Strain and bioprocess improvement of a thermophilic anaerobe for the production of ethanol from wood.

BACKGROUND: The thermophilic, anaerobic bacterium Thermoanaerobacterium saccharolyticum digests hemicellulose and utilizes the major sugars present in biomass. It was previously engineered to produce ethanol at yields equivalent to yeast. While saccharolytic anaerobes have been long studied as potential biomass-fermenting organisms, development efforts for commercial ethanol production have not been reported. RESULTS: Here, we describe the highest ethanol titers achieved from T. saccharolyticum during a 4-year project to develop it for industrial production of ethanol from pre-treated hardwood at 51-55 °C. We describe organism and bioprocess development efforts undertaken to improve ethanol production. The final strain M2886 was generated by removing genes for exopolysaccharide synthesis, the regulator perR, and re-introduction of phosphotransacetylase and acetate kinase into the methyglyoxal synthase gene. It was also subject to multiple rounds of adaptation and selection, resulting in mutations later identified by resequencing. The highest ethanol titer achieved was 70 g/L in batch culture with a mixture of cellobiose and maltodextrin. In a "mock hydrolysate" Simultaneous Saccharification and Fermentation (SSF) with Sigmacell-20, glucose, xylose, and acetic acid, an ethanol titer of 61 g/L was achieved, at 92 % of theoretical yield. Fungal cellulases were rapidly inactivated under these conditions and had to be supplemented with cellulosomes from C. thermocellum. Ethanol titers of 31 g/L were reached in a 100 L SSF of pre-treated hardwood and 26 g/L in a fermentation of a hardwood hemicellulose extract. CONCLUSIONS: This study demonstrates that thermophilic anaerobes are capable of producing ethanol at high yield and at titers greater than 60 g/L from purified substrates, but additional work is needed to produce the same ethanol titers from pre-treated hardwood.

Genome-scale resources for Thermoanaerobacterium saccharolyticum.

BACKGROUND: Thermoanaerobacterium saccharolyticum is a hemicellulose-degrading thermophilic anaerobe that was previously engineered to produce ethanol at high yield. A major project was undertaken to develop this organism into an industrial biocatalyst, but the lack of genome information and resources were recognized early on as a key limitation. RESULTS: Here we present a set of genome-scale resources to enable the systems level investigation and development of this potentially important industrial organism. Resources include a complete genome sequence for strain JW/SL-YS485, a genome-scale reconstruction of metabolism, tiled microarray data showing transcription units, mRNA expression data from 71 different growth conditions or timepoints and GC/MS-based metabolite analysis data from 42 different conditions or timepoints. Growth conditions include hemicellulose hydrolysate, the inhibitors HMF, furfural, diamide, and ethanol, as well as high levels of cellulose, xylose, cellobiose or maltodextrin. The genome consists of a 2.7 Mbp chromosome and a 110 Kbp megaplasmid. An active prophage was also detected, and the expression levels of CRISPR genes were observed to increase in association with those of the phage. Hemicellulose hydrolysate elicited a response of carbohydrate transport and catabolism genes, as well as poorly characterized genes suggesting a redox challenge. In some conditions, a time series of combined transcription and metabolite measurements were made to allow careful study of microbial physiology under process conditions. As a demonstration of the potential utility of the metabolic reconstruction, the OptKnock algorithm was used to predict a set of gene knockouts that maximize growth-coupled ethanol production. The predictions validated intuitive strain designs and matched previous experimental results. CONCLUSION: These data will be a useful asset for efforts to develop T. saccharolyticum for efficient industrial production of biofuels. The resources presented herein may also be useful on a comparative basis for development of other lignocellulose degrading microbes, such as Clostridium thermocellum.

Anaerobic detoxification of acetic acid in a thermophilic ethanologen.

BACKGROUND: The liberation of acetate from hemicellulose negatively impacts fermentations of cellulosic biomass, limiting the concentrations of substrate that can be effectively processed. Solvent-producing bacteria have the capacity to convert acetate to the less toxic product acetone, but to the best of our knowledge, this trait has not been transferred to an organism that produces ethanol at high yield. RESULTS: We have engineered a five-step metabolic pathway to convert acetic acid to acetone in the thermophilic anaerobe Thermoanaerobacterium saccharolyticum. The first steps of the pathway, a reversible conversion of acetate to acetyl-CoA, are catalyzed by the native T. saccharolyticum enzymes acetate kinase and phosphotransacetylase. ack and pta normally divert 30% of catabolic carbon flux to acetic acid; however, their re-introduction in evolved ethanologen strains resulted in virtually no acetic acid production. Conversion between acetic acid and acetyl-CoA remained active, as evidenced by rapid (13)C label transfer from exogenous acetate to ethanol. Genomic re-sequencing of six independently evolved ethanologen strains showed convergent mutations in the hfs hydrogenase gene cluster, which when transferred to wildtype T. saccharolyticum conferred a low acid production phenotype. Thus, the mutated hfs genes effectively separate acetic acid production and consumption from central metabolism, despite their intersecting at the common intermediate acetyl-CoA. To drive acetic acid conversion to a less inhibitory product, the enzymes thiolase, acetoacetate:acetate CoA-transferase, and acetoacetate decarboxylase were assembled in T. saccharolyticum with genes from thermophilic donor organisms that do not natively produce acetone. The resultant strain converted acetic acid to acetone and ethanol while maintaining a metabolic yield of 0.50 g ethanol per gram carbohydrate. CONCLUSIONS: Conversion of acetic acid to acetone results in improved ethanol productivity and titer and is an attractive low-cost solution to acetic acid inhibition.

Genome Sequences of Industrially Relevant Saccharomyces cerevisiae Strain M3707, Isolated from a Sample of Distillers Yeast and Four Haploid Derivatives.

Saccharomyces cerevisiae strain M3707 was isolated from a sample of commercial distillers yeast, and its genome sequence together with the genome sequences for the four derived haploid strains M3836, M3837, M3838, and M3839 has been determined. Yeasts have potential for consolidated bioprocessing (CBP) for biofuel production, and access to these genome sequences will facilitate their development.

Enzymatic saccharification of shrub willow genotypes with differing biomass composition for biofuel production.

In the conversion of woody biomass feedstocks into liquid fuel ethanol, the pretreatment process is the most critical and costly step. Variations in biomass composition based on genetic differences or environmental effects have a significant impact on the degree of accessibility accomplished by pretreatment and subsequent sugar release by enzymatic hydrolysis. To evaluate this, biomass from 10 genetically diverse, genotypes of shrub willow (Salix spp.) was pretreated with a hot-water process at two levels of severity, hydrolyzed using a combination of two commercial enzyme cocktails, and the release of hexose and pentose monomers was quantified by high-performance liquid chromatography. Among the genotypes selected for analysis, cellulose content ranged from 39 to 45% (w/w) and lignin content ranged from 20 to 23% (w/w) at harvest. Differences in the effectiveness of the pretreatment process were observed among the various willow genotypes. Correlations were identified between total sugar release and % cellulose and % lignin content. There was a significant effect of pretreatment severity on polysaccharide accessibility, but the response to pretreatments was different among the genotypes. At the high severity pretreatment 'SV1' was the least recalcitrant with sugar release representing as much as 60% of total biomass. These results suggest that structural, as well as chemical characteristics of the biomass may influence pretreatment and hydrolytic efficiency.

Functional heterologous expression of an engineered full length CipA from Clostridium thermocellum in Thermoanaerobacterium saccharolyticum.

BACKGROUND: Cellulose is highly recalcitrant and thus requires a specialized suite of enzymes to solubilize it into fermentable sugars. In C. thermocellum, these extracellular enzymes are present as a highly active multi-component system known as the cellulosome. This study explores the expression of a critical C. thermocellum cellulosomal component in T. saccharolyticum as a step toward creating a thermophilic bacterium capable of consolidated bioprocessing by employing heterologously expressed cellulosomes. RESULTS: We developed an inducible promoter system based on the native T. saccharolyticum xynA promoter, which was shown to be induced by xylan and xylose. The promoter was used to express the cellulosomal component cipA*, an engineered form of the wild-type cipA from C. thermocellum. Expression and localization to the supernatant were both verified for CipA*. When a ΔcipA mutant C. thermocellum strain was cultured with a CipA*-expressing T. saccharolyticum strain, hydrolysis and fermentation of 10 grams per liter SigmaCell 101, a highly crystalline cellulose, were observed. This trans-species complementation of a cipA deletion demonstrated the ability for CipA* to assemble a functional cellulosome. CONCLUSION: This study is the first example of an engineered thermophile heterologously expressing a structural component of a cellulosome. To achieve this goal we developed and tested an inducible promoter for controlled expression in T. saccharolyticum as well as a synthetic cipA. In addition, we demonstrate a high degree of hydrolysis (up to 93%) on microcrystalline cellulose.

Carbon catabolite repression in Thermoanaerobacterium saccharolyticum.

BACKGROUND: The thermophilic anaerobe Thermoanaerobacterium saccharolyticum is capable of directly fermenting xylan and the biomass-derived sugars glucose, cellobiose, xylose, mannose, galactose and arabinose. It has been metabolically engineered and developed as a biocatalyst for the production of ethanol. RESULTS: We report the initial characterization of the carbon catabolite repression system in this organism. We find that sugar metabolism in T. saccharolyticum is regulated by histidine-containing protein HPr. We describe a mutation in HPr, His15Asp, that leads to derepression of less-favored carbon source utilization. CONCLUSION: Co-utilization of sugars can be achieved by mutation of HPr in T. saccharolyticum. Further manipulation of CCR in this organism will be instrumental in achieving complete and rapid conversion of all available sugars to ethanol.

Characterization of xylan utilization and discovery of a new endoxylanase in Thermoanaerobacterium saccharolyticum through targeted gene deletions.

The economical production of fuels and commodity chemicals from lignocellulose requires the utilization of both the cellulose and hemicellulose fractions. Xylanase enzymes allow greater utilization of hemicellulose while also increasing cellulose hydrolysis. Recent metabolic engineering efforts have resulted in a strain of Thermoanaerobacterium saccharolyticum that can convert C(5) and C(6) sugars, as well as insoluble xylan, into ethanol at high yield. To better understand the process of xylan solubilization in this organism, a series of targeted deletions were constructed in the homoethanologenic T. saccharolyticum strain M0355 to characterize xylan hydrolysis and xylose utilization in this organism. While the deletion of ß-xylosidase xylD slowed the growth of T. saccharolyticum on birchwood xylan and led to an accumulation of short-chain xylo-oligomers, no other single deletion, including the deletion of the previously characterized endoxylanase XynA, had a phenotype distinct from that of the wild type. This result indicates a multiplicity of xylanase enzymes which facilitate xylan degradation in T. saccharolyticum. Growth on xylan was prevented only when a previously uncharacterized endoxylanase encoded by xynC was also deleted in conjunction with xynA. Sequence analysis of xynC indicates that this enzyme, a low-molecular-weight endoxylanase with homology to glycoside hydrolase family 11 enzymes, is secreted yet untethered to the cell wall. Together, these observations expand our understanding of the enzymatic basis of xylan hydrolysis by T. saccharolyticum.

Urease expression in a Thermoanaerobacterium saccharolyticum ethanologen allows high titer ethanol production.

Genes encoding the enzyme urease were integrated in a Thermoanaerobacterium saccharolyticum ethanologen. The engineered strain hydrolyzed urea, as evidenced by increased cellular growth and elevated final pH in urea minimal medium and urease activity in cell free extracts. Interestingly, replacement of ammonium salts with urea resulted in production of 54 g/L ethanol, one of the highest titers reported for Thermoanaerobacterium. The observed increase in ethanol titer may result from reduced pH, salt, and osmolality stresses during fermentation. Urea utilization is attractive for industrial scale fermentation, where pH control is technically challenging and increased ethanol titer is desirable.

Ethanol and anaerobic conditions reversibly inhibit commercial cellulase activity in thermophilic simultaneous saccharification and fermentation (tSSF).

BACKGROUND: A previously developed mathematical model of low solids thermophilic simultaneous saccharification and fermentation (tSSF) with Avicel was unable to predict performance at high solids using a commercial cellulase preparation (Spezyme CP) and the high ethanol yield Thermoanaerobacterium saccharolyticum strain ALK2. The observed hydrolysis proceeded more slowly than predicted at solids concentrations greater than 50 g/L Avicel. Factors responsible for this inaccuracy were investigated in this study. RESULTS: Ethanol dramatically reduced cellulase activity in tSSF. At an Avicel concentration of 20 g/L, the addition of ethanol decreased conversion at 96 hours, from 75% in the absence of added ethanol down to 32% with the addition of 34 g/L initial ethanol. This decrease is much greater than expected based on hydrolysis inhibition results in the absence of a fermenting organism. The enhanced effects of ethanol were attributed to the reduced, anaerobic conditions of tSSF, which were shown to inhibit cellulase activity relative to hydrolysis under aerobic conditions. Cellulose hydrolysis in anaerobic conditions was roughly 30% slower than in the presence of air. However, this anaerobic inhibition was reversed by exposing the cellulase enzymes to air. CONCLUSION: This work demonstrates a previously unrecognized incompatibility of enzymes secreted by an aerobic fungus with the fermentation conditions of an anaerobic bacterium and suggests that enzymes better suited to industrially relevant fermentation conditions would be valuable. The effects observed may be due to inactivation or starvation of oxygen dependent GH61 activity, and manipulation or replacement of this activity may provide an opportunity to improve biomass to fuel process efficiency.

High ethanol titers from cellulose by using metabolically engineered thermophilic, anaerobic microbes.

This work describes novel genetic tools for use in Clostridium thermocellum that allow creation of unmarked mutations while using a replicating plasmid. The strategy employed counter-selections developed from the native C. thermocellum hpt gene and the Thermoanaerobacterium saccharolyticum tdk gene and was used to delete the genes for both lactate dehydrogenase (Ldh) and phosphotransacetylase (Pta). The Δldh Δpta mutant was evolved for 2,000 h, resulting in a stable strain with 40:1 ethanol selectivity and a 4.2-fold increase in ethanol yield over the wild-type strain. Ethanol production from cellulose was investigated with an engineered coculture of organic acid-deficient engineered strains of both C. thermocellum and T. saccharolyticum. Fermentation of 92 g/liter Avicel by this coculture resulted in 38 g/liter ethanol, with acetic and lactic acids below detection limits, in 146 h. These results demonstrate that ethanol production by thermophilic, cellulolytic microbes is amenable to substantial improvement by metabolic engineering.

Marker removal system for Thermoanaerobacterium saccharolyticum and development of a markerless ethanologen.

Marker removal strategies were developed for Thermoanaerobacterium saccharolyticum to select against the pyrF gene and the pta and ack genes. The pta- and ack-based haloacetate selective strategy was subsequently used to create strain M0355, a markerless Δldh Δpta Δack strain that produces ethanol at a high yield.

Enzyme inactivation by ethanol and development of a kinetic model for thermophilic simultaneous saccharification and fermentation at 50 °C with Thermoanaerobacterium saccharolyticum ALK2.

Studies were undertaken to understand phenomena operative during simultaneous saccharification and fermentation (SSF) of a model cellulosic substrate (Avicel) at 50°C with enzymatic hydrolysis mediated by a commercial cellulase preparation (Spezyme CP) and fermentation by a thermophilic bacterium engineered to produce ethanol at high yield, Thermoanaerobacterium saccharolyticum ALK2. Thermal inactivation at 50 °C, as shown by the loss of 50% of enzyme activity over 4 days in the absence of ethanol, was more severe than at 37 °C, where only 25% of enzyme activity was lost. In addition, at 50 °C ethanol more strongly influenced enzyme stability. Enzyme activity was moderately stabilized between ethanol concentrations of 0 and 40 g/L, but ethanol concentrations above 40 g/L accelerated enzyme inactivation, leading to 75% loss of enzymatic activity in 80 g/L ethanol after 4 days. At 37 °C, ethanol did not show a strong effect on the rate of enzyme inactivation. Inhibition of cellulase activity by ethanol, measured at both temperatures, was relatively similar, with the relative rate of hydrolysis inhibited 50% at ethanol concentrations of 56.4 and 58.7 g/L at 50 and 37 °C, respectively. A mathematical model was developed to test whether the measured phenomena were sufficient to quantitatively describe system behavior and was found to have good predictive capability at initial Avicel concentrations of 20 and 50 g/L.

Deletion of the Cel48S cellulase from Clostridium thermocellum.

Clostridium thermocellum is a thermophilic anaerobic bacterium that rapidly solubilizes cellulose with the aid of a multienzyme cellulosome complex. Creation of knockout mutants for Cel48S (also known as CelS, S(S), and S8), the most abundant cellulosome subunit, was undertaken to gain insight into its role in enzymatic and microbial cellulose solubilization. Cultures of the Cel48S deletion mutant (S mutant) were able to completely solubilize 10 g/L crystalline cellulose. The cellulose hydrolysis rate of the S mutant strain was 60% lower than the parent strain, with the S mutant strain also exhibiting a 40% reduction in cell yield. The cellulosome produced by the S mutant strain was purified by affinity digestion, characterized enzymatically, and found to have a 35% lower specific activity on Avicel. The composition of the purified cellulosome was analyzed by tandem mass spectrometry with APEX quantification and no significant changes in abundance were observed in any of the major (>1% of cellulosomal protein) enzymatic subunits. Although most cellulolytic bacteria have one family 48 cellulase, C. thermocellum has two, Cel48S and Cel48Y. Cellulose solubilization by a Cel48S and Cel48Y double knockout was essentially the same as that of the Cel48S single knockout. Our results indicate that solubilization of crystalline cellulose by C. thermocellum can proceed to completion without expression of a family 48 cellulase.

Development of pyrF-based genetic system for targeted gene deletion in Clostridium thermocellum and creation of a pta mutant.

We report development of a genetic system for making targeted gene knockouts in Clostridium thermocellum, a thermophilic anaerobic bacterium that rapidly solubilizes cellulose. A toxic uracil analog, 5-fluoroorotic acid (5-FOA), was used to select for deletion of the pyrF gene. The ΔpyrF strain is a uracil auxotroph that could be restored to a prototroph via ectopic expression of pyrF from a plasmid, providing a positive genetic selection. Furthermore, 5-FOA was used to select against plasmid-expressed pyrF, creating a negative selection for plasmid loss. This technology was used to delete a gene involved in organic acid production, namely pta, which encodes the enzyme phosphotransacetylase. The C. thermocellum Δpta strain did not produce acetate. These results are the first examples of targeted homologous recombination and metabolic engineering in C. thermocellum, a microbe that holds an exciting and promising future in the biofuel industry and development of sustainable energy resources.

Natural competence in Thermoanaerobacter and Thermoanaerobacterium species.

Low-G+C thermophilic obligate anaerobes in the class Clostridia are considered among the bacteria most resistant to genetic engineering due to the difficulty of introducing foreign DNA, thus limiting the ability to study and exploit their native hydrolytic and fermentative capabilities. Here, we report evidence of natural genetic competence in 13 Thermoanaerobacter and Thermoanaerobacterium strains previously believed to be difficult to transform or genetically recalcitrant. In Thermoanaerobacterium saccharolyticum JW/SL-YS485, natural competence-mediated DNA incorporation occurs during the exponential growth phase with both replicating plasmid and homologous recombination-based integration, and circular or linear DNA. In T. saccharolyticum, disruptions of genes similar to comEA, comEC, and a type IV pilus (T4P) gene operon result in strains unable to incorporate further DNA, suggesting that natural competence occurs via a conserved Gram-positive mechanism. The relative ease of employing natural competence for gene transfer should foster genetic engineering in these industrially relevant organisms, and understanding the mechanisms underlying natural competence may be useful in increasing the applicability of genetic tools to difficult-to-transform organisms.

Identification of the [FeFe]-hydrogenase responsible for hydrogen generation in Thermoanaerobacterium saccharolyticum and demonstration of increased ethanol yield via hydrogenase knockout.

Three putative hydrogenase enzyme systems in Thermoanaerobacterium saccharolyticum were investigated at the genetic, mRNA, enzymatic, and phenotypic levels. A four-gene operon containing two [FeFe]-hydrogenase genes, provisionally termed hfs (hydrogenase-Fe-S), was found to be the main enzymatic catalyst of hydrogen production. hfsB, perhaps the most interesting gene of the operon, contains an [FeFe]-hydrogenase and a PAS sensory domain and has several conserved homologues among clostridial saccharolytic, cellulolytic, and pathogenic bacteria. A second hydrogenase gene cluster, hyd, exhibited methyl viologen-linked hydrogenase enzymatic activity, but hyd gene knockouts did not influence the hydrogen yield of cultures grown in closed-system batch fermentations. This result, combined with the observation that hydB contains NAD(P)+ and FMN binding sites, suggests that the hyd genes are specific to the transfer of electrons from NAD(P)H to hydrogen ions. A third gene cluster, a putative [NiFe]-hydrogenase with homology to the ech genes, did not exhibit hydrogenase activity under any of the conditions tested. Deletion of the hfs and hydA genes resulted in a loss of detectable methyl viologen-linked hydrogenase activity. Strains with a deletion of the hfs genes exhibited a 95% reduction in hydrogen and acetic acid production. A strain with hfs and ldh deletions exhibited an increased ethanol yield from consumed carbohydrates and represents a new strategy for engineering increased ethanol yields in T. saccharolyticum.

Metabolic engineering of a thermophilic bacterium to produce ethanol at high yield.

We report engineering Thermoanaerobacterium saccharolyticum, a thermophilic anaerobic bacterium that ferments xylan and biomass-derived sugars, to produce ethanol at high yield. Knockout of genes involved in organic acid formation (acetate kinase, phosphate acetyltransferase, and L-lactate dehydrogenase) resulted in a strain able to produce ethanol as the only detectable organic product and substantial changes in electron flow relative to the wild type. Ethanol formation in the engineered strain (ALK2) utilizes pyruvate:ferredoxin oxidoreductase with electrons transferred from ferredoxin to NAD(P), a pathway different from that in previously described microbes with a homoethanol fermentation. The homoethanologenic phenotype was stable for >150 generations in continuous culture. The growth rate of strain ALK2 was similar to the wild-type strain, with a reduction in cell yield proportional to the decreased ATP availability resulting from acetate kinase inactivation. Glucose and xylose are co-utilized and utilization of mannose and arabinose commences before glucose and xylose are exhausted. Using strain ALK2 in simultaneous hydrolysis and fermentation experiments at 50 degrees C allows a 2.5-fold reduction in cellulase loading compared with using Saccharomyces cerevisiae at 37 degrees C. The maximum ethanol titer produced by strain ALK2, 37 g/liter, is the highest reported thus far for a thermophilic anaerobe, although further improvements are desired and likely possible. Our results extend the frontier of metabolic engineering in thermophilic hosts, have the potential to significantly lower the cost of cellulosic ethanol production, and support the feasibility of further cost reductions through engineering a diversity of host organisms.