Hydrolysis and fermentation of amorphous cellulose by recombinant Saccharomyces cerevisiae

In this study, we expressed two cellulase encoding genes, an endoglucanase of Trichoderma reesei (EGI) and the beta-glucosidase of Saccharomycopsis fibuligera (BGL1), in combination in Saccharomyces cerevisiae. The resulting strain was able to grow on phosphoric acid swollen cellulose (PASC) through simultaneous production of sufficient extracellular endoglucanase and beta-glucosidase activity. Anaerobic growth (0.03h(-1)) up to 0.27gl(-1) DCW was observed on medium containing 10gl(-1) PASC as sole carbohydrate source with concomitant ethanol production of up to 1.0gl(-1). We have thus demonstrated the construction of a yeast strain capable of growth on and one-step conversion of amorphous cellulose to ethanol, representing significant progress towards realization of one-step processing of cellulosic biomass in a consolidated bioprocessing configuration. To our knowledge, this is the first report of a recombinant strain of S. cerevisiae growing on pure cellulose.     

In vitro assembly of minicellulosomes with two scaffoldins on the yeast cell surface for cellulose saccharification and bioethanol production

The present paper reports in vitro strategies for assembly of minicellulosomes with two miniscaffoldins on the Saccharomyces cerevisiae cell surface. It was carried out through incubation of the yeast cells displaying scaffoldins with Escherichia coli lysates containing recombinant cellulases, or using a four-population yeast consortium. The results showed that the display level of miniscaffoldin II was distinctly increased by moving the cellulases production into E. coli or other yeast cells, indicating that the metabolic burden of the yeast host was decreased. The yeast consortium did not show any cellulolytic activity, while the E. coli lysates-treated yeast, whose anchoring miniscaffoldin length was optimized, was able to produce ～1138 mg/L ethanol from microcrystalline cellulose within 4 days. We also confirmed that the yeast-associated minicellulosome moreover showed both higher thermal stability and lower protease accessibility than free minicellulosome. This research promotes the application of S. cerevisiae as a consolidated bioprocessing (CBP) microorganism in cellulosic bioethanol production.     

PGASO: A synthetic biology tool for engineering a cellulolytic yeast

ABSTRACT: BACKGROUND: To achieve an economical cellulosic ethanol production, a host that can do both cellulosic saccharification and ethanol fermentation is desirable. However, to engineer a non-cellulolytic yeast to be such a host requires synthetic biology techniques to transform multiple enzyme genes into its genome. RESULTS: A technique, named Promoter-based Gene Assembly and Simultaneous Overexpression (PGASO), that employs overlapping oligonucleotides for recombinatorial assembly of gene cassettes with individual promoters, was developed. PGASO was applied to engineer Kluyveromycesmarxianus KY3, which is a thermo- and toxin-tolerant yeast. We obtained a recombinant strain, called KR5, that is capable of simultaneously expressing exoglucanase and endoglucanase (both of Trichodermareesei), a beta-glucosidase (from a cow rumen fungus), a neomycin phosphotransferase, and a green fluorescent protein. High transformation efficiency and accuracy were achieved as ~63% of the transformants was confirmed to be correct. KR5 can utilize beta-glycan, cellobiose or CMC as the sole carbon source for growth and can directly convert cellobiose and beta-glycan to ethanol. CONCLUSIONS: This study provides the first example of multi-gene assembly in a single step in a yeast species other than Saccharomyces cerevisiae. We successfully engineered a yeast host with a five-gene cassette assembly and the new host is capable of co-expressing three types of cellulase genes. Our study shows that PGASO is an efficient tool for simultaneous expression of multiple enzymes in the kefir yeast KY3 and that KY3 can serve as a host for developing synthetic biology tools.     

Incorporation of Fungal Cellulases in Bacterial Minicellulosomes Yields Viable, Synergistically Acting Cellulolytic Complexes

Artificial designer minicellulosomes comprise a chimeric scaffoldin that displays an optional cellulose-binding module (CBM) and bacterial cohesins from divergent species which bind strongly to enzymes engineered to bear complementary dockerins. Incorporation of cellulosomal cellulases from Clostridium cellulolyticum into minicellulosomes leads to artificial complexes with enhanced activity on crystalline cellulose, due to enzyme proximity and substrate targeting induced by the scaffoldin-borne CBM. In the present study, a bacterial dockerin was appended to the family 6 fungal cellulase Cel6A, produced by Neocallimastix patriciarum, for subsequent incorporation into minicellulosomes in combination with various cellulosomal cellulases from C. cellulolyticum. The binding of the fungal Cel6A with a bacterial family 5 endoglucanase onto chimeric miniscaffoldins had no impact on their activity toward crystalline cellulose. Replacement of the bacterial family 5 enzyme with homologous endoglucanase Cel5D from N. patriciarum bearing a clostridial dockerin gave similar results. In contrast, enzyme pairs comprising the fungal Cel6A and bacterial family 9 endoglucanases were substantially stimulated (up to 2.6-fold) by complexation on chimeric scaffoldins, compared to the free-enzyme system. Incorporation of enzyme pairs including Cel6A and a processive bacterial cellulase generally induced lower stimulation levels. Enhanced activity on crystalline cellulose appeared to result from either proximity or CBM effects alone but never from both simultaneously, unlike minicellulosomes composed exclusively of bacterial cellulases. The present study is the first demonstration that viable designer minicellulosomes can be produced that include (i) free (noncellulosomal) enzymes, (ii) fungal enzymes combined with bacterial enzymes, and (iii) a type (family 6) of cellulase never known to occur in natural cellulosomes.     

Incorporation of fungal cellulases in bacterial minicellulosomes yields viable, synergistically acting cellulolytic complexes

Artificial designer minicellulosomes comprise a chimeric scaffoldin that displays an optional cellulose-binding module (CBM) and bacterial cohesins from divergent species which bind strongly to enzymes engineered to bear complementary dockerins. Incorporation of cellulosomal cellulases from Clostridium cellulolyticum into minicellulosomes leads to artificial complexes with enhanced activity on crystalline cellulose, due to enzyme proximity and substrate targeting induced by the scaffoldin-borne CBM. In the present study, a bacterial dockerin was appended to the family 6 fungal cellulase Cel6A, produced by Neocallimastix patriciarum, for subsequent incorporation into minicellulosomes in combination with various cellulosomal cellulases from C. cellulolyticum. The binding of the fungal Cel6A with a bacterial family 5 endoglucanase onto chimeric miniscaffoldins had no impact on their activity toward crystalline cellulose. Replacement of the bacterial family 5 enzyme with homologous endoglucanase Cel5D from N. patriciarum bearing a clostridial dockerin gave similar results. In contrast, enzyme pairs comprising the fungal Cel6A and bacterial family 9 endoglucanases were substantially stimulated (up to 2.6-fold) by complexation on chimeric scaffoldins, compared to the free-enzyme system. Incorporation of enzyme pairs including Cel6A and a processive bacterial cellulase generally induced lower stimulation levels. Enhanced activity on crystalline cellulose appeared to result from either proximity or CBM effects alone but never from both simultaneously, unlike minicellulosomes composed exclusively of bacterial cellulases. The present study is the first demonstration that viable designer minicellulosomes can be produced that include (i) free (noncellulosomal) enzymes, (ii) fungal enzymes combined with bacterial enzymes, and (iii) a type (family 6) of cellulase never known to occur in natural cellulosomes.     

β-葡萄糖苷酶在酿酒酵母表面的表达

应用表面表达技术对来自Trichodermareesei的β-葡萄糖苷酶在酿酒酵母表面的表达及后期性质进行了研究。实验结果表明酵母表面表达酶有活性,该酶的最佳诱导时间为24h,最适温度是70℃,而酶活的最适pH是5．5。使异源表面表达了Bgl1的酵母在以纤维二糖为唯一碳源的培养基中生长,发酵结果表明纤维二糖被明显利用了,但在培养186h后,发酵液中仍残留一定量的纤维二糖。这种技术对纤维素发酵系统中纤维二糖酶活性低的现状有所帮助。     

An evaluation of cellulose saccharification and fermentation with an engineered Saccharomyces cerevisiae capable of cellobiose and xylose utilization

Commercial-scale cellulosic ethanol production has been hindered by high costs associated with cellulose-to-glucose conversion and hexose and pentose co-fermentation. Simultaneous saccharification and fermentation (SSF) with a yeast strain capable of xylose and cellobiose co-utilization has been proposed as a possible avenue to reduce these costs. The recently developed DA24-16 strain of Saccharomyces cerevisiae incorporates a xylose assimilation pathway and a cellodextrin transporter (CDT) that permit rapid growth on xylose and cellobiose. In the current work, a mechanistic kinetic model of cellulase-catalyzed hydrolysis of cellulose was combined with a multi-substrate model of microbial growth to investigate the ability of DA24-16 and improved cellobiose-consuming strains to obviate the need for exogenously added β-glucosidase and to assess the impact of cellobiose utilization on SSF and separate hydrolysis and fermentation (SHF). Results indicate that improved CDT-containing strains capable of growing on cellobiose as rapidly as on glucose produced ethanol nearly as rapidly as non-CDT-containing yeast supplemented with β-glucosidase. In producing 75 g/L ethanol, SSF with any strain did not result in shorter residence times than SHF with a 12 h saccharification step. Strains with improved cellobiose utilization are therefore unlikely to allow higher titers to be reached more quickly in SSF than in SHF.     

Isobutanol production from cellobiose in Escherichia coli

Converting lignocellulosics into biofuels remains a promising route for biofuel production. To facilitate strain development for specificity and productivity of cellulosic biofuel production, a user friendly Escherichia coli host was engineered to produce isobutanol, a drop-in biofuel candidate, from cellobiose. A beta-glucosidase was expressed extracellularly by either excretion into the media, or anchoring to the cell membrane. The excretion system allowed for E. coli to grow with cellobiose as a sole carbon source at rates comparable to those with glucose. The system was then combined with isobutanol production genes in three different configurations to determine whether gene arrangement affected isobutanol production. The most productive strain converted cellobiose to isobutanol in titers of 7.64?±?0.19 g/L with a productivity of 0.16 g/L/h. These results demonstrate that efficient cellobiose degradation and isobutanol production can be achieved by a single organism, and provide insight for optimization of strains for future use in a consolidated bioprocessing system for renewable production of isobutanol.     

Cellulosic ethanol production by combination of cellulase-displaying yeast cells

As an effort to find suitable endoglucanases to generate cellulolytic yeast strains, two fungal endoglucanases, Thermoascus aurantiacus EGI and Trichoderma reesei EGII, and two bacterial endoglucanases, Clostridium thermocellum CelA and CelD, were expressed on the yeast surface, and their surface expression levels, pH- and temperature-dependent enzyme activities, and substrate specificities were analyzed. T. aurantiacus EGI showed similar patterns of pH- and temperature-dependent activities to those of T. reesei EGII which has been widely used due to its high enzyme activity. Although EGII showed higher carboxymethyl cellulose (CMC) degradation activity than EGI, EGI showed better activity toward phosphoric acid swollen cellulose (PASC). For ethanol production from PASC, we combined three types of yeast cells, each displaying T. aurantiacus EGI, T. reesei CBHII (exoglucanase) and Aspergillus aculeatus BGLI (β-glucosidase), instead of co-expressing these enzymes in a single cell. In this system, ethanol production can be easily optimized by adjusting the combination ratio of each cell type. A mixture of cells with the optimized EGI:CBHII:BGLI ratio of 6:2:1 produced 1.3 fold more ethanol (2.1 g/l) than cells composed of an equal amount of each cell type, suggesting the usefulness of this system for cellulosic ethanol production.     

The Lignicolous Fungus <Emphasis Type="Italic">Coniochaeta pulveracea</Emphasis> and Its Interactions with Syntrophic Yeasts from the Woody Phylloplane

The yeast-like fungus Coniochaeta pulveracea was studied with regard to its novel lignocellulolytic activities and the possible effect thereof on yeasts from the woody phylloplane. An enrichment procedure was used to isolate C. pulveracea from a decaying Acacia tree, and the identity of the isolate was confirmed using morphology, as well as molecular and phylogenetic techniques. This isolate, as well as strains representing C. pulveracea from different geographical regions, were compared with regard to optimum growth temperature and enzyme activity to representatives of closely related species. These include strains of Coniochaeta boothii, Coniochaeta rhopalochaeta, and Coniochaeta subcorticalis. Plate assays for cellulase and xylanase activity indicated that all representatives of the above-mentioned species were able to produce extracellular hydrolytic enzymes and were also able to degrade birchwood toothpicks during a 50-day incubation period at 30°C. To test the ability of these fungi and their enzymes to release simple sugars from complex cellulosic substrates, filtrates obtained from liquid cultures of Coniochaeta, cultivated on carboxymethyl cellulose (CMC) as sole carbon source, were analyzed using high-performance liquid chromatography analysis. Consequently, the presence of mono- and disaccharides such as glucose and cellobiose was confirmed in these culture filtrates. Two subsequent experiments were conducted to determine whether these simple sugars released from woody material by Coniochaeta may enhance growth of phylloplane yeasts. In the first experiment, representatives of Coniochaeta were co-cultured with selected yeasts suspended in agar plates containing birchwood toothpicks, followed by examination of plates for colony formation. Results indicated that Coniochaeta growth on the toothpicks enhanced growth of nearby yeast colonies in the agar plates. In the second experiment, representatives of selected yeasts and Coniochaeta species were co-cultured on CMC and xylan-containing plates where after yeast colony formation was recorded on the plates. Saccharomyces cerevisiae strains, engineered to utilize specific wood degradation products, i.e., cellobiose or xylose, as sole carbon source were used as positive controls. While it was found that cellobiose released from CMC was assimilated by the yeasts, no evidence could be obtained that xylose released from xylan was used as carbon source by the yeasts. These ambiguous results could be ascribed to secretion of nutritious metabolic end products, other than the products of fungal xylanases.     

Production of minicellulosomes for the enhanced hydrolysis of cellulosic substrates by recombinant Corynebacterium glutamicum

Although cellulosic materials of plant origin are the most abundant utilizable biomass resource, the amino acid-producing organism Corynebacterium glutamicum can not utilize these materials. Here we report the engineering of a C. glutamicum strain expressing functional minicellulosomes containing chimeric endoglucanase E bound to miniCbpA from Clostridium cellulovorans that can hydrolyze cellulosic materials. The chimeric endoglucanase E consists of the endoglucanase E catalytic backbone of Clostridium thermocellum fused with the endoglucanase B dockerin domain of C. cellulovorans. The resulting strain degraded cellulose efficiently by substrate targeting via the carbohydrate binding module. The assembly of minicellulosomes increased the activity against carboxymethyl cellulose approximately 2.8-fold compared with that for the corresponding enzymes alone. This is the first report of the formation of Clostridium minicellulosomes by C. glutamicum. The development of C. glutamicum strain that is capable of more effective cellulose hydrolysis brings about a realization of consolidated bioprocessing for the utilization of cellulosic biomass.     

Cellulosic alcoholic fermentation using recombinant Saccharomyces cerevisiae engineered for the production of Clostridium cellulovorans endoglucanase and Saccharomycopsis fibuligeraβ-glucosidase

In this study, Saccharomyces cerevisiae was engineered for simultaneous saccharification and fermentation of cellulose by the overexpression of the endoglucanase D (EngD) from Clostridium cellulovorans and the β-glucosidase (Bgl1) from Saccharomycopsis fibuligera . To promote secretion of the two enzymes, the genes were fused to the secretion signal of the S. cerevisiae α mating factor gene. The recombinant developed yeast could produce ethanol through simultaneous production of sufficient extracellular endoglucanase and β-glucosidase. When direct ethanol fermentation from 20 g L⁻¹β-glucan as a substrate was performed with our recombinant strains, the ethanol concentration reached 9.15 g L⁻¹ after 50 h of fermentation. The conversion ratio of ethanol from β-glucan was 80.3% of the theoretical ethanol concentration produced from 20 g L⁻¹β-glucan. In conclusion, we have demonstrated the construction of a yeast strain capable of conversion of a cellulosic substrate to ethanol, representing significant progress towards the realization of processing of cellulosic biomass in a consolidated bioprocessing configuration.     

Ethanol production from cellulosic materials using cellulase-expressing yeast

We demonstrate direct ethanol fermentation from amorphous cellulose using cellulase-co-expressing yeast. Endoglucanases (EG) and cellobiohydrolases (CBH) from Trichoderma reesei, and β-glucosidases (BGL) from Aspergillus aculeatus were integrated into genomes of the yeast strain Saccharomyces cerevisiae MT8-1. BGL was displayed on the yeast cell surface and both EG and CBH were secreted or displayed on the cell surface. All enzymes were successfully expressed on the cell surface or in culture supernatants in their active forms, and cellulose degradation was increased 3- to 5-fold by co-expressing EG and CBH. Direct ethanol fermentation from 10 g/L phosphoric acid swollen cellulose (PASC) was also carried out using EG-, CBH-, and BGL-co-expressing yeast. The ethanol yield was 2.1 g/L for EG-, CBH-, and BGL-displaying yeast, which was higher than that of EG- and CBH-secreting yeast (1.6 g/L ethanol). Our results show that cell surface display is more suitable for direct ethanol fermentation from cellulose.     

扣囊复膜孢酵母b-葡萄糖苷酶基因在工业酿酒酵母中的表达

本文以工业酿酒酵母菌株( Saccharomyces cerevisiae Y )为研究对象,针对其复杂的生理生化遗传特性,建立了相对应的转化体系。以pRS41H质粒为基础载体,构建了含有工业酿酒酵母自身的gpd2启动子、终止子和扣囊复膜孢酵母的b-葡萄糖苷酶基因bgl的重组质粒pRS-gb。电击转化进入工业酿酒酵母细胞,潮霉素抗性筛选,获得重组菌。该重组菌可以在以纤维二糖为唯一碳源的培养基中生长,培养36 h,b-葡萄糖苷酶酶活达到0.967 u/ml。以纤维二糖为唯一碳源的酒精发酵中,酒精度可以达到0.92 g/l。这对工业生产中利用纤维素为原料发酵生产酒精具有重要意义。     

Strain engineering of Saccharomyces cerevisiae for enhanced xylose metabolism

Efficient and rapid fermentation of all sugars present in cellulosic hydrolysates is essential for economic conversion of renewable biomass into fuels and chemicals. Xylose is one of the most abundant sugars in cellulosic biomass but it cannot be utilized by wild type Saccharomyces cerevisiae, which has been used for industrial ethanol production. Therefore, numerous technologies for strain development have been employed to engineer S. cerevisiae capable of fermenting xylose rapidly and efficiently. These include i) optimization of xylose-assimilating pathways, ii) perturbation of gene targets for reconfiguring yeast metabolism, and iii) simultaneous co-fermentation of xylose and cellobiose. In addition, the genetic and physiological background of host strains is an important determinant to construct efficient and rapid xylose-fermenting S. cerevisiae. Vibrant and persistent researches in this field for the last two decades not only led to the development of engineered S. cerevisiae strains ready for industrial fermentation of cellulosic hydrolysates, but also deepened our understanding of operational principles underlying yeast metabolism.     

Synergistic saccharification, and direct fermentation to ethanol, of amorphous cellulose by use of an engineered yeast strain codisplaying three types of cellulolytic enzyme

A whole-cell biocatalyst with the ability to induce synergistic and sequential cellulose-degradation reaction was constructed through codisplay of three types of cellulolytic enzyme on the cell surface of the yeast Saccharomyces cerevisiae. When a cell surface display system based on alpha-agglutinin was used, Trichoderma reesei endoglucanase II and cellobiohydrolase II and Aspergillus aculeatus beta-glucosidase 1 were simultaneously codisplayed as individual fusion proteins with the C-terminal-half region of alpha-agglutinin. Codisplay of the three enzymes on the cell surface was confirmed by observation of immunofluorescence-labeled cells with a fluorescence microscope. A yeast strain codisplaying endoglucanase II and cellobiohydrolase II showed significantly higher hydrolytic activity with amorphous cellulose (phosphoric acid-swollen cellulose) than one displaying only endoglucanase II, and its main product was cellobiose; codisplay of beta-glucosidase 1, endoglucanase II, and cellobiohydrolase II enabled the yeast strain to directly produce ethanol from the amorphous cellulose (which a yeast strain codisplaying beta-glucosidase 1 and endoglucanase II could not), with a yield of approximately 3 g per liter from 10 g per liter within 40 h. The yield (in grams of ethanol produced per gram of carbohydrate consumed) was 0.45 g/g, which corresponds to 88.5% of the theoretical yield. This indicates that simultaneous and synergistic saccharification and fermentation of amorphous cellulose to ethanol can be efficiently accomplished using a yeast strain codisplaying the three cellulolytic enzymes.     

Mutant selection and phenotypic and genetic characterization of ethanol-tolerant strains of <Emphasis Type="Italic">Clostridium thermocellum</Emphasis>

Clostridium thermocellum is a model microorganism for converting cellulosic biomass into fuels and chemicals via consolidated bioprocessing. One of the challenges for industrial application of this organism is its low ethanol tolerance, typically 1–2% (w/v) in wild-type strains. In this study, we report the development and characterization of mutant C. thermocellum strains that can grow in the presence of high ethanol concentrations. Starting from a single colony, wild-type C. thermocellum ATCC 27405 was sub-cultured and adapted for growth in up to 50 g/L ethanol using either cellobiose or crystalline cellulose as the growth substrate. Both the adapted strains retained their ability to grow on either substrate and displayed a higher growth rate and biomass yield than the wild-type strain in the absence of ethanol. With added ethanol in the media, the mutant strains displayed an inverse correlation between ethanol concentration and growth rate or biomass yield. Genome sequencing revealed six common mutations in the two ethanol-tolerant strains including an alcohol dehydrogenase gene and genes involved in arginine/pyrimidine biosynthetic pathway. The potential role of these mutations in ethanol tolerance phenotype is discussed.     

Characterization of the cellulolytic enzyme system from the brown-rot fungus Coniophora puteana

Summary The cellulolytic enzymes of various strains of the brown-rot fungus Coniophora puteana were studied. The organism was grown in an air-lift fermentor in mineral medium containing glucose, cellobiose or amorphous cellulose. The specific growth rate varied between 0.082 and 0.062 h^–1. On amorphous cellulose as sole carbon source, the organism secreted various proteins, some of which were characterized. The mixture contained inter alia four endocellulases, two exo-cellobiohydrolases and a cellobiose dehydrogenase. Three endocellulases (named type I) were active on soluble cellulose derivatives but inactive on p-nitrophenyllactoside (p-NPL), whereas a fourth endocellulase (named type II) was active on both. The two exo-cellobiohydrolases released cellobiose from amorphous cellulose; they were inactive on soluble cellulose derivatives but hydrolyzed p-NPL with strong cellobiose inhibition. A cellobiose dehydrogenase having spectral characteristics compatible with a flavo b-cytochrome was also identified. Neither the exo-cellobiohydrolase nor the type II endocellulase were secreted during growth on cellobiose whereas type I endocellulases and cellobiose dehydrogenase were formed at a reduced rate. No formation of cellulolytic enzymes was observed during growth on glucose alone. Correspondence to: G. Canevascini     

Synergistic Saccharification, and Direct Fermentation to Ethanol, of Amorphous Cellulose by Use of an Engineered Yeast Strain Codisplaying Three Types of Cellulolytic Enzyme

A whole-cell biocatalyst with the ability to induce synergistic and sequential cellulose-degradation reaction was constructed through codisplay of three types of cellulolytic enzyme on the cell surface of the yeast Saccharomyces cerevisiae. When a cell surface display system based on α-agglutinin was used, Trichoderma reesei endoglucanase II and cellobiohydrolase II and Aspergillus aculeatus β-glucosidase 1 were simultaneously codisplayed as individual fusion proteins with the C-terminal-half region of α-agglutinin. Codisplay of the three enzymes on the cell surface was confirmed by observation of immunofluorescence-labeled cells with a fluorescence microscope. A yeast strain codisplaying endoglucanase II and cellobiohydrolase II showed significantly higher hydrolytic activity with amorphous cellulose (phosphoric acid-swollen cellulose) than one displaying only endoglucanase II, and its main product was cellobiose; codisplay of β-glucosidase 1, endoglucanase II, and cellobiohydrolase II enabled the yeast strain to directly produce ethanol from the amorphous cellulose (which a yeast strain codisplaying β-glucosidase 1 and endoglucanase II could not), with a yield of approximately 3 g per liter from 10 g per liter within 40 h. The yield (in grams of ethanol produced per gram of carbohydrate consumed) was 0.45 g/g, which corresponds to 88.5% of the theoretical yield. This indicates that simultaneous and synergistic saccharification and fermentation of amorphous cellulose to ethanol can be efficiently accomplished using a yeast strain codisplaying the three cellulolytic enzymes.     

The ethanol pathway from Thermoanaerobacterium saccharolyticum improves ethanol production in Clostridium thermocellum

Clostridium thermocellum ferments cellulose, is a promising candidate for ethanol production from cellulosic biomass, and has been the focus of studies aimed at improving ethanol yield. Thermoanaerobacterium saccharolyticum ferments hemicellulose, but not cellulose, and has been engineered to produce ethanol at high yield and titer. Recent research has led to the identification of four genes in T. saccharolyticum involved in ethanol production: adhE, nfnA, nfnB and adhA. We introduced these genes into C. thermocellum and observed significant improvements to ethanol yield, titer, and productivity. The four genes alone, however, were insufficient to achieve in C. thermocellum the ethanol yields and titers observed in engineered T. saccharolyticum strains, even when combined with gene deletions targeting hydrogen production. This suggests that other parts of T. saccharolyticum metabolism may also be necessary to reproduce the high ethanol yield and titer phenotype in C. thermocellum.