Members of the anaerobic thermophilic bacterial genus Caldicellulosiruptor are emerging candidates for consolidated bioprocessing (CBP) because they are capable of efficiently growing on biomass without conventional pretreatment. C. bescii produces primarily lactate, acetate and hydrogen as fermentation products, and while some Caldicellulosiruptor strains produce small amounts of ethanol C. bescii does not, making it an attractive background to examine the effects of metabolic engineering. The recent development of methods for genetic manipulation has set the stage for rational engineering of this genus for improved biofuel production. Here, we report the first targeted gene deletion, the gene encoding lactate dehydrogenase (ldh), for metabolic engineering of a member of this genus.
A deletion of the C. bescii L-lactate dehydrogenase gene (ldh) was constructed on a non-replicating plasmid and introduced into the C. bescii chromosome by marker replacement. The resulting strain failed to produce detectable levels of lactate from cellobiose and maltose, instead increasing production of acetate and H2 by 21-34% relative to the wild type and ΔpyrFA parent strains. The same phenotype was observed on a real-world substrate – switchgrass (Panicum virgatum). Furthermore, the ldh deletion strain grew to a higher maximum optical density than the wild type on maltose and cellobiose, consistent with the prediction that the mutant would gain additional ATP with increased acetate production.
Deletion of ldh in C. bescii is the first use of recently developed genetic methods for metabolic engineering of these bacteria. This deletion resulted in a redirection of electron flow from production of lactate to acetate and hydrogen. New capabilities in metabolic engineering combined with intrinsic utilization of lignocellulosic materials position these organisms to provide a new paradigm for consolidated bioprocessing of fuels and other products from biomass.
ldh; Metabolic engineering; Switchgrass; Biohydrogen; Caldicellulosiruptor
Thermophilic microorganisms have special advantages for the conversion of plant biomass to fuels and chemicals. Members of the genus Caldicellulosiruptor are the most thermophilic cellulolytic bacteria known. They have the ability to grow on a variety of non-pretreated biomass substrates at or near ~80°C and hold promise for converting biomass to bioproducts in a single step. As for all such relatively uncharacterized organisms with desirable traits, the ability to genetically manipulate them is a prerequisite for making them useful. Metabolic engineering of pathways for product synthesis is relatively simple compared to engineering the ability to utilize non-pretreated biomass.
Here we report the construction of a deletion of cbeI (Cbes2438), which encodes a restriction endonuclease that is as a major barrier to DNA transformation of C. bescii. This is the first example of a targeted chromosomal deletion generated by homologous recombination in this genus and the resulting mutant, JWCB018 (ΔpyrFA ΔcbeI), is readily transformed by DNA isolated from E. coli without in vitro methylation. PCR amplification and sequencing suggested that this deletion left the adjacent methyltransferase (Cbes2437) intact. This was confirmed by the fact that DNA isolated from JWCB018 was protected from digestion by CbeI and HaeIII. Plasmid DNA isolated from C. hydrothermalis transformants were readily transformed into C. bescii. Digestion analysis of chromosomal DNA isolated from seven Caldicellulosiruptor species by using nine different restriction endonucleases was also performed to identify the functional restriction-modification activities in this genus.
Deletion of the cbeI gene removes a substantial barrier to routine DNA transformation and chromosomal modification of C. bescii. This will facilitate the functional analyses of genes as well as metabolic engineering for the production of biofuels and bioproducts from biomass. An analysis of restriction-modification activities in members of this genus suggests a way forward to eliminating restriction as a barrier to DNA transformation and efficient genetic manipulation of this important group of hyperthermophiles.
Caldicellulosiruptor species; Biomass conversion; Restriction-modification enzymes; CbeI; M.CbeI; Targeted deletion
We recently reported the isolation of a mutant of Pyrococcus furiosus, COM1, that is naturally and efficiently competent for DNA uptake. While we do not know the exact nature of this mutation, the combined transformation and recombination frequencies of this strain allow marker replacement by direct selection using linear DNA. In testing the limits of its recombination efficiency, we discovered that marker replacement was possible with as few as 40 nucleotides of flanking homology to the target region. We utilized this ability to design a strategy for selection of constructed deletions using PCR products with subsequent excision, or “pop-out,” of the selected marker. We used this method to construct a “markerless” deletion of the trpAB locus in the GLW101 (COM1 ΔpyrF) background to generate a strain (JFW02) that is a tight tryptophan auxotroph, providing a genetic background with two auxotrophic markers for further strain construction. The utility of trpAB as a selectable marker was demonstrated using prototrophic selection of plasmids and genomic DNA containing the wild-type trpAB alleles. A deletion of radB was also constructed that, surprisingly, had no obvious effect on either recombination or transformation, suggesting that its gene product is not involved in the COM1 phenotype. Attempts to construct a radA deletion mutation were unsuccessful, suggesting that this may be an essential gene. The ease and speed of this procedure will facilitate the construction of strains with multiple genetic changes and allow the construction of mutants with deletions of virtually any nonessential gene.
Thermophilic microorganisms capable of using complex substrates offer special advantages for the conversion of lignocellulosic biomass to biofuels and bioproducts. Members of the Gram-positive bacterial genus Caldicellulosiruptor are anaerobic thermophiles with optimum growth temperatures between 65°C and 78°C and are the most thermophilic cellulolytic organisms known. In fact, they efficiently use biomass non-pretreated as their sole carbon source and in successive rounds of application digest 70% of total switchgrass substrate. The ability to genetically manipulate these organisms is a prerequisite to engineering them for use in conversion of these complex substrates to products of interest as well as identifying gene products critical for their ability to utilize non-pretreated biomass. Here, we report the first example of DNA transformation of a member of this genus, C. bescii. We show that restriction of DNA is a major barrier to transformation (in this case apparently absolute) and that methylation with an endogenous unique α-class N4-Cytosine methyltransferase is required for transformation of DNA isolated from E. coli. The use of modified DNA leads to the development of an efficient and reproducible method for DNA transformation and the combined frequencies of transformation and recombination allow marker replacement between non-replicating plasmids and chromosomal genes providing the basis for rapid and efficient methods of genetic manipulation.
Transcriptional and enzymatic analyses of Pyrococcus furiosus previously indicated that three proteins play key roles in the metabolism of elemental sulfur (S0): a membrane-bound oxidoreductase complex (MBX), a cytoplasmic coenzyme A-dependent NADPH sulfur oxidoreductase (NSR), and sulfur-induced protein A (SipA). Deletion strains, referred to as MBX1, NSR1, and SIP1, respectively, have now been constructed by homologous recombination utilizing the uracil auxotrophic COM1 parent strain (ΔpyrF). The growth of all three mutants on maltose was comparable without S0, but in its presence, the growth of MBX1 was greatly impaired while the growth of NSR1 and SIP1 was largely unaffected. In the presence of S0, MBX1 produced little, if any, sulfide but much more acetate (per unit of protein) than the parent strain, demonstrating that MBX plays a critical role in S0 reduction and energy conservation. In contrast, comparable amounts of sulfide and acetate were produced by NSR1 and the parent strain, indicating that NSR is not essential for energy conservation during S0 reduction. Differences in transcriptional responses to S0 in NSR1 suggest that two sulfide dehydrogenase isoenzymes provide a compensatory NADPH-dependent S0 reduction system. Genes controlled by the S0-responsive regulator SurR were not as highly regulated in MBX1 and NSR1. SIP1 produced the same amount of acetate but more sulfide than the parent strain. That SipA is not essential for growth on S0 indicates that it is not required for detoxification of metal sulfides, as previously suggested. A model is proposed for S0 reduction by P. furiosus with roles for MBX and NSR in bioenergetics and for SipA in iron-sulfur metabolism.
We report the construction of a series of replicating shuttle vectors that consist of a low-copy-number cloning vector for Escherichia coli and functional components of the origin of replication (oriC) of the chromosome of the hyperthermophilic archaeon Pyrococcus furiosus. In the process of identifying the minimum replication origin sequence required for autonomous plasmid replication in P. furiosus, we discovered that several features of the origin predicted by bioinformatic analysis and in vitro binding studies were not essential for stable autonomous plasmid replication. A minimum region required to promote plasmid DNA replication was identified, and plasmids based on this sequence readily transformed P. furiosus. The plasmids replicated autonomously and existed in a single copy. In contrast to shuttle vectors based on a plasmid from the closely related hyperthermophile Pyrococcus abyssi for use in P. furiosus, plasmids based on the P. furiosus chromosomal origin were structurally unchanged after transformation and were stable without selection for more than 100 generations.
In attempts to develop a method of introducing DNA into Pyrococcus furiosus, we discovered a variant within the wild-type population that is naturally and efficiently competent for DNA uptake. A pyrF gene deletion mutant was constructed in the genome, and the combined transformation and recombination frequencies of this strain allowed marker replacement by direct selection using linear DNA. We have demonstrated the use of this strain, designated COM1, for genetic manipulation. Using genetic selections and counterselections based on uracil biosynthesis, we generated single- and double-deletion mutants of the two gene clusters that encode the two cytoplasmic hydrogenases. The COM1 strain will provide the basis for the development of more sophisticated genetic tools allowing the study and metabolic engineering of this important hyperthermophile.
Caldicellulosiruptor bescii DSM 6725 utilizes various polysaccharides and grows efficiently on untreated high-lignin grasses and hardwood at an optimum temperature of ∼80°C. It is a promising anaerobic bacterium for studying high-temperature biomass conversion. Its genome contains 2666 protein-coding sequences organized into 1209 operons. Expression of 2196 genes (83%) was confirmed experimentally. At least 322 genes appear to have been obtained by lateral gene transfer (LGT). Putative functions were assigned to 364 conserved/hypothetical protein (C/HP) genes. The genome contains 171 and 88 genes related to carbohydrate transport and utilization, respectively. Growth on cellulose led to the up-regulation of 32 carbohydrate-active (CAZy), 61 sugar transport, 25 transcription factor and 234 C/HP genes. Some C/HPs were overproduced on cellulose or xylan, suggesting their involvement in polysaccharide conversion. A unique feature of the genome is enrichment with genes encoding multi-modular, multi-functional CAZy proteins organized into one large cluster, the products of which are proposed to act synergistically on different components of plant cell walls and to aid the ability of C. bescii to convert plant biomass. The high duplication of CAZy domains coupled with the ability to acquire foreign genes by LGT may have allowed the bacterium to rapidly adapt to changing plant biomass-rich environments.
Very few cultivated microorganisms can degrade lignocellulosic biomass without chemical pretreatment. We show here that “Anaerocellum thermophilum” DSM 6725, an anaerobic bacterium that grows optimally at 75°C, efficiently utilizes various types of untreated plant biomass, as well as crystalline cellulose and xylan. These include hardwoods such as poplar, low-lignin grasses such as napier and Bermuda grasses, and high-lignin grasses such as switchgrass. The organism did not utilize only the soluble fraction of the untreated biomass, since insoluble plant biomass (as well as cellulose and xylan) obtained after washing at 75°C for 18 h also served as a growth substrate. The predominant end products from all growth substrates were hydrogen, acetate, and lactate. Glucose and cellobiose (on crystalline cellulose) and xylose and xylobiose (on xylan) also accumulated in the growth media during growth on the defined substrates but not during growth on the plant biomass. A. thermophilum DSM 6725 grew well on first- and second-spent biomass derived from poplar and switchgrass, where spent biomass is defined as the insoluble growth substrate recovered after the organism has reached late stationary phase. No evidence was found for the direct attachment of A. thermophilum DSM 6725 to the plant biomass. This organism differs from the closely related strain A. thermophilum Z-1320 in its ability to grow on xylose and pectin. Caldicellulosiruptor saccharolyticus DSM 8903 (optimum growth temperature, 70°C), a close relative of A. thermophilum DSM 6725, grew well on switchgrass but not on poplar, indicating a significant difference in the biomass-degrading abilities of these two otherwise very similar organisms.
“Anaerocellum thermophilum” DSM 6725 is a strictly anaerobic bacterium that grows optimally at 75°C. It uses a variety of polysaccharides, including crystalline cellulose and untreated plant biomass, and has potential utility in biomass conversion. Here we report its complete genome sequence of 2.97 Mb, which is contained within one chromosome and two plasmids (of 8.3 and 3.6 kb). The genome encodes a broad set of cellulolytic enzymes, transporters, and pathways for sugar utilization and compared to those of other saccharolytic, anaerobic thermophiles is most similar to that of Caldicellulosiruptor saccharolyticus DSM 8903.
Both morphogenesis and antibiotic production in the streptomycetes are initiated in response to starvation, and these events are coupled. We previously described a transposon-generated mutant in Streptomyces coelicolor, SE293, that resulted in a bld strain that overproduced the antibiotic actinorhodin. The SCO1135 open reading frame identified by the insertion encodes a member of the TetR family of transcriptional regulators. Here we show that a constructed deletion of the SCO1135 open reading frame resulted in the same morphological and antibiotic production phenotype as the insertion mutant. The constructed deletion also resulted in constitutive expression of SCO1135 transcript, as well as that of the gene cluster immediately adjacent to it, SCO1134-1132, which encodes a putative molybdopterin binding complex. A His6-tagged version of the SCO1135 protein product was shown to bind the intergenic region between SCO1135 and SCO1134, which contains the apparent transcription start sites for each gene mapped by primer extension analysis. Increased expression of the SCO1134-1132 transcript in the SCO1135 deletion mutant also resulted in increased expression of xanthine dehydrogenase activity, confirming the predictions about these open reading framed based on protein similarity. We have designated the SCO1134-1142 gene cluster xdhABC and the regulator encoded by SCO1135 xdhR. We speculate that the inappropriate expression of xanthine dehydrogenase affects purine salvaging pathways at the onset of development, creating artificially high concentrations of both GTP and ppGpp and perturbing the pathways these molecules participate in for the initiation of morphogenesis and antibiotic production.
The Actinomycete Actinosynnema pretiosum ssp. auranticum has commercial importance due to its production of ansamitocin P-3 (AP-3), a potent antitumor agent. One way to increase AP-3 production would be to constitutively express selected genes so as to relieve bottlenecks in the biosynthetic pathway; however, an integrative expression vector for A. pretiosum is lacking. The aim of this study was to construct a vector for heterologous gene expression in A. pretiosum.
A series of integrative expression vectors have been made with the following features: the IS117 transposase from Streptomyces coelicolor, the constitutive ermE* promoter from Saccharopolyspora erythraea, different ribosome-binding site (RBS) sequences and xylE as a translational reporter. Positive E. coli clones and A. pretiosum transconjugants were assayed by catechol. pAP42, containing an E. coli consensus RBS, and pAP43, containing an asm19 RBS, gave strong and moderate gene expression, respectively. In addition, an operon construct capable of multi-gene expression was created. Plasmid integration sites in transconjugants were investigated and four different sites were observed. Although the most common integration site was within a putative ORF with sequence similarity to NADH-flavin reductase, AP-3 levels and cell growth of transconjugants were unaffected.
A set of integrative vectors for constitutive gene expression in A. pretiosum has been constructed. Gene translation is easily determined by colorimetric assay on an agar plate. The vectors are suitable for studies relating to AP-3 biosynthesis as they do not affect AP-3 production.
We recently reported the isolation and initial characterization of a transposon-generated mutation that resulted in defects in both morphogenesis and antibiotic production in Streptomyces coelicolor. The insertion identified the SCO7168 open reading frame whose predicted product is a GntR family transcriptional regulator. Here, we show that this gene acts to repress transcription of itself as well as a series of genes immediately adjacent to it on the S. coelicolor chromosome that likely encode an ATP-binding cassette (ABC)-type transporter for carbohydrate uptake. Transcription of this transporter is strongly induced by growth on relatively poor carbon sources such as trehalose and melibiose and weakly induced by lactose and glycerol but not glucose, and induction is not repressed by the presence of glucose. Constructed deletions of the ABC transporter itself resulted in the suppression of the original transposon mutation, suggesting that inappropriate expression of the ABC transporter is responsible, at least in part, for the mutant phenotype. Because this transporter responds to the presence of α-glucosides and has similarity to two other carbohydrate transporters of this class, we have named the genes of the transporter agl3E, agl3F, and agl3G and the GntR-like protein that regulates transcription of the transporter agl3R in accordance with established nomenclature. We suggest that agl3R is one of a number of homologous proteins in Streptomyces (there are 57 putative GntR family regulators in the S. coelicolor genome) that respond to nutritional and/or environmental signals to control genes that affect morphogenesis and antibiotic production.
Products from the degradation of the branched-chain amino acids valine, leucine, and isoleucine contribute to the production of a number of important cellular metabolites, including branched-chain fatty acids, ATP and other energy production, cell-cell signaling for morphological development, and the synthesis of precursors for polyketide antibiotics. The first nonreversible reactions in the degradation of all three amino acids are catalyzed by the same branched-chain α-keto acid dehydrogenase (BCDH) complex. Actinomycetes are apparently unique among bacteria in that they contain two separate gene clusters, each of which encodes a BCDH enzyme complex. Here, we show that one of these clusters in Streptomyces coelicolor is regulated, at least in part, at the level of transcription by the product of the bkdR gene. The predicted product of this gene is a protein with similarity to a family of proteins that respond to leucine and serve to activate transcription of amino acid utilization operons. Unlike most other members of this class, however, the S. coelicolor bkdR gene product serves to repress transcription, suggesting that the branched-chain amino acids act as inducers rather than coactivators of transcription. BkdR likely responds to the presence of branched-chain amino acids. Its role in transcriptional regulation may be rationalized by the fact that transition from vegetative growth to aerial mycelium production, the first stage of morphological development in these complex bacteria, is coincident with extensive cellular lysis generating abundant amounts of protein that likely serve as the predominant source of carbon and nitrogen for metabolism. We suggest that bkdR plays a key role in the ability of Streptomyces species to sense nutrient availability and redirect metabolism for the utilization of branched-chain amino acids for energy, carbon, and perhaps even morphogen synthesis. A null mutant of bkdR is itself defective in morphogenesis and antibiotic production, suggesting that the role of the bkdR gene product may be more global than specific nutrient utilization.
We report the isolation and partial characterization of three new mutants of Streptomyces coelicolor that are defective in morphogenesis and antibiotic production. The genes identified by the mutations were located and cloned by using a combination of Tn5 in vitro mutagenesis, cotransformation, and genetic complementation. Mutant SE69 produces lower amounts of antibiotics than the wild type produces, produces spores only after prolonged incubation on rich media, and identifies a gene whose predicted protein product is similar to the GntR family of transcriptional regulators; also, production of aerial mycelia on both rich and poor media is significantly delayed in this mutant. Mutant SE293 is defective in morphogenesis, overproduces antibiotics on rich media, fails to grow on minimal media, and identifies a gene whose predicted protein product is similar to the TetR family of transcriptional regulators. Preliminary evidence suggests that the SE293 gene product may control a molybdopterin binding protein located immediately adjacent to it. Mutant SJ175 sporulates sooner and more abundantly than the wild type and overproduces antibiotics on rich media, and it identifies a gene whose predicted protein product contains regions of predominantly hydrophobic residues similar to those of integral membrane proteins.
We have demonstrated that the bldB gene of Streptomyces coelicolor is required for the formation of aerial hyphae and the synthesis of antibiotics. We also found that BldB forms a higher-order complex (most likely a dimer) and that amino acid residues 20 to 78 are important for this interaction. This region is conserved in the BldB family, suggesting that dimer formation may be a common feature of these proteins.
Xylanase activity assays were used to screen a Streptomyces coelicolor genomic library in Escherichia coli, and a xylanase gene that is 99% identical to the xylanase B gene (xlnB) of S. lividans (GenBank accession no. M64552) was identified. The promoter region of this gene was identified by using a transcriptional fusion between the upstream region of the S. coelicolor xlnB gene and the xylE reporter gene. Transcription from the xlnB promoter was found to be induced by xylan and repressed by glucose. A single apparent transcription start site was identified by both primer extension analysis and in vitro run off transcription assays. Analysis of deletions of the promoter identified a region required for glucose repression. By using the transcriptional and protein localization signals of the Streptomyces xlnB gene, an in-frame translational fusion between the end of the xlnB signal sequence and the ATG of the Thermotoga maritima xynA gene was constructed. The xynA gene encodes a thermostable xylanase that has been demonstrated to be useful in the bleaching of Kraft pulp. The xlnB-xynA gene fusion was expressed in Streptomyces, and the activity of the protein produced was thermostable and was localized to the supernatant fraction of harvested cells.
Mutants blocked at the earliest stage of morphological development in Streptomyces species are called bld mutants. These mutants are pleiotropically defective in the initiation of development, the ability to produce antibiotics, the ability to regulate carbon utilization, and the ability to send and/or respond to extracellular signals. Here we report the identification and partial characterization of a 99-amino-acid open reading frame (ORF99) that is capable of restoring morphogenesis, antibiotic production, and catabolite control to all of the bldB mutants. Of the existing bld mutants, bldB is of special interest because the phenotype of this mutant is the most pleiotropic. DNA sequence analysis of ORF99 from each of the existing bldB mutants identified base changes either within the coding region of the predicted protein or in the regulatory region of the gene. Primer extension analysis identified an apparent transcription start site. A promoter fusion to the xylE reporter gene showed that expression of bldB is apparently temporally regulated and that the bldB gene product is involved in the regulation of its own expression.