Cellobiose Metabolism Research Articles

Activation of a silent lactose utilization pathway in an evolved Listeria monocytogenes F2365 outbreak isolate

Listeria monocytogenes, a widespread food-borne pathogen, utilizes diverse growth substrates including mono- and di-saccharides via PEP-phosphotransferase (PTS) systems. We evaluated a collection of L. monocytogenes isolates of different origins for their ability to utilize lactose, a disaccharide composed of galactose and glucose and the main carbon source in milk and dairy products. Notably, the dairy-associated outbreak strain F2365 could not utilize lactose efficiently, conceivably due to a frameshift mutation (lacR887del) resulting in a truncated LacR. Transcriptional activator LacR is involved in the expression of two PTS systems, encoded by the lpo operon lmo1718-1720 in combination with lmo2708 and the lmo2683-2685 operon, and linked to lactose and/or cellobiose metabolism in L. monocytogenes. Via experimental evolution of the ancestral strain F2365, an evolved isolate F2365 EV was obtained which showed enhanced growth and metabolism of lactose. Using the lactose-positive model strain L. monocytogenes EGDe as a control, HPLC experiments showed that EGDe and F2365 EV could consume lactose and utilize the glucose moiety, while the galactose moiety was exported from the cells. Genome sequencing of F2365 EV found the original lacR887del mutation was still present but an additional point mutation lmo2766C415T had occurred, resulting in an amino acid substitution in the putative regulator Lmo2766. The lmo2766 gene is located next to operon lmo2761-2765 with putative PTS genes in the genome. Notably, comparative RNAseq analysis confirmed that the lmo2761-2765 operon was strongly upregulated in F2365 EV in the presence of lactose but not in EGDe and F2365. Conversely, the LacR-regulated lpo operon, lmo2708, and lmo2683-2685 operon were only upregulated in EGDe. Additional growth and HPLC experiments, using mutants constructed in lactose-positive L. monocytogenes EGDe, showed reduced growth of the EGDe lacR887del mutant with no utilization of lactose, while the double mutant EGDe lacR887dellmo2766C415T showed enhanced growth and lactose utilization. Hence, these results demonstrate that an amino acid substitution in the Lmo2766 regulator activates a previously silent lactose utilization pathway encoded by PTS operon lmo2761-2765, facilitating the growth and metabolism of L. monocytogenes with lactose as a substrate. This finding enhances our understanding of the metabolic capabilities and adaptability of L. monocytogenes, offering a broader view of the lactose utilization capacity of this pathogen.

Engineering of Ogataea polymorpha strains with ability for high-temperature alcoholic fermentation of cellobiose.

Successful conversion of cellulosic biomass into biofuels requires organisms capable of efficiently utilizing xylose as well as cellodextrins and glucose. Ogataea (Hansenula) polymorpha is the natural xylose-metabolizing organism and is one of the most thermotolerant yeasts known, with a maximum growth temperature above 50°C. Cellobiose-fermenting strains, derivatives of an improved ethanol producer from xylose O.polymorpha BEP/cat8∆, were constructed in this work by the introduction of heterologous genes encoding cellodextrin transporters (CDTs) and intracellular enzymes (β-glucosidase or cellobiose phosphorylase) that hydrolyze cellobiose. For this purpose, the genes gh1-1 of β-glucosidase, CDT-1m and CDT-2m of cellodextrin transporters from Neurospora crassa and the CBP gene coding for cellobiose phosphorylase from Saccharophagus degradans, were successfully expressed in O. polymorpha. Through metabolic engineering and mutagenesis, strains BEP/cat8∆/gh1-1/CDT-1m and BEP/cat8∆/CBP-1/CDT-2mAM were developed, showing improved parameters for high-temperature alcoholic fermentation of cellobiose. The study highlights the need for further optimization to enhance ethanol yields and elucidate cellobiose metabolism intricacies in O. polymorpha yeast. This is the first report of the successful development of stable methylotrophic thermotolerant strains of O.polymorpha capable of coutilizing cellobiose, glucose, and xylose under high-temperature alcoholic fermentation conditions at 45°C.

Metabolic engineering of genome-streamlined strain Pseudomonas putida KTU-U27 for medium-chain-length polyhydroxyalkanoate production from xylose and cellobiose

Bio-based plastics polyhydroxyalkanoates (PHAs) are considered as a good substitutive to traditional fossil-based plastics because PHAs outcompete chemical plastics in several important properties, such as biodegradability, biocompatibility, and renewability. However, the industrial production of PHA (especially medium-chain-length PHA, mcl-PHA) is greatly restricted by the cost of carbon sources. Currently, xylose and cellobiose derived from lignocellulose are potential substrates for mcl-PHA production. In this study, Pseudomonas putida KTU-U27, a genome-streamlined strain derived from a mcl-PHA producer P. putida KT2440, was used as the optimal chassis for the construction of microbial cell factories with the capacity to efficiently produce mcl-PHA from xylose and cellobiose by introducing the xylose and cellobiose metabolism modules and enhancing the transport of xylose and cellobiose. The lag phases of the xylose- and cellobiose-grown engineered strains were almost completely eliminated and the xylose- and cellobiose-utilizing performance was greatly improved via adaptive laboratory evolution. In shake-flask fermentation, the engineered strain 27A-P13-xylABE-Ptac-tt and 27A-P13-bglC-P13-gts had a mcl-PHA content of 41.67 wt% and 45.18 wt%, respectively, and were able to efficiently utilize xylose or cellobiose as the sole carbon source for cell growth. Herein, microbial production of mcl-PHA using xylose as the sole carbon source has been demonstrated for the first time. Meanwhile, the highest yield of mcl-PHA produced from cellobiose has been obtained in this study. Interestingly, the engineered strains derived from genome-reduced P. putida strains showed higher xylose- and cellobiose-utilizing performance and higher PHA yield than those derived from P. putida KT2440. This study highlights enormous potential of the engineered strains as promising platforms for low-cost production of mcl-PHA from xylose- and cellobiose-rich substrates.

Key roles of β-glucosidase BglA for the catabolism of both laminaribiose and cellobiose in the lignocellulolytic bacterium Clostridium thermocellum

The thermophilic bacterium Clostridium thermocellum efficiently degrades polysaccharides into oligosaccharides. The metabolism of β-1,4-linked cello-oligosaccharides is initiated by three enzymes, i.e., the cellodextrin phosphorylase (Cdp), the cellobiose phosphorylase (Cbp), and the β-glucosidase A (BglA), in C. thermocellum. In comparison, how the oligosaccharides containing other kinds of linkage are utilized is rarely understood. In this study, we found that BglA could hydrolyze the β-1,3-disaccharide laminaribiose with much higher activity than that against the β-1,4-disaccharide cellobiose. The structural basis of the substrate specificity was analyzed by crystal structure determination and molecular docking. Genetic deletions of BglA and Cbp, respectively, and enzymatic analysis of cell extracts demonstrated that BglA is the key enzyme responsible for laminaribiose metabolism. Furthermore, the deletion of BglA can suppress the expression of Cbp and the deletion of Cbp can up-regulate the expression of BglA, indicating that BglA and Cbp have cross-regulation and BglA is also critical for cellobiose metabolism. These insights pave the way for both a fundamental understanding of metabolism and regulation in C. thermocellum and emphasize the importance of the degradation and utilization of polysaccharides containing β-1,3-linked glycosidic bonds in lignocellulose biorefinery.

Comprehensive Characterization of Mutant Pichia stipitis Co-Fermenting Cellobiose and Xylose through Genomic and Transcriptomic Analyses.

The development of a yeast strain capable of fermenting mixed sugars efficiently is crucial for producing biofuels and value-added materials from cellulosic biomass. Previously, a mutant Pichia stipitis YN14 strain capable of co-fermenting xylose and cellobiose was developed through evolutionary engineering of the wild-type P. stipitis CBS6054 strain, which was incapable of cofermenting xylose and cellobiose. In this study, through genomic and transcriptomic analyses, we sought to investigate the reasons for the improved sugar metabolic performance of the mutant YN14 strain in comparison with the parental CBS6054 strain. Unfortunately, comparative wholegenome sequencing (WGS) showed no mutation in any of the genes involved in the cellobiose metabolism between the two strains. However, comparative RNA sequencing (RNA-seq) revealed that the YN14 strain had 101.2 times and 5.9 times higher expression levels of HXT2.3 and BGL2 genes involved in cellobiose metabolism, and 6.9 times and 75.9 times lower expression levels of COX17 and SOD2.2 genes involved in respiration, respectively, compared with the CBS6054 strain. This may explain how the YN14 strain enhanced cellobiose metabolic performance and shifted the direction of cellobiose metabolic flux from respiration to fermentation in the presence of cellobiose compared with the CBS6054 strain.

Transcriptome analysis revealed growth phase-associated changes of a centenarian-originated probiotic Bifidobacterium animalis subsp. lactis A6

BackgroundThe physiology and application characteristics of probiotics are closely associated with the growth phase. Bifidobacterium animalis subsp. lactis A6 is a promising probiotic strain isolated from the feces of a healthy centenarian in China. In this study, RNA-seq was carried out to investigate the metabolic mechanism between the exponential and the stationary phase in B. lactis A6.ResultsDifferential expression analysis showed that a total of 815 genes were significantly changed in the stationary phase compared to the exponential phase, which consisted of 399 up-regulated and 416 down-regulated genes. The results showed that the transport and metabolism of cellobiose, xylooligosaccharides and raffinose were enhanced at the stationary phase, which expanded carbon source utilizing profile to confront with glucose consumption. Meanwhile, genes involved in cysteine-cystathionine-cycle (CCC) pathway, glutamate dehydrogenase, branched-chain amino acids (BCAAs) biosynthesis, and Clp protease were all up-regulated in the stationary phase, which may enhance the acid tolerance of B. lactis A6 during stationary phase. Acid tolerance assay indicated that the survival rate of stationary phase cells was 51.07% after treatment by pH 3.0 for 2h, which was 730-fold higher than that of 0.07% with log phase cells. In addition, peptidoglycan biosynthesis was significantly repressed, which is comparable with the decreased growth rate during the stationary phase. Remarkably, a putative gene cluster encoding Tad pili was up-regulated by 6.5 to 12.1-fold, which is consistent with the significantly increased adhesion rate to mucin from 2.38% to 4.90% during the transition from the exponential phase to the stationary phase.ConclusionsThis study reported growth phase-associated changes of B. lactis A6 during fermentation, including expanded carbon source utilizing profile, enhanced acid tolerance, and up-regulated Tad pili gene cluster responsible for bacterial adhesion in the stationary phase. These findings provide a novel insight into the growth phase associated characteristics in B. lactis A6 and provide valuable information for further application in the food industry.

Characterization of an operon required for growth on cellobiose in Clostridioides difficile.

Cellobiose metabolism is linked to the virulence properties in numerous bacterial pathogens. Here, we characterized a putative cellobiose PTS operon of Clostridiodes difficile to investigate the role of cellobiose metabolism in C. difficile pathogenesis. Our gene knockout experiments demonstrated that the putative cellobiose operon enables uptake of cellobiose into C. difficile and allows growth when cellobiose is provided as the sole carbon source in minimal medium. Additionally, using reporter gene fusion assays and DNA pulldown experiments, we show that its transcription is regulated by CelR, a novel transcriptional repressor protein, which directly binds to the upstream region of the cellobiose operon to control its expression. We have also identified cellobiose metabolism to play a significant role in C. difficile physiology as observed by the reduction of sporulation efficiency when cellobiose uptake was compromised in the mutant strain. In corroboration to in vitro study findings, our in vivo hamster challenge experiment showed a significant reduction of pathogenicity by the cellobiose mutant strain in both the primary and the recurrent infection model - substantiating the role of cellobiose metabolism in C. difficile pathogenesis.

Characterization and virulence of Streptococcus agalactiae deficient in EIIA of the cellobiose-specific phosphotransferase system

To indicate the EIIA protein of cellobiose-specific phosphotransferase transport system in affecting the characterization and virulence of Streptococcus agalactiae. A △cel-EIIA mutant was constructed from the wild-type S. agalactiae THN0901 strain by homologous recombination. In the present study, the cellobiose metabolism and biofilm formation of the △cel-EIIA mutant was significantly decreased, relative to that of the wild-type S. agalactiae THN0901. The proliferation capacity in vitro and the invasion and colonization ability in vivo of the △cel-EIIA mutant was also significantly lower than that of S. agalactiae THN0901. Furthermore, in tilapia infection model, the lethal dose 50 % the △cel-EIIA mutant was significantly higher than that of the wild strain. Taken together, the cel-EIIA gene plays a positive role in S. agalactiae cellobiose metabolism, biofilm formation and colonization of tilapia. The results of this study provide the basic data for the cellobiose metabolism and virulence mechanism in S. agalactiae of tilapia in the future.

The influence of enzyme EII of the cellobiose-phosphotransferase system on the virulence of Streptococcus agalactiae in Nile tilapia (Oreochromis niloticus)

Streptococcus agalactiae is the main pathogen of tilapia, causing huge economic losses to the tilapia aquaculture industry in China. To elucidate the role of the EII complexes of the cellobiose-phosphotransferase system (cel-PTS) in the regulation of S. agalactiae virulence, the △cel-EII mutants of a high virulence strain (THN0901) and a low virulence strain (TFJ0901) were constructed using homologous recombination. The results showed that after the deletion of cel-EII complexes, the biological characteristics of THN0901 and TFJ0901 were consistent, including their cellobiose metabolism. Their biofilm formation abilities were significantly reduced (P < 0.01), and their ability to adhere to tilapia brain cells and compete when mixed in vitro and in vivo were enhanced. The virulence and histopathological lesions caused by △cel-EII-TFJ0901 were significantly enhanced relative to those of TFJ0901, while △cel-EII-THN0901 showed no significant differences relative to THN0901. The histopathological lesions caused by △cel-EII-TFJ0901 tend to be serious, especially in the liver, spleen, and heart. The expression levels of manA and ascA/C of PTS, ciaR and dltR of two-component signal transduction systems (TCS), and pavA, bca and spb1 of virulence genes of △cel-EII-TFJ0901 were significantly upregulated compared with TFJ0901 (P < 0.01), whereas those of △cel-EII-THN0901 were significantly downregulated (P < 0.01), or somewhat downregulated, compared with THN0901. Taken together, cel-EII complexes may have a negative effect on the virulence of TFJ0901 by regulating the expression of virulence-related genes, but the negative regulation mechanism of the cel-EII complex has no obvious effect on the virulence of THN0901.

Role of Lactic Acid Bacteria Phospho-β-Glucosidases during the Fermentation of Cereal by-Products.

Bioprocessing using lactic acid bacteria (LAB) is a powerful means to exploit plant-derived by-products as a food ingredient. LAB have the capability to metabolize a large variety of carbohydrates, but such metabolism only relies on few metabolic routes, conferring on them a high fermentation potential. One example of these pathways is that involving phospho-β-glucosidase genes, which are present in high redundancy within LAB genomes. This enzymatic activity undertakes an ambivalent role during fermentation of plant-based foods related to the release of a wide range of phenolic compounds, from their β-D-glycosylated precursors and the degradation of β-glucopyranosyl derived carbohydrates. We proposed a novel phenomic approach to characterize the metabolism drift of Lactiplantibacillus plantarum and Leuconostoc pseudomesenteroides caused by a lignocellulosic by-product, such as the brewers’ spent grain (BSG), in contrast to Rich De Man, Rogosa and Sharpe (MRS) broth. We observed an increased metabolic activity for gentiobiose, cellobiose and β-glucoside conjugates of phenolic compounds during BSG fermentation. Gene expression analysis confirmed the importance of cellobiose metabolism while a release of lignin-derived aglycones was found during BSG fermentation. We provided a comprehensive view of the important role exerted by LAB 6-phospho-β-glucosidases as well the major metabolic routes undertaken during plant-based fermentations. Further challenges will consider a controlled characterization of pbg gene expression correlated to the metabolism of β-glucosides with different aglycone moieties.

Engineering of a new Escherichia coli strain efficiently metabolizing cellobiose with promising perspectives for plant biomass-based application design

The necessity to decrease our fossil energy dependence requests bioprocesses based on biomass degradation. Cellobiose is the main product released by cellulases when acting on the major plant cell wall polysaccharide constituent, the cellulose. Escherichia coli, one of the most common model organisms for the academy and the industry, is unable to metabolize this disaccharide. In this context, the remodeling of E. coli to catabolize cellobiose should thus constitute an important progress for the design of such applications. Here, we developed a robust E. coli strain able to metabolize cellobiose by integration of a small set of modifications in its genome. Contrary to previous studies that use adaptative evolution to achieve some growth on this sugar by reactivating E. coli cryptic operons coding for cellobiose metabolism, we identified easily insertable modifications impacting the cellobiose import (expression of a gene coding a truncated variant of the maltoporin LamB, modification of the expression of lacY encoding the lactose permease) and its intracellular degradation (genomic insertion of a gene encoding either a cytosolic β-glucosidase or a cellobiose phosphorylase). Taken together, our results provide an easily transferable set of mutations that confers to E. coli an efficient growth phenotype on cellobiose (doubling time of 2.2 ​h in aerobiosis) without any prior adaptation.

Dissecting cellobiose metabolic pathway and its application in biorefinery through consolidated bioprocessing in Myceliophthora thermophila

BackgroundLignocellulosic biomass has long been recognized as a potential sustainable source for industrial applications. The costs associated with conversion of plant biomass to fermentable sugar represent a significant barrier to the production of cost-competitive biochemicals. Consolidated bioprocessing (CBP) is considered a potential breakthrough for achieving cost-efficient production of biomass-based fuels and commodity chemicals. During the degradation of cellulose, cellobiose (major end-product of cellulase activity) is catabolized by hydrolytic and phosphorolytic pathways in cellulolytic organisms. However, the details of the two intracellular cellobiose metabolism pathways in cellulolytic fungi remain to be uncovered.ResultsUsing the engineered malic acid production fungal strain JG207, we demonstrated that the hydrolytic pathway by β-glucosidase and the phosphorolytic pathway by phosphorylase are both used for intracellular cellobiose metabolism in Myceliophthora thermophila, and the yield of malic acid can benefit from the energy advantages of phosphorolytic cleavage. There were obvious differences in regulation of the two cellobiose catabolic pathways depending on whether M. thermophila JG207 was grown on cellobiose or Avicel. Disruption of Mtcpp in strain JG207 led to decreased production of malic acid under cellobiose conditions, while expression levels of all three intracellular β-glucosidase genes were significantly up-regulated to rescue the impairment of the phosphorolytic pathway under Avicel conditions. When the flux of the hydrolytic pathway was reduced, we found that β-glucosidase encoded by bgl1 was the dominant enzyme in the hydrolytic pathway and deletion of bgl1 resulted in significant enhancement of protein secretion but reduction of malate production. Combining comprehensive manipulation of both cellobiose utilization pathways and enhancement of cellobiose uptake by overexpression of a cellobiose transporter, the final strain JG412Δbgl2Δbgl3 produced up to 101.2 g/L and 77.4 g/L malic acid from cellobiose and Avicel, respectively, which corresponded to respective yields of 1.35 g/g and 1.03 g/g, representing significant improvement over the starting strain JG207.ConclusionsThis is the first report of detailed investigation of intracellular cellobiose catabolism in cellulolytic fungus M. thermophila. These results provide insights that can be applied to industrial fungi for production of biofuels and biochemicals from cellobiose and cellulose.

Characterization of the gen locus involved in β-1,6-oligosaccharide utilization by Enterococcus faecalis.

The bicistronic genBA operon (formerly named celBA) of the opportunistic pathogen Enterococcus faecalis, encodes a 6-phospho-β-glucosidase (GenA) and a phosphotransferase system permease EIIC (GenB). It resembles the cel operon of Streptococcus pyogenes, which is implicated in the metabolism of cellobiose. However, genBA mutants grew normally on cellobiose, but not (genA) or only slowly (genB) on gentiobiose and amygdalin. The two glucosides were also found to be the main inducers of the operon, confirming that the encoded proteins are involved in the utilization of β-1,6- rather than β-1,4-linked oligosaccharides. Expression of the genBA operon is regulated by the transcriptional activator GenR, which is encoded by the gene upstream from genB. Thermal shift analysis showed that it binds gentiobiose-6'-P with a Kd of 0.04mM and with lower affinity also other phospho-sugars. The GenR/gentiobiose-6'-P complex binds to the promoter region upstream from genB. The genBA promoter region contains a cre box and gel-shift experiments demonstrated that the operon is under negative control of the global carbon catabolite regulator CcpA. We also show that the orphan EIIC (GenB) protein needs the EIIA component of the putative OG1RF_10750-OG1RF_10755 operon situated elsewhere on the chromosome to form a functional PTS transporter.

Decreased metabolism and increased tolerance to extreme environments in Staphylococcus warneri during long-term spaceflight.

Many studies have shown that the space environment can affect bacteria by causing a range of mutations. However, to date, few studies have explored the effects of long‐term spaceflight (>1 month) on bacteria. In this study, a Staphylococcus warneri strain that was isolated from the Shenzhou‐10 spacecraft and had experienced a spaceflight (15 days) was carried into space again. After a 64‐day flight, combined phenotypic, genomic, transcriptomic, and proteomic analyses were performed to compare the influence of the two spaceflights on this bacterium. Compared with short‐term spaceflight, long‐term spaceflight increased the biofilm formation ability of S. warneri and the cell wall resistance to external environmental stress but reduced the sensitivity to chemical stimulation. Further analysis showed that these changes might be associated with the significantly upregulated gene expression of the phosphotransferase system, which regulates the metabolism of sugars, including glucose, mannose, fructose, and cellobiose. The mutation of S. warneri caused by the 15‐day spaceflight was limited at the phenotype and gene level after cultivation on the ground. After 79 days of spaceflight, significant changes in S. warneri were observed. The phosphotransferase system of S. warneri was upregulated by long‐term space stimulation, which resulted in a series of changes in the cell wall, biofilm, and chemical sensitivity, thus enhancing the resistance and adaptability of the bacterium to the external environment.

Bacterial cellobiose metabolism: An inquiry-driven, comprehensive undergraduate laboratory teaching approach to promote investigative learning.

Technique-centered biochemistry or molecular biology undergraduate laboratory curricula do not offer significant opportunities for thoughtful, in-depth exploration of the science to foster investigative learning. To demonstrate inclusion of inquiry-driven laboratory experiments into the undergraduate biochemistry and molecular biology curricula, a comprehensive set of laboratory experiments, covering several principles of biochemistry and molecular biology, have been developed under a single theme. The laboratory curriculum described here comprehensively investigates bacterial cellobiose metabolism using multiple biochemical, molecular biological (RNA isolation, RT-PCR, PCR, and enzyme assay), and analytical techniques (High Performance Liquid Chromatography, NMR, spectrophotometry, and thin-layer chromatography) to explore the principles of metabolomics and genomics in a single undergraduate laboratory course setting using Caulobacter crescentus as the model organism. This laboratory module serves as a model for educators to develop easy-to-implement laboratory curricula incorporating contemporary biochemistry and molecular biology concepts and techniques to provide a course-based undergraduate research experiences (CUREs) with defined learning objectives. © 2019 International Union of Biochemistry and Molecular Biology, 47(4):438-445, 2019.

Simultaneous consumption of cellobiose and xylose by Bacillus coagulans to circumvent glucose repression and identification of its cellobiose-assimilating operons

BackgroundThe use of inedible lignocellulosic biomasses for biomanufacturing provides important environmental and economic benefits for society. Efficient co-utilization of lignocellulosic biomass-derived sugars, primarily glucose and xylose, is critical for the viability of lignocellulosic biorefineries. However, the phenomenon of glucose repression prevents co-utilization of both glucose and xylose in cellulosic hydrolysates.ResultsTo circumvent glucose repression, co-utilization of cellobiose and xylose by Bacillus coagulans NL01 was investigated. During co-fermentation of cellobiose and xylose, B. coagulans NL01 simultaneously consumed the sugar mixtures and exhibited an improved lactic acid yield compared with co-fermentation of glucose and xylose. Moreover, the cellobiose metabolism of B. coagulans NL01 was investigated for the first time. Based on comparative genomic analysis, two gene clusters that encode two different operons of the cellobiose-specific phosphoenolpyruvate-dependent phosphotransferase system (assigned as CELO1 and CELO2) were identified. For CELO1, five genes were arranged as celA (encoding EIIAcel), celB (encoding EIIBcel), celC (encoding EIICcel), pbgl (encoding 6-phospho-β-glucosidase), and celR (encoding a transcriptional regulator), and these genes were found to be ubiquitous in different B. coagulans strains. Based on gene knockout results, CELO1 was confirmed to be responsible for the transport and assimilation of cellobiose. For CELO2, the five genes were arranged as celR, celB, celA, celX (encoding DUF871 domain-containing protein), and celC, and these genes were only found in some B. coagulans strains. However, through a comparison of cellobiose fermentation by NL01 and DSM1 that only possess CELO1, it was observed that CELO2 might also play an important role in the utilization of cellobiose in vivo despite the fact that no pbgl gene was found. When CELO1 or CELO2 was expressed in Escherichia coli, the recombinant strain exhibited distinct cellobiose uptake and consumption.ConclusionsThis study demonstrated the cellobiose-assimilating pathway of B. coagulans and provided a new co-utilization strategy of cellobiose and xylose to overcome the obstacles that result from glucose repression in a biorefinery system.

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, (β-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.

ClaR--a novel key regulator of cellobiose and lactose metabolism in Lactococcus lactis IL1403.

In a number of previous studies, our group has discovered an alternative pathway for lactose utilization in Lactococcus lactis that, in addition to a sugar-hydrolyzing enzyme with both P-β-glucosidase and P-β-galactosidase activity (BglS), engages chromosomally encoded components of cellobiose-specific PTS (PTSCel-Lac), including PtcA, PtcB, and CelB. In this report, we show that this system undergoes regulation via ClaR, a novel activator protein from the RpiR family of transcriptional regulators. Although RpiR proteins are widely distributed among lactic acid bacteria, their roles have yet to be confirmed by functional assays. Here, we show that ClaR activity depends on intracellular cellobiose-6-phosphate availability, while other sugars such as glucose or galactose have no influence on it. We also show that ClaR is crucial for activation of the bglS and celB expression in the presence of cellobiose, with some limited effects on ptcA and ptcB activation. Among 190 of carbon sources tested, the deletion of claR reduces L. lactis growth only in lactose- and/or cellobiose-containing media, suggesting a narrow specificity of this regulator within the context of sugar metabolism.

Leveraging transcription factors to speed cellobiose fermentation by Saccharomyces cerevisiae.

BackgroundSaccharomyces cerevisiae, a key organism used for the manufacture of renewable fuels and chemicals, has been engineered to utilize non-native sugars derived from plant cell walls, such as cellobiose and xylose. However, the rates and efficiencies of these non-native sugar fermentations pale in comparison with those of glucose. Systems biology methods, used to understand biological networks, hold promise for rational microbial strain development in metabolic engineering. Here, we present a systematic strategy for optimizing non-native sugar fermentation by recombinant S. cerevisiae, using cellobiose as a model.ResultsDifferences in gene expression between cellobiose and glucose metabolism revealed by RNA deep sequencing indicated that cellobiose metabolism induces mitochondrial activation and reduces amino acid biosynthesis under fermentation conditions. Furthermore, glucose-sensing and signaling pathways and their target genes, including the cAMP-dependent protein kinase A pathway controlling the majority of glucose-induced changes, the Snf3-Rgt2-Rgt1 pathway regulating hexose transport, and the Snf1-Mig1 glucose repression pathway, were at most only partially activated under cellobiose conditions. To separate correlations from causative effects, the expression levels of 19 transcription factors perturbed under cellobiose conditions were modulated, and the three strongest promoters under cellobiose conditions were applied to fine-tune expression of the heterologous cellobiose-utilizing pathway. Of the changes in these 19 transcription factors, only overexpression of SUT1 or deletion of HAP4 consistently improved cellobiose fermentation. SUT1 overexpression and HAP4 deletion were not synergistic, suggesting that SUT1 and HAP4 may regulate overlapping genes important for improved cellobiose fermentation. Transcription factor modulation coupled with rational tuning of the cellobiose consumption pathway significantly improved cellobiose fermentation.ConclusionsWe used systems-level input to reveal the regulatory mechanisms underlying suboptimal metabolism of the non-glucose sugar cellobiose. By identifying key transcription factors that cause suboptimal cellobiose fermentation in engineered S. cerevisiae, and by fine-tuning the expression of a heterologous cellobiose consumption pathway, we were able to greatly improve cellobiose fermentation by engineered S. cerevisiae. Our results demonstrate a powerful strategy for applying systems biology methods to rapidly identify metabolic engineering targets and overcome bottlenecks in performance of engineered strains.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-014-0126-6) contains supplementary material, which is available to authorized users.

Leveraging transcription factors to speed cellobiose fermentation by

Saccharomyces cerevisiae, a key organism used for the manufacture of renewable fuels and chemicals, has been engineered to utilize non-native sugars derived from plant cell walls, such as cellobiose and xylose. However, the rates and efficiencies of these non-native sugar fermentations pale in comparison with those of glucose. Systems biology methods, used to understand biological networks, hold promise for rational microbial strain development in metabolic engineering. Here, we present a systematic strategy for optimizing non-native sugar fermentation by recombinant S. cerevisiae, using cellobiose as a model. Differences in gene expression between cellobiose and glucose metabolism revealed by RNA deep sequencing indicated that cellobiose metabolism induces mitochondrial activation and reduces amino acid biosynthesis under fermentation conditions. Furthermore, glucose-sensing and signaling pathways and their target genes, including the cAMP-dependent protein kinase A pathway controlling the majority of glucose-induced changes, the Snf3-Rgt2-Rgt1 pathway regulating hexose transport, and the Snf1-Mig1 glucose repression pathway, were at most only partially activated under cellobiose conditions. To separate correlations from causative effects, the expression levels of 19 transcription factors perturbed under cellobiose conditions were modulated, and the three strongest promoters under cellobiose conditions were applied to fine-tune expression of the heterologous cellobiose-utilizing pathway. Of the changes in these 19 transcription factors, only overexpression of SUT1 or deletion of HAP4 consistently improved cellobiose fermentation. SUT1 overexpression and HAP4 deletion were not synergistic, suggesting that SUT1 and HAP4 may regulate overlapping genes important for improved cellobiose fermentation. Transcription factor modulation coupled with rational tuning of the cellobiose consumption pathway significantly improved cellobiose fermentation. We used systems-level input to reveal the regulatory mechanisms underlying suboptimal metabolism of the non-glucose sugar cellobiose. By identifying key transcription factors that cause suboptimal cellobiose fermentation in engineered S. cerevisiae, and by fine-tuning the expression of a heterologous cellobiose consumption pathway, we were able to greatly improve cellobiose fermentation by engineered S. cerevisiae. Our results demonstrate a powerful strategy for applying systems biology methods to rapidly identify metabolic engineering targets and overcome bottlenecks in performance of engineered strains.