Regioselective chemoenzymatic syntheses of ferulate conjugates as chromogenic substrates for feruloyl esterases.

Generally, carbohydrate-active enzymes are studied using chromogenic substrates that provide quick and easy color-based detection of enzyme-mediated hydrolysis. For feruloyl esterases, commercially available chromogenic ferulate derivatives are both costly and limited in terms of their experimental application. In this study, we describe solutions for these two issues, using a chemoenzymatic approach to synthesize different ferulate compounds. The overall synthetic routes towards commercially available 5-bromo-4-chloro-3-indolyl and 4-nitrophenyl 5-O-feruloyl-α-ʟ-arabinofuranosides were significantly shortened (from 7 or 8 to 4-6 steps), and the transesterification yields were enhanced (from 46 to 73% and from 47 to 86%, respectively). This was achieved using enzymatic (immobilized Lipozyme® TL IM from Thermomyces lanuginosus) transesterification of unprotected vinyl ferulate to the primary hydroxy group of α-ʟ-arabinofuranosides. Moreover, a novel feruloylated 4-nitrocatechol-1-yl-substituted butanetriol analog, containing a cleavable hydroxylated linker, was also synthesized in 32% overall yield in 3 steps (convergent synthesis). The latter route combined the regioselective functionalization of 4-nitrocatechol and enzymatic transferuloylation. The use of this strategy to characterize type A feruloyl esterase from Aspergillus niger reveals the advantages of this substrate for the characterizations of feruloyl esterases.

Redirecting substrate regioselectivity using engineered ΔN123-GBD-CD2 branching sucrases for the production of pentasaccharide repeating units of S. flexneri 3a, 4a and 4b haptens.

The (chemo-)enzymatic synthesis of oligosaccharides has been hampered by the lack of appropriate enzymatic tools with requisite regio- and stereo-specificities. Engineering of carbohydrate-active enzymes, in particular targeting the enzyme active site, has notably led to catalysts with altered regioselectivity of the glycosylation reaction thereby enabling to extend the repertoire of enzymes for carbohydrate synthesis. Using a collection of 22 mutants of ΔN123-GBD-CD2 branching sucrase, an enzyme from the Glycoside Hydrolase family 70, containing between one and three mutations in the active site, and a lightly protected chemically synthesized tetrasaccharide as an acceptor substrate, we showed that altered glycosylation product specificities could be achieved compared to the parental enzyme. Six mutants were selected for further characterization as they produce higher amounts of two favored pentasaccharides compared to the parental enzyme and/or new products. The produced pentasaccharides were shown to be of high interest as they are precursors of representative haptens of Shigella flexneri serotypes 3a, 4a and 4b. Furthermore, their synthesis was shown to be controlled by the mutations introduced in the active site, driving the glucosylation toward one extremity or the other of the tetrasaccharide acceptor. To identify the molecular determinants involved in the change of ΔN123-GBD-CD2 regioselectivity, extensive molecular dynamics simulations were carried out in combination with in-depth analyses of amino acid residue networks. Our findings help to understand the inter-relationships between the enzyme structure, conformational flexibility and activity. They also provide new insight to further engineer this class of enzymes for the synthesis of carbohydrate components of bacterial haptens.

Multimodularity of a GH10 Xylanase Found in the Termite Gut Metagenome.

The functional screening of a Pseudacanthotermes militaris termite gut metagenomic library revealed an array of xylan-degrading enzymes, including P. militaris 25 (Pm25), a multimodular glycoside hydrolase family 10 (GH10). Sequence analysis showed details of the unusual domain organization of this enzyme. It consists of one catalytic domain, which is intercalated by two carbohydrate binding modules (CBMs) from family 4. The genes upstream of the genes encoding Pm25 are susC-susD-unk, suggesting Pm25 is a Xyn10C-like enzyme belonging to a polysaccharide utilization locus. The majority of Xyn10C-like enzymes shared the same interrupted domain architecture and were vastly distributed in different xylan utilization loci found in gut Bacteroidetes, indicating the importance of this enzyme in glycan acquisition for gut microbiota. To understand its unusual multimodularity and the possible role of the CBMs, a detailed characterization of the full-length Pm25 and truncated variants was performed. Results revealed that the GH10 catalytic module is specific toward the hydrolysis of xylan. Ligand binding results indicate that the GH10 module and the CBMs act independently, whereas the tandem CBM4s act synergistically with each other and improve enzymatic activity when assayed on insoluble polysaccharides. In addition, we show that the UNK protein upstream of Pm25 is able to bind arabinoxylan. Altogether, these findings contribute to a better understanding of the potential role of Xyn10C-like proteins in xylan utilization systems of gut bacteria.IMPORTANCE Xylan is the major hemicellulosic polysaccharide in cereals and contributes to the recalcitrance of the plant cell wall toward degradation. Members of the Bacteroidetes, one of the main phyla in rumen and human gut microbiota, have been shown to encode polysaccharide utilization loci dedicated to the degradation of xylan. Here, we present the biochemical characterization of a xylanase encoded by a Bacteroidetes strain isolated from the termite gut metagenome. This xylanase is a multimodular enzyme, the sequence of which is interrupted by the insertion of two CBMs from family 4. Our results show that this enzyme resembles homologues that were shown to be important for xylan degradation in rumen or human diet and show that the CBM insertion in the middle of the sequence seems to be a common feature in xylan utilization systems. This study shed light on our understanding of xylan degradation and plant cell wall deconstruction, which can be applied to several applications in food, feed, and bioeconomy.

Corrigendum: The Role of the FMN-Domain of Human Cytochrome P450 Oxidoreductase in Its Promiscuous Interactions With Structurally Diverse Redox Partners.

[This corrects the article DOI: 10.3389/fphar.2020.00299.].

The Role of the FMN-Domain of Human Cytochrome P450 Oxidoreductase in Its Promiscuous Interactions With Structurally Diverse Redox Partners.

NADPH cytochrome P450 oxidoreductase (CPR) is the obligatory electron supplier that sustains the activity of microsomal cytochrome P450 (CYP) enzymes. The variant nature of the isoform-specific proximal interface of microsomal CYPs indicates that CPR is capable of multiple degenerated interactions with CYPs for electron transfer, through different binding mechanisms, and which are still not well-understood. Recently, we showed that CPR dynamics allows formation of open conformations that can be sampled by its structurally diverse redox partners in a CYP-isoform dependent manner. To further investigate the role of the CPR FMN-domain in effective binding of CPR to its diverse acceptors and to clarify the underlying molecular mechanisms, five different CPR-FMN-domain random mutant libraries were created. These libraries were screened for mutants with increased activity when combined with specific CYP-isoforms. Seven CPR-FMN-domain mutants were identified, supporting a gain in activity for CYP1A2 (P117H, G144C, A229T), 2A6 (P117L/L125V, G175D, H183Y), or 3A4 (N151D). Effects were evaluated using extended enzyme kinetic analysis, cytochrome b 5 competition, ionic strength effect on CYP activity, and structural analysis. Mutated residues were located either in or adjacent to several acidic amino acid stretches - formerly indicated to be involved in CPR:CYP interactions - or close to two tyrosine residues suggested to be involved in FMN binding. Several of the identified positions co-localize with mutations found in naturally occurring CPR variants that were previously shown to cause CYP-isoform-dependent effects. The mutations do not seem to significantly alter the geometry of the FMN-domain but are likely to cause very subtle alterations leading to improved interaction with a specific CYP. Overall, these data suggest that CYPs interact with CPR using an isoform specific combination of several binding motifs of the FMN-domain.

High-Level Heterologous Expression of Endo-1,4-ß-Xylanase from Penicillium citrinum in Pichia pastoris X-33 Directed through Codon Optimization and Optimized Expression.

Most common industrial xylanases are produced from filamentous fungi. In this study, the codon-optimized xynA gene encoding xylanase A from the fungus Penicilium citrinum was successfully synthesized and expressed in the yeast Pichia pastoris. The levels of secreted enzyme activity under the control of glyceraldehyde-3-phosphate dehydrogenase (PGAP) and alcohol oxidase 1 (PAOX1) promoters were compared. The Pc Xyn11A was produced as a soluble protein and the total xylanase activity under the control of PGAP and PAOX1 was 34- and 193-fold, respectively, higher than that produced by the native strain of P. citrinum. The Pc Xyn11A produced under the control of the PAOX1 reached a maximum activity of 676 U/mL when induced with 1% (v/v) methanol every 24 h for 5 days. The xylanase was purified by ion exchange chromatography and then characterized. The enzyme was optimally active at 55 °C and pH 5.0 but stable over a broad pH range (3.0-9.0), retaining more than 80% of the original activity after 24 h or after pre-incubation at 40 °C for 1 h. With birchwood xylan as a substrate, Pc Xyn11A showed a Km(app) of 2.8 mg/mL, and a kcat of 243 s-1. The high level of secretion of Pc Xyn11A and its stability over a wide range of pH and moderate temperatures could make it useful for a variety of biotechnological applications.

Efficient expression and secretion of endo-1,4-ß-xylanase from Penicillium citrinum in non-conventional yeast Yarrowia lipolytica directed by the native and the preproLIP2 signal peptides.

Filamentous fungi are the most common industrial xylanase producers. In this study, the xynA gene encoding xylanase A of Penicilium citrinum was successfully synthesized and expressed in Yarrowia lipolytica under the control of the strong constitutive TEF promoter. Native and preproLIP2 secretion signals were used for comparison of the expression and secretion level. The recombinant xylanase was produced as a soluble protein, and the total activity production reached 11 and 52 times higher than the level of activity produced by the fungus P. citrinum native strain, respectively. Maximum activity was observed with the preproLIP2 secretion signal at 180 U/mL. Post translational glycosylation affected the molecular mass of the recombinant xylanase, resulting in an apparent molecular weight larger than 60 kDa, whereas after deglycosylation, the recombinant XynA displayed a molecular mass of 20 kDa. The deglycosylated xylanase was purified by ion exchange chromatography and reached 185-fold of purification. The enzyme was optimally active at 55 °C and pH 5 and stable over a broad pH range (3-9). It retained more than 80% of the original activity after 24 h. It conserved around 80% of the original activity after pre-incubation at 40 °C for 6 h. With birchwood xylan as substrate, the enzyme showed a Km of 5.2 mg/mL, and kcat of 245 per s. The high level of secretion and the stability over a wide range of pH and at moderate temperatures of the re-XynA could be useful for variety of biotechnological applications.

Directed evolution of the type C feruloyl esterase from Fusarium oxysporum FoFaeC and molecular docking analysis of its improved variants.

The need to develop competitive and eco-friendly processes in the cosmetic industry leads to the search for new enzymes with improved properties for industrial bioconversions in this sector. In the present study, a complete methodology to generate, express and screen diversity for the type C feruloyl esterase from Fusarium oxysporium FoFaeC was set up in a high-throughput fashion. A library of around 30,000 random mutants of FoFaeC was generated by error prone PCR of fofaec cDNA and expressed in Yarrowia lipolytica. Screening for enzymatic activity towards the substrates 5-bromo-4-chloroindol-3-yl and 4-nitrocatechol-1-yl ferulates allowed the selection of 96 enzyme variants endowed with improved enzymatic activity that were then characterized for thermo- and solvent- tolerance. The five best mutants in terms of higher activity, thermo- and solvent- tolerance were selected for analysis of substrate specificity. Variant L432I was shown to be able to hydrolyze all the tested substrates, except methyl sinapate, with higher activity than wild type FoFaeC towards methyl p-coumarate, methyl ferulate and methyl caffeate. Moreover, the E455D variant was found to maintain completely its hydrolytic activity after two hour incubation at 55 °C, whereas the L284Q/V405I variant showed both higher thermo- and solvent- tolerance than wild type FoFaeC. Small molecule docking simulations were applied to the five novel selected variants in order to examine the binding pattern of substrates used for enzyme characterization of wild type FoFaeC and the evolved variants.

Human cytochrome P450 expression in bacteria: Whole-cell high-throughput activity assay for CYP1A2, 2A6 and 3A4.

Cytochrome P450s (CYPs) are key enzymes involved in drug and xenobiotic metabolism. A wide array of in vitro methodologies, including recombinant sources, are currently been used to assess CYP catalysis, to identify the metabolic profile of compounds, potential drug-drug interactions, protein-protein interactions in the CYP enzyme complex and the role of polymorphic enzymes. We report here on a bacterial whole-cells high-throughput method for the activity evaluation of human CYP1A2, 2A6, and 3A4, when sustained by NADPH cytochrome P450 oxidoreductase (CPR), in the absence or presence of cytochrome b5 (CYB5). This new assay consists of a microplate real-time fluorometric method, with direct measurement of metabolite formation, in a suspension of Escherichia coli BTC-CYP bacteria, a human CYP competent tester strain when incubated with specific fluorogenic substrates. Overall, the maximum turnover (kcat) velocities of the three human CYPs resulting from the whole-BTC cells assays were similar to those obtained when applying the corresponding standard reference membrane fractions assays. CYP activity screening with co-expression of CYB5 suggests an enhancing effect of CYB5 on the kcat of specific isoforms, when using the whole-BTC cells assay. Our results demonstrate that this new approach can offer an efficient high-throughput method for screening of CYP1A2, 2A6 and 3A4 activity and can be potentially applicable for other human CYPs. This can be of particular use for timely and efficient screening of chemical libraries or mutant libraries of CYP enzyme complex proteins, without the necessity for labor intensive isolation of subcellular fractions.

Correction: The Hinge Segment of Human NADPH-Cytochrome P450 Reductase in Conformational Switching: The Critical Role of Ionic Strength.

[This corrects the article on p. 755 in vol. 8, PMID: 29163152.].

Expressing accessory proteins in cellulolytic Yarrowia lipolytica to improve the conversion yield of recalcitrant cellulose.

BACKGROUND: A recently constructed cellulolytic Yarrowia lipolytica is able to grow efficiently on an industrial organosolv cellulose pulp, but shows limited ability to degrade crystalline cellulose. In this work, we have further engineered this strain, adding accessory proteins xylanase II (XYNII), lytic polysaccharide monooxygenase (LPMO), and swollenin (SWO) from Trichoderma reesei in order to enhance the degradation of recalcitrant substrate. RESULTS: The production of EG I was enhanced using a promoter engineering strategy. This provided a new cellulolytic Y. lipolytica strain, which compared to the parent strain, exhibited higher hydrolytic activity on different cellulosic substrates. Furthermore, three accessory proteins, TrXYNII, TrLPMOA and TrSWO, were individually expressed in cellulolytic and non-cellulolytic Y. lipolytica. The amount of rhTrXYNII and rhTrLPMOA secreted by non-cellulolytic Y. lipolytica in YTD medium during batch cultivation in flasks was approximately 62 and 52 mg/L, respectively. The purified rhTrXYNII showed a specific activity of 532 U/mg-protein on beechwood xylan, while rhTrLPMOA exhibited a specific activity of 14.4 U/g-protein when using the Amplex Red/horseradish peroxidase assay. Characterization of rhTrLPMOA revealed that this protein displays broad specificity against ß-(1,4)-linked glucans, but is inactive on xylan. Further studies showed that the presence of TrLPMOA synergistically enhanced enzymatic hydrolysis of cellulose by cellulases, while TrSWO1 boosted cellulose hydrolysis only when it was applied before the action of cellulases. The presence of rTrXYNII enhanced enzymatic hydrolysis of an industrial cellulose pulp and of wheat straw. Co-expressing TrXYNII and TrLPMOA in cellulolytic Y. lipolytica with enhanced EG I production procured a novel engineered Y. lipolytica strain that displayed enhanced ability to degrade both amorphous (CIMV-cellulose) and recalcitrant crystalline cellulose in complex biomass (wheat straw) by 16 and 90%, respectively. CONCLUSIONS: This study has provided a potent cellulose-degrading Y. lipolytica strain that co-expresses a core set of cellulolytic enzymes and some accessory proteins. Results reveal that the tuning of cellulase production and the production of accessory proteins leads to optimized performance. Accordingly, the beneficial effect of accessory proteins for cellulase-mediated degradation of cellulose is underlined, especially when crystalline cellulose and complex biomass are used as substrates. Findings specifically underline the benefits and specific properties of swollenin. Although in our study swollenin clearly promoted cellulase action, its use requires process redesign to accommodate its specific mode of action.

The Hinge Segment of Human NADPH-Cytochrome P450 Reductase in Conformational Switching: The Critical Role of Ionic Strength.

NADPH-cytochrome P450 reductase (CPR) is a redox partner of microsomal cytochromes P450 and is a prototype of the diflavin reductase family. CPR contains 3 distinct functional domains: a FMN-binding domain (acceptor reduction), a linker (hinge), and a connecting/FAD domain (NADPH oxidation). It has been demonstrated that the mechanism of CPR exhibits an important step in which it switches from a compact, closed conformation (locked state) to an ensemble of open conformations (unlocked state), the latter enabling electron transfer to redox partners. The conformational equilibrium between the locked and unlocked states has been shown to be highly dependent on ionic strength, reinforcing the hypothesis of the presence of critical salt interactions at the interface between the FMN and connecting FAD domains. Here we show that specific residues of the hinge segment are important in the control of the conformational equilibrium of CPR. We constructed six single mutants and two double mutants of the human CPR, targeting residues G240, S243, I245 and R246 of the hinge segment, with the aim of modifying the flexibility or the potential ionic interactions of the hinge segment. We measured the reduction of cytochrome c at various salt concentrations of these 8 mutants, either in the soluble or membrane-bound form of human CPR. All mutants were found capable of reducing cytochrome c yet with different efficiency and their maximal rates of cytochrome c reduction were shifted to lower salt concentration. In particular, residue R246 seems to play a key role in a salt bridge network present at the interface of the hinge and the connecting domain. Interestingly, the effects of mutations, although similar, demonstrated specific differences when present in the soluble or membrane-bound context. Our results demonstrate that the electrostatic and flexibility properties of the hinge segment are critical for electron transfer from CPR to its redox partners.

Conferring cellulose-degrading ability to Yarrowia lipolytica to facilitate a consolidated bioprocessing approach.

BACKGROUND: Yarrowia lipolytica, one of the most widely studied "nonconventional" oleaginous yeast species, is unable to grow on cellulose. Recently, we identified and overexpressed two endogenous ß-glucosidases in Y. lipolytica, thus enabling this yeast to use cello-oligosaccharides as a carbon source for growth. Using this engineered yeast platform, we have now gone further toward building a fully cellulolytic Y. lipolytica for use in consolidated bioprocessing of cellulose. RESULTS: Initially, different essential enzyme components of a cellulase cocktail (i.e,. cellobiohydrolases and endoglucanases) were individually expressed in Y. lipolytica in order to ascertain the viability of the strategy. Accordingly, the Trichoderma reesei endoglucanase I (TrEG I) and II (TrEG II) were secreted as active proteins in Y. lipolytica, with the secretion yield of EG II being twice that of EG I. Characterization of the purified His-tagged recombinant EG proteins (rhTrEGs) revealed that rhTrEG I displayed higher specific activity than rhTrEG II on both cellotriose and insoluble cellulosic substrates, such as Avicel, ß-1, 3 glucan, ß-1, 4 glucan, and PASC. Similarly, cellobiohydrolases, such as T. reesei CBH I and II (TrCBH I and II), and the CBH I from Neurospora crassa (NcCBH I) were successfully expressed in Y. lipolytica. However, the yield of the expressed TrCBH I was low, so work on this was not pursued. Contrastingly, rhNcCBH I was not only well expressed, but also highly active on PASC and more active on Avicel (0.11 U/mg) than wild-type TrCBH I (0.065 U/mg). Therefore, work was pursued using a combination of NcCBH I and TrCBH II. The quantification of enzyme levels in culture supernatants revealed that the use of a hybrid promoter instead of the primarily used TEF promoter procured four and eight times more NcCBH I and TrCBH II expressions, respectively. Finally, the coexpression of the previously described Y. lipolytica ß-glucosidases, the CBH II, and EG I and II from T. reesei, and the N. crassa CBH I procured an engineered Y. lipolytica strain that was able to grow both on model cellulose substrates, such as highly crystalline Avicel, and on industrial cellulose pulp, such as that obtained using an organosolv process. CONCLUSIONS: A Y. lipolytica strain coexpressing six cellulolytic enzyme components has been successfully developed. In addition, the results presented show how the recombinant strain can be optimized, for example, using artificial promoters to tailor expression levels. Most significantly, this study has provided a demonstration of how the strain can grow on a sample of industrial cellulose as sole carbon source, thus revealing the feasibility of Yarrowia-based consolidated bioprocess for the production of fuel and chemical precursors. Further, enzyme and strain optimization, coupled to appropriate process design, will undoubtedly lead to much better performances in the future.

Characterization and mutagenesis of two novel iron-sulphur cluster pentonate dehydratases.

We describe here the identification and characterization of two novel enzymes belonging to the IlvD/EDD protein family, the D-xylonate dehydratase from Caulobacter crescentus, Cc XyDHT, (EC 4.2.1.82), and the L-arabonate dehydratase from Rhizobium leguminosarum bv. trifolii, Rl ArDHT (EC 4.2.1.25), that produce the corresponding 2-keto-3-deoxy-sugar acids. There is only a very limited amount of characterization data available on pentonate dehydratases, even though the enzymes from these oxidative pathways have potential applications with plant biomass pentose sugars. The two bacterial enzymes share 41 % amino acid sequence identity and were expressed and purified from Escherichia coli as homotetrameric proteins. Both dehydratases were shown to accept pentonate and hexonate sugar acids as their substrates and require Mg(2+) for their activity. Cc XyDHT displayed the highest activity on D-xylonate and D-gluconate, while Rl ArDHT functioned best on D-fuconate, L-arabonate and D-galactonate. The configuration of the OH groups at C2 and C3 position of the sugar acid were shown to be critical, and the C4 configuration also contributed substantially to the substrate recognition. The two enzymes were also shown to contain an iron-sulphur [Fe-S] cluster. Our phylogenetic analysis and mutagenesis studies demonstrated that the three conserved cysteine residues in the aldonic acid dehydratase group of IlvD/EDD family members, those of C60, C128 and C201 in Cc XyDHT, and of C59, C127 and C200 in Rl ArDHT, are needed for coordination of the [Fe-S] cluster. The iron-sulphur cluster was shown to be crucial for the catalytic activity (kcat) but not for the substrate binding (Km) of the two pentonate dehydratases.

Functional Metagenomics: Construction and High-Throughput Screening of Fosmid Libraries for Discovery of Novel Carbohydrate-Active Enzymes.

Activity-based metagenomics is one of the most efficient approaches to boost the discovery of novel biocatalysts from the huge reservoir of uncultivated bacteria. In this chapter, we describe a highly generic procedure of metagenomic library construction and high-throughput screening for carbohydrate-active enzymes. Applicable to any bacterial ecosystem, it enables the swift identification of functional enzymes that are highly efficient, alone or acting in synergy, to break down polysaccharides and oligosaccharides.

Salivary Composition Is Associated with Liking and Usual Nutrient Intake.

Salivary flow and composition have an impact on flavor perception. However, very few studies have explored the relationship between saliva, individual liking and usual dietary intake. The aim of our study was to evaluate the association of salivary flow and composition with both a liking for fat, saltiness and sweetness and the usual nutrient intake in an adult French population. Liking for fat, saltiness, and sweetness were inferred from liking scores obtained during hedonic tests on 32 food products among 282 French adults participating in the Nutrinet-Santé Study. Before assessing liking, resting saliva was collected. Standard biochemical analyses were performed to assess specific component concentrations and enzymatic activities. Dietary data were collected using three web-based 24 h records. Relationships between salivary flow and composition, sensory liking and nutrient intake were assessed using linear regression. Total antioxidant capacity was positively associated with simple carbohydrate intake (ß = 31.3, 95% CI = 1.58; 60.99) and inversely related to complex carbohydrate consumption (ß = -52.4, 95% CI = -87.51; -19.71). Amylolysis was positively associated with both total (ß = 0.20, 95% CI = 0.01; 0.38) and simple carbohydrate intake (ß = 0.21, 95% CI = 0.01; 0.39). Salivary flow was positively associated with liking for fat (ß = 0.14, 95% CI = 0.03; 0.25). Proteolysis was positively associated with liking for saltiness and for fat (ß = 0.31, 95% CI = 0.02; 0.59; ß = 0.28, 95% CI = 0.01; 0.56, respectively). Amylolysis was inversely associated with liking for sweetness (ß = -10.13, 95% CI = -19.51; -0.75). Carbonic anhydrase 6 was inversely associated with liking for saltiness (ß = -46.77, 95% CI = -86.24; -7.30). Saliva does not substantially vary according to a usual diet, except for carbohydrate intake, whereas the specific association between salivary flow/composition and sensory liking suggests the influence of saliva characteristics in food acceptance.

Development of cellobiose-degrading ability in Yarrowia lipolytica strain by overexpression of endogenous genes.

BACKGROUND: Yarrowia lipolytica, one of the most widely studied "nonconventional" oleaginous yeast species, is unable to grow on cellobiose. Engineering cellobiose-degrading ability into this yeast is a vital step towards the development of cellulolytic biocatalysts suitable for consolidated bioprocessing. RESULTS: In the present work, we identified six genes encoding putative ß-glucosidases in the Y. lipolytica genome. To study these, homologous expression was attempted in Y. lipolytica JMY1212 Zeta. Two strains overexpressing BGL1 (YALI0F16027g) and BGL2 (YALI0B14289g) produced ß-glucosidase activity and were able to degrade cellobiose, while the other four did not display any detectable activity. The two active ß-glucosidases, one of which was mainly cell-associated while the other was present in the extracellular medium, were purified and characterized. The two Bgls were most active at 40-45°C and pH 4.0-4.5, and exhibited hydrolytic activity on various ß-glycoside substrates. Specifically, Bgl1 displayed 12.5-fold higher catalytic efficiency on cellobiose than Bgl2. Significantly, in experiments where cellobiose or cellulose (performed in the presence of a ß-glucosidase-deficient commercial cellulase cocktail produced by Trichoderma reseei) was used as carbon source for aerobic cultivation, Y. lipolytica ∆pox co-expressing BGL1 and BGL2 grew better than the Y. lipolytica strains expressing single BGLs. The specific growth rate and biomass yield of Y. lipolytica JMY1212 co-expressing BGL1 and BGL2 were 0.15 h(-1) and 0.50 g-DCW/g-cellobiose, respectively, similar to that of the control grown on glucose. CONCLUSIONS: We conclude that the bi-functional Y. lipolytica developed in the current study represents a vital step towards the creation of a cellulolytic yeast strain that can be used for lipid production from lignocellulosic biomass. When used in combination with commercial cellulolytic cocktails, this strain will no doubt reduce enzyme requirements and thus costs.

Efficient anaerobic transformation of raw wheat straw by a robust cow rumen-derived microbial consortium.

A rumen-derived microbial consortium was enriched on raw wheat straw as sole carbon source in a sequential batch-reactor (SBR) process under strict mesophilic anaerobic conditions. After five cycles of enrichment the procedure enabled to select a stable and efficient lignocellulolytic microbial consortium, mainly constituted by members of Firmicutes and Bacteroidetes phyla. The enriched community, designed rumen-wheat straw-derived consortium (RWS) efficiently hydrolyzed lignocellulosic biomass, degrading 55.5% w/w of raw wheat straw over 15days at 35°C and accumulating carboxylates as main products. Cellulolytic and hemicellulolytic activities, mainly detected on the cell bound fraction, were produced in the earlier steps of degradation, their production being correlated with the maximal lignocellulose degradation rates. Overall, these results demonstrate the potential of RWS to convert unpretreated lignocellulosic substrates into useful chemicals.

Investigating the function of an arabinan utilization locus isolated from a termite gut community.

Biocatalysts are essential for the development of bioprocesses efficient for plant biomass degradation. Previously, a metagenomic clone containing DNA from termite gut microbiota was pinpointed in a functional screening that revealed the presence of arabinofuranosidase activity. Subsequent genetic and bioinformatic analysis revealed that the DNA fragment belonged to a member of the genus Bacteroides and encoded 19 open reading frames (ORFs), and annotation suggested the presence of hypothetical transporter and regulator proteins and others involved in the catabolism of pentose sugar. In this respect and considering the phenotype of the metagenomic clone, it was noted that among the ORFs, there are four putative arabinose-specific glycoside hydrolases, two from family GH43 and two from GH51. In this study, a thorough bioinformatics analysis of the metagenomic clone gene cluster has been performed and the four aforementioned glycoside hydrolases have been characterized. Together, the results provide evidence that the gene cluster is a polysaccharide utilization locus dedicated to the breakdown of the arabinan component in pectin and related substrates. Characterization of the two GH43 and the two GH51 glycoside hydrolases has revealed that each of these enzymes displays specific catalytic capabilities and that when these are combined the enzymes act synergistically, increasing the efficiency of arabinan degradation.

Construction of a highly active xylanase displaying oleaginous yeast: comparison of anchoring systems.

Three Yarrowia lipolytica cell wall proteins (YlPir, YlCWP1 and YlCBM) were evaluated for their ability to display the xylanase TxXYN from Thermobacillus xylanilyticus on the cell surface of Y. lipolytica. The fusion proteins were produced in Y. lipolytica JMY1212, a strain engineered for mono-copy chromosomal insertion, and enabling accurate comparison of anchoring systems. The construction using YlPir enabled cell bound xylanase activity to be maximised (71.6 U/g). Although 48% of the activity was released in the supernatant, probably due to proteolysis at the fusion zone, this system is three times more efficient for the anchoring of TxXYN than the YlCWP1 system formerly developed for Y. lipolytica. As far as we know it represents the best displayed xylanase activity ever published. It could be an attractive alternative anchoring system to display enzymes in Y. lipolytica.