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Engineering cellulases for conversion of lignocellulosic biomass.

ABSTRACT: Lignocellulosic biomass is a renewable source of energy, chemicals and materials. Many applications of this resource require the depolymerization of one or more of its polymeric constituents. Efficient enzymatic depolymerization of cellulose to glucose by cellulases and accessory enzymes such as lytic polysaccharide monooxygenases is a prerequisite for economically viable exploitation of this biomass. Microbes produce a remarkably diverse range of cellulases, which consist of glycoside hydrolase (GH) catalytic domains and, although not in all cases, substrate-binding carbohydrate-binding modules (CBMs). As enzymes are a considerable cost factor, there is great interest in finding or engineering improved and robust cellulases, with higher activity and stability, easy expression, and minimal product inhibition. This review addresses relevant engineering targets for cellulases, discusses a few notable cellulase engineering studies of the past decades and provides an overview of recent work in the field.

SUBMITTER: Chaudhari YB

PROVIDER: S-EPMC10394125 | biostudies-literature | 2023 Jan

REPOSITORIES: biostudies-literature

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Engineering cellulases for conversion of lignocellulosic biomass.

Chaudhari Yogesh B YB Várnai Anikó A Sørlie Morten M Horn Svein J SJ Eijsink Vincent G H VGH

Protein engineering, design & selection : PEDS 20230101

Lignocellulosic biomass is a renewable source of energy, chemicals and materials. Many applications of this resource require the depolymerization of one or more of its polymeric constituents. Efficient enzymatic depolymerization of cellulose to glucose by cellulases and accessory enzymes such as lytic polysaccharide monooxygenases is a prerequisite for economically viable exploitation of this biomass. Microbes produce a remarkably diverse range of cellulases, which consist of glycoside hydrolase ...[more]

PMID: 36892404

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Project description:Lignocellulosic biomass conversion inhibitors, furfural and HMF, inhibit microbial growth and interfere with subsequent fermentation of ethanol, posing significant challenges for a sustainable cellulosic ethanol conversion industry. Numerous yeast genes were found to be associated with the inhibitor tolerance. However, limited knowledge is available about mechanisms of the tolerance and the detoxification of the biomass conversion inhibitors. Using a robust standard for absolute mRNA quantification assay and a recently developed tolerant ethanologenic yeast Saccharomyces cerevisiae NRRL Y-50049, we investigate pathway-based transcription profiles relevant to the yeast tolerance and the inhibitor detoxification. Under the synergistic inhibitory challenges by furfural and HMF, Y-50049 was able to withstand the inhibitor stress, in situ detoxify furfural and HMF, and produce ethanol, while its parental control Y-12632 failed to function till 65 h after incubation. The tolerant strain Y-50049 displayed enriched genetic background with significantly higher abundant of transcripts for at least 16 genes than a non-tolerant parental strain Y-12632. The enhanced expression of ZWF1 appeared to drive glucose metabolism in favor of pentose phosphate pathway over glycolysis at earlier steps of glucose metabolisms. Cofactor NAD(P)H generation steps were likely accelerated by enzymes encoded by ZWF1, GND1, GND2, TDH1, and ALD4. NAD(P)H-dependent aldehyde reductions including conversion of furfural and HMF, in return, provided sufficient NAD(P)(+) for NAD(P)H regeneration in the yeast detoxification pathways. Enriched genetic background and a well maintained redox balance through reprogrammed expression responses of Y-50049 were accountable for the acquired tolerance and detoxification of furfural to furan methanol and HMF to furan dimethanol. We present significant gene interactions and regulatory networks involved in NAD(P)H regenerations and functional aldehyde reductions under the inhibitor stress.

Project description:BACKGROUND:High enzyme loading is a major economic bottleneck for the commercial processing of pretreated lignocellulosic biomass to produce fermentable sugars. Optimizing the enzyme cocktail for specific types of pretreated biomass allows for a significant reduction in enzyme loading without sacrificing hydrolysis yield. This is especially important for alkaline pretreatments such as Ammonia fiber expansion (AFEX) pretreated corn stover. Hence, a diverse set of hemicellulases supplemented along with cellulases is necessary for high recovery of monosaccharides. RESULTS:The core fungal cellulases in the optimal cocktail include cellobiohydrolase I [CBH I; glycoside hydrolase (GH) family 7A], cellobiohydrolase II (CBH II; GH family 6A), endoglucanase I (EG I; GH family 7B) and ?-glucosidase (?G; GH family 3). Hemicellulases tested along with the core cellulases include xylanases (LX1, GH family 10; LX2, GH family 10; LX3, GH family 10; LX4, GH family 11; LX5, GH family 10; LX6, GH family 10), ?-xylosidase (L?X; GH family 52), ?-arabinofuranosidase (LArb, GH family 51) and ?-glucuronidase (L?Gl, GH family 67) that were cloned, expressed and/or purified from different bacterial sources. Different combinations of these enzymes were tested using a high-throughput microplate based 24 h hydrolysis assay. Both family 10 (LX3) and family 11 (LX4) xylanases were found to most efficiently hydrolyze AFEX pretreated corn stover in a synergistic manner. The optimal mass ratio of xylanases (LX3 and LX4) to cellulases (CBH I, CBH II and EG I) is 25:75. L?X (0.6 mg/g glucan) is crucial to obtaining monomeric xylose (54% xylose yield), while LArb (0.6 mg/g glucan) and L?Gl (0.8 mg/g glucan) can both further increase xylose yield by an additional 20%. Compared with Accellerase 1000, a purified cocktail of cellulases supplemented with accessory hemicellulases will not only increase both glucose and xylose yields but will also decrease the total enzyme loading needed for equivalent yields. CONCLUSIONS:A diverse set of accessory hemicellulases was found necessary to enhance the synergistic action of cellulases hydrolysing AFEX pretreated corn stover. High glucose (around 80%) and xylose (around 70%) yields were achieved with a moderate enzyme loading (~20 mg protein/g glucan) using an in-house developed cocktail compared to commercial enzymes.

Project description:BackgroundMitigation 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.ResultsThis 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.ConclusionsThis work brings R. toruloides closer to being able to make industrially relevant quantities of terpene from lignocellulosic biomass.

Dataset Information

Engineering cellulases for conversion of lignocellulosic biomass.

Publications

Engineering cellulases for conversion of lignocellulosic biomass.

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