Physicochemical and Biological Treatments for Enzymatic/Microbial Conversion of Lignocellulosic Biomass

Abstract Background Mitigation of climate change requires that new routes for the production of fuels and chemicals be as oil-independent as possible. The microbial conversion of lignocellulosic feedstocks into terpene-based biofuels and bioproducts represents one such route. This work builds upon previous demonstrations that the single-celled carotenogenic basidiomycete, Rhodosporidium toruloides, is a promising host for the production of terpenes from lignocellulosic hydrolysates. Results This study focuses on the optimization of production of the monoterpene 1,8-cineole and the sesquiterpene α-bisabolene in R. toruloides. The α-bisabolene titer attained in R. toruloides was found to be proportional to the copy number of the bisabolene synthase (BIS) expression cassette, which in turn influenced the expression level of several native mevalonate pathway genes. The addition of more copies of BIS under a stronger promoter resulted in production of α-bisabolene at 2.2 g/L from lignocellulosic hydrolysate in a 2-L fermenter. Production of 1,8-cineole was found to be limited by availability of the precursor geranylgeranyl pyrophosphate (GPP) and expression of an appropriate GPP synthase increased the monoterpene titer fourfold to 143 mg/L at bench scale. Targeted mevalonate pathway metabolite analysis suggested that 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGR), mevalonate kinase (MK) and phosphomevalonate kinase (PMK) may be pathway bottlenecks are were therefore selected as targets for overexpression. Expression of HMGR, MK, and PMK orthologs and growth in an optimized lignocellulosic hydrolysate medium increased the 1,8-cineole titer an additional tenfold to 1.4 g/L. Expression of the same mevalonate pathway genes did not have as large an impact on α-bisabolene production, although the final titer was higher at 2.6 g/L. Furthermore, mevalonate pathway intermediates accumulated in the mevalonate-engineered strains, suggesting room for further improvement. Conclusions This work brings R. toruloides closer to being able to make industrially relevant quantities of terpene from lignocellulosic biomass.

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Experimental evolution reveals an effective avenue to release catabolite repression via mutations in XylR

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1700345114 ◽

2017 ◽

Vol 114 (28) ◽

pp. 7349-7354 ◽

Cited By ~ 17

Author(s):

Christian Sievert ◽

Lizbeth M. Nieves ◽

Larry A. Panyon ◽

Taylor Loeffler ◽

Chandler Morris ◽

...

Keyword(s):

Amino Acid ◽

Lignocellulosic Biomass ◽

Catabolite Repression ◽

High Efficiency ◽

Xylose Fermentation ◽

Amino Acid Substitutions ◽

Xylose Utilization ◽

Microbial Conversion ◽

E Coli ◽

Product Titer

Microbial production of fuels and chemicals from lignocellulosic biomass provides promising biorenewable alternatives to the conventional petroleum-based products. However, heterogeneous sugar composition of lignocellulosic biomass hinders efficient microbial conversion due to carbon catabolite repression. The most abundant sugar monomers in lignocellulosic biomass materials are glucose and xylose. Although industrialEscherichia colistrains efficiently use glucose, their ability to use xylose is often repressed in the presence of glucose. Here we independently evolved threeE. colistrains from the same ancestor to achieve high efficiency for xylose fermentation. Each evolved strain has a point mutation in a transcriptional activator for xylose catabolic operons, either CRP or XylR, and these mutations are demonstrated to enhance xylose fermentation by allelic replacements. Identified XylR variants (R121C and P363S) have a higher affinity to their DNA binding sites, leading to a xylose catabolic activation independent of catabolite repression control. Upon introducing these amino acid substitutions into theE. coliD-lactate producer TG114, 94% of a glucose–xylose mixture (50 g⋅L−1each) was used in mineral salt media that led to a 50% increase in product titer after 96 h of fermentation. The two amino acid substitutions in XylR enhance xylose utilization and release glucose-induced repression in differentE. colihosts, including wild type, suggesting its potential wide application in industrialE. colibiocatalysts.

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Cellulose solvent-based pretreatment for enhanced second-generation biofuel production: a review

Sustainable Energy & Fuels ◽

10.1039/c8se00287h ◽

2019 ◽

Vol 3 (1) ◽

pp. 11-62 ◽

Cited By ~ 58

Author(s):

Behzad Satari ◽

Keikhosro Karimi ◽

Rajeev Kumar

Keyword(s):

Lignocellulosic Biomass ◽

Second Generation ◽

Biofuel Production ◽

Microbial Conversion ◽

Cellulose Solvent ◽

Fuels And Chemicals ◽

Generation Biofuel

Cellulose solvent-based fractionation technologies can prove to be economical to enhance lignocellulosic biomass microbial conversion to fuels and chemicals.

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Engineering of Saccharomyces cerevisiae for efficient fermentation of cellulose

FEMS Yeast Research ◽

10.1093/femsyr/foz089 ◽

2020 ◽

Vol 20 (1) ◽

Cited By ~ 2

Author(s):

Eun Joong Oh ◽

Yong-Su Jin

Keyword(s):

Saccharomyces Cerevisiae ◽

Lignocellulosic Biomass ◽

Biofuel Production ◽

Cellulolytic Enzymes ◽

Microbial Conversion ◽

Biomass Hydrolysis ◽

Yeast Strains ◽

Attractive Option ◽

Engineered Yeast ◽

Efficient Fermentation

ABSTRACT Conversion of lignocellulosic biomass to biofuels using microbial fermentation is an attractive option to substitute petroleum-based production economically and sustainably. The substantial efforts to design yeast strains for biomass hydrolysis have led to industrially applicable biological routes. Saccharomyces cerevisiae is a robust microbial platform widely used in biofuel production, based on its amenability to systems and synthetic biology tools. The critical challenges for the efficient microbial conversion of lignocellulosic biomass by engineered S. cerevisiae include heterologous expression of cellulolytic enzymes, co-fermentation of hexose and pentose sugars, and robustness against various stresses. Scientists developed many engineering strategies for cellulolytic S. cerevisiae strains, bringing the application of consolidated bioprocess at an industrial scale. Recent advances in the development and implementation of engineered yeast strains capable of assimilating lignocellulose will be reviewed.

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Experimental evolution reveals a novel avenue to release catabolite repression via mutations in XylR

10.1101/100602 ◽

2017 ◽

Author(s):

Christian Sievert ◽

Lizbeth M. Nieves ◽

Larry A. Panyon ◽

Taylor Loeffler ◽

Chandler Morris ◽

...

Keyword(s):

Amino Acid ◽

Lignocellulosic Biomass ◽

Catabolite Repression ◽

High Efficiency ◽

Xylose Fermentation ◽

Amino Acid Substitutions ◽

Xylose Utilization ◽

Microbial Conversion ◽

E Coli ◽

Product Titer

AbstractMicrobial production of fuels and chemicals from lignocellulosic biomass provides promising bio-renewable alternatives to the conventional petroleum-based products. However, heterogeneous sugar composition of lignocellulosic biomass hinders efficient microbial conversion due to carbon catabolite repression. The most abundant sugar monomers in lignocel-lulosic biomass materials are glucose and xylose. While industrialEscherichia colistrains efficiently utilize glucose, their ability to utilize xylose is often repressed in the presence of glucose. Here we independently evolved threeE. colistrains from the same ancestor to achieve high efficiency for xylose fermentation. Each evolved strain has a point mutation in a transcriptional activator for xylose catabolic operons, either CRP or XylR, and these mutations are demonstrated to enhance xylose fermentation by allelic replacements. Identified XylR variants (R121C and P363S) have a higher affinity to their DNA binding sites, leading to a xylose catabolic activation independent of catabolite repression control. Upon introducing these amino acid substitutions into theE. coliD-lactate producer TG114, 94 % of a glucose-xylose mixture (50 g L-1each) was utilized in mineral salt media that led to a 50 % increase in product titer after 96 h of fermentation. The two amino acid substitutions in XylR enhance xylose utilization and release glucose-induced repression in differentE. colihosts, including wild-type, suggesting its potential wide application in industrialE. colibiocatalysts.

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Further Engineering of R. Toruloides for the Production of Terpenes From Lignocellulosic Biomass

10.21203/rs.3.rs-118273/v1 ◽

2020 ◽

Author(s):

James Kirby ◽

Gina M. Geiselman ◽

Junko Yaegashi ◽

Joonhoon Kim ◽

Xun Zhuang ◽

...

Keyword(s):

Lignocellulosic Biomass ◽

Copy Number ◽

Mevalonate Pathway ◽

Mevalonate Kinase ◽

Metabolite Analysis ◽

Lignocellulosic Hydrolysate ◽

Microbial Conversion ◽

Lignocellulosic Hydrolysates ◽

Lignocellulosic Feedstocks ◽

Fuels And Chemicals

Abstract Background: Mitigation of climate change requires that new routes for the production of fuels and chemicals be as oil-independent as possible. The microbial conversion of lignocellulosic feedstocks into terpene-based biofuels and bioproducts represents one such route. This work builds upon previous demonstrations that the single-celled carotenogenic basidiomycete, Rhodosporidium toruloides, is a promising host for the production of terpenes from lignocellulosic hydrolysates. Results:This study focuses on the optimization of production of the monoterpene 1,8-cineole and the sesquiterpene α-bisabolene in R. toruloides. The α-bisabolene titer attained in R. toruloides was found to be proportional to the copy number of the bisabolene synthase (BIS) expression cassette, which in turn influenced the expression level of several native mevalonate pathway genes. The addition of more copies of BIS under a stronger promoter resulted in production of α-bisabolene at 2.2 g/L from lignocellulosic hydrolysate in a 2 L fermenter. Production of 1,8-cineole was found to be limited by availability of the precursor geranylgeranyl pyrophosphate (GPP) and expression of an appropriate GPP synthase increased the monoterpene titer 4-fold to 143 mg/L at bench scale. Targeted mevalonate pathway metabolite analysis suggested that 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGR), mevalonate kinase (MK) and phosphomevalonate kinase (PMK) may be pathway bottlenecks are were therefore selected as targets for overexpression. Expression of HMGR, MK, and PMK orthologs and growth in an optimized lignocellulosic hydrolysate medium increased the 1,8-cineole titer an additional 10-fold to 1.4 g/L. Expression of the same mevalonate pathway genes did not have as large an impact on α-bisabolene production, although the final titer was higher at 2.6 g/L. Furthermore, mevalonate pathway intermediates accumulated in the mevalonate-engineered strains, suggesting room for further improvement. Conclusions:This work brings R. toruloides closer to being able to make industrially relevant quantities of terpene from lignocellulosic biomass.

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