丙酮丁醇发酵菌的分子遗传改造

丙酮丁醇梭菌及拜氏梭菌是重要的ABE（丙酮、丁醇和乙醇）工业生产菌株,其发酵产物中的丙酮和丁醇均为重要的化工原料,汽车发动机试验证明丁醇还是一种性能优于乙醇的极具潜力的生物燃料和燃料添加剂。随着新生物技术的不断发展及工业生产的需求,遗传工程改造不断应用于丙酮丁醇生产菌株。在前人研究及工业实践的基础上,对丙酮丁醇生产菌株的遗传特性及其分子遗传改造取得的进展进行了详细概述。     

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.     

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.     

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).     

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.     

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。     

Characterization of the cellulolytic complex (cellulosome) of Clostridium acetobutylicum

A large cellulosomal gene cluster was identified in the recently sequenced genome of Clostridium acetobutylicum ATCC 824. Sequence analysis revealed that this cluster contains the genes for the scaffolding protein CipA, the processive endocellulase Cel48A, several endoglucanases of families 5 and 9, the mannanase Man5G, and a hydrophobic protein, OrfXp. Surprisingly, genetic organization of this large cluster is very similar to that of Clostridium cellulolyticum, the model of mesophilic clostridial cellulosomes. As C. acetobutylicum is unable to grow on cellulosic substrates, the existence of a cellulosomal gene cluster in the genome raises questions about its expression, function and evolution. Biochemical evidence for the expression of a cellulosomal protein complex was investigated. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, N-terminal sequencing and Western blotting with antibodies against specific components of the C. cellulolyticum cellulosome suggest that at least four major cellulosomal proteins are present. In addition, despite the fact that no cellulolytic activities were detected, we report here the evidence for the production of a high molecular mass cellulosomal complex in C. acetobutylicum.     

Plasmid localization of a type E botulinal neurotoxin gene homologue in toxigenic Clostridium butyricum strains, and absence of this gene in non-toxigenic C. butyricum strains

It has been shown recently that two Clostridium butyricum strains (ATCC 43181 and ATCC 43755) contain a botulinal neurotoxin type E (BoNT/E) gene closely related to that of C. botulinum type E. In this study, we show that this gene is located on a large plasmid in the two toxigenic C. butyricum strains and is absent in 18 non-toxigenic C. butyricum and C. beijerinckii strains. Interestingly, the 230 bp upstream and the 1260 bp downstream of the neurotoxin coding sequence are not present in either the non-toxigenic C. butyricum or C. beijerinckii strains. Our data suggest a BoNT/E gene transfer from C. botulinum E to originally non-toxigenic C. butyricum strains.     

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.     

丙酮丁醇梭菌的遗传操作系统

丙酮丁醇梭菌是极具潜力的替代燃料——生物丁醇的合成菌,受到各国研究者的普遍关注。丙酮丁醇梭菌菌株改造是生物丁醇产业化进程中的一项重要工作,其中遗传操作是核心内容之一。以下对丙酮丁醇梭菌的遗传操作系统的发展历史、种类和原理进行了综述,分析了目前几种遗传操作系统的局限性,并对其发展进行了展望。     

D-2,3-butanediol production due to heterologous expression of an acetoin reductase in Clostridium acetobutylicum

Acetoin reductase (ACR) catalyzes the conversion of acetoin to 2,3-butanediol. Under certain conditions, Clostridium acetobutylicum ATCC 824 (and strains derived from it) generates both d- and l-stereoisomers of acetoin, but because of the absence of an ACR enzyme, it does not produce 2,3-butanediol. A gene encoding ACR from Clostridium beijerinckii NCIMB 8052 was functionally expressed in C. acetobutylicum under the control of two strong promoters, the constitutive thl promoter and the late exponential adc promoter. Both ACR-overproducing strains were grown in batch cultures, during which 89 to 90% of the natively produced acetoin was converted to 20 to 22 mM d-2,3-butanediol. The addition of a racemic mixture of acetoin led to the production of both d-2,3-butanediol and meso-2,3-butanediol. A metabolic network that is in agreement with the experimental data is proposed. Native 2,3-butanediol production is a first step toward a potential homofermentative 2-butanol-producing strain of C. acetobutylicum.     

Host-plasmid interactions in recombinant strains of Clostridium acetobutylicum ATCC 824

Abstract Plasmid-containing strains of Clostridium acetobutylicum produced higher levels of solvents and lower levels of acids than wild-type cells in controlled pH 4.5 batch fermentations. This effect was observed regardless of whether or not the plasmids contained C. acetobutylicum genes. The effect was less prevalent in higher pH fermentations and apparently independent of the actual DNA sequences contained on these plasmids. The plasmid-containing strains were found to have lower growth-rates and higher solventogenic enzyme activities than wild-type cells. However, similar activity levels were found for both butyrate-pathway enzymes.     

Complete activity profile of Clostridium acetobutylicum [FeFe]-hydrogenase and kinetic parameters for endogenous redox partners

In Clostridium acetobutylicum, [FeFe]-hydrogenase is involved in hydrogen production in vivo by transferring electrons from physiological electron donors, ferredoxin and flavodoxin, to protons. In this report, by modifications of the purification procedure, the specific activity of the enzyme has been improved and its complete catalytic profile in hydrogen evolution, hydrogen uptake, proton/deuterium exchange and para-H2/ortho-H2 conversion has been determined. The major ferredoxin expressed in the solvent-producing C. acetobutylicum cells was purified and identified as encoded by ORF CAC0303. Clostridium acetobutylicum recombinant holoflavodoxin CAC0587 was also purified. The kinetic parameters of C. acetobutylicum [FeFe]-hydrogenase for both physiological partners, ferredoxin CAC0303 and flavodoxin CAC0587, are reported for hydrogen uptake and hydrogen evolution activities.     

The regulation of thiomethylgalactoside transport in Clostridium acetobutylicum P262 by inducer exclusion and inducer expulsion mechanisms

Abstract Clostridium acetobutylicum P262 had phosphotransferase systems for glucose and lactose, and the lactose system was inducible. When C. acetobutylicum P262 was provided with glucose and lactose, the cultures grew in a diauxic fashion, and glucose was used preferentially. Cells grown on lactose took up thiomethylgalactoside, and retained this non-metabolizable lactose analog for long periods of time. Because glucose inhibited thiomethylgalactoside uptake and caused the efflux of thiomethylgalactoside that had already been taken up, it appeared that C. acetobutylicum P262 had inducer exclusion and inducer expulsion mechanisms similar to those found in lactic acid bacteria.     

Genes encoding homologues of three consecutive enzymes in the butyrate/butanol-producing pathway of Clostridium acetobutylicum are clustered on the Clostridium difficile chromosome

Abstract Screening of a Clostridium difficile ψEMBL3 gene library with antisera raised against C. difficile culture supernatant identified several clones expressing a 31-kDa protein. A 1.8-kb Hin dIII fragment subcloned from one of the clones was sufficient for expression of the 31-kDa polypeptide. Southern blot analysis showed a region homologous to this fragment to be present in all of 13 different C. difficile strains tested. Sequence analysis of the 1.8-kb fragment revealed three adjacent open reading frames. A database search showed that these three open reading frames appeared to encode homologues of three consecutive enzymes in the butanol/butyrate-producing pathway of Clostridium acetobutylicum (crotonase, β-hydroxybutyryl coenzyme A dehydrogenase and thiolase).     

Rapid expansion of the physical and genetic map of the chromosome of Clostridium perfringens CPN50.

The physical map of the 3.6-megabase chromosome of Clostridium perfringens CPN50 was extended by positioning sites for the endonucleases SfiI and I-CeuI, and in parallel, the gene map was expanded by using a genome scanning strategy. This involved the cloning and sequencing of random chromosomal fragments, identification of the functions of the putative genes by database searches, and then hybridization analysis. The current gene map comprises almost 100 markers, many of which encode housekeeping functions while others are involved in sporulation or pathogenesis. Strikingly, most of the virulence genes were found to be confined to a 1,200-kb segment of the chromosome near oriC, while the pleiotropic regulatory locus, virRS, was situated toward the putative replication terminus. A comparison of the gene maps of three endospore-forming bacilli, C. perfringens, Clostridium beijerinckii, and Bacillus subtilis, revealed a similar order and distribution of key sporulation and heat shock genes which might reflect an ancient evolutionary relationship.