Introducing a single secondary alcohol dehydrogenase into butanol-tolerant Clostridium acetobutylicum Rh8 switches ABE fermentation to high level IBE fermentation

ABSTRACT: BACKGROUND: Previously we have developed a butanol tolerant mutant of Clostridium acetobutylicum, Rh8, from the wild type strain DSM 1731. Strain Rh8 can tolerate up to 19 g/L butanol, with solvent titer improved accordingly, thus exhibiting industrial application potential. To test if strain Rh8 can be used for production of high level mixed alcohols, a single secondary alcohol dehydrogenase from Clostridium beijerinckii NRRL B593 was overexpressed in strain Rh8 under the control of constitutive thl promoter. RESULTS: The heterogenous gene sADH was functionally expressed in C. acetobutylicum Rh8. This simple, one-step engineering approach led to the complete conversion of acetone into isopropanol, achieving a total alcohol titer of 23.88 g/l (7.6 g/l isopropanol, 15 g/l butanol, and 1.28 g/l ethanol) with a yield to glucose of 31.42%. The acid (butyrate and acetate) assimilation rate in isopropanol producing strain Rh8(psADH) was increased. CONCLUSIONS: The improved butanol tolerance and the enhanced solvent biosynthesis machinery in strain Rh8 is beneficial for production of high concentration of mixed alcohols. Strain Rh8 thus can be considered as a good host for further engineering of solvent/alcohol production.     

Complete genome sequence of Clostridium acetobutylicum DSM 1731, a solvent-producing strain with multireplicon genome architecture

Clostridium acetobutylicum is an important microorganism for solvent production. We report the complete genome sequence of C. acetobutylicum DSM 1731, a genome with multireplicon architecture. Comparison with the sequenced type strain C. acetobutylicum ATCC 824, the genome of strain DSM1731 harbors a 1.7-kb insertion and a novel 11.1-kb plasmid, which might have been acquired during evolution.     

Comparative analysis on the membrane proteome of Clostridium acetobutylicum wild type strain and its butanol-tolerant mutant

The solventogenic bacterium Clostridium acetobutylicum is the most important species of Clostridium used in the fermentation industry. However, the intolerance to butanol hampers the efficient production of solvents. Butanol toxicity has been attributed to the chaotropic effect on the cell membrane, but the knowledge on the effect of butanol on membrane associated proteins is quite limited. Using 2-DE combined with MALDI-TOF MS/MS and 1-DE integrated with LC-MS/MS, 341 proteins in the membrane fractions of cell lysate were identified, thus establishing the first comprehensive membrane proteome of C. acetobutylicum. The identified proteins are mainly involved in transport, cellular membrane/wall machinery, formation of surface coat and flagella, and energy metabolism. Comparative analysis on the membrane proteomes of the wild type strain DSM 1731 and its butanol-tolerant mutant Rh8 revealed 73 differentially expressed proteins. Hierarchical clustering analysis suggested that mutant Rh8 may have evolved a more stabilized membrane structure, and have developed a cost-efficient energy metabolism strategy, to cope with the butanol challenge. This comparative membrane proteomics study, together with our previous published work on comparative cytoplasmic proteomics, allows us to obtain a systemic understanding of the effect of butanol on cellular physiology of C. acetobutylicum.     

Formic acid triggers the "Acid Crash" of acetone-butanol-ethanol fermentation by Clostridium acetobutylicum

Solvent production by Clostridium acetobutylicum collapses when cells are grown in pH-uncontrolled glucose medium, the so-called "acid crash" phenomenon. It is generally accepted that the fast accumulation of acetic acid and butyric acid triggers the acid crash. We found that addition of 1 mM formic acid into corn mash medium could trigger acid crash, suggesting that formic acid might be related to acid crash. When it was grown in pH-uncontrolled glucose medium or glucose-rich medium, C. acetobutylicum DSM 1731 containing the empty plasmid pIMP1 failed to produce solvents and was found to accumulate 0.5 to 1.24 mM formic acid intracellularly. In contrast, recombinant strain DSM 1731 with formate dehydrogenase activity did not accumulate formic acid intracellularly and could produce solvent as usual. We therefore conclude that the accumulation of formic acid, rather than acetic acid and butyric acid, is responsible for the acid crash of acetone-butanol-ethanol fermentation.     

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.     

Over-expression of stress protein-encoding genes helps Clostridium acetobutylicum to rapidly adapt to butanol stress

The toxicity of n-butanol in microbial fermentations limits its formation. The stress response of Clostridium acetobutylicum involves various stress proteins and therefore, over-expression of genes encoding stress proteins constitutes an option to improve solvent tolerance. Over-expression of groESL, grpE and htpG, significantly improved butanol tolerance of C. acetobutylicum. Whereas the wild type and vector control strain did not survive 2?% (v/v) butanol for 2?h, the recombinant strains showed 45?% (groESL), 25?% (grpE) and 56?% (htpG), respectively, of the initial c.f.u. after 2?h of butanol exposure. As previously, over-expression of groESL led to higher butanol production rates, but the novel strains over-expressing grpE or htpG produced only 51 and 68?%, respectively, of the wild type butanol concentrations after 72?h clearly differentiating butanol tolerance and production. Not only butanol tolerance but also the adaptation to butanol in successive stress experiments was significantly facilitated by increased levels of GroESL, GrpE and HtpG. Re-transformation and sequence analyses of the plasmids confirmed that not the plasmids, but the host cells evolved to a more robust phenotype.     

Acetone, butanol and ethanol production from domestic organic waste by solventogenic clostridia

Domestic organic waste (DOW) was washed and dried to 85 % dryness by VAM (The Netherlands). This material contained 25.1 g glucose, 8.4 g xylose and 5.8 g other monosaccharides/100 g dry matter. Using Mansonite steam explosion and enzymatic hydrolysis, a hydrolysate containing 15.4 g glucose, 2.2 g xylose and 0.8 g other monosaccharides per l was made. Clostridium acetobutylicum DSM 1731 produced 1.5 and C. beijerinckii B-592 0.9 g/l ABE and Clostridium LMD 84.48 1.9 g/l IBE, respectively, from this hydrolysate without further supplementation. Incubation with 2 fold concentrated hydrolysate completely impaired ABE production. After removal of unspecific inhibiting components, the yield of ABE production by Clostridium acetobutylicum DSM 1731 increased about 3 fold as compared to the nontreated hydrolysate. From 4 fold concentrated, partially purified, hydrolysate containing 34.2 g glucose/l, ABE production was 9.3 g/l after 120 h as compared to 3.2 g ABE/I from non-concentrated hydrolysate which contained 12.0 g glucose/l after elution over the same column. The concentration of butyric acid in the fermented hydrolysates was 2.2 and 0.4 g/l, respectively. This reasonably low amount of butyric acid showed that the fermentation had proceeded quite well.     

Identification and characterization of two functionally unknown genes involved in butanol tolerance of Clostridium acetobutylicum

Solvents toxicity is a major limiting factor hampering the cost-effective biotechnological production of chemicals. In Clostridium acetobutylicum, a functionally unknown protein (encoded by SMB_G1518) with a hypothetical alcohol interacting domain was identified. Disruption of SMB_G1518 and/or its downstream gene SMB_G1519 resulted in increased butanol tolerance, while overexpression of SMB_G1518-1519 decreased butanol tolerance. In addition, SMB_G1518-1519 also influences the production of pyruvate:ferredoxin oxidoreductase (PFOR) and flagellar protein hag, the maintenance of cell motility. We conclude that the system of SMB_G1518-1519 protein plays a role in the butanol sensitivity/tolerance phenotype of C. acetobutylicum, and can be considered as potential targets for engineering alcohol tolerance.     

Alcohol dehydrogenase: multiplicity and relatedness in the solvent-producing clostridia

Abstract: Alcohol dehydrogenase (ADH) is a key enzyme for the production of butanol, ethanol, and isopropanol by the solvent-producing clostridia. Initial studies of ADH in extracts of several strains of Clostridium acetobutylicum and C. beijerinckii gave conflicting molecular properties. A more coherent picture has emerged because of the following results: (i) identification of ADHs with different coenzyme specificities in these species; (ii) discovery of structurally conserved ADHs (type 3) in three solvent-producing species; (iii) isolation of mutants with deficiencies in butanol production and restoration of butanol production with a cloned alcohol/aldehyde dehydrogenase gene; and (iv) resolution of various ' C. acetobutylicum ' cultures into four species. The three ADH isozymes of C. beijerinckii NRRL B592 have high sequence similarities to ADH-1 of Clostridium sp. NCP 262 (formerly C. acetobutylicum P262) and to the ADH domain of the alcohol/aldehyde dehydrogenase of C. acetobutylicum ATCC 824/DSM 792. The NADH-dependent activity of the ADHs from C. beijerinckii NRRL B592 and the BDHs from C. acetobutylicum ATCC 824 is profoundly affected by the pH of the assay, and the relative importance of NADH and NADPH to butanol production may be misappraised when NAD(P)H-dependent activities were measured at different pH values. The primary/secondary ADH of isopropanol-producing C. beijerinckii is a type-1 enzyme and is highly conserved in Thermoanaerobacter brockii (formerly Thermoanaerobium brockii ) and Entamoeba histolytica . Several solvent-forming enzymes (primary ADH, aldehyde dehydrogenase, and 3-hydroxybutyryl-CoA dehydrogenase) are very similar between C. beijerinckii and the species represented by Clostridium sp. NCP 262 and NRRL B643. The realization of such relationships will facilitate the elucidation of the roles of different ADHs because each type of ADH can now be studied in an organism most amenable to experimental manipulations.     

Phenotypic and genotypic differences between certain strains of Clostridium acetobutylicum

Abstract It has become evident that several of the strains of Clostridium acetobutylicum that have been employed in physiological studies of the acetone-butanol fermentation, are heterogeneous. Studies of the phenotypic and genotypic characteristics of several of these strains (involving inter alia both pyrolysis mass spectrometry and 16S rRNA sequence determinations) demonstrated that the type strain obtained from ATCC was not identical with that supplied by NCIMB, and that NCIMB 8052T is in fact Clostridium beijerinckii . We therefore suggest that the name Clostridium acetobutylicum should be restricted to those strains that are genetically closely related to ATCC 824T (which include strains DSM 792 and DSM 1731 but not strain P262).     

提高丙酮丁醇梭菌产溶剂稳定性及防止退化的研究

在丙酮丁醇梭菌连续传代过程中,添加乙酸钠可增强其稳定性,同时在未添加乙酸钠的发酵液中分离获得溶剂产量明显降低的退化菌株DNU83,其丁醇产量为2.33 g·L-1,仅为初始菌株的1/6.培养基中添加乙酸钠、丁酸钠或K2 HPO4等弱酸盐均可恢复退化菌株的产溶剂能力,如同时添加苄基紫精,可显著促进丁醇合成.7％玉米培养基中添加4 g·L -1 K2 HPO4和30 mg·L-1苄基紫精,丁醇产量可达18.01 g·L-1,总溶剂21.59 g·L-1,丁醇比为83.43％,丁醇产量较未退化菌株NU22提高24.09％.     

Stability of solvent production by Clostridium acetobutylicum in continuous culture: strain differences

W oolley , R.C. & M orris , J.G. 1990. Stability of solvent production by Clostridium acetobutylicum in continuous culture: strain differences. Journal of Applied Bacteriology 69 , 718–728.
Several strains of Clostridium acetobutylicum , including strains ATCC 824 and DSM 1731, continue to produce solvents during prolonged periods of chemostat culture. In such cultures, dominance is established by asporogenous mutant(s) that retain the ability to produce solvents. Strain NCIB 8052 (which is not identical with ATCC 824) behaved differently in that its chemostat cultures invariably became acidogenic due to ultimate selection of asporogenous mutant(s) unable to produce solvents, incapable of synthesizing granulose, and demonstrating enhanced sensitivity to environmental stresses of various types. These mutants spontaneously reverted, at a low but measurable frequency, to the parental phenotype, indicating thai their multiple loss of capacities was the pleiotropic consequence of a lesion in some global regulatory gene. Their resemblance to previously described cls mutants of strain P262 and the possible nature of the affected regulatory gene are discussed. A simple tetrazolium blue plate assay procedure is described which allows visual discrimination between solvent-producing and non-solventogenic colonies of Cl. ocetobutylicum .     

高浓度丁醇浸泡及N＋束注入诱变双重作用筛选丁醇高产菌

通过高浓度丁醇浸泡处理丙酮丁醇梭菌（Clostridiumacetobutylicum）CL-2,筛选得到一株丁醇耐受能力提高并溶剂产量增加的菌株BR30—2,丁醇产量达11．77g/L,比CL-2提高了16．65％。以BR30—2作为出发菌株,进行N＋束注入诱变,筛选得到高产菌株BH．9,丁醇产量达14．5g/L,总溶剂为23．14g／L。在BH-9发酵过程中添加0．1％丁酸钠,丁醇产量达到16．59g/L,丁醇比例提高至67．38％。     

Molecular cloning of an alcohol (butanol) dehydrogenase gene cluster from Clostridium acetobutylicum ATCC 824.

In Clostridium acetobutylicum, conversion of butyraldehyde to butanol is enzymatically achieved by butanol dehydrogenase (BDH). A C. acetobutylicum gene that encodes this protein was identified by using an oligonucleotide designed on the basis of the N-terminal amino acid sequence of purified C. acetobutylicum NADH-dependent BDH. Enzyme assays of cell extracts of Escherichia coli harboring the clostridial gene demonstrated 15-fold-higher NADH-dependent BDH activity than untransformed E. coli, as well as an additional NADPH-dependent BDH activity. Kinetic, sequence, and isoelectric focusing analyses suggest that the cloned clostridial DNA contains two or more distinct C. acetobutylicum enzymes with BDH activity.     

Switching Clostridium acetobutylicum to an ethanol producer by disruption of the butyrate/butanol fermentative pathway

Solventogenic clostridia are well-known since almost a century due to their unique capability to biosynthesize the solvents acetone and butanol. Based on recently developed genetic engineering tools, a targeted 3-hydroxybutyryl-CoA dehydrogenase (Hbd)-negative mutant of Clostridium acetobutylicum was generated. Interestingly, the entire butyrate/butanol (C₄) metabolic pathway of C. acetobutylicum could be inactivated without a severe growth limitation and indicated the general feasibility to manipulate the central fermentative metabolism for product pattern alteration. Cell extracts of the mutant C. acetobutylicum hbd::int(69) revealed clearly reduced thiolase, Hbd and crotonase but increased NADH-dependent alcohol dehydrogenase enzyme activities as compared to the wildtype strain. Neither butyrate nor butanol were detected in cultures of C. acetobutylicum hbd::int(69), and the formation of molecular hydrogen was significantly reduced. Instead up to 16 and 20 g/l ethanol were produced in glucose and xylose batch cultures, respectively. Further sugar addition in glucose fed-batch fermentations increased the ethanol production to a final titer of 33 g/l, resulting in an ethanol to glucose yield of 0.38 g/g.     

The ald gene, encoding a coenzyme A-acylating aldehyde dehydrogenase, distinguishes Clostridium beijerinckii and two other solvent-producing clostridia from Clostridium acetobutylicum.

The coenzyme A (CoA)-acylating aldehyde dehydrogenase (ALDH) catalyzes a key reaction in the acetone- and butanol (solvent)-producing clostridia. It reduces acetyl-CoA and butyryl-CoA to the corresponding aldehydes, which are then reduced by alcohol dehydrogenase (ADH) to form ethanol and 1-butanol. The ALDH of Clostridium beijerinckii NRRL B593 was purified. It had no ADH activity, was NAD(H) specific, and was more active with butyraldehyde than with acetaldehyde. The N-terminal amino acid sequence of the purified ALDH was determined. The open reading frame preceding the ctfA gene (encoding a subunit of the solvent-forming CoA transferase) of C. beijerinckii NRRL B593 was identified as the structural gene (ald) for the ALDH. The ald gene encodes a polypeptide of 468 amino acid residues with a calculated M(r) of 51, 353. The position of the ald gene in C. beijerinckii NRRL B593 corresponded to that of the aad/adhE gene (encoding an aldehyde-alcohol dehydrogenase) of Clostridium acetobutylicum ATCC 824 and DSM 792. In Southern analyses, a probe derived from the C. acetobutylicum aad/adhE gene did not hybridize to restriction fragments of the genomic DNAs of C. beijerinckii and two other species of solvent-producing clostridia. In contrast, a probe derived from the C. beijerinckii ald gene hybridized to restriction fragments of the genomic DNA of three solvent-producing species but not to those of C. acetobutylicum, indicating a key difference among the solvent-producing clostridia. The amino acid sequence of the ALDH of C. beijerinckii NRRL B593 was most similar (41% identity) to those of the eutE gene products (CoA-acylating ALDHs) of Salmonella typhimurium and Escherichia coli, whereas it was about 26% identical to the ALDH domain of the aldehyde-alcohol dehydrogenases of C. acetobutylicum, E. coli, Lactococcus lactis, and amitochondriate protozoa. The predicted secondary structure of the C. beijerinckii ALDH suggests the presence of an atypical Rossmann fold for NAD(+) binding. A comparison of the proposed catalytic pockets of the CoA-dependent and CoA-independent ALDHs identified 6 amino acids that may contribute to interaction with CoA.     

Aldehyde-alcohol dehydrogenase and/or thiolase overexpression coupled with CoA transferase downregulation lead to higher alcohol titers and selectivity in Clostridium acetobutylicum fermentations

Metabolic engineering (ME) of Clostridium acetobutylicum has led to increased solvent (butanol, acetone, and ethanol) production and solvent tolerance, thus demonstrating that further efforts have the potential to create strains of industrial importance. With recently developed ME tools, it is now possible to combine genetic modifications and thus implement more advanced ME strategies. We have previously shown that antisense RNA (asRNA)-based downregulation of CoA transferase (CoAT, the first enzyme in the acetone-formation pathway) results in increased butanol to acetone selectivity, but overall reduced butanol yields and titers. In this study the alcohol/aldehyde dehydrogenase (aad) gene (encoding the bifunctional protein AAD responsible for butanol and ethanol production from butyryl-CoA and acetyl-CoA, respectively) was expressed from the phosphotransbutyrylase (ptb) promoter to enhance butanol formation and selectivity, while CoAT downregulation was used to minimize acetone production. This led to early production of high alcohol (butanol plus ethanol) titers, overall solvent titers of 30 g/L, and a higher alcohol/acetone ratio. Metabolic flux analysis revealed the likely depletion of butyryl-CoA. In order to increase then the flux towards butyryl-CoA, we examined the impact of thiolase (THL, thl) overexpression. THL converts acetyl-CoA to acetoacetyl-CoA, the first step of the pathway from acetyl-CoA to butyryl-CoA, and thus, combining thl overexpression with aad overexpression decreased, as expected, acetate and ethanol production while increasing acetone and butyrate formation. thl overexpression in strains with asRNA CoAT downregulation did not significantly alter product formation thus suggesting that a more complex metabolic engineering strategy is necessary to enhance the intracellular butyryl-CoA pool and reduce the acetyl-CoA pool in order to achieve improved butanol titers and selectivity.     

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.     

高丁醇比丙酮丁醇梭菌的选育与应用

设计了专一性分离方法,从土样中分离了多株能产生溶剂的梭苗,经多次单细胞分离、纯化,再经亚硝基胍和甲基磺酸乙酯诱变和抗性筛选,获得几株高丁醇的丙酮丁醇梭菌。对高产菌株的性状稳定性、发酵过程、混合原料应用、温度的影响进行了研究。结果证明菌株性状稳定,丁醇产量为总溶剂的７０％;过程为典型的丙酮丁醇发酵,对温度可耐受到３９－４０℃;能利用玉米和薯干,玉米和高梁进行正常发酵。菌株已在百吨生产罐,连续应用一年