.适应性进化使得敲除细胞生长恢复,并解析机制是由于LPL1和IZH3缺失引起; 2. 1 L生物反应器	[137]
	蓝藻 (Synechocystis sp. PCC 6803)	CO₂		脂肪酸甲酯 (120 mg/L)		1.Δaas; 2.过表达硫酯酶(UcFatB1),O-甲基转移酶(DmJHAMT); 3.引入S-腺苷甲硫氨酸(SAM)循环供应甲基;	1.不利用甲醇作为甲基供体,利用SAM合成酶供应甲基; 2.可能产生的脂肪酸甲酯类物质:C12:0,C14:0,C16:0	[138]
	解脂耶氏酵母(Yarrowia lipolytica)	CO₂	酵母合成培养基,酵母提取物	脂肪酸 (10.7 g/L)		1.增强脂肪酸合成:过表达生物素羧化酶(BC); 2.引入CO₂利用途径:过表达碳酸酐酶(CA);	1.循环利用CO₂生产脂肪酸; 2. 250 ml摇瓶发酵	[139]
萜类物质	扭脱甲基杆菌AM1(Methylobacteriumextorquens)	甲醇	无机盐基础培养基	甲羟戊酸 (2.22 g/L)	丝氨酸循环,EMC途径,类异戊二烯途径	1.表达HMG-CoA合成酶(Enterococcus faecalistiters),HMG-CoA还原酶(Enterococcus faecalistiters),乙酰乙酰CoA硫解酶(Ralstonia eutropha)	1.产率:28.4 mg/g甲醇; 2. 5 L-生物反应器	[121]
	类黄色噬氢菌DSM1 084 (Hydrogenophaga pseudoflava)	CO₂, 合成气	醋酸盐, 果糖, 蔗糖等, 无机盐基础培养基	α-红没药烯 (59.0±7.9 μg)	卡尔文循环,Wood-Ljungdahl途径(WL),类异戊二烯途径	1. 表达没药烯合成酶( TPS, Abies grandis)	1.自养和异养条件皆可生长:自养条件下利用合成气作为碳源,异养条件下可以利用果糖、蔗糖等作为碳源; 2.自养条件利用卡尔文循环和WL途径,异养条件利用MEP途径;	[140]
	富养罗尔斯通氏菌 (Cupriavidus necator)	CO₂,H₂,O₂	果糖,无机盐基础培养基	α-蛇麻烯(10.8±2.5 mg/g DCW or 17 mg/g DCW)	类异戊二烯途径	1.表达MVA途径:焦磷酸法尼合成酶(ERG20),IPP异构酶,α-蛇麻烯合酶(ZSSI)	1.化学自养和电自养均可:化能自养主要利用CO₂等,电自养需要电极和水辅助;	[141]
萜类物质	嗜甲烷菌20Z(Methylomicrobium alcaliphilum)	50%甲烷	硝酸矿物盐培养基(NMS)	α-蛇麻烯 (0.75 mg/g DCW, 835 μg/L)	类异戊二烯途径	1.表达α-蛇麻烯合成酶(ZSS1); 2.表达1-脱氧木酮糖-5-磷酸合酶(DXS),HMBPP合成酶(ISPG),FPP合成酶(ISPA); 3.Δpgi; 4.提高NADPH:表达转氢酶(PNTAB),葡萄糖6-磷酸脱氢酶(ZWF),磷酸葡糖酸脱氢酶(PGD)	1.优化MEP途径;	[125]

途径	前体物质	主要中间物	关键酶	合成产物	主要应用	备注	参考文献
酮酸途径	丙酮酸	2-酮酸	2-酮酸脱羧酶,醇脱氢酶	1-丙醇,异丁醇,1-丁醇, 2-甲基-1-丁醇,3-甲基-1-丁醇,2-苯乙醇	生物汽油	1.丙酮酸经过氨基酸生物合成途径转化为不同长度碳链的2-酮酸; 2. 2-酮酸经过延伸酶、脱羧酶、水解酶形成终产物;	[65]
类异戊二烯途径	乙酰CoA, 3-磷酸甘油醛(G3P),丙酮酸	异戊烯焦磷酸(IPP), 二甲基丙烯焦磷酸酯(DMAPP), 香叶基焦磷酸酯(GPP), 法呢基焦磷酸酯(FPP)	萜烯合成酶	异戊醇,异戊烯醇,3-甲基- 2-丁烯醇,松萜,柠烯,红没药烯,法呢烯	生物汽油,航空用油,发动机燃料	1.乙酰CoA经MVA途径合成IPP 和DMAPP; 2.G3P和丙酮酸经MEP途径合成IPP和DMAPP	[58]
逆β氧化途径	乙酰CoA,CoA	酰基-CoA	酰基转移酶	1-丁醇,丁酸,异丙醇, 1-乙醇,1-辛醇	生物汽油,生物柴油,航空燃料	1.CoA直接作为供体; 2.不同类型的硫酯酶可形成不同类型的化合物;	[47]
脂肪酸生物合成途径	乙酰CoA, 酰基载体蛋白(ACP)	酰基-ACP	脂肪酸合成酶,硫酯酶,还原酶	脂肪酸,脂肪醇,脂肪酸甲酯,脂肪酸乙酯,烷烃	生物汽油,生物柴油	1.CoA供体为ACP;	[98]
聚酮生物合成途径	乙酰CoA, 酰基载体蛋白(ACP)	β-酮酰基-ACP	聚酮合成酶	1-丁烯,1-己烯,1-己醇, 乙基酮,甲基酮,支链醇	生物汽油,航空用油,发动机燃料	1.CoA供体为ACP;	[72]

Advances in microbial production of liquid biofuels

液体生物燃料合成与炼制的研究进展

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