• 特约评述 •
邵明威1, 孙思勉1, 杨时茂1, 陈国强1,2,3
收稿日期:
2024-02-04
修回日期:
2024-04-27
出版日期:
2024-04-28
通讯作者:
陈国强
作者简介:
基金资助:
Mingwei SHAO1, Simian SUN1, Shimao YANG1, Guo-Qiang CHEN1,2,3
Received:
2024-02-04
Revised:
2024-04-27
Online:
2024-04-28
Contact:
Guo-Qiang CHEN
摘要:
以微生物或酶为基础的生物制造,正以其绿色、环保、可持续等优势逐渐替代以化石燃料为原料的传统化工生产模式。然而,传统工业生物技术在易染菌、设备复杂、难以连续发酵等劣势。相较而言,“下一代工业生物技术(NGIB)”利用以嗜盐菌、嗜热菌和嗜酸碱菌等极端微生物作为底盘细胞,使用廉价底物生产多种高附加值产品,具有开放、无需灭菌、连续发酵等优点。本文介绍了嗜盐菌、嗜热菌和嗜酸碱菌极端微生物的定义以及在高盐、高温极度酸碱等极端环境下快速生长的特性。随后总结了目前极端微生物的基因工程手段例如启动子工程、以CRISPR为代表的基因编辑技术、命运共同体策略、稳定质粒载体等,代谢工程手段例如增加碳源前体、敲除旁路代谢、减少副产物、提高转运等,以及极端微生物生产的多种产品例如PHA、蛋白质、氨基酸及小分子衍生物等。同时概况了目前在极端微生物底盘细胞改造过程中仍存在的问题,如缺乏多种优秀的质粒载体、质粒转化效率低、缺乏高效基因编辑技术以及其他非嗜盐菌生长发酵周期较长等,并提出了相应的解决策略。最后展望了如何充分利用不同类型极端微生物的特性生产优势产品,推动下一代工业生物技术的发展与完善,实现绿色、环保、可持续的生物制造。
中图分类号:
邵明威, 孙思勉, 杨时茂, 陈国强. 基于极端微生物的生物制造[J]. 合成生物学, DOI: 10.12211/2096-8280.2024-016.
Mingwei SHAO, Simian SUN, Shimao YANG, Guo-Qiang CHEN. Bioproduction based on extremophiles[J]. Synthetic Biology Journal, DOI: 10.12211/2096-8280.2024-016.
图1 基于以盐单胞菌为代表的极端微生物下一代工业生物技术示意图[12](使用重组盐单胞菌作为底盘细胞,在塑料或其他低成本生物反应器中利用海水和廉价的底物进行开放、无需灭菌和连续发酵,生产各种类型的PHA、蛋白质和胞外小分子产物。在塑料或其他低成本生物反应器中进行的开放、无需灭菌和连续发酵,可以在海水中低底物上生长的重组卤单胞菌生产细胞内 PHA 产物或蛋白质和细胞外小分子)
Fig. 1 Next generation industrial biotechnology based on extremophiles represented by Halomonas spp.(Production of intracellular PHA products or proteins and extracelullar small molecules by recombinant Halomonas grown on low-cost substrates in seawater under open unsterile and continuous processes conducted in plastic or other low cost bioreactors)
图2 极端微生物的基因工程与代谢工程[88](A)基因表达调控元件和工具。基因元件包括启动子、绝缘子、核糖体结合位点和终止子。基因调控包括静态调控例如旁路敲除,通路过表达以及动态调控例如化学诱导系统,生物传感器;(B)基因编辑技术包括同源重组、TALENs、CRISPR/Cas9和特异位点重组;(C)代谢工程包括形态学工程、膜工程、调控NAD(P)+/NAD(P)H比例、表达血红蛋白;(D)基因组挖掘。利用组学数据构建代谢模型预测新的元件、酶和通路。
Fig. 2 Genetic and metabolic engineering of extremophiles.(A) Gene expression regulatory elements and tools. Genetic elements include promoters, insulators, ribosome binding sites, and terminators. Gene regulation includes static regulation such as bypass knockout, pathway overexpression, and dynamic regulation such as chemical-induction system, biosensors; (B) Gene editing tools include homologous recombination, TALENs, CRISPR/Cas9 and specific-site recombination; (C) Metabolic engineering includes morphological engineering, membrane engineering, regulation of NAD(P)+/NAD(P)H ratio, and expression of hemoglobin; (D) Genome mining. The applications of omics data to build metabolic models for prediction of new elements, enzymes, and pathways.
类型 | 生长环境 | 代表微生物 | 生产应用 | 参考 文献 |
---|---|---|---|---|
嗜盐菌 | 高盐高碱 (NaCl>30 g/l, pH 8-10) | 盐单胞菌:H. bluephagenesis TD01,H. campaniensis LS21,H. smyrnensis AAD6 | 生产PHA(以H. bluephagenesis为例:细胞干重大于80 g/L,PHB百分比大于90 wt%),四氢嘧啶(以H. bluephagenesis为例,生产滴度达85 g/L),生产速率达1 g/L/h)等 | [ |
嗜热菌 | 高温(>45 ℃) | 嗜热细菌:Fervidobacterium thermophilum, Thermoanaerobacterium saccharolyticum | 生产生物燃料(以Thermoanaerobacter sp.X514为例,正丁醇生产滴度达357 mg/L,生产速率达2.975 g/L/h),分离热稳定酶(纤维素酶,角质溶解酶)等 | [ |
嗜酸/碱菌 | 极酸或极碱 (PH<3,PH>10) | 嗜酸性铁硫杆菌Acidithiobacillus ferrooxidans, 嗜碱细菌Clostridium alkalicellulosi | 生产有机酸(以Issatchenkia orientalis SD108为例,琥珀酸生产滴度达到11.63 g/L) | [ |
表1 各种类型极端微生物的特性与生产应用
Table 1 Characteristics and production applications of various types of extremophiles
类型 | 生长环境 | 代表微生物 | 生产应用 | 参考 文献 |
---|---|---|---|---|
嗜盐菌 | 高盐高碱 (NaCl>30 g/l, pH 8-10) | 盐单胞菌:H. bluephagenesis TD01,H. campaniensis LS21,H. smyrnensis AAD6 | 生产PHA(以H. bluephagenesis为例:细胞干重大于80 g/L,PHB百分比大于90 wt%),四氢嘧啶(以H. bluephagenesis为例,生产滴度达85 g/L),生产速率达1 g/L/h)等 | [ |
嗜热菌 | 高温(>45 ℃) | 嗜热细菌:Fervidobacterium thermophilum, Thermoanaerobacterium saccharolyticum | 生产生物燃料(以Thermoanaerobacter sp.X514为例,正丁醇生产滴度达357 mg/L,生产速率达2.975 g/L/h),分离热稳定酶(纤维素酶,角质溶解酶)等 | [ |
嗜酸/碱菌 | 极酸或极碱 (PH<3,PH>10) | 嗜酸性铁硫杆菌Acidithiobacillus ferrooxidans, 嗜碱细菌Clostridium alkalicellulosi | 生产有机酸(以Issatchenkia orientalis SD108为例,琥珀酸生产滴度达到11.63 g/L) | [ |
工程化改造思路 | 具体策略 | 参考文献 |
---|---|---|
基因表达元件工程 | porin启动子,类T7表达系统,响应十种小分子诱导物的多重诱导系统 | [ |
基因编辑技术 | CRISPR/Cas9系统,CRISPRi系统,CRISPR/AID系统,sRNA调控系统 | [ |
生物传感器与动态调控 | 感知油酸、群体感应信号分子AHL调控系统,荧光定量PHA含量(qPHA) | [ |
命运共同体策略 | 将PHA合成基因插入必须基因ompW启动子后共表达 | [ |
形态学工程 | 抑制细胞骨架基因MreB,ftsZ的表达 | [ |
稳定质粒表达载体 | 基于盐单胞菌内源性质粒中的hbpB/hbpC毒素-抗毒素系统 | [ |
表2 盐单胞菌的基因工程改造技术
Tab. 2 Genetic engineering of Halomonas spp.
工程化改造思路 | 具体策略 | 参考文献 |
---|---|---|
基因表达元件工程 | porin启动子,类T7表达系统,响应十种小分子诱导物的多重诱导系统 | [ |
基因编辑技术 | CRISPR/Cas9系统,CRISPRi系统,CRISPR/AID系统,sRNA调控系统 | [ |
生物传感器与动态调控 | 感知油酸、群体感应信号分子AHL调控系统,荧光定量PHA含量(qPHA) | [ |
命运共同体策略 | 将PHA合成基因插入必须基因ompW启动子后共表达 | [ |
形态学工程 | 抑制细胞骨架基因MreB,ftsZ的表达 | [ |
稳定质粒表达载体 | 基于盐单胞菌内源性质粒中的hbpB/hbpC毒素-抗毒素系统 | [ |
产物 | 碳源/底物 | 改造策略 | 产量 | 文献 |
---|---|---|---|---|
PHB | 葡萄糖,淀粉水解物,厨余垃圾混合碳源 | 限氧,限氮;平衡NADH+/NAD比例,补充乙酸 | 细胞干重大于80 g/L,PHB百分比大于90 wt % | [ |
3-羟基丁酸与4-羟基丁酸共聚物 (P34HB) | 葡萄糖,葡萄糖酸盐废弃物,废弃玉米浆,γ-丁内酯 | 构建两条相互关联的4-羟基丁酸(4HB)生物合成途径,表达4-羟基丁酸转移酶orfZ;敲除琥珀酸半醛脱氢酶gadD;构建数学模型与理性计算辅助设计;敲除外膜相关基因lpxL和lpxM | 7L发酵罐产生26.3 g/L细胞干重,包含60.5wt%的 P(3HB-co-17.04mol%4HB);5L发酵罐中产生100 g/L细胞干重包含60.4wt%的P(3HB-co-13.5 mol %4HB);敲除外膜菌H. bluephagenesis WZY254在7L发酵罐产生84 g/L细胞干重包含含81wt%的P(3HB-co-26 mol %4HB) | [ |
3-羟基丁酸与3-羟基戊酸共聚物(PHBV) | 葡萄糖,葡萄糖酸钠 | 敲低或敲除2-甲基柠檬酸合成酶prpC基因;敲除TCA循环相关基因sdhE和icl;在染色体上表达编码磷酸烯醇式丙酮酸羧化酶ppc基因 | 摇瓶中6.3 g/L细胞干重,包含65 wt %的PHBV,其中3HV摩尔比达到35% | [ |
3-羟基丁酸与3-羟基己酸共聚物(PHBHHx) | 葡萄糖,己酸钠 | 表达来自Aeromonas caviae FA440的 PHA合成酶PhaCac基因和烯酰辅酶- A水合酶PhaJac基因 | 7L发酵罐产生细胞干重33.1 g/L细胞干重,包含50.32wt%的P(3HB-co-37.23 mol % 3HHx),3HHx比例可以在0-37 mol%范围调控 | [ |
PHA颗粒相蛋白(PhaP) | 葡萄糖 | 敲除phaC基因,在基因组上过表达phaP基因 | PhaP累积量占比19%,产量为1.86 g/L | [ |
淀粉酶, 葡萄糖苷酶,PHAs, 小分子 | 淀粉 | 筛选合适的信号肽和连接子将过表达的α淀粉酶和葡萄糖苷酶分泌到胞外 | 以淀粉作为唯一碳源生产PHA、四氢嘧啶、苏氨酸等多种产品 | [ |
氨基酸 (L-苏氨酸,L-赖氨酸) | 葡萄糖 | 异源表达5个L-苏氨酸合成基因和外排转运蛋白,敲除内运转运蛋白和L-苏氨酸脱氢酶;过表达L-赖氨酸合成相关基因,解除底物抑制效应,增强L-赖氨酸外排能力 | 7L发酵罐生产33 g/L的苏氨酸;7升发酵罐生产22.59 g/L的赖氨酸 | [ |
戊二胺 | 赖氨酸 | 异源表达赖氨酸脱羧酶CadA, LdcC基因 | 7L发酵罐生产118 g/L的戊二胺 | [ |
四氢嘧啶 | 葡萄糖 | 理性调控和四氢嘧啶合成相关的ectABC,lysC和asd三个基因簇,提高前体供应,增强产物转运系统,优化培养条件 | 7L发酵罐生产85 g/L的四氢嘧啶 | ⋅ [ |
5-氨基戊酸 | 赖氨酸 | 敲除gabT基因,在基因组上表达三个拷贝的dvaBA基因 | 7L发酵罐生产67.4 g/L的5-氨基戊酸 | [ |
3-羟基丙酸 | 葡萄糖, 1,3-丙二醇 | 理性调控3-羟基丙酸合成相关的AldDHb和AdhP基因,敲除降解基因DddA | 7L发酵罐生产154 g/L的3-羟基丙酸 | [ |
乙偶姻 | 丙酮酸 | 异源表达枯草芽孢杆菌α-乙酰乳酸合酶alsS基因和α-乙酰乳酸脱羧酶 alsD基因 | 全细胞催化生产85.84 g/L的乙偶姻 | [ |
衣康酸 | 柠檬酸 | 表达顺乌头酸脱羧酶编码基因cadA和顺乌头酸酶编码基因acn;表达分子伴侣GroESL;增加编码限速酶acn基因的拷贝数以及弱化竞争途径 | 摇瓶中生产63.60 g/L的衣康酸 | [ |
甲羟戊酸 | 葡萄糖 | 敲除PhaB和PhaC基因;异源表达甲羟戊酸合成基因HMG-CoA合成酶和HMG-CoA-还原酶;CIRSPRi技术敲低50个候选基因;引入非氧糖酵解通路(NOG通路)减少碳损失 | 5L发酵罐中生产121 g/L的甲羟戊酸 | [ |
表3 基于盐单胞菌生产的各种产物
Table 3 Various products based on the production of Halomonas spp.
产物 | 碳源/底物 | 改造策略 | 产量 | 文献 |
---|---|---|---|---|
PHB | 葡萄糖,淀粉水解物,厨余垃圾混合碳源 | 限氧,限氮;平衡NADH+/NAD比例,补充乙酸 | 细胞干重大于80 g/L,PHB百分比大于90 wt % | [ |
3-羟基丁酸与4-羟基丁酸共聚物 (P34HB) | 葡萄糖,葡萄糖酸盐废弃物,废弃玉米浆,γ-丁内酯 | 构建两条相互关联的4-羟基丁酸(4HB)生物合成途径,表达4-羟基丁酸转移酶orfZ;敲除琥珀酸半醛脱氢酶gadD;构建数学模型与理性计算辅助设计;敲除外膜相关基因lpxL和lpxM | 7L发酵罐产生26.3 g/L细胞干重,包含60.5wt%的 P(3HB-co-17.04mol%4HB);5L发酵罐中产生100 g/L细胞干重包含60.4wt%的P(3HB-co-13.5 mol %4HB);敲除外膜菌H. bluephagenesis WZY254在7L发酵罐产生84 g/L细胞干重包含含81wt%的P(3HB-co-26 mol %4HB) | [ |
3-羟基丁酸与3-羟基戊酸共聚物(PHBV) | 葡萄糖,葡萄糖酸钠 | 敲低或敲除2-甲基柠檬酸合成酶prpC基因;敲除TCA循环相关基因sdhE和icl;在染色体上表达编码磷酸烯醇式丙酮酸羧化酶ppc基因 | 摇瓶中6.3 g/L细胞干重,包含65 wt %的PHBV,其中3HV摩尔比达到35% | [ |
3-羟基丁酸与3-羟基己酸共聚物(PHBHHx) | 葡萄糖,己酸钠 | 表达来自Aeromonas caviae FA440的 PHA合成酶PhaCac基因和烯酰辅酶- A水合酶PhaJac基因 | 7L发酵罐产生细胞干重33.1 g/L细胞干重,包含50.32wt%的P(3HB-co-37.23 mol % 3HHx),3HHx比例可以在0-37 mol%范围调控 | [ |
PHA颗粒相蛋白(PhaP) | 葡萄糖 | 敲除phaC基因,在基因组上过表达phaP基因 | PhaP累积量占比19%,产量为1.86 g/L | [ |
淀粉酶, 葡萄糖苷酶,PHAs, 小分子 | 淀粉 | 筛选合适的信号肽和连接子将过表达的α淀粉酶和葡萄糖苷酶分泌到胞外 | 以淀粉作为唯一碳源生产PHA、四氢嘧啶、苏氨酸等多种产品 | [ |
氨基酸 (L-苏氨酸,L-赖氨酸) | 葡萄糖 | 异源表达5个L-苏氨酸合成基因和外排转运蛋白,敲除内运转运蛋白和L-苏氨酸脱氢酶;过表达L-赖氨酸合成相关基因,解除底物抑制效应,增强L-赖氨酸外排能力 | 7L发酵罐生产33 g/L的苏氨酸;7升发酵罐生产22.59 g/L的赖氨酸 | [ |
戊二胺 | 赖氨酸 | 异源表达赖氨酸脱羧酶CadA, LdcC基因 | 7L发酵罐生产118 g/L的戊二胺 | [ |
四氢嘧啶 | 葡萄糖 | 理性调控和四氢嘧啶合成相关的ectABC,lysC和asd三个基因簇,提高前体供应,增强产物转运系统,优化培养条件 | 7L发酵罐生产85 g/L的四氢嘧啶 | ⋅ [ |
5-氨基戊酸 | 赖氨酸 | 敲除gabT基因,在基因组上表达三个拷贝的dvaBA基因 | 7L发酵罐生产67.4 g/L的5-氨基戊酸 | [ |
3-羟基丙酸 | 葡萄糖, 1,3-丙二醇 | 理性调控3-羟基丙酸合成相关的AldDHb和AdhP基因,敲除降解基因DddA | 7L发酵罐生产154 g/L的3-羟基丙酸 | [ |
乙偶姻 | 丙酮酸 | 异源表达枯草芽孢杆菌α-乙酰乳酸合酶alsS基因和α-乙酰乳酸脱羧酶 alsD基因 | 全细胞催化生产85.84 g/L的乙偶姻 | [ |
衣康酸 | 柠檬酸 | 表达顺乌头酸脱羧酶编码基因cadA和顺乌头酸酶编码基因acn;表达分子伴侣GroESL;增加编码限速酶acn基因的拷贝数以及弱化竞争途径 | 摇瓶中生产63.60 g/L的衣康酸 | [ |
甲羟戊酸 | 葡萄糖 | 敲除PhaB和PhaC基因;异源表达甲羟戊酸合成基因HMG-CoA合成酶和HMG-CoA-还原酶;CIRSPRi技术敲低50个候选基因;引入非氧糖酵解通路(NOG通路)减少碳损失 | 5L发酵罐中生产121 g/L的甲羟戊酸 | [ |
图3 利用盐单胞菌生产各类化合物[107]利用盐单胞菌目生产各种类型的化合物例如蛋白质,氨基酸及衍生物,有机酸。ALAS:5-氨基乙酰丙酸合酶;lysC:编码天冬氨酸酶;thrA*:编码来自大肠杆菌MG1655的G433R高丝氨酸脱氢酶突变体;thrB:编码高丝氨酸激酶;thrC:编码L-苏氨酸合酶;ectA:编码L-2,4-二氨基丁酸乙酰转移酶;ectB:编码L-2,4-丁二酸转氨酶;ectC:编码四氢嘧啶合酶;ACN:乌头酸酶;CAD:顺乌头酸脱羧酶;DhaTPp和AdhP:醇脱氢酶和醛脱氢酶;phaA:编码3-酮硫醇酶;mvaS:编码HMG-CoA合酶;mvaE:编码HMG-CoA还原酶。
Fig. 3 Diverse chemical compounds produced by Halomonas spp.H. bluephagenesis has been engineered to produce non-PHA chemicals including proteins, amino acids, and their derivates, organic acids. ALAS: 5-aminolevulinic acid synthase; lysC: gene of aspartokinase; thrA*: gene of homoserine dehydrogenase mutant at G433R from Escherichia coli MG1655; thrB: gene of homoserine kinase; thrC: gene of L-threonine synthase; ectA: encoding L-2,4-diaminobutyrate acetyltransferase; ectB: encoding L-2,4-diaminobutyrate transaminase; ectC: encoding ectoine synthase; ACN: aconitase; CAD: cis-aconitate decarboxylase; DhaTPp and AdhP: alcohol dehydrogenase and aldehyde dehydrogenase; phaA: encoding 3-ketothiolase; mvaS: encoding HMG-CoA synthase; mvaE: encoding HMG-CoA reductase.
类型 | 未来产品 | 优势 |
---|---|---|
嗜冷菌 | 蛋白质或酶 | 在胞内不容易形成包涵体 |
嗜热菌 | 挥发性小分子化合物 | 直接蒸馏提纯产品, 简化处理工艺 |
嗜酸菌 | 酸性化合物(例如有机酸) | 耐受高浓度酸性产物 |
嗜碱菌 | 碱性化合物(例如赖氨酸) | 耐受高浓度碱性产物 |
嗜盐菌 | 饲料蛋白与 酸性小分子化合物 | 开放式发酵,无需灭菌, 耐渗透压 |
表4 未来基于极端微生物生产的产品
Table 4 Products based on extremophiles in the future
类型 | 未来产品 | 优势 |
---|---|---|
嗜冷菌 | 蛋白质或酶 | 在胞内不容易形成包涵体 |
嗜热菌 | 挥发性小分子化合物 | 直接蒸馏提纯产品, 简化处理工艺 |
嗜酸菌 | 酸性化合物(例如有机酸) | 耐受高浓度酸性产物 |
嗜碱菌 | 碱性化合物(例如赖氨酸) | 耐受高浓度碱性产物 |
嗜盐菌 | 饲料蛋白与 酸性小分子化合物 | 开放式发酵,无需灭菌, 耐渗透压 |
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