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  • 1
    Online Resource
    Online Resource
    Overexpression of Oxidoreductase YghA Confers Tolerance of Furfural in Ethanologenic Escherichia coli Strain SSK42
    American Society for Microbiology ; 2021
    In:  Applied and Environmental Microbiology Vol. 87, No. 23 ( 2021-11-10)
    Details
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 87, No. 23 ( 2021-11-10)
    Abstract: Furfural is a common furan inhibitor formed due to dehydration of pentose sugars, like xylose, and acts as an inhibitor of microbial metabolism. Overexpression of NADH-specific FucO and deletion of NADPH-specific YqhD had been a successful strategy in the past in conferring tolerance against furfural in Escherichia coli , which highlights the importance of oxidoreductases in conferring tolerance against furfural. In a screen consisting of various oxidoreductases, dehydrogenases, and reductases, we identified the yghA gene as an overexpression target to confer tolerance against furfural. YghA preferably used NADH as a cofactor and had an apparent K m value of 0.03 mM against furfural. In the presence of 1 g liter −1 furfural and 10% xylose (wt/vol), yghA overexpression in an ethanologenic E. coli strain SSK42 resulted in an ethanol efficiency of ∼97%, with a 5.3-fold increase in ethanol titers compared to the control. YghA also exhibited activity against the less toxic inhibitor 5-hydroxymethyl furfural, which is formed due to dehydration of hexose sugars, and thus is a formidable target for overexpression in ethanologenic strain for fermentation of sugars in biomass hydrolysate. IMPORTANCE Lignocellulosic biomass represents an inexhaustible source of carbon for second-generation biofuels. Thermo-acidic pretreatment of biomass is performed to loosen the lignocellulosic fibers and make the carbon bioavailable for microbial metabolism. The pretreatment process also results in the formation of inhibitors that inhibit microbial metabolism and increase production costs. Furfural is a potent furan inhibitor that increases the toxicity of other inhibitors present in the hydrolysate. Thus, it is desirable to engineer furfural tolerance in E. coli for efficient fermentation of hydrolysate sugars.
    Type of Medium: Online Resource
    ISSN: 0099-2240 , 1098-5336
    URL: Article
    DOI: 10.1128/AEM.01855-21
    RVK:
    WA 15000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2021
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  • 2
    Online Resource
    Online Resource
    Synthesis and Characterization of Chimeric Proteins Based on Cellulase and Xylanase from an Insect Gut Bacterium
    American Society for Microbiology ; 2011
    In:  Applied and Environmental Microbiology Vol. 77, No. 14 ( 2011-07-15), p. 4859-4866
    Details
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 77, No. 14 ( 2011-07-15), p. 4859-4866
    Abstract: Insects living on wood and plants harbor a large variety of bacterial flora in their guts for degrading biomass. We isolated a Paenibacillus strain, designated ICGEB2008, from the gut of a cotton bollworm on the basis of its ability to secrete a variety of plant-hydrolyzing enzymes. In this study, we cloned, expressed, and characterized two enzymes, β-1,4-endoglucanase (Endo5A) and β-1,4-endoxylanase (Xyl11D), from the ICGEB2008 strain and synthesized recombinant bifunctional enzymes based on Endo5A and Xyl11D. The gene encoding Endo5A was obtained from the genome of the ICGEB2008 strain by shotgun cloning. The gene encoding Xyl11D was obtained using primers for conserved xylanase sequences, which were identified by aligning xylanase sequences in other species of Paenibacillus . Endo5A and Xyl11D were overexpressed in Escherichia coli , and their optimal activities were characterized. Both Endo5A and Xyl11D exhibited maximum specific activity at 50°C and pH 6 to 7. To take advantage of this feature, we constructed four bifunctional chimeric models of Endo5A and Xyl11D by fusing the encoding genes either end to end or through a glycine-serine (GS) linker. We predicted three-dimensional structures of the four models using the I-TASSER server and analyzed their secondary structures using circular dichroism (CD) spectroscopy. The chimeric model Endo5A-GS-Xyl11D, in which a linker separated the two enzymes, yielded the highest C-score on the I-TASSER server, exhibited secondary structure properties closest to the native enzymes, and demonstrated 1.6-fold and 2.3-fold higher enzyme activity than Endo5A and Xyl11D, respectively. This bifunctional enzyme could be effective for hydrolyzing plant biomass owing to its broad substrate range.
    Type of Medium: Online Resource
    ISSN: 0099-2240 , 1098-5336
    URL: Article
    DOI: 10.1128/AEM.02808-10
    RVK:
    WA 15000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2011
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  • 3
    Online Resource
    Online Resource
    Specific Fusion of β-1,4-Endoglucanase and β-1,4-Glucosidase Enhances Cellulolytic Activity and Helps in Channeling of Intermediates
    American Society for Microbiology ; 2012
    In:  Applied and Environmental Microbiology Vol. 78, No. 20 ( 2012-10-15), p. 7447-7454
    Details
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 78, No. 20 ( 2012-10-15), p. 7447-7454
    Abstract: Identification and design of new cellulolytic enzymes with higher catalytic efficiency are a key factor in reducing the production cost of lignocellulosic bioalcohol. We report here identification of a novel β-glucosidase (Gluc1C) from Paenibacillus sp. strain MTCC 5639 and construction of bifunctional chimeric proteins based on Gluc1C and Endo5A, a β-1,4-endoglucanase isolated from MTCC 5639 earlier. The 448-amino-acid-long Gluc1C contained a GH superfamily 1 domain and hydrolyzed cellodextrin up to a five-sugar chain length, with highest efficiency toward cellobiose. Addition of Gluc1C improved the ability of Endo5A to release the reducing sugars from carboxymethyl cellulose. We therefore constructed six bifunctional chimeric proteins based on Endo5A and Gluc1C varying in the positions and sizes of linkers. One of the constructs, EG5, consisting of Endo5A-(G 4 S) 3 -Gluc1C, demonstrated 3.2- and 2-fold higher molar specific activities for β-glucosidase and endoglucanase, respectively, than Gluc1C and Endo5A alone. EG5 also showed 2-fold higher catalytic efficiency than individual recombinant enzymes. The thermal denaturation monitored by circular dichroism (CD) spectroscopy demonstrated that the fusion of Gluc1C with Endo5A resulted in increased thermostability of both domains by 5°C and 9°C, respectively. Comparative hydrolysis experiments done on alkali-treated rice straw and CMC indicated 2-fold higher release of product by EG5 than that by the physical mixture of Endo5A and Gluc1C, providing a rationale for channeling of intermediates. Addition of EG5 to a commercial enzyme preparation significantly enhanced release of reducing sugars from pretreated biomass, indicating its commercial applicability.
    Type of Medium: Online Resource
    ISSN: 0099-2240 , 1098-5336
    URL: Article
    DOI: 10.1128/AEM.01386-12
    RVK:
    WA 15000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2012
    detail.hit.zdb_id: 223011-2
    detail.hit.zdb_id: 1478346-0
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  • 4
    Online Resource
    Online Resource
    Profiling of the β-glucosidases identified in the genome of Penicillium funiculosum : insights from genomics, transcriptomics, proteomics, and homology-modeling studies
    American Society for Microbiology ; 2023
    In:  Applied and Environmental Microbiology Vol. 89, No. 9 ( 2023-09-28)
    Details
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 89, No. 9 ( 2023-09-28)
    Abstract: Commercially available cellulases are primarily produced from Trichoderma reesei . However, external supplementation of the cellulase cocktail from this host with exogenous β-glucosidase is often required to achieve the desired optimal saccharification of cellulosic feedstocks. This challenge has led to the exploration of other cellulase-producing strains. The nonmodel hypercellulolytic fungus Penicillium funiculosum has been studied in recent times and identified as a promising source of industrial cellulases mainly due to its ability to produce a balanced concoction of cellulolytic enzymes, including β-glucosidases. Various genetic interventions targeted at strain improvement for cellulase production have been performed; however, the β-glucosidases of this strain have remained largely understudied. This study, therefore, reports profiling of all eight β-glucosidases of P. funiculosum via molecular and computational approaches. The results of this study provide useful insights that will establish the background for future engineering strategies to transform this fungus into an industrial workhorse.
    Type of Medium: Online Resource
    ISSN: 0099-2240 , 1098-5336
    URL: Article
    DOI: 10.1128/aem.00704-23
    RVK:
    WA 15000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2023
    detail.hit.zdb_id: 223011-2
    detail.hit.zdb_id: 1478346-0
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  • 5
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    Online Resource
    Synergistic Action of a Lytic Polysaccharide Monooxygenase and a Cellobiohydrolase from Penicillium funiculosum in Cellulose Saccharification under High-Level Substrate Loading
    American Society for Microbiology ; 2020
    In:  Applied and Environmental Microbiology Vol. 86, No. 23 ( 2020-11-10)
    Details
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 86, No. 23 ( 2020-11-10)
    Abstract: Lytic polysaccharide monooxygenases (LPMOs) are crucial industrial enzymes required in the biorefinery industry as well as in the natural carbon cycle. These enzymes, known to catalyze the oxidative cleavage of glycosidic bonds, are produced by numerous bacterial and fungal species to assist in the degradation of cellulosic biomass. In this study, we annotated and performed structural analysis of an uncharacterized LPMO from Penicillium funiculosum (PfLPMO9) based on computational methods in an attempt to understand the behavior of this enzyme in biomass degradation. PfLPMO9 exhibited 75% and 36% sequence identities with LPMOs from Thermoascus aurantiacus (TaLPMO9A) and Lentinus similis (LsLPMO9A), respectively. Furthermore, multiple fungal genetic manipulation tools were employed to simultaneously overexpress LPMO and cellobiohydrolase I (CBH1) in a catabolite-derepressed strain of Penicillium funiculosum , Pf Mig1 88 (an engineered variant of P. funiculosum ), to improve its saccharification performance toward acid-pretreated wheat straw (PWS) at 20% substrate loading. The resulting transformants showed improved LPMO and CBH1 expression at both the transcriptional and translational levels, with ∼200% and ∼66% increases in ascorbate-induced LPMO and Avicelase activities, respectively. While the secretome of Pf Mig 88 overexpressing LPMO or CBH1 increased the saccharification of PWS by 6% or 13%, respectively, over the secretome of Pf Mig1 88 at the same protein concentration, the simultaneous overexpression of these two genes led to a 20% increase in saccharification efficiency over that observed with Pf Mig1 88 , which accounted for 82% saccharification of PWS under 20% substrate loading. IMPORTANCE The enzymatic hydrolysis of cellulosic biomass by cellulases continues to be a significant bottleneck in the development of second-generation biobased industries. While increasing efforts are being made to obtain indigenous cellulases for biomass hydrolysis, the high production cost of this enzyme remains a crucial challenge affecting its wide availability for the efficient utilization of cellulosic materials. This is because it is challenging to obtain an enzymatic cocktail with balanced activity from a single host. This report describes the annotation and structural analysis of an uncharacterized lytic polysaccharide monooxygenase (LPMO) gene in Penicillium funiculosum and its impact on biomass deconstruction upon overexpression in a catabolite-derepressed strain of P. funiculosum . Cellobiohydrolase I (CBH1), which is the most important enzyme produced by many cellulolytic fungi for the saccharification of crystalline cellulose, was further overexpressed simultaneously with LPMO. The resulting secretome was analyzed for enhanced LPMO and exocellulase activities and the corresponding improvement in saccharification performance (by ∼20%) under high-level substrate loading using a minimal amount of protein.
    Type of Medium: Online Resource
    ISSN: 0099-2240 , 1098-5336
    URL: Article
    DOI: 10.1128/AEM.01769-20
    RVK:
    WA 15000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2020
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    detail.hit.zdb_id: 1478346-0
    SSG: 12
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  • 6
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    Online Resource
    Fermentative Utilization of Glycerol by Escherichia coli and Its Implications for the Production of Fuels and Chemicals
    American Society for Microbiology ; 2008
    In:  Applied and Environmental Microbiology Vol. 74, No. 4 ( 2008-02-15), p. 1124-1135
    Details
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 74, No. 4 ( 2008-02-15), p. 1124-1135
    Abstract: Availability, low prices, and a high degree of reduction make glycerol an ideal feedstock to produce reduced chemicals and fuels via anaerobic fermentation. Although glycerol metabolism in Escherichia coli had been thought to be restricted to respiratory conditions, we report here the utilization of this carbon source in the absence of electron acceptors. Cells grew fermentatively on glycerol and exhibited exponential growth at a maximum specific growth rate of 0.040 ± 0.003 h −1 . The fermentative nature of glycerol metabolism was demonstrated through studies in which cell growth and glycerol utilization were observed despite blocking several respiratory processes. The incorporation of glycerol in cellular biomass was also investigated via nuclear magnetic resonance analysis of cultures in which either 50% U- 13 C-labeled or 100% unlabeled glycerol was used. These studies demonstrated that about 20% of the carbon incorporated into the protein fraction of biomass originated from glycerol. The use of U- 13 C-labeled glycerol also allowed the unambiguous identification of ethanol and succinic, acetic, and formic acids as the products of glycerol fermentation. The synthesis of ethanol was identified as a metabolic determinant of glycerol fermentation; this pathway fulfills energy requirements by generating, in a redox-balanced manner, 1 mol of ATP per mol of glycerol converted to ethanol. A fermentation balance analysis revealed an excellent closure of both carbon (∼95%) and redox (∼96%) balances. On the other hand, cultivation conditions that prevent H 2 accumulation were shown to be an environmental determinant of glycerol fermentation. The negative effect of H 2 is related to its metabolic recycling, which in turn generates an unfavorable internal redox state. The implications of our findings for the production of reduced chemicals and fuels were illustrated by coproducing ethanol plus formic acid and ethanol plus hydrogen from glycerol at yields approaching their theoretical maximum.
    Type of Medium: Online Resource
    ISSN: 0099-2240 , 1098-5336
    URL: Article
    DOI: 10.1128/AEM.02192-07
    RVK:
    WA 15000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2008
    detail.hit.zdb_id: 223011-2
    detail.hit.zdb_id: 1478346-0
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