巴氏梭菌作为工业底盘细胞高效生产1，3-丙二醇——从代谢工程和菌种进化到过程工程和产品分离

doi:10.12211/2096-8280.2024-030

合成生物学 ›› 2024, Vol. 5 ›› Issue (6): 1386-1403.DOI: 10.12211/2096-8280.2024-030

巴氏梭菌作为工业底盘细胞高效生产1，3-丙二醇——从代谢工程和菌种进化到过程工程和产品分离

刘建明¹^,²^,³^,⁴, 张炽坚⁵, 张冰¹^,²^,³, 曾安平¹^,²^,³^,⁴

^1.西湖大学合成生物学与生物智造中心，浙江杭州 310030
^2.西湖大学工学院，浙江杭州 310030
^3.浙江省全省智能低碳生物合成重点实验室，浙江杭州 310030
^4.西湖大学未来产业研究中心，浙江杭州 310030
^5.广东恒碳科技有限公司，广东广州 510630

收稿日期:2024-03-27 修回日期:2024-06-18 出版日期:2024-12-31 发布日期:2025-01-10
通讯作者: 曾安平
作者简介:刘建明（1985—），男，研究员。研究方向为合成生物学，低碳生物制造，代谢工程等。 E-mail：liujianming@westlake.edu.cn
曾安平（1963—），男，讲席教授，博士生导师，德国工程院院士，西湖大学合成生物学与生物智造中心创始主任。研究方向为生物化工、合成生物学、新型催化软物质和功能材料等。 E-mail：zenganping@westlake.edu.cn
基金资助:
国家重点研发计划(2023YFA09014003)

Clostridium pasteurianum as an industrial chassis for efficient production of 1,3-propanediol: from metabolic engineering to fermentation and product separation

Jianming LIU¹^,²^,³^,⁴, Chijian ZHANG⁵, Bing ZHANG¹^,²^,³, Anping ZENG¹^,²^,³^,⁴

^1.Center for Synthetic Biology and Biomanufacturing，Westlake University，Hangzhou 310030，Zhejiang，China
^2.Engineering institute，Westlake University，Hangzhou 310030，Zhejiang，China
^3.Zhejiang Key Laboratory of Low-Carbon Intelligent Synthetic Biology，Hangzhou 310030，Zhejiang，China
^4.Research Center for Industries of the Future，Westlake University，Hangzhou 310030，Zhejiang，China
^5.Guangdong C1 Life Biotech Co. ，Ltd. ，Guangzhou 510630，Guangdong，China

Received:2024-03-27 Revised:2024-06-18 Online:2024-12-31 Published:2025-01-10
Contact: Anping ZENG

摘要/Abstract

摘要：

1，3-丙二醇（PDO）是一种重要的化工原料，广泛应用于材料和化妆品等领域。生物制造PDO具有原料可再生性和环境友好性等众多优点和广阔的发展前景。由于巴氏梭菌（Clostridium pasteurianum）菌株安全、非致病、代谢甘油速率快、生长快、不依赖昂贵的培养基组分、天然具备高效生产PDO代谢途径等条件，利用和改造巴氏梭菌作为工业底盘生产PDO呈现出得天独厚的优势。本文首先回顾了PDO的生物制造现状和挑战，随后深入探讨了采用巴氏梭菌生产PDO的方法，特别关注于巴氏梭菌的甘油代谢机制、甘油发酵的策略和发酵过程的工艺设计。值得一提的是，本文作者研究团队筛选到的巴氏梭菌突变体和随之开发的鲁棒性发酵工艺在一定程度上突破了传统巴氏梭菌对环境的要求，特别是对铁离子浓度的敏感性；在电辅助甘油发酵过程中，PDO产量达到120.7 g/L，生产强度达到4.8 g/（L·h），收率达到理论值；并进一步阐述了巴氏梭菌基因工程改造方面的天然屏障，围绕着理性基因组改造和定向进化等几个方面进行了详细讨论；由于PDO产品纯度（>99.9%）通常有较高要求，因此开发高效的下游处理技术对于实现利用可再生资源发酵生产PDO的工业化应用至关重要，本文在分离工艺方面主要讨论了基于蒸发和蒸馏的PDO纯化技术以及基于萃取的PDO纯化技术。通过对代谢工程、菌种进化、发酵过程优化以及产品分离等多个维度全方位分析，全面地解析了巴氏梭菌生产PDO的特点和优势，以及巴氏梭菌作为新型工业底盘微生物在未来发展过程中值得关注的问题。

关键词: 生物制造, 巴氏梭菌, 1, 3-丙二醇, 粗甘油发酵, 下游分离纯化

Abstract:

1,3-Propanediol (PDO) is an important chemical extensively used in material science and the cosmetics industry. The biomanufacturing of PDO offers numerous advantages, such as the renewability of raw materials and environmental friendliness. Among various microorganisms, Clostridium pasteurianum emerges as an ideal choice for industrial PDO production due to its safety, non-pathogenic nature, rapid glycerol metabolism, fast growth rate, independence from expensive culture medium components, and its inherent efficient metabolic pathway for PDO production. This review begins by introducing the current state and challenges of PDO biomanufacturing, followed by an in-depth discussion of the methods for producing PDO using C. pasteurianum. Special attention is paid to the glycerol metabolism mechanism, strategies for glycerol fermentation, and the design of the fermentation process. Notably, our research group has identified C. pasteurianum mutant strains and developed robust processes that have largely addressed the organism’s traditional sensitivities to environmental conditions, especially regarding iron concentration and impurities of raw glycerol. In an electricity-aided fermentation process, PDO concentration as high as 120.6 g/L was achieved, with a productivity of 4.8 g/(L·h) and a yield reaching the theoretical maximum. We further discuss the natural limitations of genetic engineering in C. pasteurianum, exploring strategies based on rational genomic modification and directed evolution. Finally, the development of efficient downstream processing technologies is emphasized as crucial for realizing the cost-effective microbial production of PDO from renewable resources, since the industrial application of PDO requires a very high purity (>99.9%). The discussion on PDO downstream processing mainly focuses on evaporation, distillation, and extraction-based purification techniques. Through a comprehensive coverage of metabolic engineering, strain evolution, fermentation optimization, and product separation technologies, this review discusses about the characteristics and advantages of PDO production from C. pasteurianum, highlighting key considerations for advancing this microorganism as a new industrial chassis.

Key words: biomanufacturing, Clostridium pasteurianum, 1,3-propanediol, crude glycerol fermentation, downstream separation and purification

中图分类号:

Q816

刘建明, 张炽坚, 张冰, 曾安平. 巴氏梭菌作为工业底盘细胞高效生产1，3-丙二醇——从代谢工程和菌种进化到过程工程和产品分离[J]. 合成生物学, 2024, 5(6): 1386-1403.

Jianming LIU, Chijian ZHANG, Bing ZHANG, Anping ZENG. Clostridium pasteurianum as an industrial chassis for efficient production of 1,3-propanediol: from metabolic engineering to fermentation and product separation[J]. Synthetic Biology Journal, 2024, 5(6): 1386-1403.

图/表 5

图1 利用不同原料生物合成PDO的代谢途径C6—葡萄糖；C3—甘油；C2—乙醇；C1—二氧化碳和甲醇；GPD—甘油-3-磷酸脱氢酶；GPP—甘油-3-磷酸水解酶；GDHt—甘油脱水酶；YqhD—非特异性醇脱氢酶；DhaT—1，3-丙二醇脱氢酶；GDH—谷氨酸脱氢酶；KDC—酮酸脱羧酶；ACC—乙酰辅酶A羧化酶；MCR—丙二酸辅酶A还原酶；ydfG—3-羟基酸脱氢酶；Mdh2—甲醇脱氢酶；KHB—2-酮-4-羟基丁酸醛缩酶；PDC—丙酮酸脱羧酶；ADH—乙醇脱氢酶；DERA—脱氧核糖-5-磷酸醛缩酶

Fig. 1 Metabolic pathways for PDO bioproduction from various substratesC6—glucose; C3—glycerin; C2—ethanol; C1—CO2 and methanol; GPD—glycerol-3-phosphate dehydrogenase; GPP—glycerol-3-phosphate phosphohydrolase; GDHt—glycerol dehydratase; YqhD—nonspecific alcohol dehydrogenase; DhaT—1,3-propanediol dehydrogenase; GDH—glutamate dehydrogenase; KDC—keto acid decarboxylase; ACC—acetyl-CoA carboxylase; MCR—malonyl-CoA reductase; ydfG—3-hydroxy acid dehydrogenase; Mdh2—methanol dehydrogenase; KHB—2-keto-4-hydroxybutyrate aldolase; PDC—pyruvate decarboxylase; ADH—alcohol dehydrogenase; DERA—deoxyribose-5-phosphate aldolase

图2 巴氏梭菌核心代谢途径：以葡萄糖和甘油为原料［39］参与代谢途径的酶编号如下：1—丙二醇脱水酶；2—1，3-丙二醇脱氢酶；3—甘油-3-磷酸脱氢酶；4—二羟基丙酮激酶；5—三磷酸甘油醛异构酶；6—六碳激酶；7—磷酸葡萄糖异构酶；8—磷酸果糖激酶；9—甘油醛-3-磷酸脱氢酶；10—丙酮酸激酶；11—乳酸脱氢酶；12—丙酮酸甲酸裂解酶；13—丙酮酸-黄弗氏蛋白氧化还原酶；14—NADH依赖的还原铁硫蛋白：NADP+氧化还原酶；15—铁硫蛋白氢化酶；16—乙醛脱氢酶；17—醇脱氢酶；18—磷酸乙酰转移酶；19—乙酸激酶；20—乙酰辅酶A乙酰转移酶；21—3-羟基丁酰辅酶A脱氢酶；22—克罗托酶；23—2，4-二烯酰辅酶A还原酶；24—铁硫蛋白依赖的丁酰辅酶A脱氢酶/电子转移黄素蛋白复合物（BCdH-ETF）；25—磷酸丁酰转移酶；26—丁酸激酶；27—醛脱氢酶；28—丁醇脱氢酶

Fig. 2 Pathways of glucose and glycerol metabolism in C. pasteurianum[39]Enzymes involved in the metabolic pathways are numbered as follow: 1—Propanediol dehydratase; 2—1,3-Propanediol dehydrogenase; 3—Glycerol-3-phosphate dehydrogenase; 4—Dihydroxyacetone kinase; 5—Triosephosphate isomerase; 6—Hexokinase; 7—Phosphoglucose isomerase; 8—Phosphofructokinase; 9—Glyceraldehyde-3-phosphate dehydrogenase; 10—Pyruvate kinase; 11—Lactate dehydrogenase; 12—Pyruvate formate-lyase; 13—Pyruvate-flavodoxin oxidoreductase; 14—NADH-dependent reduced ferredoxin: NADP+ oxidoreductase; 15—Ferredoxin hydrogenase; 16—Acetaldehyde dehydrogenase; 17—Alcohol dehydrogenase; 18—Phosphate acetyltransferase; 19—Acetate kinase; 20—Acetyl-CoA acetyltransferase; 21—3-Hydroxybutyryl-CoA dehydrogenase; 22—Crotonase; 23—2,4-dienoyl-CoA reductase; 24—Ferredoxin dependent butyryl-CoA dehydrogenase/electron transferring flavoprotein complex (BCdH-ETF); 25—Phosphate butyryltransferase; 26—Butyrate kinase; 27—Aldehyde dehydrogenase; 28—Butanol dehydrogenase

表1 巴氏梭菌的能量、还原当量和产品平衡

Table 1 Energy, reducing equivalents and product balances in C. pasteurianum

Substrate	End-product	Energy, reducing equivalents and product balances
glycerol	PDO	glycerol+NADH→PDO
	lactate	glycerol→lactate+NADH+ATP
	acetate	glycerol→acetate+CO₂+2ATP+2NADH+FdH₂
	ethanol	glycerol→ethanol+CO₂+ATP+FdH₂
	butyrate	2glycerol→butyrate+2CO₂+3ATP+2NADH+2FdH₂
	butyrate	*2glycerol→butyrate+2CO₂+3ATP+NADH+3FdH₂
	butanol	2glycerol→butanol+2CO₂+2ATP+2FdH₂
	butanol	*2glycerol+NADH→butanol+2CO₂+2ATP+3FdH₂
glucose	lactate	glucose→2lactate+2ATP
	acetate	glucose→2acetate+2CO₂+4ATP+2NADH+2FdH₂
	ethanol	glucose+2NADH→2ethanol+2CO₂+2ATP+2FdH₂
	butyrate	glucose→butyrate+2CO₂+3ATP+2FdH₂
	butyrate	*glucose+NADH→butyrate+2CO₂+3ATP+3FdH₂
	butanol	glucose+2NADH→butanol+2CO₂+2ATP+2FdH₂
	butanol	*glucose+3NADH→butanol+2CO₂+2ATP+3FdH₂

表2 基于蒸发和蒸馏的PDO纯化方法

Table 2 PDO purification based on evaporation and distillation

分离步骤	作用与功能	存在的问题	分离物质
蒸馏	液态混合物中各组分沸点不同，去除残留甘油		残余甘油
离心和微滤	有效去除所有微生物细胞（生物量）		生物质
低分子截留	有效去除高含量的可溶性蛋白质，避免其在水分蒸发过程中严重起泡从而导致过程效率降低		可溶性蛋白
壳聚糖絮凝
活性炭吸附
纳滤	分离去除葡萄糖，避免葡萄糖在蒸发和蒸馏过程中发生沉淀		葡萄糖
离子交换吸附	有效去除发酵液中的有机酸盐和无机盐，因为盐在水分蒸发过程中部分结晶，结晶盐的沉积导致蒸发器底部形成黏稠的浆液，进而导致高能耗和目标产品的低产量	树脂的快速饱和，需要大量的NaOH和HCl溶液进行再生	有机酸盐和无机盐
电渗析		电渗析的能源和材料成本通常非常高，阻碍了其在廉价大宗化学品的商业生产中的实际应用
醇沉淀和稀释结晶		不足以有效去除在醇类中溶解度高的有机酸盐
蒸发	蒸发去除水分		水

表3 基于萃取的PDO纯化方法

Table 3 PDO purification based on extraction

分离方法		作用与功能	存在的问题	提取率
液-液萃取	使用疏水有机溶剂乙酸乙酯	使用乙酸乙酯对生物合成的PDO进行溶剂萃取	从实际发酵液中回收PDO的最高分配系数仅达到0.14	12%
	基于化学反应的反应萃取	将PDO转化为疏水性PDO衍生物，有机溶剂萃取后，通过逆反应以获得PDO	反应物和萃取剂都有毒，发酵液中与PDO具有相似化学结构的其他物质（如2,3-丁二醇、甘油、乙醇等）也会与反应物发生反应	91%
	基于生物催化转化的反应萃取	利用与辛酸的酯化反应在脂肪酶催化下，将PDO转化为疏水酯	PDO和脂肪酸之间的反应效率低，需要重复三次脂肪酶催化的酯化反应	90%
两相盐析萃取	“K₂CO₃+K₂HPO₄”异丙醇盐析萃取系统	使用亲水有机溶剂的盐析萃取在从发酵液中回收亲水产品方面表现出显著更高的回收率	盐析萃取系统中形成两相所需的盐量大，且分离过程造成大量的废水排放	98%
	由乙醇和碳酸钠组成的盐析萃取系统	使用亲水有机溶剂的盐析萃取在从发酵液中回收亲水产品方面表现出显著更高的回收率		97%
	两步盐析萃取	第一步中，使用疏水性的正丁酸乙酯高效提丁酸，在第二步中，使用乙醇并添加NaH₂PO₄作为盐析剂，回收PDO		95%

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巴氏梭菌作为工业底盘细胞高效生产1，3-丙二醇——从代谢工程和菌种进化到过程工程和产品分离

Clostridium pasteurianum as an industrial chassis for efficient production of 1,3-propanediol: from metabolic engineering to fermentation and product separation

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