Janet Westpheling

Efficient Degradation of Lignocellulosic Plant Biomass, without Pretreatment, by the Thermophilic Anaerobe “Anaerocellum thermophilum” DSM 6725

ABSTRACT 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.

Natural Competence in the Hyperthermophilic Archaeon Pyrococcus furiosus Facilitates Genetic Manipulation: Construction of Markerless Deletions of Genes Encoding the Two Cytoplasmic Hydrogenases

ABSTRACT 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.

Direct conversion of plant biomass to ethanol by engineered Caldicellulosiruptor bescii

Significance The ever-increasing demand for transportation fuels, the decrease in global petroleum reserves, and the negative impact of greenhouse gases resulting from burning petroleum make renewable and sustainable biofuels an imperative for the future. First-generation biofuels produced from food crops are limited by cost and competition with food supply. Considerable effort has been made to produce fuels from lignocellulosic biomass, but the need for chemical and enzymatic pretreatment to solubilize the biomass prior to microbial bioconversion is a major economic barrier to the development of an industrial process. Here we report the metabolic engineering of a bacterium, Caldicellulosiruptor bescii, that is capable of using unprocessed switchgrass, an abundant, environmentally desirable, and economically sustainable lignocellulosic plant biomass, as feedstock to produce ethanol. Ethanol is the most widely used renewable transportation biofuel in the United States, with the production of 13.3 billion gallons in 2012 [John UM (2013) Contribution of the Ethanol Industry to the Economy of the United States]. Despite considerable effort to produce fuels from lignocellulosic biomass, chemical pretreatment and the addition of saccharolytic enzymes before microbial bioconversion remain economic barriers to industrial deployment [Lynd LR, et al. (2008) Nat Biotechnol 26(2):169–172]. We began with the thermophilic, anaerobic, cellulolytic bacterium Caldicellulosiruptor bescii, which efficiently uses unpretreated biomass, and engineered it to produce ethanol. Here we report the direct conversion of switchgrass, a nonfood, renewable feedstock, to ethanol without conventional pretreatment of the biomass. This process was accomplished by deletion of lactate dehydrogenase and heterologous expression of a Clostridium thermocellum bifunctional acetaldehyde/alcohol dehydrogenase. Whereas wild-type C. bescii lacks the ability to make ethanol, 70% of the fermentation products in the engineered strain were ethanol [12.8 mM ethanol directly from 2% (wt/vol) switchgrass, a real-world substrate] with decreased production of acetate by 38% compared with wild-type. Direct conversion of biomass to ethanol represents a new paradigm for consolidated bioprocessing, offering the potential for carbon neutral, cost-effective, sustainable fuel production.

The bld mutants of Streptomyces coelicolor are defective in the regulation of carbon utilization, morphogenesis and cell–cell signalling

Mutants of Streptomyces coelicolor blocked at the earliest visible stage of morphological differentiation are called bld mutants. These mutants fail to form aerial hyphae on rich medium and most are defective in antibiotic production. One striking feature of these mutants is that, with the exception of bldB, their morphological defect is carbon‐source dependent. In our investigation of catabolite control in Streptomyces, we identified mutants that were resistant to glucose repression and were also bld. The existence of these new bld mutants led us to examine the catabolite control phenotype of the previously described bld mutants which were not known to contain defects in carbon regulation. We report here that all of the characterized bld mutants of S.coelicolor are defective in the regulation of galP1, and that at least one of the bld mutants, bldB, is globally deregulated for carbon utilization. Complementation of the morphological defect of bldA and bldB mutants with a cloned copy of the wild‐type bld gene simultaneously restored normal regulation of galP1, indicating that both aspects of the mutant phenotype are caused by the same lesion. We suggest a new interpretation for the role of the bld genes in development in Streptomyces. We suggest that the primary defect in bld mutants is in the regulation of carbon utilization, not specifically in the activation of genes whose products regulate the development pathway as previously suggested. We speculate that the inability of bld mutants to initiate morphogenesis is a secondary consequence of their inability to sense and/or signal starvation.

Structure and function of the Bacillus SpoIIE protein and its localization to sites of sporulation septum assembly

Functioning of the spoIIE locus of Bacillus subtilis is required for formation of a normal polar septum during sporulation and for activation of the transcription factor σF, which directs early forespore‐specific gene expression. We have determined the DNA sequence of the wild type and several mutant alleles of the spoIIE gene of B. subtilis and sequenced a substantial portion of its presumptive homologue in Bacillus megaterium. We show that the spoIIE locus encodes a single large protein with a predicted molecular mass of 92 kDa. Each of five point‐mutation alleles, which have traditionally defined the locus, and two transposon‐generated mutations were shown to fall within the coding sequence for the 92 kDa gene product or within sequences expected to be required for its expression. The amino‐terminal portion of the predicted SpoIIE gene product, comprising approximately 40% of the protein, is extremely hydrophobic and is expected to contain up to 12 membrane‐spanning segments. The remainder of the protein contains no hydrophobic segments long enough to span a lipid bilayer and is therefore presumed to comprise one or more globular, aqueous‐phase exposed domains. An in‐frame fusion joining the 3′ end of the B. megaterium spoIIE coding sequence to the 5′ end of gfp, a gene encoding the green fluorescent protein (GFP) of Aquorea victoria, resulted in a strong, sporulation‐specific fluorescent signal localized to the sites of sporulation septum assembly. We speculate that SpoIIE plays a role in assembling the sporulation septum, perhaps determining the special properties of the structure that permit intercompartment signalling during development.

Metabolic engineering of Caldicellulosiruptor bescii yields increased hydrogen production from lignocellulosic biomass

BackgroundMembers 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.ResultsA 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.ConclusionsDeletion 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.

A New GntR Family Transcriptional Regulator in Streptomyces coelicolor Is Required for Morphogenesis and Antibiotic Production and Controls Transcription of an ABC Transporter in Response to Carbon Source

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 alpha-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.

Methylation by a Unique α-class N4-Cytosine Methyltransferase Is Required for DNA Transformation of Caldicellulosiruptor bescii DSM6725

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.

Release of lignin from kraft pulp by a hyperthermophilic xylanase from Thermatoga maritima

Abstract The cloning, sequencing, and expression in E. coli of a gene from the hyperthermophile, Thermatoga maritima , encoding a thermostable xylanase is reported. The enzyme is active at 100°C for several hours and efficient in releasing lignin from kraft pulp. Comparison of the T. maritima recombinant enzyme with a commercially available xylanase, Pulpzyme, indicated that the hyperthermophilic enzyme has several advantages that make it an attractive biotechnological reagent. In studies of the release of reducing sugars and lignin from hardwood and softwood kraft pulp, the specific activity for the partially purified enzyme was 131 U mg −1 . The enzyme released reducing sugars and aromatic materials from the pulp suspensions over a pH range from 3.5–10.

Deletion Strains Reveal Metabolic Roles for Key Elemental Sulfur-Responsive Proteins in Pyrococcus furiosus

Transcriptional and enzymatic analyses of Pyrococcus furiosus previously indicated that three proteins play key roles in the metabolism of elemental sulfur (S(0)): 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 S(0), 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 S(0), 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 S(0) 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 S(0) reduction. Differences in transcriptional responses to S(0) in NSR1 suggest that two sulfide dehydrogenase isoenzymes provide a compensatory NADPH-dependent S(0) reduction system. Genes controlled by the S(0)-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 S(0) indicates that it is not required for detoxification of metal sulfides, as previously suggested. A model is proposed for S(0) reduction by P. furiosus with roles for MBX and NSR in bioenergetics and for SipA in iron-sulfur metabolism.