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.     

Molecular genetics and the initiation of solventogenesis in Clostridium beijerinckii (formerly Clostridium acetobutylicum) NCIMB 8052

Abstract: A physical map of the Clostridium beijerinckii (formerly Clostridium acetobutylicum ) NCIMB 8052 chromosome has been constructed, encompassing about 90 rare restriction sites. The 14 rrn operons together with 40 genes have been assigned positions on the map. Genetic analysis and gene transfer have been developed in this organism to enable in vivo analysis of the roles of cloned genes using marker replacement technology. Experiments using the available genetic tools have shown that spo0A plays a cardinal role in controlling several aspects of the transition from exponential growth to stationary phase in C. beijerinckii . These include initiation of sporulation, accumulation of the storage polysaccharide, granulose, and production of acetone and butanol. Several C. beijerinckii and C. acetobutylicum genes concerned with fermentative metabolism, whose expression is modulated at the onset of solventogenesis, contain sequence motifs resembling 0A boxes in their 5' regulatory regions. This invites the speculation that they are under direct control of Spo0A, and additional data are now required to test this prediction.     

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)).     

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).     

Degeneration of solventogenic Clostridium strains monitored by Fourier transform infrared spectroscopy of bacterial cells

Strain degeneration in solventogenic clostridia is a known problem in the technical acetone–butanol fermentation bioprocess, especially in the continuous process mode. Clostridial strain degeneration was studied by Fourier transform infrared (FT-IR) spectroscopy of the bacterial cells. Degenerative variant formation in two strains, Clostridium beijerinckii NCIMB 8052 and Clostridium species AA332, was detected spectroscopically. Colonies on solid media were sampled, or assayed directly in situ by IR microscopy. It has previously been shown that the distinctive acidogenic and solventogenic physiological phases of Clostridium acetobutylicum in liquid medium can be discriminated by FT-IR spectroscopy. This was confirmed here for C. beijerinckii NCIMB 8052. The proportion of degenerate cells in a mixed population in liquid medium could be quantified, as the spectral features change in different ways during the normal growth cycle of wild type organisms and degenerate variants in batch culture. This opens a new perspective for physiology-based process monitoring and control, especially of the continuous acetone–butanol fermentation. Journal of Industrial Microbiology & Biotechnology (2001) 27, 314–321. Received 06 October 2000/ Accepted in revised form 20 April 2001     

Chemotherapeutic tumour targeting using clostridial spores

Abstract: The toxicity associated with conventional cancer chemotherapy is primarily due to a lack of specificity for tumour cells. In contrast, intravenously injected clostridial spores exhibit a remarkable specificity for tumours. This is because, following their administration, clostridial spores become exclusively localised to, and germinate in, the hypoxic/necrotic tissue of tumours. This unique property could be exploited to deliver therapeutic agents to tumours. In particular, genetic engineering could be used to endow a suitable clostridial host with the capacity to produce an enzyme within the tumour which can metabolise a systematically introduced, non-toxic prodrug into a toxic metabolite. The feasibility of this strategy (clostridial-directed enzyme prodrug therapy, CDEPT) has been demonstrated by cloning the Escherichia coli B gene encoding nitroreductase (an enzyme which converts the prodrug CB1954 to a highly toxic bifunctional alkylating agent) into a clostridial expression vector and introducing the resultant plasmid into Clostridium beijerinckii (formerly C. acetobutylicum ) NCIMB 8052. The gene was efficiently expressed, with recombinant nitroreductase representing 8% of the cell soluble protein. Following the intravenous injection of the recombinant spores into mice, tumour lysates have been shown, by Western blots, to contain the E. coli -derived enzyme.     

Isolation of a Degeneration-Resistant Mutant of Clostridium acetobutylicum NCIMB 8052

Unless periodically grown from germinated spores, Clostridium acetobutylicum tends to degenerate (that is, to spontaneously lose the capacity both to produce solvents and to develop into spores). To obtain mutants that are deficient in degeneration, C. acetobutylicum NCIMB 8052 was mated with Enterococcus faecalis BM4110 harboring transposon Tn1545. We developed a degeneration resistance assay based on a secondary effect of degeneration, the production of toxic levels of acetic and butyric acids. Erythromycin-resistant transconjugant clones were tested individually for longevity by repeated and timely subculturing. One long-lived mutant, A10, survived 18 ± 3 transfers (mean ± standard deviation; n = 20) before extinction, while the wild type (parental cells) survived 6.6 ± 1.5 transfers (n = 11). The three-fold difference in longevity is statistically significant. In a batch culture in a rich medium, the wild-type cells degenerated within 24 h after inoculation with 1% of an overnight culture derived from germinated spores. In contrast, A10 cells were able to switch to solventogenesis and to sporulate. In a minimal medium with greater buffering capacity, both cell types produced solvents and spores. Southern blots of EcoRI and HindIII restriction digests of A10 chromosomal DNA (but not parental DNA) showed that only one copy of Tn1545 was inserted into the clostridial chromosome. Our findings are consistent with the hypothesis that there was an alteration at a regulatory locus that was effected by the insertion of the transposon.     

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.     

Effect of Acetate on Molecular and Physiological Aspects of Clostridium beijerinckii NCIMB 8052 Solvent Production and Strain Degeneration

The addition of sodium acetate to chemically defined MP2 medium was found to increase and stabilize solvent production and also increase glucose utilization by Clostridium beijerinckii NCIMB 8052. RNA and enzyme analyses indicated that coenzyme A (CoA) transferase was highly expressed and has higher activity in C. beijerinckii NCIMB 8052 grown in MP2 medium containing added sodium acetate than in the microorganism grown without sodium acetate. RNA analysis suggested the existence of a sol operon and confirmed the presence of a ptb-buk operon in C. beijerinckii NCIMB 8052. In addition to CoA transferase, C. beijerinckii NCIMB 8052 grown in MP2 medium containing added acetate demonstrated higher acetate kinase- and butyrate kinase-specific activity than when the culture was grown in MP2 medium containing no added acetate. Southern blot analysis with chromosomal DNA isolated from solventogenic and degenerated C. beijerinckii NCIMB 8052 indicated that C. beijerinckii NCIMB 8052 strain degeneration does not involve loss of the CoA transferase genes. The addition of acetate to MP2 medium may induce the expression of the sol operon, which ensures solvent production and prevents strain degeneration in C. beijerinckii NCIMB 8052.     

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。     

Note: Sucrose transport and metabolism in Clostridium beijerinckii NCIMB 8052

Sucrose is the major carbon source in molasses, the traditional substrate employed in the industrial acetone-butanol-ethanol (ABE) fermentation by solventogenic clostridia. The utilization of sucrose by Clostridium beijerinckii NCIMB 8052 was investigated. Extracts prepared from cultures grown on sucrose (but not xylose or fructose) as the sole carbon source possessed sucrose phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) activity. Extract fractionation and reconstitution experiments revealed that the entire sucrose Enzyme II complex resides within the membrane in this organism. Sucrose-6-phosphate hydrolase and fructokinase activities were also detected in sucrose grown cultures. The fructokinase activity, which is required specifically during growth on sucrose, was shown to be inducible under these conditions. A pathway for sucrose metabolism in this organism is proposed.     

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.     

Butanol production by hypersolvent-producing mutant Clostridium beijerinckii BA101 in corn steep water medium containing maltodextrin

Clostridium beijerinckii NCIMB 8052 parent strain and BA101, a hypersolvent-producing mutant, fermented 6% (w/v) glucose, maltodextrin, maltose or xylose in a medium containing corn steep water (CSW) to produce butanol. Batch fermentation in an unoptimized 6% (w/v) maltodextrin plus 1.6% solids CSW medium demonstrated that C. beijerinckii NCIMB 8052 and BA101 produced 10.7 g butanol/L and 14.5 g butanol/L, respectively.     

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.     

A new insertion sequence, ISCb1, from Clostridium beijernickii NCIMB 8052

The NCIMB 8052 strain of Clostridium beijerinckii contains nine copies of a novel insertion sequence, ISCb1, belonging to the IS4 family. The 1764 bp element has 18 bp inverted repeats at its extremities, and generates 11 bp target repeats upon insertion. It contains a 1365 bp ORF whose predicted product (455 amino acids) resembles bacterial transposases. The highly conserved DD(35)E motif is present, as are signatures characteristic of the N3 and C1 domains of bacterial transposases. Codon usage of the ORF is somewhat different from that of other C. beijerinckii genes, suggesting that ISCb1 may have been acquired from another organism by horizontal gene transfer in the evolutionary past. One ISCb1 copy lies close to the site of insertion of Tn 1545 in a mutant strain, C10, which shows a reduced tendency to degenerate (i.e. loss of the potential to form solvents) compared with the wild type. In the C10 strain, the characteristic pattern of DNA fragments detected by an IS-specific probe was altered, but this was due to the Tn1545 insertion itself, rather than an ISCb1-mediated genome re-arrangement. There is currently no evidence that the element is involved in strain degeneration, since 12 independently isolated spontaneous mutants that had lost the ability to form solvents had the same ISCb1 profile as that of the wild type strain. The element is apparently restricted to a series of closely related solvent-forming clostridia.