Cross-utilization of ß-galactosides and cellobiose in Geobacillus stearothermophilus.

Strains of the Gram-positive, thermophilic bacterium Geobacillus stearothermophilus possess elaborate systems for the utilization of hemicellulolytic polysaccharides, including xylan, arabinan, and galactan. These systems have been studied extensively in strains T-1 and T-6, representing microbial models for the utilization of soil polysaccharides, and many of their components have been characterized both biochemically and structurally. Here, we characterized routes by which G. stearothermophilus utilizes mono- and disaccharides such as galactose, cellobiose, lactose, and galactosyl-glycerol. The G. stearothermophilus genome encodes a phosphoenolpyruvate carbohydrate phosphotransferase system (PTS) for cellobiose. We found that the cellobiose-PTS system is induced by cellobiose and characterized the corresponding GH1 6-phospho-ß-glucosidase, Cel1A. The bacterium also possesses two transport systems for galactose, a galactose-PTS system and an ABC galactose transporter. The ABC galactose transport system is regulated by a three-component sensing system. We observed that both systems, the sensor and the transporter, utilize galactose-binding proteins that also bind glucose with the same affinity. We hypothesize that this allows the cell to control the flux of galactose into the cell in the presence of glucose. Unexpectedly, we discovered that G. stearothermophilus T-1 can also utilize lactose and galactosyl-glycerol via the cellobiose-PTS system together with a bifunctional 6-phospho-ß-gal/glucosidase, Gan1D. Growth curves of strain T-1 growing in the presence of cellobiose, with either lactose or galactosyl-glycerol, revealed initially logarithmic growth on cellobiose and then linear growth supported by the additional sugars. We conclude that Gan1D allows the cell to utilize residual galactose-containing disaccharides, taking advantage of the promiscuity of the cellobiose-PTS system.

A reassessment of flocculosin-mediated biocontrol activity of Pseudozyma flocculosa through CRISPR/Cas9 gene editing.

Pseudozyma flocculosa is an epiphytic yeast with powerful antagonistic activity against powdery mildews. This activity has been associated with the production of a rare antifungal glycolipid, flocculosin. In spite of the discovery of a specific gene cluster for flocculosin synthesis, attempts to ascribe a functional role to the molecule have been hampered by the inability to efficiently transform P. flocculosa. In this study, two different approaches, target gene replacement by homologous recombination (HR) and CRISPR-Cas9 based genome-editing, were utilized to decipher the role of flocculosin in the biocontrol activity of P.flocculosa. It was possible to alter the production of flocculosin through edition of fat1 by HR, but such mutants displayed abnormal phenotypes and the inability to produce sporidia. Sequencing analyses revealed that transformation by HR led to multiple insertions in the genome explaining the pleiotrophic effects of the approach. On the other hand, CRISPR-Cas9 transformation yielded one mutant that was altered specifically in the proper synthesis of flocculosin. Notwithstanding the loss of flocculosin production, such mutant was phenotypically similar to the wild-type, and when tested for its biocontrol activity against powdery mildew, displayed the same efficacy. These results offer strong evidence that flocculosin-mediated antibiosis is not responsible for the mode of action of P. flocculosa and highlight the potential of CRISPR-Cas9 for functional studies of otherwise difficult-to-transform fungi such as P. flocculosa.

Enhanced glycolic acid yield through xylose and cellobiose utilization by metabolically engineered Escherichia coli.

Microbial biorefinery is a promising route toward sustainable production of glycolic acid (GA), a valuable raw material for various industries. However, inherent microbial GA production has limited substrate consumption using either D-xylose or D-glucose as carbon catabolite repression (CCR) averts their co-utilization. To bypass CCR, a GA-producing strain using D-xylose via Dahms pathway was engineered to allow cellobiose uptake. Unlike glucose, cellobiose was assimilated and intracellularly degraded without repressing D-xylose uptake. The final GA-producing E. coli strain (CLGA8) has an overexpressed cellobiose phosphorylase (cep94A) from Saccharophagus degradans 2-40 and an activated glyoxylate shunt pathway. Expression of cep94A improved GA production reaching the maximum theoretical yield (0.51 g GA g-1 xylose), whereas activation of glyoxylate shunt pathway enabled GA production from cellobiose, which further increased the GA titer (2.25 g GA L-1). To date, this is the highest reported GA yield from D-xylose through Dahms pathway in an engineered E. coli with cellobiose as co-substrate.

Synthetic microbial consortium with specific roles designated by genetic circuits for cooperative chemical production.

Synthetic microbial consortia consisting of microorganisms with different synthetic genetic circuits or divided synthetic metabolic pathway components can exert functions that are beyond the capacities of single microorganisms. However, few consortia of microorganisms with different synthetic genetic circuits have been developed. We designed and constructed a synthetic microbial consortium composed of an enzyme-producing strain and a target chemical-producing strain using Escherichia coli for chemical production with efficient saccharification. The enzyme-producing strain harbored a synthetic genetic circuit to produce beta-glucosidase, which converts cellobiose to glucose, destroys itself via the lytic genes, and release the enzyme when the desired cell density is reached. The target chemical-producing strain was programmed by a synthetic genetic circuit to express enzymes in the synthetic metabolic pathway for isopropanol production when the enzyme-producing strain grows until release of the enzyme. Our results demonstrate the benefits of synthetic microbial consortia with distributed tasks for effective chemical production from biomass.

Refactoring the upper sugar metabolism of Pseudomonas putida for co-utilization of cellobiose, xylose, and glucose.

Given its capacity to tolerate stress, NAD(P)H/ NAD(P) balance, and increased ATP levels, the platform strain Pseudomonas putida EM42, a genome-edited derivative of the soil bacterium P. putida KT2440, can efficiently host a suite of harsh reactions of biotechnological interest. Because of the lifestyle of the original isolate, however, the nutritional repertoire of P. putida EM42 is centered largely on organic acids, aromatic compounds and some hexoses (glucose and fructose). To enlarge the biochemical network of P. putida EM42 to include disaccharides and pentoses, we implanted heterologous genetic modules for D-cellobiose and D-xylose metabolism into the enzymatic complement of this strain. Cellobiose was actively transported into the cells through the ABC complex formed by native proteins PP1015-PP1018. The knocked-in ß-glucosidase BglC from Thermobifida fusca catalyzed intracellular cleavage of the disaccharide to D-glucose, which was then channelled to the default central metabolism. Xylose oxidation to the dead end product D-xylonate was prevented by deleting the gcd gene that encodes the broad substrate range quinone-dependent glucose dehydrogenase. Intracellular intake was then engineered by expressing the Escherichia coli proton-coupled symporter XylE. The sugar was further metabolized by the products of E. coli xylA (xylose isomerase) and xylB (xylulokinase) towards the pentose phosphate pathway. The resulting P. putida strain co-utilized xylose with glucose or cellobiose to complete depletion of the sugars. These results not only show the broadening of the metabolic capacity of a soil bacterium towards new substrates, but also promote P. putida EM42 as a platform for plug-in of new biochemical pathways for utilization and valorization of carbohydrate mixtures from lignocellulose processing.

Solution conformation of a cohesin module and its scaffoldin linker from a prototypical cellulosome.

Bacterial cellulases are drawing increased attention as a means to obtain plentiful chemical feedstocks and fuels from renewable lignocellulosic biomass sources. Certain bacteria deploy a large extracellular multi-protein complex, called the cellulosome, to degrade cellulose. Scaffoldin, a key non-catalytic cellulosome component, is a large protein containing a cellulose-specific carbohydrate-binding module and several cohesin modules which bind and organize the hydrolytic enzymes. Despite the importance of the structure and protein/protein interactions of the cohesin module in the cellulosome, its structure in solution has remained unknown to date. Here, we report the backbone 1H, 13C and 15N NMR assignments of the Cohesin module 5 from the highly stable and active cellulosome from Clostridium thermocellum. These data reveal that this module adopts a tightly packed, well folded and rigid structure in solution. Furthermore, since in scaffoldin, the cohesin modules are connected by linkers we have also characterized the conformation of a representative linker segment using NMR spectroscopy. Analysis of its chemical shift values revealed that this linker is rather stiff and tends to adopt extended conformations. This suggests that the scaffoldin linkers act to minimize interactions between cohesin modules. These results pave the way towards solution studies on cohesin/dockerin's fascinating dual-binding mode.

Transcriptional regulator XYR1 activates the expression of cellobiose synthase to promote the production of cellulase from glucose.

OBJECTIVE: To investigate the transcriptional regulation of cellobiose synthase (CBS) in Rhizopus stolonifer. RESULTS: Transcription factor XYR1 was identified as responsible for the activation of cbs. In comparison with wild-type R. stolonifer, the deletion of XYR1 resulted in transcriptional down-regulation of cbs by approximately 40%, while XYR1 over-expression increased cbs transcription up to 175%. The highest FPA activity (1.8 IU/ml) was obtained in the XYR1-overexpressing strain OExyr1 cultivated in a 2% (m/V) glucose media, corresponding to a 96% increase compared with that of the parent strain (0.92 IU/ml). Moreover, cellulase synthesis was inhibited after cbs-inactivation mutation in OExyr1. CONCLUSION: XYR1 directly activates the transcription of cbs to promote cellulase production in R. stolonifer utilizing glucose as a substrate.

Lactic acid production from cellobiose and xylose by engineered Saccharomyces cerevisiae.

Efficient and rapid production of value-added chemicals from lignocellulosic biomass is an important step toward a sustainable society. Lactic acid, used for synthesizing the bioplastic polylactide, has been produced by microbial fermentation using primarily glucose. Lignocellulosic hydrolysates contain high concentrations of cellobiose and xylose. Here, we constructed a recombinant Saccharomyces cerevisiae strain capable of fermenting cellobiose and xylose into lactic acid. Specifically, genes (cdt-1, gh1-1, XYL1, XYL2, XYL3, and ldhA) coding for cellobiose transporter, ß-glucosidase, xylose reductase, xylitol dehydrogenase, xylulokinase, and lactate dehydrogenase were integrated into the S. cerevisiae chromosomes. The resulting strain produced lactic acid from cellobiose or xylose with high yields. When fermenting a cellulosic sugar mixture containing 10 g/L glucose, 40 g/L xylose, and 80 g/L cellobiose, the engineered strain produced 83 g/L of lactic acid with a yield of 0.66 g lactic acid/g sugar (66% theoretical maximum). This study demonstrates initial steps toward the feasibility of sustainable production of lactic acid from lignocellulosic sugars by engineered yeast.

Two major facilitator superfamily sugar transporters from Trichoderma reesei and their roles in induction of cellulase biosynthesis.

Proper perception of the extracellular insoluble cellulose is key to initiating the rapid synthesis of cellulases by cellulolytic Trichoderma reesei. Uptake of soluble oligosaccharides derived from cellulose hydrolysis represents a potential point of control in the induced cascade. In this study, we identified a major facilitator superfamily sugar transporter Stp1 capable of transporting cellobiose by reconstructing a cellobiose assimilation system in Saccharomyces cerevisiae. The absence of Stp1 in T. reesei resulted in differential cellulolytic response to Avicel versus cellobiose. Transcriptional profiling revealed a different expression profile in the Δstp1 strain from that of wild-type strain in response to Avicel and demonstrated that Stp1 somehow repressed induction of the bulk of major cellulase and hemicellulose genes. Two other putative major facilitator superfamily sugar transporters were, however, up-regulated in the profiling. Deletion of one of them identified Crt1 that was required for growth and enzymatic activity on cellulose or lactose, but was not required for growth or hemicellulase activity on xylan. The essential role of Crt1 in cellulase induction did not seem to rely on its transporting activity because the overall uptake of cellobiose or sophorose by T. reesei was not compromised in the absence of Crt1. Phylogenetic analysis revealed that orthologs of Crt1 exist in the genomes of many filamentous ascomycete fungi capable of degrading cellulose. These data thus shed new light on the mechanism by which T. reesei senses and transmits the cellulose signal and offers potential strategies for strain improvement.

Loop motions important to product expulsion in the Thermobifida fusca glycoside hydrolase family 6 cellobiohydrolase from structural and computational studies.

Cellobiohydrolases (CBHs) are typically major components of natural enzyme cocktails for biomass degradation. Their active sites are enclosed in a tunnel, enabling processive hydrolysis of cellulose chains. Glycoside hydrolase Family 6 (GH6) CBHs act from nonreducing ends by an inverting mechanism and are present in many cellulolytic fungi and bacteria. The bacterial Thermobifida fusca Cel6B (TfuCel6B) exhibits a longer and more enclosed active site tunnel than its fungal counterparts. Here, we determine the structures of two TfuCel6B mutants co-crystallized with cellobiose, D274A (catalytic acid), and the double mutant D226A/S232A, which targets the putative catalytic base and a conserved serine that binds the nucleophilic water. The ligand binding and the structure of the active site are retained when compared with the wild type structure, supporting the hypothesis that these residues are directly involved in catalysis. One structure exhibits crystallographic waters that enable construction of a model of the α-anomer product after hydrolysis. Interestingly, the product sites of TfuCel6B are completely enclosed by an "exit loop" not present in fungal GH6 CBHs and by an extended "bottom loop". From the structures, we hypothesize that either of the loops enclosing the product subsites in the TfuCel6B active site tunnel must open substantially for product release. With simulation, we demonstrate that both loops can readily open to allow product release with equal probability in solution or when the enzyme is engaged on cellulose. Overall, this study reveals new structural details of GH6 CBHs likely important for functional differences among enzymes from this important family.

Laboratory evolution and multi-platform genome re-sequencing of the cellulolytic actinobacterium Thermobifida fusca.

Biological utilization of cellulose is a complex process involving the coordinated expression of different cellulases, often in a synergistic manner. One possible means of inducing an organism-level change in cellulase activity is to use laboratory adaptive evolution. In this study, evolved strains of the cellulolytic actinobacterium, Thermobifida fusca, were generated for two different scenarios: continuous exposure to cellobiose (strain muC) or alternating exposure to cellobiose and glucose (strain muS). These environmental conditions produced a phenotype specialized for growth on cellobiose (muC) and an adaptable, generalist phenotype (muS). Characterization of cellular phenotypes and whole genome re-sequencing were conducted for both the muC and muS strains. Phenotypically, the muC strain showed decreased cell yield over the course of evolution concurrent with decreased cellulase activity, increased intracellular ATP concentrations, and higher end-product secretions. The muS strain increased its cell yield for growth on glucose and exhibited a more generalist phenotype with higher cellulase activity and growth capabilities on different substrates. Whole genome re-sequencing identified 48 errors in the reference genome and 18 and 14 point mutations in the muC and muS strains, respectively. Among these mutations, the site mutation of Tfu_1867 was found to contribute the specialist phenotype and the site mutation of Tfu_0423 was found to contribute the generalist phenotype. By conducting and characterizing evolution experiments on Thermobifida fusca, we were able to show that evolutionary changes balance ATP energetic considerations with cellulase activity. Increased cellulase activity is achieved in stress environments (switching carbon sources), otherwise cellulase activity is minimized to conserve ATP.

XlnR-independent signaling pathway regulates both cellulase and xylanase genes in response to cellobiose in Aspergillus aculeatus.

The expression levels of the cellulase and xylanase genes between the host strain and an xlnR disruptant were compared by quantitative RT-PCR (qPCR) to identify the genes controlled by XlnR-independent signaling pathway. The cellulose induction of the FI-carboxymethyl cellulase (cmc1) and FIb-xylanase (xynIb) genes was controlled by XlnR; in contrast, the cellulose induction of the FIII-avicelase (cbhI), FII-carboxymethyl cellulase (cmc2), and FIa-xylanase (xynIa) genes was controlled by an XlnR-independent signaling pathway. To gain deeper insight into the XlnR-independent signaling pathway, the expression profile of cbhI was analyzed as a representative target gene. Cellobiose together with 1-deoxynojirimycin (DNJ), a glucosidase inhibitor, induced cbhI the most efficiently among disaccharides composed of ß-glucosidic bonds. Furthermore, cellobiose with DNJ induced the transcription of cmc2 and xynIa, whereas cmc1 and xynIb were not induced. GUS reporter fusion analyses of truncated and mutated cbhI promoters revealed that three regions were necessary for effective cellulose-induced transcription, all of which contained the conserved sequence 5'-CCGN(2)CCN(7)G(C/A)-3' within the CeRE, which has been identified as the upstream activating element essential for expression of eglA in A. nidulans (Endo et al. 2008). The data therefore delineate a pathway in which A. aculeatus perceives the presence of cellobiose, thereby activating a signaling pathway that drives cellulase and hemicellulase gene expression under the control of the XlnR-independent regulation through CeRE.

Effects of Engineered Saccharomyces cerevisiae Fermenting Cellobiose through Low-Energy-Consuming Phosphorolytic Pathway in Simultaneous Saccharification and Fermentation.

Until recently, four types of cellobiose-fermenting Saccharomyces cerevisiae strains have been developed by introduction of a cellobiose metabolic pathway based on either intracellular ß-glucosidase (GH1-1) or cellobiose phosphorylase (CBP), along with either an energy-consuming active cellodextrin transporter (CDT-1) or a non-energy-consuming passive cellodextrin facilitator (CDT-2). In this study, the ethanol production performance of two cellobiose-fermenting S. cerevisiae strains expressing mutant CDT-2 (N306I) with GH1-1 or CBP were compared with two cellobiose-fermenting S. cerevisiae strains expressing mutant CDT-1 (F213L) with GH1-1 or CBP in the simultaneous saccharification and fermentation (SSF) of cellulose under various conditions. It was found that, regardless of the SSF conditions, the phosphorolytic cellobiose-fermenting S. cerevisiae expressing mutant CDT-2 with CBP showed the best ethanol production among the four strains. In addition, during SSF contaminated by lactic acid bacteria, the phosphorolytic cellobiose-fermenting S. cerevisiae expressing mutant CDT-2 with CBP showed the highest ethanol production and the lowest lactate formation compared with those of other strains, such as the hydrolytic cellobiose-fermenting S. cerevisiae expressing mutant CDT-1 with GH1-1, and the glucose-fermenting S. cerevisiae with extracellular ß-glucosidase. These results suggest that the cellobiose-fermenting yeast strain exhibiting low energy consumption can enhance the efficiency of the SSF of cellulosic biomass.

Biochemical characterization of a thermophilic cellobiose 2-epimerase from a thermohalophilic bacterium, Rhodothermus marinus JCM9785.

Cellobiose 2-epimerase (CE) reversibly converts glucose residue to mannose residue at the reducing end of ß-1,4-linked oligosaccharides. It efficiently produces epilactose carrying prebiotic properties from lactose, but the utilization of known CEs is limited due to thermolability. We focused on thermoholophilic Rhodothermus marinus JCM9785 as a CE producer, since a CE-like gene was found in the genome of R. marinus DSM4252. CE activity was detected in the cell extract of R. marinus JCM9785. The deduced amino acid sequence of the CE gene from R. marinus JCM9785 (RmCE) was 94.2% identical to that from R. marinus DSM4252. The N-terminal amino acid sequence and tryptic peptide masses of the native enzyme matched those of RmCE. The recombinant RmCE was most active at 80 °C at pH 6.3, and stable in a range of pH 3.2-10.8 and below 80 °C. In contrast to other CEs, RmCE demonstrated higher preference for lactose over cellobiose.

Modulating Pathway Performance by Perturbing Local Genetic Context.

Combinatorial engineering is a preferred strategy for attaining optimal pathway performance. Previous endeavors have been concentrated on regulatory elements (e.g., promoters, terminators, and ribosomal binding sites) and/or open reading frames. Accumulating evidence indicates that noncoding DNA sequences flanking a transcriptional unit on the genome strongly impact gene expression. Here, we sought to mimic the effect imposed on expression cassettes by the genome. We created variants of the model yeast Saccharomyces cerevisiae with significantly improved fluorescence or cellobiose consumption rate by randomizing the sequences adjacent to the GFP expression cassette or the cellobiose-utilization pathway, respectively. Interestingly, nucleotide specificity was observed at certain positions and showed to be essential for achieving optimal cellobiose assimilation. Further characterization suggested that the modulation effects of the short sequences flanking the expression cassettes could be potentially mediated by remodeling DNA packaging and/or recruiting transcription factors. Collectively, these results indicate that the often-overlooked contiguous DNA sequences can be exploited to rapidly achieve balanced pathway expression, and the corresponding approach could be easily stacked with other combinatorial engineering strategies.

Transcriptional regulation of the cellobiose operon of Streptococcus mutans.

The ability of Streptococcus mutans to catabolize cellobiose, a beta-linked glucoside generated during the hydrolysis of cellulose, is shown to be regulated by a transcriptional regulator, CelR, which is encoded by an operon with a phospho-beta-glucosidase (CelA) and a cellobiose-specific sugar phosphotransferase system (PTS) permease (EII(Cel)). The roles of CelR, EII(Cel) components, and certain fructose/mannose-PTS permeases in the transcriptional regulation of the cel locus were analyzed. The results revealed that (i) the celA and celB (EIIB(Cel)) gene promoters require CelR for transcriptional activation in response to cellobiose, but read-through from the celA promoter contributes to expression of the EII(Cel) genes; (ii) the EII(Cel) subunits were required for growth on cellobiose and for transcriptional activation of the cel genes; (iii) CcpA plays little direct role in catabolite repression of the cel regulon, but loss of specific PTS permeases alleviated repression of cel genes in the presence of preferred carbohydrates; and (iv) glucose could induce transcription of the cel regulon when transported by EII(Cel). CelR derivatives containing amino acid substitutions for five conserved histidine residues in two PTS regulatory domains and an EIIA-like domain also provided important insights regarding the function of this regulator. Based on these data, a model for the involvement of PTS permeases and the general PTS proteins enzyme I and HPr was developed that reveals a critical role for the PTS in CcpA-independent catabolite repression and induction of cel gene expression in S. mutans.

Origin of Lactose Fermentation in Kluyveromyces lactis by Interspecies Transfer of a Neo-functionalized Gene Cluster during Domestication.

Humans have used yeasts to make cheese and kefir for millennia, but the ability to ferment the milk sugar lactose is found in only a few yeast species, of which the foremost is Kluyveromyces lactis [1]. Two genes, LAC12 (lactose permease) and LAC4 (lactase), are sufficient for lactose uptake and hydrolysis to glucose and galactose [2]. Here, we show that these genes have a complex evolutionary history in the genus Kluyveromyces that is likely the result of human activity during domestication. We show that the ancestral Lac12 was bifunctional, able to import both lactose and cellobiose into the cell. These disaccharides were then hydrolyzed by Lac4 in the case of lactose or Cel2 in the case of cellobiose. A second cellobiose transporter, Cel1, was also present ancestrally. In the K. lactis lineage, the ancestral LAC12 and LAC4 were lost and a separate upheaval in the sister species K. marxianus resulted in loss of CEL1 and quadruplication of LAC12. One of these LAC12 genes became neofunctionalized to encode an efficient lactose transporter capable of supporting fermentation, specifically in dairy strains of K. marxianus, where it formed a LAC4-LAC12-CEL2 gene cluster, although another remained a cellobiose transporter. Then, the ability to ferment lactose was acquired very recently by K. lactis var. lactis by introgression of LAC12 and LAC4 on a 15-kb subtelomeric region from a dairy strain of K. marxianus. The genomic history of the LAC genes shows that strong selective pressures were imposed on yeasts by early dairy farmers.

Improved ethanol production by engineered Saccharomyces cerevisiae expressing a mutated cellobiose transporter during simultaneous saccharification and fermentation.

Although simultaneous saccharification and fermentation (SSF) of cellulosic biomass can offer efficient hydrolysis of cellulose through alleviating feed-back inhibition of cellulases by glucose, supplementation of ß-glucosidase is necessary because most fermenting microorganisms cannot utilize cellobiose. Previously, we observed that SSF of cellulose by an engineered Saccharomyces cerevisiae expressing a cellobiose transporter (CDT-1) and an intracellular ß-glucosidase (GH1-1) without ß-glucosidase could not be performed as efficiently as the traditional SSF with extracellular ß-glucosidase. However, we improved the ethanol production from SSF of cellulose by employing a further engineered S. cerevisiae expressing a mutant cellobiose transporter [CDT-1 (F213L) exhibiting higher VMAX than CDT-1] and GH1-1 in this study. Furthermore, limitation of cellobiose formation by reducing the amounts of cellulases mixture in SSF could lead the further engineered strain to produce ethanol considerably better than the parental strain with ß-glucosidase. Probably, better production of ethanol by the further engineered strain seemed to be due to a higher affinity to cellobiose, which might be attributed to not only 2-times lower Monod constant (KS) for cellobiose than KS of the parental strain for glucose but also 5-times lower KS than Michaelis-Menten constant (KM) of the extracellular ß-glucosidase for glucose. Our results suggest that modification of the cellobiose transporter in the engineered yeast to transport lower level of cellobiose enables a more efficient SSF for producing ethanol from cellulose.

Two New Native ß-Glucosidases from Clavispora NRRL Y-50464 Confer Its Dual Function as Cellobiose Fermenting Ethanologenic Yeast.

Yeast strain Clavispora NRRL Y-50464 is able to produce cellulosic ethanol from lignocellulosic materials without addition of external ß-glucosidase by simultaneous saccharification and fermentation. A ß-glucosidase BGL1 protein from this strain was recently reported supporting its cellobiose utilization capability. Here, we report two additional new ß-glucosidase genes encoding enzymes designated as BGL2 and BGL3 from strain NRRL Y-50464. Quantitative gene expression was analyzed and the gene function of BGL2 and BGL3 was confirmed by heterologous expression using cellobiose as a sole carbon source. Each gene was cloned and partially purified protein obtained separately for direct enzyme assay using varied substrates. Both proteins showed the highest specific activity at pH 5 and relatively strong affinity with a Km of 0.08 and 0.18 mM for BGL2 and BGL3, respectively. The optimum temperature was found to be 50°C for BGL2 and 55°C for BGL3. Both proteins were able to hydrolyze 1,4 oligosaccharides evaluated in this study. They also showed a strong resistance to glucose product inhibition with a Ki of 61.97 and 38.33 mM for BGL2 and BGL3, respectively. While BGL3 was sensitive showing a significantly reduced activity to 4% ethanol, BGL2 demonstrated tolerance to ethanol. Its activity was enhanced in the presence of ethanol but reduced at concentrations greater than 16%. The presence of the fermentation inhibitors furfural and HMF did not affect the enzyme activity. Our results suggest that a ß-glucosidase gene family exists in Clavispora NRRL Y-50464 with at least three members in this group that validate its cellobiose hydrolysis functions for lower-cost cellulosic ethanol production. Results of this study confirmed the cellobiose hydrolysis function of strain NRRL Y-50464, and further supported this dual functional yeast as a candidate for lower-cost cellulosic ethanol production and next-generation biocatalyst development in potential industrial applications.

Novel Functions and Regulation of Cryptic Cellobiose Operons in Escherichia coli.

Presence of cellobiose as a sole carbon source induces mutations in the chb and asc operons of Escherichia coli and allows it to grow on cellobiose. We previously engineered these two operons with synthetic constitutive promoters and achieved efficient cellobiose metabolism through adaptive evolution. In this study, we characterized two mutations observed in the efficient cellobiose metabolizing strain: duplication of RBS of ascB gene, (ß-glucosidase of asc operon) and nonsense mutation in yebK, (an uncharacterized transcription factor). Mutations in yebK play a dominant role by modulating the length of lag phase, relative to the growth rate of the strain when transferred from a rich medium to minimal cellobiose medium. Mutations in ascB, on the other hand, are specific for cellobiose and help in enhancing the specific growth rate. Taken together, our results show that ascB of the asc operon is controlled by an internal putative promoter in addition to the native cryptic promoter, and the transcription factor yebK helps to remodel the host physiology for cellobiose metabolism. While previous studies characterized the stress-induced mutations that allowed growth on cellobiose, here, we characterize the adaptation-induced mutations that help in enhancing cellobiose metabolic ability. This study will shed new light on the regulatory changes and factors that are needed for the functional coupling of the host physiology to the activated cryptic cellobiose metabolism.