Clostridium beijerinckii Cells Expressing Neocallimastix patriciarum Glycoside Hydrolases Show Enhanced Lichenan Utilization and Solvent Production

Growth and the production of acetone, butanol, and ethanol by Clostridium beijerinckii NCIMB 8052 on several polysaccharides and sugars were analyzed. On crystalline cellulose, growth and solvent production were observed only when a mixture of fungal cellulases was added to the medium. On lichenan growth and solvent production occurred, but this polymer was only partially utilized. To increase utilization of these polymers and subsequent solvent production, the genes for two new glycoside hydrolases, celA and celD from the fungus Neocallimastix patriciarum, were cloned separately into C. beijerinckii. To do this, a secretion vector based on the pMTL500E shuttle vector and containing the promoter and signal sequence coding region of the Clostridium saccharobutylicum NCP262 eglA gene was constructed and fused either to the celA gene or the celD gene. Stable C. beijerinckii transformants were obtained with the resulting plasmids, pWUR3 (celA) and pWUR4 (celD). The recombinant strains showed clear halos on agar plates containing carboxymethyl cellulose upon staining with Congo red. In addition, their culture supernatants had significant endoglucanase activities (123 U/mg of protein for transformants harboring celA and 78 U/mg of protein for transformants harboring celD). Although C. beijerinckii harboring either celA or celD was not able to grow, separately or in mixed culture, on carboxymethyl cellulose or microcrystalline cellulose, both transformants showed a significant increase in solvent production during growth on lichenan and more extensive degradation of this polymer than that exhibited by the wild-type strain.     

Cloning and expression of two cellulase genes of Clostridium cellulolyticum in Escherichia coli

Two cellulase genes isolated from Clostridium cellulolyticum strain ATCC3519 were cloned in Escherichia coli using plasmid pACYC184. Plasmids pB52 and pB43 were isolated from the transformants producing carboxymethylcellulase (CMCase) and the two cloned CMCase-coding genes were found to be included in two EcoRI fragments of 5.7 kb and 2.6 kb, respectively. These two genes showed no homology. The CMCase-coding genes were found to be contained in a 1.8-kb KpnI-HindIII fragment and a 2.05-kb HindIII-PvuII fragment of the DNA donor strain. Expression of these genes in E. coli was found not to depend on their orientation in the cloning vector. Hybridization experiments between these two fragments and Clostridium thermocellum NCIB10682 DNA fragments carrying genes celA, celB, celC and celD were carried out and some homologies were detected.     

Substrate-induced production and secretion of cellulases by Clostridium acetobutylicum

Clostridium acetobutylicum ATCC 824 is a solventogenic bacterium that grows heterotrophically on a variety of carbohydrates, including glucose, cellobiose, xylose, and lichenan, a linear polymer of beta-1,3- and beta-1,4-linked beta-D-glucose units. C. acetobutylicum does not degrade cellulose, although its genome sequence contains several cellulase-encoding genes and a complete cellulosome cluster of cellulosome genes. In the present study, we demonstrate that a low but significant level of induction of cellulase activity occurs during growth on xylose or lichenan. The celF gene, located in the cellulosome-like gene cluster and coding for a unique cellulase that belongs to glycoside hydrolase family 48, was cloned in Escherichia coli, and antibodies were raised against the overproduced CelF protein. A Western blot analysis suggested a possible catabolite repression by glucose or cellobiose and an up-regulation by lichenan or xylose of the extracellular production of CelF by C. acetobutylicum. Possible reasons for the apparent inability of C. acetobutylicum to degrade cellulose are discussed.     

Isolation of mesophilic solvent-producing clostridia from Colombian sources: physiological characterization, solvent production and polysaccharide hydrolysis

One hundred and seventy-eight new butanol-acetone producing bacteria related to saccharolytic clostridia were isolated from agricultural sources in Colombia and their fermentation potential was evaluated. Thirteen isolates produced more total solvents from glucose than Clostridium acetobutylicum ATCC 824. The isolates with the highest single solvent production were IBUN 125C and IBUN 18A with 0.46 mol butanol and 0.96 mol ethanol formed from 1 mol glucose, yielding 25. 2 and 29.1 g l(-1) total solvents, respectively, which is close to the maximum values described to date. Most of the new isolates produced exoenzymes for the hydrolysis of starch, carboxymethyl cellulose, xylan, polygalacturonic acid, inulin and chitosan. Together with the high efficiency of solvent production, these hydrolytic isolates may be useful for the direct fermentation of biomass. According to their physiological profile, the most solvent-productive isolates could be classified as strains of C. acetobutylicum, Clostridium beijerinckii, and Clostridium NCP262.     

New integration vector using a cellulase gene as a screening marker for Lactobacillus

The new integration vector for Lactobacillus, pJC4, was developed using the extracellular endoglucanase A gene (celA) of Clostridium thermocellum as a screening marker. pJC4 was transformed into four Lactobacillus species, Lb. johnsonii, Lb. gasseri, Lb. bulgaricus, and Lb. plantarum. In each species, the pJC4 integrants were easily and accurately detected by the appearance of a clear halo on a cellulase screening plate without any false transformants. Polymerase chain reaction and Southern hybridization indicated that all transformants with clear halos contained pJC4 in their chromosomal DNAs. The celA gene could be a useful screening marker for other lactic acid bacteria.     

Genetic systems development in the clostridia

Abstract: This review describes recent developments in the genetic manipulation of the solventogenic clostridia, Clostridium acetobutylicum and C. beijerinckii . It is to be noted that our laboratory stock of C. acetobutylicum ATCC 824, which was obtained from the American Type Culture Collection, has recently been re-identified as C. beijerinckii NCIMB 8052 based on DNA similarity studies using the S1 nuclease method (personal communication, Dr. Jiann-Shin Chen, Virginia Polytechnic Institute and State University). Reference to our laboratory 824 culture has been changed to C. beijerinckii NCIMB 8052 throughout this paper in order to be consistent with this finding. The focus of this review specifically involves the characterization of an M13-like genetic system for the clostridia based on the pCAK1 phagemid, as well as preliminary work on development of a plasmid-based vector based on the indigenous pDM11 plasmid recovered from C. acetobutylicum NCIB 6443. The construction of a C. beijerinckii strain with amplified endoglucanase activity was achieved by inserting the engB gene from C. cellulovorans into C. beijerinckii . The successful expression of a heterologous engB gene from C. cellulovorans in C. beijerinckii NCIMB 8052 has important industrial significance for the eventual utilization of cellulose by this acetone-butanol-ethanol fermentation microorganism.     

Enhanced Butanol Production by Clostridium beijerinckii BA101 Grown in Semidefined P2 Medium Containing 6 Percent Maltodextrin or Glucose

Dramatically elevated levels of butanol and acetone resulted in higher butanol and total solvent yields for hyperamylolytic Clostridium beijerinckii BA101 relative to the NCIMB 8052 parent strain grown in semidefined P2 medium containing either 6% glucose or STAR-DRI 5 maltodextrin. C. beijerinckii BA101 consistently produced on the order of 19 g of butanol per liter in 20-liter batch fermentations. This represents a greater than 100% increase in butanol concentration by the BA101 strain compared to the parent NCIMB 8052 strain. The kinetics of butanol production over time also indicate a more rapid rate of butanol production by BA101 in semidefined P2 medium containing glucose or maltodextrin. The lower levels of butyric and acetic acids produced over the course of the fermentation carried out by BA101 are consistent with an enhanced capacity for uptake and recycling of these acids. C. beijerinckii BA101 appears to more completely utilize carbohydrate compared to the 8052 strain. Carbon balance following fermentation by C. beijerinckii 8052 and BA101 indicates that sufficient carbon is available for the twofold increase in butanol concentration observed during BA101 fermentations. C. beijerinckii BA101 also has superior solvent production capacity during continuous culture fermentation in P2 medium containing 6% glucose. Volumetric solvent yields of 0.78 and 1.74 g/liter/h for BA101 and 0.34 and 1.17 g/liter/h for NCIMB 8052 were obtained at dilution rates of 0.05 and 0.20 h(sup-1), respectively. No drift towards acid synthesis (strain degeneration) was observed for up to 200 h (d = 0.05 h(sup-1)) and 100 h (d = 0.20 h(sup-1)).     

嗜热子囊菌JCM12803来源的双功能木聚糖/纤维素酶

纤维素和木聚糖的充分利用对于生物燃料的生产是非常重要的。文中利用PCR的方法从嗜热子囊菌Thermoascus crustaceus JCM12803中克隆到一个新颖的双功能木聚糖/纤维素酶基因Tcxyn10a,并将其在毕赤酵母Pichia pastoris GS115中实现高效异源表达。经过蛋白纯化和酶学性质研究分析,TcXyn10A的最适pH值和最适温度分别为5.0和65-70℃,能够在酸性至碱性(pH 3.0-11.0)条件下和60℃下保持稳定;对榉木木聚糖、小麦阿拉伯木聚糖、羧甲基纤维素钠和地衣多糖均有降解活性,比活分别为(1 480±26)U/mg、(2 055±28)U/mg、(7.4±0.2)U/mg和(10.9±0.4)U/mg;同源建模结构以及分子对接试验表明,双功能酶TcXyn10A只含有单一催化结构域,且木聚糖底物与纤维素底物共用一条催化通道。文中为探索双功能酶结构与其功能的关系提供了很好的素材。     

Cloning and sequencing of a cellobiose phosphotransferase system operon from Bacillus stearothermophilus XL-65-6 and functional expression in Escherichia coli.

Cellulolytic strains of Bacillus stearothermophilus were isolated from nature and screened for the presence of activities associated with the degradation of plant cell walls. One isolate (strain XL-65-6) which exhibited strong activities with 4-methylumbelliferyl-beta-D-glucopyranoside (MUG) and 4-methylumbelliferyl-beta-D-cellobiopyranoside (MUC) was used to construct a gene library in Escherichia coli. Clones degrading these model substrates were found to encode the cellobiose-specific genes of the phosphoenolpyruvate-dependent phosphotransferase system (PTS). Both MUG and MUC activities were present together, and both activities were lost concurrently during subcloning experiments. A functional E. coli ptsI gene was required for MUC and MUG activities (presumably a ptsH gene also). The DNA fragment from B. stearothermophilus contained four open reading frames which appear to form a cel operon. Intergenic stop codons for celA, celB, and celC overlapped the ribosomal binding sites of the respective downstream genes. Frameshift mutations or deletions in celA, celB, and celD were individually shown to result in a loss of MUC and MUG activities. On the basis of amino acid sequence homology and hydropathy plots of translated sequences, celA and celB were identified as encoding PTS enzyme II and celD was identified as encoding PTS enzyme III. These translated sequences were remarkably similar to their respective E. coli homologs for cellobiose transport. No reported sequences exhibited a high level of homology with the celC gene product. The predicted carboxy-terminal region for celC was similar to the corresponding region of E. coli celF, a phospho-beta-glucosidase. An incomplete regulatory gene (celR) and proposed promoter sequence were located 5' to the proposed cel operon. A stem-loop resembling a rho-independent terminator was present immediately downstream from celD. These results indicate that B. stearothermophilus XL-65-6 contains a cellobiose-specific PTS for cellobiose uptake. Similar systems may be present in other gram-positive bacteria.     

Metabolic engineering of d-xylose pathway in Clostridium beijerinckii to optimize solvent production from xylose mother liquid

Clostridium beijerinckii is an attractive butanol-producing microbe for its advantage in co-fermenting hexose and pentose sugars. However, this Clostridium strain exhibits undesired efficiency in utilizing d-xylose, one of the major building blocks contained in lignocellulosic materials. Here, we reported a useful metabolic engineering strategy to improve d-xylose consumption by C. beijerinckii. Gene cbei2385, encoding a putative d-xylose repressor XylR, was first disrupted in the C. beijerinckii NCIMB 8052, resulting in a significant increase in d-xylose consumption. A d-xylose proton-symporter (encoded by gene cbei0109) was identified and then overexpressed to further optimize d-xylose utilization, yielding an engineered strain 8052xylR-xylT(ptb) (xylR inactivation plus xylT overexpression driven by ptb promoter). We investigated the strain 8052xylR-xylT(ptb) in fermenting xylose mother liquid, an abundant by-product from industrial-scale xylose preparation from corncob and rich in d-xylose, finally achieving a 35% higher Acetone, Butanol and Ethanol (ABE) solvent titer (16.91g/L) and a 38% higher yield (0.29g/g) over those of the wild-type strain. The strategy used in this study enables C. beijerinckii more suitable for butanol production from lignocellulosic materials.     

Acetone, Isopropanol, and Butanol Production by Clostridium beijerinckii (syn. Clostridium butylicum) and Clostridium aurantibutyricum

Thirty-four strains representing 15 species of anaerobic bacteria were screened for acetone, isopropanol, and n-butanol (solvent) production. Under our culture conditions, several strains of Clostridium beijerinckii and C. aurantibutyricum produced at least 40 mM n-butanol (C. acetobutylicum strains produced up to 41 mM n-butanol under similar conditions). Both solvent-producing and non-solvent-producing strains of C. beijerinckii have high DNA homology with a reference strain of C. beijerinckii. Strains labeled “Clostridium butylicum” are phenotypically similar to C. beijerinckii and showed at least 78% DNA homology to a reference strain of C. beijerinckii. Therefore, these “C. butylicum” strains are members of C. beijerinckii. An earlier DNA homology study has shown that C. beijerinckii, C. aurantibutyricum, and C. acetobutylicum are distinct species.     

Cloning and expression of Clostridium thermocellum genes coding for thermostable exoglucanases (cellobiohydrolases) in Escherichia coli cells

By special screening approach two independent Cl. thermocellum genes directing the synthesis of thermostable glucanases with an exo-mode of action have been isolated from pUC19-based gene bank in E. coli TG1. The genes are located on 3.4 and 11.3 kb DNA fragments showing no homology. E. coli-derived exoglucanases, presumably, cellobiohydrolases, are able to cleave lichenan, carboxymethyl cellulose, xylan and p-nitrophenyl derivatives of cellobioside and lactoside. Cellobiose is the main degradation product of carboxymethyl cellulose, treated with the identified exoglucanases. With p-nitrophenil-beta-D-cellobioside as substrate the enzymes had a pH optimum around 6.5 and a temperature optimum at 65 degrees C. The identified and expressed enzymes differ from all other Cl. thermocellum proteins known to date.     

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.     

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.     

Transcriptional analysis of Clostridium beijerinckii NCIMB 8052 and the hyper-butanol-producing mutant BA101 during the shift from acidogenesis to solventogenesis

Clostridium beijerinckii is an anaerobic bacterium used for the fermentative production of acetone and butanol. The recent availability of genomic sequence information for C. beijerinckii NCIMB 8052 has allowed for an examination of gene expression during the shift from acidogenesis to solventogenesis over the time course of a batch fermentation using a ca. 500-gene set DNA microarray. The microarray was constructed using a collection of genes which are orthologs of members of gene families previously found to be important to the physiology of C. acetobutylicum ATCC 824. Similar to the onset of solventogenesis in C. acetobutylicum 824, the onset of solventogenesis in C. beijerinckii 8052 was concurrent with the initiation of sporulation. However, forespores and endospores developed more rapidly in C. beijerinckii 8052 than in C. acetobutylicum 824, consistent with the accelerated expression of the sigE- and sigG-regulated genes in C. beijerinckii 8052. The comparison of gene expression patterns and morphological changes in C. beijerinckii 8052 and the hyper-butanol-producing C. beijerinckii strain BA101 indicated that BA101 was less efficient in sporulation and phosphotransferase system-mediated sugar transport than 8052 but that it exhibited elevated expression of several primary metabolic genes and chemotaxis/motility genes.     

Clostridial strain degeneration

Abstract: Strain degeneration, the loss of the capacity to produce solvents and form spores, typically occurs when Clostridium acetobutylicum and related clostridia are repeatedly subcultured in batch culture or grown in continuous culture, as opposed to being grown from germinated, heat-treated spores. Several mechanisms for degeneration have been identified thus far. (i) Degeneration can be caused by excessive acidification of the culture during exponential growth. We present data interpreted to mean that C. beijerinckii (formerly C. acetobutylicum ) NCIMB 8052 cells ferment glucose to acetic and butyric acids at an uncontrolled rate, so that, during rapid growth, the rate of acid production can exceed the rate of induction of the solventogenic pathway enzymes. As a result, the medium pH drops to bactericical levels, and the cells cannot switch to solventogenesis and sporulation. The clostridia seem to be poised either to produce excess acids, or to initiate solventogenesis, depending on small differences in the rates of growth. (ii) We have isolated transposon-insertion mutants of C. beijerinckii NCIMB 8052 that are resistant to degeneration, suggesting the involvement of a regulatory region of the clostridial chromosome. (iii) Involvement of a global regulatory gene has been inferred in C. beijerinckii NCIMB 8052 which degenerates irreversibly in chemostat culture. (iv) Impairment of butanol formation due to a defect in NADH generation has been reported in an oligosporogenous strain which can revert to the non-degenerate phenotype. (v) In continuous culture, degenerate cells may be selected because they continue to divide, while the non-degenerate cells stop dividing and start differentiating.     

Nutrient limitation of two saccharolytic clostridia; secretion, sporulation, and solventogenesis

Abstract Growth of the cellulolytic acidogen Clostridium strain C7 undergoing progressive nutrient limitation has been compared with that of the solventogen Clostridium beijerinckii . On the basis of cellulase secretion, differentiation of dissimilatory metabolism, and sporulation, different survival strategies by the two clostridia in progressive nutrient limitation can be discerned. In addition, the metabolic differentiation to butanol production in Clostridium beijerinckii can be specifically associated with the sporulation stage in which the forespore is enclosed by double membranes but not by a spore coat.