Gene essentiality in the solventogenic Clostridium acetobutylicum DSM 792.

Clostridium acetobutylicum is a solventogenic, anaerobic, gram-positive bacterium that is commonly considered the model organism for studying acetone-butanol-ethanol fermentation. The need to produce these chemicals sustainably and with a minimal impact on the environment has revived the interest in research on this bacterium. The recent development of efficient genetic tools allows to better understand the physiology of this micro-organism, aiming at improving its fermentation capacities. Knowledge about gene essentiality would guide the future genetic editing strategies and support the understanding of crucial cellular functions in this bacterium. In this work, we applied a transposon insertion site sequencing method to generate large mutant libraries containing millions of independent mutants that allowed us to identify a core group of 418 essential genes needed for in vitro development. Future research on this significant biocatalyst will be guided by the data provided in this work, which will serve as a valuable resource for the community. IMPORTANCE: Clostridium acetobutylicum is a leading candidate to synthesize valuable compounds like three and four carbons alcohols. Its ability to convert carbohydrates into a mixture of acetone, butanol, and ethanol as well as other chemicals of interest upon genetic engineering makes it an advantageous organism for the valorization of lignocellulose-derived sugar mixtures. Since, genetic optimization depends on the fundamental insights supplied by accurate gene function assignment, gene essentiality analysis is of great interest as it can shed light on the function of many genes whose functions are still to be confirmed. The data obtained in this study will be of great value for the research community aiming to develop C. acetobutylicum as a platform organism for the production of chemicals of interest.

Alleviation of Carbon Catabolite Repression through araR and xylR Inactivation in Clostridium acetobutylicum DSM 792.

Efficient bioconversion processes of lignocellulose-derived carbohydrates into chemicals have received increasing interest in the last decades since they represent a promising alternative to petro-based processes. Despite efforts to adapt microorganisms to the use of such substrates, one of their major limitations remains their inability to consume multiple sugars simultaneously. In particular, the solventogenic model organism Clostridium acetobutylicum struggles to efficiently use second generation (2G) substrates because of carbon catabolite repression mechanisms that prevent the assimilation of xylose and arabinose in the presence of glucose. In this study, we addressed this issue by inactivating genes encoding transcriptional repressors involved in such mechanisms in the C. acetobutylicum strain DSM 792. Our results showed that the deletion of the two putative copies of xylR (CA_C2613 and CA_C3673) had little or no effect on the ability of the strain to consume xylose. Unlikely, the deletion of araR (CA_C1340) led to a 2.5-fold growth rate increase on xylose. The deletion of both araR and xylR genes resulted in the coassimilation of arabinose together with glucose, while xylose consumption remained inefficient. Transcriptional analyses of the wild-type strain and mutants grown on glucose, arabinose, xylose, and combinations of them provided a crucial, global overview of regulations triggered by the products of both araR and xylR in C. acetobutylicum. As suggested by these data, overexpression of xylA and xylB led to further improvement of pentose assimilation. Those results represent a step forward in the development of genetically modified strains of C. acetobutylicum able to coassimilate lignocellulosic-derived sugars. IMPORTANCE C. acetobutylicum is a strong candidate to produce chemicals of interest such as C3 and C4 alcohols. Used for more than a century for its capacity to produce a mixture of acetone, butanol, and ethanol from first generation (1G) substrates, its natural ability to assimilate a wide variety of monoosides also predisposes it as an auspicious organism for the valorization of lignocellulose-derived sugar mixtures. To achieve this purpose, a better understanding of carbon catabolite repression mechanisms is essential. The work done here provides critical knowledge on how these mechanisms occur during growth on glucose, arabinose, and xylose mixtures, as well as strategies to tackle them.

Enzymatic breakdown of biofilm matrix to allow flow cytometry viability analysis of Clostridium beijerinckii cells.

AIMS: Flow cytometry (FC) is a good way to enumerate the number of viable cells in suspension but is not adapted to mature biofilm analysis. The aim of this study is to investigate the effect of mechanical treatment coupled with enzymatic hydrolysis of biofilm matrix on FC viability analysis of biofilm cells. METHODS AND RESULTS: Biofilm was grown for 300 h of continuous fermentation on polyurethane foams. Fermentation was stopped, and the biofilm was detached by agitating the foams in PBS buffer with vortex agitation for 2 min. The best enzymatic hydrolysis consisted of sequential use of DNase I and proteinase K incubated for 1 h at 34°C. Biofilm cells detached from polyurethane foams were stained with both propidium iodide (PI) and carboxyfluoresceine diacetate and analyzed by FC. FC analysis performed after vortex agitation revealed the presence of high non-fluorescent events (78.9% ± 3.3%). After enzymatic treatment, a cell population was extracted from background noise and could be observed on FSC-SSC profile. The non-fluorescent events of this cell population decreased drastically to 41.9% ± 6.6%, and the percentage of viable cells was enhanced from 2.6% ± 0.9% to 38.2% ± 4.0% compared to analysis performed after mechanical treatment alone. CONCLUSIONS: Consequently, protease and nuclease activity are essential to hydrolyze extra polymeric substances prior to FC viability analysis in mature biofilm formed by Clostridium beijerinckii.

Adaptation and application of a two-plasmid inducible CRISPR-Cas9 system in Clostridium beijerinckii.

Recent developments in CRISPR technologies have opened new possibilities for improving genome editing tools dedicated to the Clostridium genus. In this study we adapted a two-plasmid tool based on this technology to enable scarless modification of the genome of two reference strains of Clostridium beijerinckii producing an Acetone/Butanol/Ethanol (ABE) or an Isopropanol/Butanol/Ethanol (IBE) mix of solvents. In the NCIMB 8052 ABE-producing strain, inactivation of the SpoIIE sporulation factor encoding gene resulted in sporulation-deficient mutants, and this phenotype was reverted by complementing the mutant strain with a functional spoIIE gene. Furthermore, the fungal cellulase-encoding celA gene was inserted into the C. beijerinckii NCIMB 8052 chromosome, resulting in mutants with endoglucanase activity. A similar two-plasmid approach was next used to edit the genome of the natural IBE-producing strain C. beijerinckii DSM 6423, which has never been genetically engineered before. Firstly, the catB gene conferring thiamphenicol resistance was deleted to make this strain compatible with our dual-plasmid editing system. As a proof of concept, our dual-plasmid system was then used in C. beijerinckii DSM 6423 ΔcatB to remove the endogenous pNF2 plasmid, which led to a sharp increase of transformation efficiencies.

A CRISPR/Anti-CRISPR Genome Editing Approach Underlines the Synergy of Butanol Dehydrogenases in Clostridium acetobutylicum DSM 792.

Although Clostridium acetobutylicum is the model organism for the study of acetone-butanol-ethanol (ABE) fermentation, its characterization has long been impeded by the lack of efficient genome editing tools. In particular, the contribution of alcohol dehydrogenases to solventogenesis in this bacterium has mostly been studied with the generation of single-gene deletion strains. In this study, the three butanol dehydrogenase-encoding genes located on the chromosome of the DSM 792 reference strain were deleted iteratively by using a recently developed CRISPR-Cas9 tool improved by using an anti-CRISPR protein-encoding gene, acrIIA4 Although the literature has previously shown that inactivation of either bdhA, bdhB, or bdhC had only moderate effects on the strain, this study shows that clean deletion of both bdhA and bdhB strongly impaired solvent production and that a triple mutant ΔbdhA ΔbdhB ΔbdhC was even more affected. Complementation experiments confirmed the key role of these enzymes and the capacity of each bdh copy to fully restore efficient ABE fermentation in the triple deletion strain.IMPORTANCE An efficient CRISPR-Cas9 editing tool based on a previous two-plasmid system was developed for Clostridium acetobutylicum and used to investigate the contribution of chromosomal butanol dehydrogenase genes during solventogenesis. Thanks to the control of cas9 expression by inducible promoters and of Cas9-guide RNA (gRNA) complex activity by an anti-CRISPR protein, this genetic tool allows relatively fast, precise, markerless, and iterative modifications in the genome of this bacterium and potentially of other bacterial species. As an example, scarless mutants in which up to three genes coding for alcohol dehydrogenases are inactivated were then constructed and characterized through fermentation assays. The results obtained show that in C. acetobutylicum, other enzymes than the well-known AdhE1 are crucial for the synthesis of alcohol and, more globally, to perform efficient solventogenesis.

Correction to: Genome and transcriptome of the natural isopropanol producer Clostridium beijerinckii DSM6423.

Following the publication of this article [1], the authors noticed that Figs. 2, 3 and 4 were in the incorrect order and thus had incorrect captions.

Genome and transcriptome of the natural isopropanol producer Clostridium beijerinckii DSM6423.

BACKGROUND: There is a worldwide interest for sustainable and environmentally-friendly ways to produce fuels and chemicals from renewable resources. Among them, the production of acetone, butanol and ethanol (ABE) or Isopropanol, Butanol and Ethanol (IBE) by anaerobic fermentation has already a long industrial history. Isopropanol has recently received a specific interest and the best studied natural isopropanol producer is C. beijerinckii DSM 6423 (NRRL B-593). This strain metabolizes sugars into a mix of IBE with only low concentrations of ethanol produced (< 1 g/L). However, despite its relative ancient discovery, few genomic details have been described for this strain. Research efforts including omics and genetic engineering approaches are therefore needed to enable the use of C. beijerinckii as a microbial cell factory for production of isopropanol. RESULTS: The complete genome sequence and a first transcriptome analysis of C. beijerinckii DSM 6423 are described in this manuscript. The combination of MiSeq and de novo PacBio sequencing revealed a 6.38 Mbp chromosome containing 6254 genomic objects. Three Mobile Genetic Elements (MGE) were also detected: a linear double stranded DNA bacteriophage (ϕ6423) and two plasmids (pNF1 and pNF2) highlighting the genomic complexity of this strain. A first RNA-seq transcriptomic study was then performed on 3 independent glucose fermentations. Clustering analysis allowed us to detect some key gene clusters involved in the main life cycle steps (acidogenesis, solvantogenesis and sporulation) and differentially regulated among the fermentation. These putative clusters included some putative metabolic operons comparable to those found in other reference strains such as C. beijerinckii NCIMB 8052 or C. acetobutylicum ATCC 824. Interestingly, only one gene was encoding for an alcohol dehydrogenase converting acetone into isopropanol, suggesting a single genomic event occurred on this strain to produce isopropanol. CONCLUSIONS: We present the full genome sequence of Clostridium beijerinckii DSM 6423, providing a complete genetic background of this strain. This offer a great opportunity for the development of dedicated genetic tools currently lacking for this strain. Moreover, a first RNA-seq analysis allow us to better understand the global metabolism of this natural isopropanol producer, opening the door to future targeted engineering approaches.

A filamentous bacteriophage targeted to carcinoembryonic antigen induces tumor regression in mouse models of colorectal cancer.

Colorectal cancer is a deadly disease, which is frequently diagnosed at advanced stages, where conventional treatments are no longer effective. Cancer immunotherapy has emerged as a new form to treat different malignancies by turning-on the immune system against tumors. However, tumors are able to evade antitumor immune responses by promoting an immunosuppressive microenvironment. Single-stranded DNA containing M13 bacteriophages are highly immunogenic and can be specifically targeted to the surface of tumor cells to trigger inflammation and infiltration of activated innate immune cells, overcoming tumor-associated immunosuppression and promoting antitumor immunity. Carcinoembryonic antigen (CEA) is highly expressed in colorectal cancers and has been shown to promote several malignant features of colorectal cancer cells. In this work, we targeted M13 bacteriophage to CEA, a tumor-associated antigen over-expressed in a high proportion of colorectal cancers but largely absent in normal cells. The CEA-targeted M13 bacteriophage was shown to specifically bind to purified CEA and CEA-expressing tumor cells in vitro. Both intratumoral and systemic administration of CEA-specific bacteriophages significantly reduced tumor growth of mouse models of colorectal cancer, as compared to PBS and control bacteriophage administration. CEA-specific bacteriophages promoted tumor infiltration of neutrophils and macrophages, as well as maturation dendritic cells in tumor-draining lymph nodes, suggesting that antitumor T-cell responses were elicited. Finally, we demonstrated that tumor protection provided by CEA-specific bacteriophage particles is mediated by CD8+ T cells, as depletion of circulating CD8+ T cells completely abrogated antitumor protection. In summary, we demonstrated that CEA-specific M13 bacteriophages represent a potential immunotherapy against colorectal cancer.

Mechanistic modeling of enzymatic hydrolysis of cellulose integrating substrate morphology and cocktail composition.

A mechanistic model of enzymatic hydrolysis taking into account the morphology of the cellulosic particles and its evolution with time was developed. The individual behavior of the main enzymes involved in the reaction (cellobiohydrolases, endoglucanases, and ß-glucosidases), as well as synergy effects, were also included. A large panel of experimental tests was done to fit and validate the model. This database included different enzymes mixtures and operating conditions and allowed to determine and compare with accuracy the adsorption and kinetic parameters of the different enzymes. Model predictions on short hydrolysis times were very satisfactory. On longer times, a deactivation constant was added to represent the hydrolysis slowdown. The model also allowed to predict the impact of enzymes ratios and initial substrate parameters (chain length distribution, polymerization degree) on hydrolysis, and to follow the evolution of these parameters with time. This model revealed general trends on the impact of cellulose morphology on hydrolysis. It is a useful tool to better understand the mechanisms involved in enzymatic hydrolysis of cellulose and to determine optimal cellulolytic cocktails for process design.

Targeted mutagenesis of the ß-strand DNA binding region of African swine fever virus histone-like protein (pA104R) impairs DNA-binding activity and antibody recognition.

African Swine Fever (ASF) is a highly contagious disease caused by a double-stranded DNA virus (ASFV). Despite significant advances made over the last decade, issues such as residual virulence and absence of differentiating infected from vaccinated animals (DIVA) capacity remain an obstacle in the development of live attenuated vaccines (LAVs) against ASFV. It is, therefore, necessary to identify novel strategies to improve vaccine safety, by rational mutagenesis of virulence associated genes and generation of DIVA markers. ASFV encodes a HU (histone-like protein from E. coli strain U93) homolog protein, pA104R, which is involved in viral genome assembly and host immune recognition. A phylogenetic analysis revealed that pA104R is highly conserved among ASFV isolates, suggesting that it can be a good target for vaccine design. Thus, we selectively mutated the ß-strand DNA binding region (BDR) of pA104R to attenuate its enzymatic activity, and identified and mutated several B-cell epitopes present in pA104R to generate a negative marker. Residues K64, K66, and R69 in the BDR were identified as relevant for pA104R activity, with double mutation of the first two showing additive attenuation. pA104R-reactive IgM and IgG epitopes were also identified in the bottom of the BDR, with selective mutagenesis drastically reducing antibody recognition and, when combined with mutations in the arm of the BDR, leading to a further reduction of DNA-binding activity. Interestingly, the immunodominant pA104R-reactive IgG epitope was mainly recognized by IgG1 suggesting that pA104R induces a dominant Th2 response. In sum, the rational mutagenesis can reduce pA104R-DNA binding activity and immune reactivity, providing a rationale for the development of an ASFV pA104R-based DIVA vaccine.

Development and characterization of a bacteriophage cocktail with high lytic efficacy against field-isolated Salmonella enterica.

Salmonella spp. is a prevalent pathogen that causes great public health concern worldwide. Bacteriophage-based cocktails have arisen as an alternative to antibiotics to inhibit the growth of Salmonella. However, the bactericidal effect of bacteriophage cocktails in vivo largely differs from their observed effect in vitro. This is partly because in vitro developments of cocktails do not always consider the bacterial diversity nor the environmental conditions where bacteriophages will have to replicate. Here, we isolated and sequenced 47 bacteriophages that showed variable degrees of lytic activity against 258 Salmonella isolates from a commercial broiler company in Brazil. Three of these bacteriophages were characterized and selected to assemble a cocktail. In vitro quantitative assays determined the cocktail to be highly effective against multiple serovars of Salmonella, including Minnesota and Heidelberg. Remarkably, the in vitro lytic activity of the cocktail was retained or improved in conditions that more closely resembled the chicken gut, such as anaerobiosis, 42°C, and Salmonella mono-strain biofilms. Analysis of bacterial cross-resistance between the 3 bacteriophages composing the cocktail revealed limited or no generation of cross-resistance. Our results highlight the relevance of an optimized flux of work to develop bacteriophage cocktails against Salmonella with high lytic efficacy and strong potential to be applied in vivo in commercial broiler farms.

Genome-Wide TSS Distribution in Three Related Clostridia with Normalized Capp-Switch Sequencing.

Transcription initiation is a tightly regulated process that is crucial for many aspects of prokaryotic physiology. High-throughput transcription start site (TSS) mapping can shed light on global and local regulation of transcription initiation, which in turn may help us understand and predict microbial behavior. In this study, we used Capp-Switch sequencing to determine the TSS positions in the genomes of three model solventogenic clostridia: Clostridium acetobutylicum ATCC 824, C. beijerinckii DSM 6423, and C. beijerinckii NCIMB 8052. We first refined the approach by implementing a normalization pipeline accounting for gene expression, yielding a total of 12,114 mapped TSSs across the species. We further compared the distributions of these sites in the three strains. Results indicated similar distribution patterns at the genome scale, but also some sharp differences, such as for the butyryl-CoA synthesis operon, particularly when comparing C. acetobutylicum to the C. beijerinckii strains. Lastly, we found that promoter structure is generally poorly conserved between C. acetobutylicum and C. beijerinckii. A few conserved promoters across species are discussed, showing interesting examples of how TSS determination and comparison can improve our understanding of gene expression regulation at the transcript level. IMPORTANCE Solventogenic clostridia have been employed in industry for more than a century, initially being used in the acetone-butanol-ethanol (ABE) fermentation process for acetone and butanol production. Interest in these bacteria has recently increased in the context of green chemistry and sustainable development. However, our current understanding of their genomes and physiology limits their optimal use as industrial solvent production platforms. The gene regulatory mechanisms of solventogenesis are still only partly understood, impeding efforts to increase rates and yields. Genome-wide mapping of transcription start sites (TSSs) for three model solventogenic Clostridium strains is an important step toward understanding mechanisms of gene regulation in these industrially important bacteria.

Optical flow sensor as a reference for reduction of motion artefacts in photoplethysmographic measurements.

Methods commonly used for reduction of motion artefacts in photoplethysmography employ accelerometry as a reference for adaptive filtering and signal processing. In this paper, we propose the use of an optical flow sensor to measure the relative displacement between a photoplethysmographic sensor and the measurement site. In order to evaluate the performances of this novel method, a wrist-worn device that enables simultaneous acquisition of physiological information and relative motion has been developed. The optical flow sensor provides a two-dimensional information source correlated with artefacts contained in the cardiac frequency band. Preliminary results show a clear correlation between motion recorded by the sensor and artefacts contained in the photoplethysmographic signal. In association with adaptive filtering, the proposed technique shows efficient reduction of motion artefacts during physical activity.

σ54 (σL) plays a central role in carbon metabolism in the industrially relevant Clostridium beijerinckii.

The solventogenic C. beijerinckii DSM 6423, a microorganism that naturally produces isopropanol and butanol, was previously modified by random mutagenesis. In this work, one of the resulting mutants was characterized. This strain, selected with allyl alcohol and designated as the AA mutant, shows a dominant production of acids, a severely diminished butanol synthesis capacity, and produces acetone instead of isopropanol. Interestingly, this solvent-deficient strain was also found to have a limited consumption of two carbohydrates and to be still able to form spores, highlighting its particular phenotype. Sequencing of the AA mutant revealed point mutations in several genes including CIBE_0767 (sigL), which encodes the σ54 sigma factor. Complementation with wild-type sigL fully restored solvent production and sugar assimilation and RT-qPCR analyses revealed its transcriptional control of several genes related to solventogensis, demonstrating the central role of σ54 in C. beijerinckii DSM 6423. Comparative genomics analysis suggested that this function is conserved at the species level, and this hypothesis was further confirmed through the deletion of sigL in the model strain C. beijerinckii NCIMB 8052.

A two-plasmid inducible CRISPR/Cas9 genome editing tool for Clostridium acetobutylicum.

CRISPR/Cas-based genetic engineering has revolutionised molecular biology in both eukaryotes and prokaryotes. Several tools dedicated to the genomic transformation of the Clostridium genus of Gram-positive bacteria have been described in the literature; however, the integration of large DNA fragments still remains relatively limited. In this study, a CRISPR/Cas9 genome editing tool using a two-plasmid strategy was developed for the solventogenic strain Clostridium acetobutylicum ATCC 824. Codon-optimised cas9 from Streptococcus pyogenes was placed under the control of an anhydrotetracycline-inducible promoter on one plasmid, while the gRNA expression cassettes and editing templates were located on a second plasmid. Through the sequential introduction of these vectors into the cell, we achieved highly accurate genome modifications, including nucleotide substitution, gene deletion and cassette insertion up to 3.6kb. To demonstrate its potential, this genome editing tool was used to generate a marker-free mutant of ATCC 824 that produced an isopropanol-butanol-ethanol mixture. Whole-genome sequencing confirmed that no off-target modifications were present in the mutants. Such a tool is a prerequisite for efficient metabolic engineering in this solventogenic strain and provides an alternative editing strategy that might be applicable to other Clostridium strains.

Draft Genome Sequence of the Butyric Acid Producer Clostridium tyrobutyricum Strain CIP I-776 (IFP923).

Here, we report the draft genome sequence of Clostridium tyrobutyricum CIP I-776 (IFP923), an efficient producer of butyric acid. The genome consists of a single chromosome of 3.19 Mb and provides useful data concerning the metabolic capacities of the strain.

Significado e considerações sobre a osteoporose por mulheres com e sem diagnóstico da doença / Meaning and considerations about osteoporosis by women with and without diagnosis of the disease

INTRODUÇÃO: Entre pacientes diagnosticados e em tratamento para osteoporose, acredita-se que há desconhecimento geral sobre a doença. Entre os pacientes em faixa etária de risco, não diagnosticados, crê-se que o desconhecimento é maior. OBJETIVO: Avaliar as considerações de mulheres sobre o conceito da osteoporose e sua prevenção. MÉTODOS: Estudo qualitativo, descritivo, realizado no Hospital das Clínicas da Universidade Federal de Minas Gerais, Belo Horizonte, com 10 pacientes portadores de osteoporose, e 10 pacientes sem. A tabulação dos dados ocorreu por meio da utilização de três figuras metodológicas: ideia central, expressões chave e o discurso sujeito coletivo. O estudo foi aprovado pelo Comitê de Ética e Pesquisa com Seres Humanos. RESULTADOS: Dentre as pacientes com diagnóstico, destacou-se como ideias centrais a vulnerabilidade às fraturas e os conceitos de apresentação da doença como osteopenia e osteoporose. No grupo sem diagnóstico, 40% desconhecia a doença. Na prevenção, observou-se maior conhecimento no grupo portador da doença, sendo as ideias centrais destacadas: os exercícios físicos, alimentação rica em cálcio e vitamina D, além da exposição solar. CONCLUSÃO: Evidenciou-se um conhecimento limitado sobre a osteoporose e suas formas de prevenção, sobretudo no grupo sem diagnóstico, porém em faixa etária de risco. Assim, conclui-se que, tratando-se de uma doença de elevada prevalência, de altos custos orçamentários para a saúde pública, e com riscos significativos uma vez não diagnosticados e não tratada, medidas resolutivas de maior esclarecimento sobre a doença devem ser praticadas em todas as esferas da saúde pública.

INTRODUCTION: Among patients diagnosed and under treatment for osteoporosis, it is believed that there is general ignorance about the disease. Among undiagnosed patients at risk, it is believed that the lack of knowledge is larger. OBJECTIVE: To evaluate the considerations of women about the concept of osteoporosis and its prevention. METHODS: A qualitative, descriptive study was carried out at Hospital das Clínicas da Universidade Federal de Minas Gerais, Belo Horizonte, Brazil, with 10 patients with osteoporosis and 10 patients without the disease. Data tabulation occurred using three methodological figures: central idea, key expressions and collective subject discourse. The study was approved by the Committee of Ethics and Research with Human Beings. RESULTS: Among the diagnosed patients, the central ideas were vulnerability to fractures and the concepts of disease presentation like osteopenia and osteoporosis. In the undiagnosed group, 40% were unaware of the disease. About prevention, greater knowledge was observed in the group with the disease, with the main ideas being highlighted: physical exercises, calcium and vitamin D rich food, as well as sun exposure. CONCLUSION: There was limited knowledge about osteoporosis and its prevention, especially in the group without diagnosis, but in the age group at risk. Thus, it is concluded that, in the case of a disease of high prevalence, of high budgetary costs for public health, and with significant risks once it is undiagnosed and untreated, resolute measures of greater clarity about the disease should be practiced in all spheres of public health.

Optimization of a synthetic mixture composed of major Trichoderma reesei enzymes for the hydrolysis of steam-exploded wheat straw.

BACKGROUND: An efficient hydrolysis of lignocellulosic substrates to soluble sugars for biofuel production necessitates the interplay and synergistic interaction of multiple enzymes. An optimized enzyme mixture is crucial for reduced cost of the enzymatic hydrolysis step in a bioethanol production process and its composition will depend on the substrate and type of pretreatment used. In the present study, an experimental design was used to determine the optimal composition of a Trichoderma reesei enzyme mixture, comprising the main cellulase and hemicellulase activities, for the hydrolysis of steam-exploded wheat straw. METHODS: Six enzymes, CBH1 (Cel7a), CBH2 (Cel6a), EG1 (Cel7b), EG2 (Cel5a), as well as the xyloglucanase Cel74a and the xylanase XYN1 (Xyl11a) were purified from a T. reesei culture under lactose/xylose-induced conditions. Sugar release was followed in milliliter-scale hydrolysis assays for 48 hours and the influence of the mixture on initial conversion rates and final yields is assessed. RESULTS: The developed model could show that both responses were strongly correlated. Model predictions suggest that optimal hydrolysis yields can be obtained over a wide range of CBH1 to CBH2 ratios, but necessitates a high proportion of EG1 (13% to 25%) which cannot be replaced by EG2. Whereas 5% to 10% of the latter enzyme and a xylanase content above 6% are required for highest yields, these enzymes are predicted to be less important in the initial stage of hydrolysis. CONCLUSIONS: The developed model could reliably predict hydrolysis yields of enzyme mixtures in the studied domain and highlighted the importance of the respective enzyme components in both the initial and the final hydrolysis phase of steam-exploded wheat straw.

Effect of pretreatment and enzymatic hydrolysis of wheat straw on cell wall composition, hydrophobicity and cellulase adsorption.

The present study aimed to determine the impact of cell wall composition and lignin content on enzyme adsorption and degradability. Thioacidolysis analysis of residual lignins in wheat straw after steam-explosion or organosolv pretreatment revealed an increase in lignin condensation degree of 27% and 33%, respectively. Surface hydrophobicity assessed through wettability tests decreased after the pretreatments (contact angle decrease of 20-50%), but increased with enzymatic conversion (30% maximum contact angle increase) and correlatively to lignin content. Adsorption of the three major cellulases Cel7A, Cel6A and Cel7B from Trichoderma reesei decreased with increasing hydrolysis time, down to 7%, 31% and 70% on the sample with the highest lignin content, respectively. The fraction of unspecifically bound enzymes was dependent both on the enzyme and the lignin content. Adsorption and specific activity were shown to be inversely proportional to lignin content and hydrophobicity, suggesting that lignin is one of the factors restricting enzymatic hydrolysis.

Comparative kinetic analysis of two fungal beta-glucosidases.

BACKGROUND: The enzymatic hydrolysis of cellulose is still considered as one of the main limiting steps of the biological production of biofuels from lignocellulosic biomass. It is a complex multistep process, and various kinetic models have been proposed. The cellulase enzymatic cocktail secreted by Trichoderma reesei has been intensively investigated. beta-glucosidases are one of a number of cellulolytic enzymes, and catalyze the last step releasing glucose from the inhibitory cellobiose. beta-glucosidase (BGL1) is very poorly secreted by Trichoderma reesei strains, and complete hydrolysis of cellulose often requires supplementation with a commercial beta-glucosidase preparation such as that from Aspergillus niger (Novozymes SP188). Surprisingly, kinetic modeling of beta-glucosidases lacks reliable data, and the possible differences between native T. reesei and supplemented beta-glucosidases are not taken into consideration, possibly because of the difficulty of purifying BGL1. RESULTS: A comparative kinetic analysis of beta-glucosidase from Aspergillus niger and BGL1 from Trichoderma reesei, purified using a new and efficient fast protein liquid chromatography protocol, was performed. This purification is characterized by two major steps, including the adsorption of the major cellulases onto crystalline cellulose, and a final purification factor of 53. Quantitative analysis of the resulting beta-glucosidase fraction from T. reesei showed it to be 95% pure. Kinetic parameters were determined using cellobiose and a chromogenic artificial substrate. A new method allowing easy and rapid determination of the kinetic parameters was also developed. beta-Glucosidase SP188 (Km = 0.57 mM; Kp = 2.70 mM) has a lower specific activity than BGL1 (Km = 0.38 mM; Kp = 3.25 mM) and is also more sensitive to glucose inhibition. A Michaelis-Menten model integrating competitive inhibition by the product (glucose) has been validated and is able to predict the beta-glucosidase activity of both enzymes. CONCLUSIONS: This article provides a useful comparison between the activity of beta-glucosidases from two different fungi, and shows the importance of fully characterizing both enzymes. A Michaelis-Menten model was developed, including glucose inhibition and kinetic parameters, which were accurately determined and compared. This model can be further integrated into a cellulose hydrolysis model dissociating beta-glucosidase activity from that of other cellulases. It can also help to define the optimal enzymatic cocktails for new beta-glucosidase activities.