Mutagenesis of Clostridium acetobutylicum

Mutagenesis of the obligate anaerobe Clostridium acetobutylicum was best accomplished using agents (e.g. ethyl methane sulphonate or N -methyl- N '-nitro- N -nitrosoguanidine) which are believed to act by a direct mutagenic mechanism. Other agents (e.g. u.v. radiation) whose effectiveness relies on misrepair of damaged DNA via an error-prone pathway, were poor mutagens of this organism. Procedures are described which readily yielded a variety of auxotrophic and other useful mutant strains of Cl. acetobutylicum and related saccharolytic clostridia.     

Use of immobilized microbial membrane fragments to remove oxygen and favor the acetone-butanol fermentation.

Oxygen-reducing membrane fragments obtained from Escherichia coli were used with Clostridium acetobutylicum (C. acetobutylicum) to provide an oxygen-free microenvironment for the conversion of glucose to acetone, butanol, and ethanol (ABE). The batch fermentation of suspended C. acetobutylicum NRRL-B-643 and its ability to produce solvents in the presence of membranes as the oxygen-elimination agent are described and compared with the conventional sparging technique used to maintain anaerobiosis. The use of membrane fragments to remove oxygen for fermentation by C. acetobutylicum was successful and gave slightly improved results over the use of sparing with regard to lag, biomass, and solvent production (e.g., final butanol concentration of 3.25 and 2.7 g/L, respectively). Solvent production is also reported for a continuous columnar reactor with coimmobilized cells and membranes in kappa-carrageenan gel beads and air-saturated liquid feed.     

Reaction engineering studies of acetone-butanol-ethanol fermentation with Clostridium acetobutylicum

Acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum has been extensively studied in recent years because the organism is recognized as an excellent butanol producer. A parallel bioreactor system with 48 stirred-tank bioreactors on a 12 mL scale was evaluated for batch cultivations of the strictly anaerobic, butanol-producing C. acetobutylicum ATCC 824. Continuous gassing with nitrogen gas was applied to control anaerobic conditions. Process performances of ABE batch fermentations on a milliliter scale were identical to the liter-scale stirred-tank reactor if reaction conditions were identical on the different scales (e.g., initial medium, pH, temperature, specific evaporation rates, specific power input by the stirrers). The effects of varying initial ammonia concentrations (0.1-4.4 g L(-1) ) were studied in parallel with respect to glucose consumption and butanol production of C. acetobutylicum ATCC 824 as a first application example. The highest butanol yield of 33% (mol mol(-1) ) was observed at initial ammonia concentrations of 0.5 and 1.1 g L(-1) . This is the first report on the successful application of a 48 parallel stirred-tank bioreactor system for reaction engineering studies of strictly anaerobic microorganisms at the milliliter scale.     

Phosphoketolase Pathway for Xylose Catabolism in Clostridium acetobutylicum Revealed by 13C Metabolic Flux Analysis

Solvent-producing clostridia are capable of utilizing pentose sugars, including xylose and arabinose; however, little is known about how pentose sugars are catabolized through the metabolic pathways in clostridia. In this study, we identified the xylose catabolic pathways and quantified their fluxes in Clostridium acetobutylicum based on [1-(13)C]xylose labeling experiments. The phosphoketolase pathway was found to be active, which contributed up to 40% of the xylose catabolic flux in C. acetobutylicum. The split ratio of the phosphoketolase pathway to the pentose phosphate pathway was markedly increased when the xylose concentration in the culture medium was increased from 10 to 20 g liter(-1). To our knowledge, this is the first time that the in vivo activity of the phosphoketolase pathway in clostridia has been revealed. A phosphoketolase from C. acetobutylicum was purified and characterized, and its activity with xylulose-5-P was verified. The phosphoketolase was overexpressed in C. acetobutylicum, which resulted in slightly increased xylose consumption rates during the exponential growth phase and a high level of acetate accumulation.     

限制葡萄糖、葡萄糖/乙酸双底物条件下自由控制丙丁梭菌ABE发酵丙酮浓度和丙酮/丁醇比

提出一种可以提高和自由控制丙丁梭菌ABE发酵丙酮浓度与丙酮/丁醇比的方法。（1）通过控制糖化酶用量、反应时间和温度调节玉米培养基初始葡萄糖浓度,使发酵进入到产溶剂期后,残留葡萄糖浓度降至接近于0 g/L的水平;（2）在葡萄糖受限的条件下,诱导丙丁梭菌合成分泌糖化酶,分解寡糖,将葡萄糖维持于低浓度,进而限制梭菌胞内糖酵解途径的碳代谢和NADH生成速度。与此同时,外添乙酸形成葡萄糖/乙酸双底物环境。在能量代谢基本不受破坏、丁醇未达到抑制浓度的条件下,适度抑制丁醇生产,有效地利用外添乙酸强化丙酮合成;（3）在外添乙酸的基础上,添加适量酿酒酵母,形成丙丁梭菌/酿酒酵母混合发酵体系,提高梭菌对高丁醇浓度的耐受能力。整个发酵体系可以将丙酮浓度和丙酮/丁醇比自由控制在5~12 g/L和0.5~1.0的水平,最大丙酮浓度和丙酮/丁醇比达到11.74 g/L和1.02,并可维持丁醇浓度于10~14 g/L的正常水平,充分满足工业ABE发酵对于丙酮和丁醇产品的不同需求。     

Biotransformation of furfural and 5-hydroxymethyl furfural (HMF) by Clostridium acetobutylicum ATCC 824 during butanol fermentation

The ability of fermenting microorganisms to tolerate furan aldehyde inhibitors (furfural and 5-hydroxymethyl furfural (HMF)) will enhance efficient bioconversion of lignocellulosic biomass hydrolysates to fuels and chemicals. The effect of furfural and HMF on butanol production by Clostridium acetobutylicum 824 was investigated. Whereas specific growth rates, μ, of C. acetobutylicum in the presence of furfural and HMF were in the range of 15-85% and 23-78%, respectively, of the uninhibited Control, μ increased by 8-15% and 23-38% following exhaustion of furfural and HMF in the bioreactor. Using high performance liquid chromatography and spectrophotometric assays, batch fermentations revealed that furfural and HMF were converted to furfuryl alcohol and 2,5-bis-hydroxymethylfuran, respectively, with specific conversion rates of 2.13g furfural and 0.50g HMF per g (biomass) per hour, by exponentially growing C. acetobutylicum. Biotransformation of these furans to lesser inhibitory compounds by C. acetobutylicum will probably enhance overall fermentation of lignocellulosic hydrolysates to butanol.     

Metabolic engineering of Clostridium acetobutylicum ATCC 824 for isopropanol-butanol-ethanol fermentation

Clostridium acetobutylicum naturally produces acetone as well as butanol and ethanol. Since acetone cannot be used as a biofuel, its production needs to be minimized or suppressed by cell or bioreactor engineering. Thus, there have been attempts to disrupt or inactivate the acetone formation pathway. Here we present another approach, namely, converting acetone to isopropanol by metabolic engineering. Since isopropanol can be used as a fuel additive, the mixture of isopropanol, butanol, and ethanol (IBE) produced by engineered C. acetobutylicum can be directly used as a biofuel. IBE production is achieved by the expression of a primary/secondary alcohol dehydrogenase gene from Clostridium beijerinckii NRRL B-593 (i.e., adh(B-593)) in C. acetobutylicum ATCC 824. To increase the total alcohol titer, a synthetic acetone operon (act operon; adc-ctfA-ctfB) was constructed and expressed to increase the flux toward isopropanol formation. When this engineering strategy was applied to the PJC4BK strain lacking in the buk gene (encoding butyrate kinase), a significantly higher titer and yield of IBE could be achieved. The resulting PJC4BK(pIPA3-Cm2) strain produced 20.4 g/liter of total alcohol. Fermentation could be prolonged by in situ removal of solvents by gas stripping, and 35.6 g/liter of the IBE mixture could be produced in 45 h.     

Isolation and characterization of Clostridium acetobutylicum mutants with enhanced amylolytic activity.

Clostridium acetobutylicum mutants BA 101 (hyperamylolytic) and BA 105 (catabolite depressed) were isolated by using N-methyl-N'-nitro-N-nitrosoguanidine together with selective enrichment on the glucose analog 2-deoxyglucose. Amylolytic enzyme production by C. acetobutylicum BA 101 was 1.8- and 2.5-fold higher than that of the ATCC 824 strain grown in starch and glucose, respectively. C. acetobutylicum BA 105 produced 6.5-fold more amylolytic activity on glucose relative to that of the wild-type strain. The addition of glucose at time zero to starch-based P2 medium reduced the total amylolytic activities of C. acetobutylicum BA 101 and BA 105 by 82 and 25%, respectively, as compared with the activities of the same strains grown on starch alone. Localization studies demonstrated that the amylolytic activities of C. acetobutylicum BA 101 and BA 105 were primarily extracellular on all carbohydrates tested.     

Isolation and characterization of Clostridium acetobutylicum mutants with enhanced amylolytic activity.

Clostridium acetobutylicum mutants BA 101 (hyperamylolytic) and BA 105 (catabolite depressed) were isolated by using N-methyl-N'-nitro-N-nitrosoguanidine together with selective enrichment on the glucose analog 2-deoxyglucose. Amylolytic enzyme production by C. acetobutylicum BA 101 was 1.8- and 2.5-fold higher than that of the ATCC 824 strain grown in starch and glucose, respectively. C. acetobutylicum BA 105 produced 6.5-fold more amylolytic activity on glucose relative to that of the wild-type strain. The addition of glucose at time zero to starch-based P2 medium reduced the total amylolytic activities of C. acetobutylicum BA 101 and BA 105 by 82 and 25%, respectively, as compared with the activities of the same strains grown on starch alone. Localization studies demonstrated that the amylolytic activities of C. acetobutylicum BA 101 and BA 105 were primarily extracellular on all carbohydrates tested.     

可发酵糖为底物不同菌种丁醇生产能力的研究

随着新一代生物质能源的研发,利用梭菌的发酵生产丁醇已成为热点。选用能生产丁醇的Clostridium acetobutylicum AS1.7,Clostridium acetobutylicum AS1.132,Clostridium acetobutylicumAS1.134和Clostridium beijerinckii NCMIB 8052,在多种糖源下进行发酵培养,通过比较其在不同糖源条件下的生长情况、糖利用率、丁醇及副产物产量、对丁醇、木糖耐受能力等,综合筛选出了最适用于发酵生产丁醇的备选菌种。NCMIB8052因具有最高产量、相对优良的耐受性及可利用多种糖源的特点,而被确定为发酵能力最强的菌种。     

Cloning, nucleotide sequence and structural analysis of the Clostridium acetobutylicum dnaJ gene

Abstract The complete dnaJ gene of Clostridium acetobutylicum was isolated by chromosome walking using the previously cloned 5' end of the gene as a probe. Nucleotide sequencing of a positively reacting 2.2-kb Hin cII fragment, contained in the recombinant plasmid pKG4, revealed that the reading frame of the dnaJ gene of C. acetobutylicum consists of 1125 bp, encoding a protein of 374 amino acids with a calculated M _r of 40376 and an isoelectric points of 9.54. The deduced amino acid sequence showed high similarity to the DnaJ proteins of other bacteria (e.g. Escherichia coli, Bacillus subtilis ) as well as of an archaeon ( Methanosarcina mazei ) and to the corresponding proteins of eukaryotes ( Saccharomyces cerevisiae, Homo sapiens ). The areas of similarity included a conserved N-terminal domain of about 70 amino acids, a glycine-rich region of about 30 residues, and a central domain containing four repeats of a CXXCXGXG motif, whereas the C-terminal domain was less conserved. Northern (RNA) blot analysis indicated that dnaJ is induced by heat shock and that it is part of the dnaK operon of C. acetobutylicum . The 5' end (901 bp) of another gene ( orfB ), downstream of dnaJ and not heat-inducible, showed no significant similarity to other sequences available in EMBL and GenBank databases.     

Adaptive responses to oxygen stress in obligatory anaerobes Clostridium acetobutylicum and Clostridium aminovalericum

Clostridium acetobutylicum and Clostridium aminovalericum, both obligatory anaerobes, grow normally after growth conditions are changed from anoxic to microoxic, where the cells consume oxygen proficiently. In C. aminovalericum, a gene encoding a previously characterized H2O-forming NADH oxidase, designated noxA, was cloned and sequenced. The expression of noxA was strongly upregulated within 10 min after the growth conditions were altered to a microoxic state, indicating that C. aminovalericum NoxA is involved in oxygen metabolism. In C. acetobutylicum, genes suggested to be involved in oxygen metabolism and genes for reactive oxygen species (ROS) scavenging were chosen from the genome database. Although no clear orthologue of C. aminovalericum NoxA was found, Northern blot analysis identified many O2-responsive genes (e.g., a gene cluster [CAC2448 to CAC2452] encoding an NADH rubredoxin oxidoreductase-A-type flavoprotein-desulfoferrodoxin homologue-MerR family-like protein-flavodoxin, an operon [CAC1547 to CAC1549] encoding a thioredoxin-thioredoxin reductase-glutathione peroxidase-like protein, an operon [CAC1570 and CAC1571] encoding two glutathione peroxidase-like proteins, and genes encoding thiol peroxidase, bacterioferritin comigratory proteins, and superoxide dismutase) whose expression was quickly and synchronously upregulated within 10 min after flushing with 5% O2. The corresponding enzyme activities, such as NAD(P)H-dependent peroxide (H2O2 and alkyl hydroperoxides) reductase, were highly induced, indicating that microoxic growth of C. acetobutylicum is associated with the expression of a number of genes for oxygen metabolism and ROS scavenging.     

Identification of two genes encoding putative new members of the ECF subfamily of eubacterial RNA polymerase sigma factors in Clostridium acetobutylicum

Two genes from Clostridium acetobutylicum DSM 792 were identified which are predicted to encode new members of the ECF subfamily of eubacterial RNA polymerase sigma factors. The sigX gene has the potential to encode a 184-amino acid protein with a molecular mass of 21,870 Da and with the highest overall similarity to Fecl of Escherichia coli (27 % identical residues). The second gene, which is predicted to encode an alternative sigma factor of the ECF subfamily, is the previously described orf2 gene (Gerischer and Dürre, 1990) located in the adc gene region of C. acetobutylicum. The deduced protein of orf2 has significant similarity to SigX of C. acetobutylicum (22 % identical residues) and shares structural features with other alternative sigma factors. Therefore, it is proposed to rename orf2 as sigY. Analysis of the phylogenetic relationship revealed that SigX from C. acetobutylicum, together with sigmaE from Streptomyces coelicolor and SigX from Bacillus subtilis, form a gram-positive cluster within the ECF subfamily and that SigY from C. acetobutylicum together with UviA from Clostridium perfringens, form a separate cluster located between the gram-positive cluster and the sporulation sigma factor sigmaH from B. subtilis.     

纤维二糖发酵生产丙酮丁醇

分别考察C.acetobutylicum 810705、810706以不同浓度的麸皮和玉米粉添加物作为营养元素,纤维二糖直接进行丙酮丁醇(ABE)发酵的结果,发现2株菌对于玉米粉和麸皮的浓度变化趋势一致,C.acetobutylicum 810706转化率较高。纤维二糖ABE发酵工艺条件表明:玉米粉添加量为总糖含量的30%、底物糖质量浓度60 g/L,pH 6.5、温度35℃时,C.acetobutylicum 810706转化率达到37.38%,总溶剂质量浓度22.43 g/L,比葡萄糖、木糖ABE发酵转化率高。模拟纤维素酶水解产物配制混合糖培养基,其溶剂转化率较单独的葡萄糖、木糖发酵的转化率高,为34.95%。对比纤维素酶水解条件,C.acetobutylicum 810706具有优良的纤维素酶水解同步糖化ABE发酵能力。     

Development of an anhydrotetracycline-inducible gene expression system for solvent-producing Clostridium acetobutylicum: A useful tool for strain engineering

Clostridium acetobutylicum is an important solvent (acetone-butanol-ethanol) producing bacterium. However, a stringent, effective, and convenient-to-use inducible gene expression system that can be used for regulating the gene expression strength in C. acetobutylicum is currently not available. Here, we report an anhydrotetracycline-inducible gene expression system for solvent-producing bacterium C. acetobutylicum. This system consists of a functional chloramphenicol acetyltransferase gene promoter containing tet operators (tetO), Pthl promoter (thiolase gene promoter from C. acetobutylicum) controlling TetR repressor expression cassette, and the chemical inducer anhydrotetracycline (aTc). The optimized system, designated as pGusA2-2tetO1, allows gene regulation in an inducer aTc concentration-dependent way, with an inducibility of over two orders of magnitude. The stringency of TetR repression supports the introduction of the genes encoding counterselective marker into C. acetobutylicum, which can be used to increase the mutant screening efficiency. This aTc-inducible gene expression system will thus increase the genetic manipulation capability for engineering C. acetobutylicum.     

Heterologous expression of endo-beta-1,4-D-glucanase from Clostridium cellulovorans in Clostridium acetobutylicum ATCC 824 following transformation of the engB gene.

Heterologous expression of the Clostridium cellulovorans engB gene by Clostridium acetobutylicum BKW-1 was detected as zones of hydrolysis on carboxymethyl cellulose (CMC) Trypticase glucose yeast plates stained with Congo red. The extracellular cellulase preparation from C. acetobutylicum BKW-1 has a specific activity towards CMC which is more than fourfold that present in C. acetobutylicum ATCC 824. Western blot (immunoblot) analysis using the C. cellulovorans anti-EngB primary antibody demonstrated that an additional 44-kDa protein band was present in the supernatant derived from C. acetobutylicum BKW-1 but was not present in ATCC 824 or ATCC 824(pMTL500E).     

电子载体对丁醇发酵的影响

研究在培养基中加入不同电子载体对丁醇发酵的影响。结果表明:添加微量的苄基紫精可以促进丁醇的产生,同时可强烈抑制丙酮的合成,丁醇体积分数由66.92%提高到82.35%。苄基紫精可促进菌株快速进入产溶剂期,发酵周期明显缩短,丁醇生产强度显著提高。7%玉米培养基中加入40 mg/L苄基紫精,丁醇产量最高达16.10 g/L,生产强度为0.37 g/(L.h),分别较对照提高10.96%和60.87%。在初始丁醇体积分数较低的条件下,苄基紫精对丁醇合成的促进作用更明显。     

Identification of the gene encoding NADH-rubredoxin oxidoreductase in Clostridium acetobutylicum.

NADH-rubredoxin oxidoreductase (NROR), a flavoprotein from the obligately anaerobe Clostridium acetobutylicum is encoded by an ORF (nror) of 1140 nucleotides. Whereas primary structure analysis reveals that NROR has amino acid sequence patterns homologous with those involved in FAD and NAD-binding, the enzyme is distantly related to other flavoproteins in the databank. NROR is highly active for reducing clostridial rubredoxin (Rd) especially against C. acetobutylicum Rd with an efficiency (k(cat)/K(m)) of 400,000 mM(-1)s(-1). These results suggest that Rd from C. acetobutylicum, C. pasteurianum, C. butyricum, and C. cellulolyticum can be interchanged with each other. Since C. acetobutylicum is the sole Clostridium strain that possesses such an enzyme, possible functions are discussed with regard to Desulfovibrio gigas and Pyrococcus furiosus, the only two other anaerobic systems for which a similar activity was reported, but no gene isolated.     

Effects of pH and added metabolites on bioconversions by immobilized non-growing Clostridium acetobutylicum

The bioconversion activity of calcium alginate-immobilized Clostridium acetobutylicum ATCC 824 was investigated in a continuous reactor system utilizing a defined feed medium which did not support cell growth. The changes in biocatalytic activity with time were studied at different pH values as well as when different metabolites (butyric, acetic, and acetoacetic acids) were present in the feed stream. Although the nongrowing cells were metabolically active, the product distribution was shifted from solvent production to acidogenesis. Overall activity losses occurred due to cell lysis, sporulation, and the effects of nitrogen level on macromolecular turnover. These effects were minimized under some operating conditions (e.g., pH 6), resulting in significantly longer productivity lifetimes.     

Heterologous production, assembly, and secretion of a minicellulosome by Clostridium acetobutylicum ATCC 824

The gene man5K encoding the mannanase Man5K from Clostridium cellulolyticum was cloned alone or as an operon with the gene cipC1 encoding a truncated scaffoldin (miniCipC1) of the same origin in the solventogenic Clostridium acetobutylicum. The expression of the heterologous gene(s) was under the control of a weakened thiolase promoter Pthl. The recombinant strains of the solventogenic bacterium were both found to secrete active Man5K in the range of milligrams per liter. In the case of the strain expressing only man5K, a large fraction of the recombinant enzyme was truncated and lost the N-terminal dockerin domain, but it remained active towards galactomannan. When man5K was coexpressed with cipC1 in C. acetobutylicum, the recombinant strain secreted almost exclusively full-length mannanase, which bound to the scaffoldin miniCipC1, thus showing that complexation to the scaffoldin stabilized the enzyme. The secreted heterologous complex was found to be functional: it binds to crystalline cellulose via the carbohydrate binding module of the miniscaffoldin, and the complexed mannanase is active towards galactomannan. Taken together, these data show that C. acetobutylicum is a suitable host for the production, assembly, and secretion of heterologous minicellulosomes.