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Abstract Curdlan is a commercial polysaccharide made by fermentation of Agrobacterium sp. Its anticipated expansion to larger volume markets demands improvement in its production efficiency. Metabolic engineering for strain improvement has so far been limited due to the lack of genetic tools. This research aimed to identify strong promoters and to engineer a strain that converts cellobiose efficiently to curdlan. Three strong promoters were identified and were used to install an energy-efficient cellobiose phosphorolysis mechanism in a curdlan-producing strain. The engineered strains were shown with enhanced ability to utilize cellobiose, resulting in a 2.5-fold increase in titer. The availability of metabolically engineered strain capable of producing β-glucan from cellobiose paves the way for its production from cellulose. The identified native promoters from Agrobacterium open up opportunities for further metabolic engineering for improved production of curdlan and other products. The success shown here marks the first such metabolic engineering effort in this microbe.

Escherichia coli normally cannot utilize the β-glucoside sugar cellobiose as a carbon and energy source unless a stringent selection pressure for survival is present. The cellobiose-utilization phenotype can be conferred by mutations in the two cryptic operons, chb and asc. In this study, the cellobiose-utilization phenotype was conferred to E. coli by replacing the cryptic promoters of these endogenous operons with a constitutive promoter. Evolutionary adaptation of the engineered strain CP12CHBASC by repeated subculture in cellobiose-containing minimal medium led to an increase in the rate of cellobiose uptake and cell growth on cellobiose. An efficient cellobiose-metabolizing E. coli strain would be of great importance over glucose-metabolizing E. coli for a simultaneous saccharification and fermentation process, as the cost of the process would be reduced by eliminating one of the three enzymes needed to hydrolyze cellulose into simple sugars. [PUBLICATION ABSTRACT]

Cellobiose lipids (CBLs) are glycolipid biosurfactants that are widely applicable across diverse industries.CBLs have remarkable gelling, surface-active, and antifungal properties, making them ideal compounds for developing novel biotechnologies (e.g., biomaterials, detergents, and emulsifiers) with commercial potential.Various production methodologies have been developed via an interplay between producing organisms, media, and production approaches, each with its unique advantages and limitations toward scalability.Significant exploration is still required regarding downstream processing of CBLs because an optimised and scalable downstream technique is needed to complete the biotechnological cycle of CBL production.Finally, the industrialisation of CBL production requires the implementation of techno-economic and life-cycle assessments, which are currently understudied. Cellobiose lipids (CBLs) are glycolipid biosurfactants that have garnered attention due to their potential applications in diverse industries. Here, we review the current state of CBL research, from production and purification, to the potential applications of CBLs. We elucidate CBL functionality and consider some commercial applications, as well as how operating conditions (e.g., media and organism, or production approaches) impact productivity. Methodologies based on enzymatic synthesis or postproduction chemical modification of CBL variants are also presented. Given the importance of purity in current CBL applications, we discuss CBL separation and purification techniques. Finally, we highlight the importance of techno-economic and life-cycle assessments for the industrialisation of CBLs, while suggesting potential future routes for investigation. Cellobiose lipids (CBLs) are glycolipid biosurfactants that have garnered attention due to their potential applications in diverse industries. Here, we review the current state of CBL research, from production and purification, to the potential applications of CBLs. We elucidate CBL functionality and consider some commercial applications, as well as how operating conditions (e.g., media and organism, or production approaches) impact productivity. Methodologies based on enzymatic synthesis or postproduction chemical modification of CBL variants are also presented. Given the importance of purity in current CBL applications, we discuss CBL separation and purification techniques. Finally, we highlight the importance of techno-economic and life-cycle assessments for the industrialisation of CBLs, while suggesting potential future routes for investigation.

Plant cell-wall polysaccharides constitute the most abundant but recalcitrant organic carbon source in nature. Microbes residing in the digestive tract of herbivorous bilaterians are particularly efficient at depolymerizing polysaccharides into fermentable sugars and play a significant support role towards their host’s lifestyle. Here, we combine large-scale functional screening of fosmid libraries, shotgun sequencing, and biochemical assays to interrogate the gut microbiota of the wood-feeding “higher” termite Globitermes brachycerastes . A number of putative polysaccharide utilization gene clusters were identified with multiple fibrolytic genes. Our large-scale functional screening of 50,000 fosmid clones resulted in 464 clones demonstrating plant polysaccharide-degrading activities, including 267 endoglucanase-, 24 exoglucanase-, 72 β-glucosidase-, and 101 endoxylanase-positive clones. We sequenced 173 functionally active clones and identified ~219 genes encoding putative carbohydrate-active enzymes (CAZymes) targeting cellulose, hemicellulose and pectin. Further analyses revealed that 68 of 154 contigs encode one or more CAZyme, which includes 35 examples of putative saccharolytic operons, suggesting that clustering of CAZymes is common in termite gut microbial inhabitants. Biochemical characterization of a representative xylanase cluster demonstrated that constituent enzymes exhibited complementary physicochemical properties and saccharolytic capabilities. Furthermore, diverse cellobiose-metabolizing enzymes include β-glucosidases, cellobiose phosphorylases, and phopho-6-β-glucosidases were identified and functionally verified, indicating that the termite gut micro-ecosystem utilizes diverse metabolic pathways to interconnect hydrolysis and central metabolism. Collectively, these results provide an in-depth view of the adaptation and digestive strategies employed by gut microbiota within this tiny-yet-efficient host-associated ecosystem.

Neurospora crassa colonizes burnt grasslands in the wild and metabolizes both cellulose and hemicellulose from plant cell walls. When switched from a favored carbon source such as sucrose to cellulose, N. crassa dramatically upregulates expression and secretion of a wide variety of genes encoding lignocellulolytic enzymes. However, the means by which N. crassa and other filamentous fungi sense the presence of cellulose in the environment remains unclear. Here, we show that an N. crassa mutant carrying deletions of two genes encoding extracellular β-glucosidase enzymes and one intracellular β-glucosidase lacks β-glucosidase activity, but efficiently induces cellulase gene expression in the presence of cellobiose, cellotriose, or cellotetraose as a sole carbon source. These data indicate that cellobiose, or a modified version of cellobiose, functions as an inducer of lignocellulolytic gene expression in N. crassa. Furthermore, the inclusion of a deletion of the catabolite repressor gene, cre-1, in the triple β-glucosidase mutant resulted in a strain that produces higher concentrations of secreted active cellulases on cellobiose. Thus, the ability to induce cellulase gene expression using a common and soluble carbon source simplifies enzyme production and characterization, which could be applied to other cellulolytic filamentous fungi.

The plant cell wall, often the site of initial encounters between plants and their microbial pathogens, is composed of a complex mixture of cellulose, hemicellulose, and pectin polysaccharides as well as proteins. The concept of damage-associated molecular patterns (DAMPs) was proposed to describe plant elicitors like oligogalacturonides (OGs), which can be derived by the breakdown of the pectin homogalacturon by pectinases. OGs act via many of the same signaling steps as pathogen- or microbe-associated molecular patterns (PAMPs) to elicit defenses and provide protection against pathogens. Given both the complexity of the plant cell wall and the fact that many pathogens secrete a wide range of cell wall-degrading enzymes, we reasoned that the breakdown products of other cell wall polymers may be similarly biologically active as elicitors and may help to reinforce the perception of danger by plant cells. Our results indicate that oligomers derived from cellulose are perceived as signal molecules in Arabidopsis (Arabidopsis thaliana), triggering a signaling cascade that shares some similarities to responses to well-known elicitors such as chitooligomers and OGs. However, in contrast to other known PAMPs/DAMPs, cellobiose stimulates neither detectable reactive oxygen species production nor callose deposition. Confirming our idea that both PAMPs and DAMPs are likely to cooccur at infection sites, cotreatments of cellobiose with flg22 or chitooligomers led to synergistic increases in gene expression. Thus, the perception of cellulose-derived oligomers may participate in cell wall integrity surveillance and represents an additional layer of signaling following plant cell wall breakdown during cell wall remodeling or pathogen attack.

Rational engineering of filamentous fungi for improved cellulase production is hampered by our incomplete knowledge of transcriptional regulatory networks. We therefore used the model filamentous fungus Neurospora crassa to search for uncharacterized transcription factors associated with cellulose deconstruction. A screen of a N. crassa transcription factor deletion collection identified two uncharacterized zinc binuclear cluster transcription factors (clr-1 and clr-2) that were required for growth and enzymatic activity on cellulose, but were not required for growth or hemicellulase activity on xylan. Transcriptional profiling with next-generation sequencing methods refined our understanding of the N. crassa transcriptional response to cellulose and demonstrated that clr-1 and clr-2 were required for the bulk of that response, including induction of all major cellulase and some major hemicellulase genes. Functional CLR-1 was necessary for expression of clr-2 and efficient cellobiose utilization. Phylogenetic analyses showed that CLR-1 and CLR-2 are conserved in the genomes of most filamentous ascomycete fungi capable of degrading cellulose. In Aspergillus nidulans, a strain carrying a deletion of the clr-2 homolog (clrB) failed to induce cellulase gene expression and lacked cellulolytic activity on Avicel. Further manipulation of this control system in industrial production strains may significantly improve yields of cellulases for cellulosic biofuel production.

A deeper mechanistic understanding of the saccharification of cellulosic biomass could enhance the efficiency of biofuels development. We report here the real-time visualization of crystalline cellulose degradation by individual cellulase enzymes through use of an advanced version of high-speed atomic force microscopy. Trichoderma reesei cellobiohydrolase I (TrCel7A) molecules were observed to slide unidirectionally along the crystalline cellulose surface but at one point exhibited collective halting analogous to a traffic jam. Changing the crystalline polymorphic form of cellulose by means of an ammonia treatment increased the apparent number of accessible lanes on the crystalline surface and consequently the number of moving cellulase molecules. Treatment of this bulky crystalline cellulose simultaneously or separately with T. reesei cellobiohydrolase II (TrCel6A) resulted in a remarkable increase in the proportion of mobile enzyme molecules on the surface. Cellulose was completely degraded by the synergistic action between the two enzymes.

Caldimonas thermodepolymerans DSM 15344, a moderately thermophilic bacterium, has emerged as a promising candidate for next-generation industrial biotechnology (NGIB) due to its ability to utilize lignocellulose-derived sugars for polyhydroxyalkanoate (PHA) production. This study assesses its metabolic potential by evaluating the utilization of various plant-derived sugars and their mixtures, with a focus on xylose, glucose, and cellobiose. The results indicate that C. thermodepolymerans exhibits a strong preference for xylose (3.97 g/L PHB) over glucose (2.28 g/L PHB) but demonstrates even greater efficiency in metabolizing cellobiose (4.96 g/L PHB). However, extracellular hydrolysis of cellobiose leads to glucose accumulation, which constrains overall productivity. Our findings suggest that the primary limitation in glucose metabolism is inefficient glucose transport rather than intracellular catabolism. To address this bottleneck, the glf glucose facilitator gene from the mesophilic bacterium Zymomonas mobilis was introduced into C. thermodepolymerans , enhancing its glucose utilization capacity. The engineered strain (Cald_GLF3) exhibited significantly improved PHA productivity, particularly when cultivated on sugar mixtures containing cellobiose. Despite being grown at suboptimal temperatures due to the thermal instability of Glf from Z. mobilis , Cald_GLF3 outperformed the wild-type strain, achieving notably high PHA yields when cultivated with cellobiose as the sole carbon source (9.26 g/L PHB). These findings highlight the critical role of glucose transport in the metabolism of C. thermodepolymerans and suggest that targeted engineering can further enhance its biotechnological potential. This study establishes C. thermodepolymerans as a promising thermophilic chassis for PHA production from lignocellulosic sugars, contributing to sustainable biopolymer synthesis. Key points C. thermodepolymerans DSM 15344 produces PHA from lignocellulose-derived sugars Xylose and cellobiose are preferred substrates, while glucose is poorly utilized Deficient glucose transport in DSM 15344 restored by Zymomonas mobilis glf gene

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. Strains of xylose-fermenting yeast Ogataea polymorpha have been constructed capable of efficient cellobiose utilization and fermentation at elevated temperature (45°C).