Dataset Information

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

ABSTRACT: Background:Lignocellulosic 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. Results:Using the engineered malic acid production fungal strain JG207, we demonstrated that the hydrolytic pathway by ?-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 ?-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 ?-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 JG412?bgl2?bgl3 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. Conclusions:This 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.

SUBMITTER: Li J

PROVIDER: S-EPMC6852783 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Dissecting cellobiose metabolic pathway and its application in biorefinery through consolidated bioprocessing in <i>Myceliophthora thermophila</i>.

Li Jingen J Gu Shuying S Zhao Zhen Z Chen Bingchen B Liu Qian Q Sun Tao T Sun Wenliang W Tian Chaoguang C

Fungal biology and biotechnology 20191113

<h4>Background</h4>Lignocellulosic 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 (m ...[more]

PMID: 31754437

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Project description:Background:Cellulosic biomass is a promising resource for bioethanol production. However, various sugars in plant biomass hydrolysates including cellodextrins, cellobiose, glucose, xylose, and arabinose, are poorly fermented by microbes. The commonly used ethanol-producing microbe Saccharomyces cerevisiae can usually only utilize glucose, although metabolically engineered strains that utilize xylose have been developed. Direct fermentation of cellobiose could avoid glucose repression during biomass fermentation, but applications of an engineered cellobiose-utilizing S. cerevisiae are still limited because of its long lag phase. Bioethanol production from biomass-derived sugars by a cellulolytic filamentous fungus would have many advantages for the biorefinery industry. Results:We selected Myceliophthora thermophila, a cellulolytic thermophilic filamentous fungus for metabolic engineering to produce ethanol from glucose and cellobiose. Ethanol production was increased by 57% from glucose but not cellobiose after introduction of ScADH1 into the wild-type (WT) strain. Further overexpression of a glucose transporter GLT-1 or the cellodextrin transport system (CDT-1/CDT-2) from N. crassa increased ethanol production by 131% from glucose or by 200% from cellobiose, respectively. Transcriptomic analysis of the engineered cellobiose-utilizing strain and WT when grown on cellobiose showed that genes involved in oxidation-reduction reactions and the stress response were downregulated, whereas those involved in protein biosynthesis were upregulated in this effective ethanol production strain. Turning down the expression of pyc gene results the final engineered strain with the ethanol production was further increased by 23%, reaching up to 11.3 g/L on cellobiose. Conclusions:This is the first attempt to engineer the cellulolytic fungus M. thermophila to produce bioethanol from biomass-derived sugars such as glucose and cellobiose. The ethanol production can be improved about 4 times up to 11 grams per liter on cellobiose after a couple of genetic engineering. These results show that M. thermophila is a promising platform for bioethanol production from cellulosic materials in the future.

Project description:BackgroundClostridial co-culture containing cellulolytic and solventogenic species is a potential consolidated bioprocessing (CBP) approach for producing biochemicals and biofuels from cellulosic biomass. It has been demonstrated that the rate of cellulose utilization in the co-culture of Clostridium acetobutylicum and Clostridium cellulolyticum is improved compared to the mono-culture of C. cellulolyticum (BL 5:119-124, 1983). However, the metabolic interactions in this co-culture are not well understood. To investigate the metabolic interactions in this co-culture we dynamically characterized the physiology and microbial composition using qPCR.ResultsThe qPCR data suggested a higher growth rate of C. cellulolyticum in the co-culture compared to its mono-culture. Our results also showed that in contrast to the mono-culture of C. cellulolyticum, which did not show any cellulolytic activity under conditions similar to those of co-culture, the co-culture did show cellulolytic activity even superior to the C. cellulolyticum mono-culture at its optimal pH of 7.2. Moreover, experiments indicated that the co-culture cellulolytic activity depends on the concentration of C. acetobutylicum in the co-culture, as no cellulolytic activity was observed at low concentration of C. acetobutylicum, and thus confirming the essential role of C. acetobutylicum in improving C. cellulolyticum growth in the co-culture. Furthermore, butanol concentration of 350 mg/L was detected in the co-culture batch experiments.ConclusionThese results suggest the presence of synergism between these two species, while C. acetobutylicum metabolic activity significantly improves the cellulolytic activity in the co-culture, and allows C. cellulolyticum to survive under harsh co-culture conditions, which do not allow C. cellulolyticum to grow and metabolize cellulose independently. It is likely that C. acetobutylicum improves the cellulolytic activity of C. cellulolyticum in the co-culture through exchange of metabolites such as pyruvate, enabling it to grow and metabolize cellulose under harsh co-culture conditions.

Project description:BackgroundWith D-xylose being the second most abundant sugar in nature, its conversion into products could significantly improve biomass-based process economy. There are two well-studied phosphorylative pathways for D-xylose metabolism. One is isomerase pathway mainly found in bacteria, and the other one is oxo-reductive pathway that always exists in fungi. Except for these two pathways, there are also non-phosphorylative pathways named xylose oxidative pathways and they have several advantages over traditional phosphorylative pathways. In Myceliophthora thermophila, D-xylose can be metabolized through oxo-reductive pathway after plant biomass degradation. The survey of non-phosphorylative pathways in this filamentous fungus will offer a potential way for carbon-efficient production of fuels and chemicals using D-xylose.ResultsIn this study, an alternative for utilization of D-xylose, the non-phosphorylative Weimberg pathway was established in M. thermophila. Growth on D-xylose of strains whose D-xylose reductase gene was disrupted, was restored after overexpression of the entire Weimberg pathway. During the construction, a native D-xylose dehydrogenase with highest activity in M. thermophila was discovered. Here, M. thermophila was also engineered to produce 1,2,4-butanetriol using D-xylose through non-phosphorylative pathway. Afterwards, transcriptome analysis revealed that the D-xylose dehydrogenase gene was obviously upregulated after deletion of D-xylose reductase gene when cultured in a D-xylose medium. Besides, genes involved in growth were enriched in strains containing the Weimberg pathway.ConclusionsThe Weimberg pathway was established in M. thermophila to support its growth with D-xylose being the sole carbon source. Besides, M. thermophila was engineered to produce 1,2,4-butanetriol using D-xylose through non-phosphorylative pathway. To our knowledge, this is the first report of non-phosphorylative pathway recombinant in filamentous fungi, which shows great potential to convert D-xylose to valuable chemicals.

Project description:Background:Fumaric acid is widely used in food and pharmaceutical industries and is recognized as a versatile industrial chemical feedstock. Increasing concerns about energy and environmental problems have resulted in a focus on fumaric acid production by microbial fermentation via bioconversion of renewable feedstocks. Filamentous fungi are the predominant microorganisms used to produce organic acids, including fumaric acid, and most studies to date have focused on Rhizopus species. Thermophilic filamentous fungi have many advantages for the production of compounds by industrial fermentation. However, no previous studies have focused on fumaric acid production by thermophilic fungi. Results:We explored the feasibility of producing fumarate by metabolically engineering Myceliophthora thermophila using the CRISPR/Cas9 system. Screening of fumarases suggested that the fumarase from Candida krusei was the most suitable for efficient production of fumaric acid in M. thermophila. Introducing the C. krusei fumarase into M. thermophila increased the titer of fumaric acid by threefold. To further increase fumarate production, the intracellular fumarate digestion pathway was disrupted. After deletion of the two fumarate reductase and the mitochondrial fumarase genes of M. thermophila, the resulting strain exhibited a 2.33-fold increase in fumarate titer. Increasing the pool size of malate, the precursor of fumaric acid, significantly increased the final fumaric acid titer. Finally, disruption of the malate-aspartate shuttle increased the intracellular malate content by 2.16-fold and extracellular fumaric acid titer by 42%, compared with that of the parental strain. The strategic metabolic engineering of multiple genes resulted in a final strain that could produce up to 17 g/L fumaric acid from glucose in a fed-batch fermentation process. Conclusions:This is the first metabolic engineering study on the production of fumaric acid by the thermophilic filamentous fungus M. thermophila. This cellulolytic fungal platform provides a promising method for the sustainable and efficient-cost production of fumaric acid from lignocellulose-derived carbon sources in the future.

Project description:BackgroundAlthough the biocatalytic conversion of cellulosic biomass could replace fossil oil for the production of various compounds, it is often not economically viable due to the high costs of cellulolytic enzymes. One possibility to reduce costs is consolidated bioprocessing (CBP), integrating cellulase production, hydrolysis of cellulose, and the fermentation of the released sugars to the desired product into one process step. To establish such a process, the most suitable cellulase-producing organism has to be identified. Thereby, it is crucial to evaluate the candidates under target process conditions. In this work, the chosen model process was the conversion of cellulose to the platform chemical itaconic acid by a mixed culture of a cellulolytic fungus with Aspergillus terreus as itaconic acid producer. Various cellulase producers were analyzed by the introduced freeze assay that measures the initial carbon release rate, quantifying initial cellulase activity under target process conditions. Promising candidates were then characterized online by monitoring their respiration activity metabolizing cellulose to assess the growth and enzyme production dynamics.ResultsThe screening of five different cellulase producers with the freeze assay identified Trichoderma reesei and Penicillium verruculosum as most promising. The measurement of the respiration activity revealed a retarded induction of cellulase production for P. verruculosum but a similar cellulase production rate afterwards, compared to T. reesei. The freeze assay measurement depicted that P. verruculosum reaches the highest initial carbon release rate among all investigated cellulase producers. After a modification of the cultivation procedure, these results were confirmed by the respiration activity measurement. To compare both methods, a correlation between the measured respiration activity and the initial carbon release rate of the freeze assay was introduced. The analysis revealed that the different initial enzyme/cellulose ratios as well as a discrepancy in cellulose digestibility are the main differences between the two approaches.ConclusionsWith two complementary methods to quantify cellulase activity and the dynamics of cellulase production for CBP applications, T. reesei and P. verruculosum were identified as compatible candidates for the chosen model process. The presented methods can easily be adapted to screen for suitable cellulose degrading organisms for various other applications.

Dataset Information

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

Publications

Dissecting cellobiose metabolic pathway and its application in biorefinery through consolidated bioprocessing in <i>Myceliophthora thermophila</i>.

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