图1 微生物正丁醇合成途径（1—磷酸烯醇丙酮酸羧化酶；2—天冬氨酸转氨酶；3—柠苹酸合酶；4—3-异丙基苹果酸脱氢酶/异构酶；5—苏氨酸脱水酶；6—2-异丙基苹果酸合酶；7，8—3-异丙基苹果酸异构酶；9—3-异丙基苹果酸脱氢酶；10—2-酮酸脱羧酶；11—丙酮酸激酶；12—丙酮酸脱氢酶复合体/丙酮酸甲酸裂解酶/丙酮酸黄素蛋白氧化还原酶；13—硫解酶/乙酰辅酶A转乙酰基酶；14—乙酰辅酶A羧化酶；15—乙酰乙酰辅酶A合成酶；16—3-羟丁酰辅酶A脱氢酶/羟烷基辅酶A脱氢酶；17—巴豆酸酶；18—丁酰辅酶A脱氢酶和电子转移黄素蛋白 AB/反式-2-烯酰辅酶A还原酶；19—乙醛/丁醛脱氢酶； 20—乙醇/丁醇脱氢酶；21—乙酰CoA转酰酶；22—丙二酸单酰CoA转酰酶；23—β-酮酰-ACP合酶；24—β-酮酰-ACP还原酶；25—β-酮酰-ACP脱水酶；26—烯酰-ACP还原酶；27—酰基-ACP硫酯酶；28—羧酸还原酶；29—甘氨酸氧化酶；30—苹果酸合酶；DHAP—磷酸二羟丙酮）

Fig. 1 n-Butanol biosynthesis pathway in microorganism（1—Phosphoenolpyruvate carboxylase; 2—Aspartate transaminase; 3—Citramalate synthase; 4—3-isopropylmalate dehydrogenase/isomerase; 5—Threonine dehydratase; 6—2-isopropylmalate synthase; 7,8—3-isopropylmalate isomerase; 9—3-isopropylmalate dehydrogenase; 10—2-ketoacid decarboxylase 11—pyruvate kinase; 12—pyruvate dehydrogenase complex/pyruvate formate lyase/ pyruvate-ferredoxin oxidoreductase; 13—thiolase/acetyl-CoA transacetylase; 14—acetyl CoA carboxylase; 15—acetoacetyl-CoA synthase; 16—3-hydroxybutyryl-CoA dehydrogenase/hydroxyalkyl-CoA dehydrogenase; 17—crotonase; 18—butyryl-CoA dehydrogenase/trans-2-enoyl-CoA reductase; 19—acetaldehyde/butyraldehyde dehydrogenase; 20— ethanol/butanol dehydrogenase; 21—acetyl-CoA transacylase; 22—malonyl-CoA transacylase; 23—β-ketoacyl-ACP synthase; 24—β-ketoacyl-ACP reductase; 25—β-ketoacyl-ACP dehydratase; 26—enoyl-ACP reductase; 27—acyl-ACP thioesterase; 28—carboxylic acid reductase; 29—glycine oxidase; 30—malate synthase; DHAP—dihydroxyacetone phosphate）

图2 梭菌遗传操作工具开发的里程碑式进展（蓝色字体代表基于同源重组的遗传操作工具，红色字体代表不依赖于同源重组的遗传操作工具）

Fig. 2 Milestones in the development of genetic manipulation tools for clostridia(Blue font represents genetic manipulation tools based on homologous recombination, and red represents genetic manipulation tools independent on homologous recombination)

图3 梭菌正丁醇合成途径代谢工程改造（图中所示基因表达的酶：pfk—6-磷酸果糖激酶；fba—果糖-二磷酸醛缩酶；pyk—丙酮酸激酶；ilvB—乙酰乳酸合酶；aldc—乙酰乳酸脱羧酶；acr—乙偶姻还原酶；pfor—丙酮酸-铁氧还蛋白氧化酶；hyd—氢酶；pta—乙酰基磷酸转移酶；ack—乙酸激酶；thl—硫解酶；hbd—3-羟基丁酰CoA脱氢酶；crt—巴豆酸酶；bcd—丁酰CoA脱氢酶；ter—反式烯酰辅酶A还原酶；ptb—磷酸丁酰转移酶；buk—丁酸激酶；ctfAB—乙酰乙酰CoA：乙酸/丁酸：CoA转移酶；adc—乙酰乙酸脱羧酶；sadh—异丙醇脱氢酶；adh—乙醇脱氢酶；bdh/edh—丁醇/乙醇脱氢酶；adhE—醇/醛脱氢酶）

Fig. 3 Metabolic engineering of clostridiafor n-butanol production(pfk—6-phosphofructokinase; fba—fructose-bisphosphate aldolase; pyk—pyruvate kinase; ilvB—acetolactate synthase; aldc—acetolactate decarboxylase; acr—acetoin reductase; pfor—pyruvate-ferredoxin oxidoreductase; hyd—hydrogenase; pta—phosphate acyltransferase; ack—acetate kinase; thl—thiolase; hbd—3-hydroxybutyryl-CoA dehydrogenase; crt—crotonase; bcd—butyryl-CoA dehydrogenase; ter—trans-enoyl-CoA reductase; ptb—phosphobutyryl transferase; buk—butyrate kinase; ctfAB—acetoacetyl-CoA: acetate/butyrate: CoA transferase; adc—acetoacetate decarboxylase; sadh—isopropanol dehydrogenase; adh/edh—alcohol/ehanol dehydrogenase; bdh/edh—butanol/ehanol dehydrogenase; adhE—alcohol/aldehyde dehydrogenase)

图4 梭菌的正丁醇合成途径重构[绿色代表梭菌的糖类代谢途径；黄色代表梭菌的甘油代谢途径；湖蓝色代表食气梭菌的合成气代谢途径；正蓝色代表梭菌内的正丁醇代谢途径（部分梭菌途径并不完整）。图中所示基因表达的酶：dhaBCE—甘油脱水酶；dhaT—1,3-丙二醇脱氢酶；gldA—甘油-3-磷酸脱氢酶；dhaKL—磷酸二羟丙酮激酶；mgsA—甲基乙二醛合酶；yqhD—醇脱氢酶；fucO—1,2-丙二醇脱氢酶；hk—己糖激酶；gpi—果糖-6-磷酸异构酶；gapdh—甘油醛-3-磷酸脱氢酶；codh—一氧化碳脱氢酶；acs—乙酰辅酶A合酶；fdh—甲酸脱氢酶；fhs—甲酰-四氢叶酸合成酶；folD—亚甲基四氢叶酸环化酶/脱氢酶；metF—亚甲基四氢叶酸还原酶；metTr—甲基转移酶；cat1—辅酶A转移酶（图3已出现的基因代表的含义此处不再列出）]

Fig. 4 Reconstruction of n-butanol synthesis pathway in clostridia[Green lines represent carbohydrate metabolic pathway; the yellow lines represent the glycerol metabolic pathway; the lake blue represents the syngas metabolic pathway in gas-fermenting clostridia; the positive blue lines represent the synthesis pathway of n-butanol in Clostridium. dhaBCE—glycerol dehydratase; dhaT—1,3-propanediol dehydrogenase; gldA—glycerol-3-phosphate dehydrogenase; dhaKL—dihydroxyacetone phosphate kinase; mgsA—methylglyoxal synthase; yqhD—alcohol dehydrogenase; fucO—1,2-propanediol dehydrogenase; hk—hexokinase; gpi—fructose-6-phosphate isomerase; gapdh—glyceraldehyde-3-phosphate dehydrogenase; codh—carbon monoxide dehydrogenase; acs—acetyl-CoA synthase; fdh—formate dehydrogenase; fhs—formyl-tetrahydrofolate synthase; folD—methylenetetrahydrofolate cyclase/dehydrogenase; metF—methylenetetrahydrofolate reductase; metTr—methyltransferase; cat1—CoA transferase (Genes appeared in Figure 3 is not listed here)]

图5 产正丁醇梭菌的戊糖代谢途径及其调控蛋白(XylT/XylFGH—木糖特异性转运蛋白；AraE/AraFGH—阿拉伯糖特异性转运蛋白；PTS—己糖的磷酸转移酶系统；GlcG—PTS的组成酶II，与碳源代谢抑制效应密切相关；G6P—葡萄糖-6-磷酸；F6P—果糖-6-磷酸；FBP—果糖-1，6-二磷酸；E4P—赤藓糖-4-磷酸；S7P—景天庚酮糖-7-磷酸；Ru5P—核酮糖-5-磷酸；Xu5P—木酮糖-5-磷酸；G3P—3-磷酸甘油醛；DHA—二羟丙酮；DHAP—磷酸二羟丙酮；PEP—磷酸烯醇式丙酮酸图中所示基因表达的酶：xylA-I/II—木糖异构酶；xylB—木酮糖激酶；araA—阿拉伯糖异构酶；araB/K—核酮糖激酶；araD—核酮糖-5-磷酸异构酶；rpi—5-磷酸核糖异构酶；tal—转醛酶；tkt—转酮酶；pk—磷酸酮醇酶）

Fig. 5 Pentose metabolism pathway and involved regulatory proteins in n-butanol-producing clostridia[XylT/XylFGH—xylose-specific transporter; AraE/AraFGH—arabinose-specific transporter; PTS—phosphotransferase system; GlcG—enzyme II of PTS, closely related to the CCR; G6P—glucose-6-phosphate; F6P—fructose-6-phosphate; FBP—fructose-1,6-Diphosphate; E4P—erythrose-4-phosphate; S7P—sedum heptulose-7-phosphate; Ru5P—ribulose-5-phosphate; Xu5P—xylulose-5-phosphate; G3P—3-Glyceraldehyde phosphate; DHA—dihydroxyacetone; DHAP—dihydroxyacetone phosphate; PEP—phosphoenolpyruvate； xylA-I/II—xylose isomerase; xylB—xylulose kinase; araA—arabinose isomerase; araB/K—ribulose kinase; araD—ribulose-5-phosphate isomerase; rpi—5-phosphoribose isomerase; tal—transaldolase; tkt—transketolase; pk—phosphoketolase (Genes appeared in Fig. 2~4 are not listed here)]