Recent Advances in Food Waste Transformations into Essential Bioplastic Materials.

Lignocellulose is a major biopolymer in plant biomass with a complex structure and composition. It consists of a significant amount of high molecular aromatic compounds, particularly vanillin, syringeal, ferulic acid, and muconic acid, that could be converted into intracellular metabolites such as polyhydroxyalkanoates (PHA) and hydroxybutyrate (PHB), a key component of bioplastic production. Several pre-treatment methods were utilized to release monosaccharides, which are the precursors of the relevant pathway. The consolidated bioprocessing of lignocellulose-capable microbes for biomass depolymerization was discussed in this study. Carbon can be stored in a variety of forms, including PHAs, PHBs, wax esters, and triacylglycerides. From a biotechnology standpoint, these compounds are quite adaptable due to their precursors' utilization of hydrogen energy. This study lays the groundwork for the idea of lignocellulose valorization into value-added products through several significant dominant pathways.

Construction of an amylolytic Saccharomyces cerevisiae strain with high copies of α-amylase and glucoamylase genes integration for bioethanol production from sweet potato residue.

Sweet potato residue (SPR) is the by-product of starch extraction from fresh sweet potatoes and is rich in carbohydrates, making it a suitable substrate for bioethanol production. An amylolytic industrial yeast strain with co-expressing α-amylase and glucoamylase genes would combine enzyme production, SPR hydrolysis, and glucose fermentation into a one-step process. This consolidated bioprocessing (CBP) shows great application potential in the economic production of bioethanol. In this study, a convenient heterologous gene integration method was developed. Eight copies of a Talaromyces emersonii α-amylase expression cassette and eight copies of a Saccharomycopsis fibuligera glucoamylase expression cassette were integrated into the genome of industrial diploid Saccharomyces cerevisiae strain 1974. The resulting recombinant strains exhibited clear transparent zones in the iodine starch plates, and SDS-PAGE analysis indicated that α-amylase and glucoamylase were secreted into the culture medium. Enzymatic activity analysis demonstrated that the optimal temperature for α-amylase and glucoamylase was 60-70°C, and the pH optima for α-amylase and glucoamylase was 4.0 and 5.0, respectively. Initially, soluble corn starch with a concentration of 100 g/L was initially used to evaluate the ethanol production capability of recombinant amylolytic S. cerevisiae strains. After 7 days of CBP fermentation, the α-amylase-expressing strain 1974-temA and the glucoamylase-expressing strain 1974-GA produced 33.03 and 28.37 g/L ethanol, respectively. However, the 1974-GA-temA strain, which expressed α-amylase and glucoamylase, produced 42.22 g/L ethanol, corresponding to 70.59% of the theoretical yield. Subsequently, fermentation was conducted using the amylolytic strain 1974-GA-temA without the addition of exogenous α-amylase and glucoamylase, which resulted in the production of 32.15 g/L ethanol with an ethanol yield of 0.30 g/g. The addition of 20% glucoamylase (60 U/g SPR) increased ethanol concentration to 50.55 g/L, corresponding to a theoretical yield of 93.23%, which was comparable to the ethanol production observed with the addition of 100% α-amylase and glucoamylase. The recombinant amylolytic strains constructed in this study will facilitate the advancement of CBP fermentation of SPR for the production of bioethanol.

Consolidated bioprocessing of lignocellulosic wastes in Northwest China for D-glucaric acid production by an artificial microbial consortium.

D-glucaric acid is a platform chemical of great importance and the consolidated bioprocessing (CBP) of lignocellulose by the microbial consortium of Trichoderma reesei C10 and Saccharomyces cerevisiae LGA-1C3S2 features prospects in biomanufacturing it. Here we compared some representative lignocelluloses in Northwest China including corn stover, wheat straw and switchgrass, and the leading pretreatments including steam explosion, subcritical water pretreatment, sodium hydroxide pretreatment, aqueous ammonia pretreatment, lime pretreatment, and diluted sulfuric acid pretreatment. It was found that sodium hydroxide pretreated switchgrass (SHPSG) was the best substrate for D-glucaric acid production, resulting in the highest D-glucaric acid titers, 11.69 ± 0.73 g/L in shake flask and 15.71 ± 0.80 g/L in 10L airlift fermenter, respectively. To the best of our knowledge, this is the highest D-glucaric acid production titer from lignocellulosic biomass. This work offers a paradigm of producing low-cost D-glucaric acid for low-carbon polyethylene 2,5-furandicarboxylate (PEF) and a reference on developing biorefinery in Northwest China.

Industrial biotechnology goes thermophilic: Thermoanaerobes as promising hosts in the circular carbon economy.

Transitioning away from fossil feedstocks is imperative to mitigate climate change, and necessitates the utilization of renewable, alternative carbon and energy sources to foster a circular carbon economy. In this context, lignocellulosic biomass and one-carbon compounds emerge as promising feedstocks that could be renewably upgraded by thermophilic anaerobes (thermoanaerobes) via gas fermentation or consolidated bioprocessing to value-added products. In this review, the potential of thermoanaerobes for cost-efficient, effective and sustainable bioproduction is discussed. Metabolic and bioprocess engineering approaches are reviewed to draw a comprehensive picture of current developments and future perspectives for the conversion of renewable feedstocks to chemicals and fuels of interest. Selected bioprocessing scenarios are outlined, offering practical insights into the applicability of thermoanaerobes at a large scale. Collectively, the potential advantages of thermoanaerobes regarding process economics could facilitate an easier transition towards sustainable bioprocesses with renewable feedstocks.

Characterization of sugarcane bagasse solubilization and utilization by thermophilic cellulolytic and saccharolytic bacteria at increasing solid loadings.

In Brazil the main feedstock used for ethanol production is sugarcane juice, resulting in large amounts of bagasse. Bagasse has high potential for cellulosic ethanol production, and consolidated bioprocessing (CBP) has potential for lowering costs. However, economic feasibility requires bioprocessing at high solids loadings, entailing engineering and biological challenges. This study aims to document and characterize carbohydrate solubilization and utilization by defined cocultures of Clostridium thermocellum and Thermoanaerobacterium thermosaccharolyticum at increasing loadings of sugarcane bagasse. Results show that fractional carbohydrate solubilization decreases as solids loading increases from 10 g/L to 80 g/L. Cocultures enhance solubilization and carbohydrate utilization compared to monocultures, irrespective of initial solids loading. Rinsing bagasse before fermentation slightly decreases solubilization. Experiments studying inhibitory effects using spent media and dilution of broth show that negative effects are temporary or reversible. These findings highlight the potential of converting sugarcane bagasse via CBP, pointing out performance limitations that must be addressed.

Adaptive evolution of Clostridium thermocellum ATCC 27405 on alternate carbon sources leads to altered fermentation profiles.

Consolidated bioprocessing candidate, Clostridium thermocellum, is a cellulose hydrolysis specialist, with the ability to ferment the released sugars to produce bioethanol. C. thermocellum is generally studied with model substrates Avicel and cellobiose to understand the metabolic pathway leading to ethanol. In the present study, adaptive laboratory evolution, allowing C. thermocellum DSM 1237 to adapt to growth on glucose, fructose, and sorbitol, with the prospect that some strains will adapt their metabolism to yield more ethanol. Adaptive growth on glucose and sorbitol resulted in an approximately 1 mM and 2 mM increase in ethanol yield per millimolar glucose equivalent, respectively, accompanied by a shift in the production of the other expected fermentation end products. The increase in ethanol yield observed for sorbitol adapted cells was due to the carbon source being more reduced compared to cellobiose. Glucose and cellobiose have similar oxidation states thus the increase in ethanol yield is due to the rerouting of electrons from other reduced metabolic products excluding H2 which did not decrease in yield. There was no increase in ethanol yield observed for fructose adapted cells, but there was an unanticipated elimination of formate production, also observed in sorbitol adapted cells suggesting that fructose has regulatory implications on formate production either at the transcription or protein level.

Engineering the cellulolytic bacterium, Clostridium thermocellum, to co-utilize hemicellulose.

Consolidated bioprocessing (CBP) of lignocellulosic biomass holds promise to realize economic production of second-generation biofuels/chemicals, and Clostridium thermocellum is a leading candidate for CBP due to it being one of the fastest degraders of crystalline cellulose and lignocellulosic biomass. However, CBP by C. thermocellum is approached with co-cultures, because C. thermocellum does not utilize hemicellulose. When compared with a single-species fermentation, the co-culture system introduces unnecessary process complexity that may compromise process robustness. In this study, we engineered C. thermocellum to co-utilize hemicellulose without the need for co-culture. By evolving our previously engineered xylose-utilizing strain in xylose, an evolved clonal isolate (KJC19-9) was obtained and showed improved specific growth rate on xylose by ∼3-fold and displayed comparable growth to a minimally engineered strain grown on the bacteria's naturally preferred substrate, cellobiose. To enable full xylan deconstruction to xylose, we recombinantly expressed three different ß-xylosidase enzymes originating from Thermoanaerobacterium saccharolyticum into KJC19-9 and demonstrated growth on xylan with one of the enzymes. This recombinant strain was capable of co-utilizing cellulose and xylan simultaneously, and we integrated the ß-xylosidase gene into the KJC19-9 genome, creating the KJCBXint strain. The strain, KJC19-9, consumed monomeric xylose but accumulated xylobiose when grown on pretreated corn stover, whereas the final KJCBXint strain showed significantly greater deconstruction of xylan and xylobiose. This is the first reported C. thermocellum strain capable of degrading and assimilating hemicellulose polysaccharide while retaining its cellulolytic capabilities, unlocking significant potential for CBP in advancing the bioeconomy.

Improved consolidated bioprocessing for itaconic acid production by simultaneous optimization of cellulase and metabolic pathway of Neurospora crassa.

Lignocellulose was directly used in itaconic acid production by a model filamentous fungus Neurospora crassa. The promoters of two clock control genes and cellobiohydrolase 1 gene were selected for heterologous genes expression by evaluating different types of promoters. The effect of overexpression of different cellulase was compared, and it was found that expression of cellobiohydrolase 2 from Trichoderma reesei increased the filter paper activity by 2 times, the cellobiohydrolase activity by 4.5 times, and that the itaconic acid titer was also significantly improved. A bidirectional cis-aconitic acid accumulation strategy was established by constructing the reverse glyoxylate shunt and expressing the transporter MTTA, which increased itaconic acid production to 637.2 mg/L. The simultaneous optimization of cellulase and metabolic pathway was more conducive to the improvement of cellulase activity than that of cellulase alone, so as to further increase itaconic acid production. Finally, through the combination of fermentation by optimized strains and medium optimization, the titers of itaconic acid using Avicel and corn stover as substrate were 1165.1 mg/L and 871.3 mg/L, respectively. The results prove the potential of the consolidated bioprocessing that directly converts lignocellulose to itaconic acid by a model cellulase synthesizing strain.

Engineering Saccharomyces cerevisiae for targeted hydrolysis and fermentation of glucuronoxylan through CRISPR/Cas9 genome editing.

BACKGROUND: The abundance of glucuronoxylan (GX) in agricultural and forestry residual side streams positions it as a promising feedstock for microbial conversion into valuable compounds. By engineering strains of the widely employed cell factory Saccharomyces cerevisiae with the ability to directly hydrolyze and ferment GX polymers, we can avoid the need for harsh chemical pretreatments and costly enzymatic hydrolysis steps prior to fermentation. However, for an economically viable bioproduction process, the engineered strains must efficiently express and secrete enzymes that act in synergy to hydrolyze the targeted polymers. RESULTS: The aim of this study was to equip the xylose-fermenting S. cerevisiae strain CEN.PK XXX with xylanolytic enzymes targeting beechwood GX. Using a targeted enzyme approach, we matched hydrolytic enzyme activities to the chemical features of the GX substrate and determined that besides endo-1,4-ß-xylanase and ß-xylosidase activities, α-methyl-glucuronidase activity was of great importance for GX hydrolysis and yeast growth. We also created a library of strains expressing different combinations of enzymes, and screened for yeast strains that could express and secrete the enzymes and metabolize the GX hydrolysis products efficiently. While strains engineered with BmXyn11A xylanase and XylA ß-xylosidase could grow relatively well in beechwood GX, strains further engineered with Agu115 α-methyl-glucuronidase did not display an additional growth benefit, likely due to inefficient expression and secretion of this enzyme. Co-cultures of strains expressing complementary enzymes as well as external enzyme supplementation boosted yeast growth and ethanol fermentation of GX, and ethanol titers reached a maximum of 1.33 g L- 1 after 48 h under oxygen limited condition in bioreactor fermentations. CONCLUSION: This work underscored the importance of identifying an optimal enzyme combination for successful engineering of S. cerevisiae strains that can hydrolyze and assimilate GX. The enzymes must exhibit high and balanced activities, be compatible with the yeast's expression and secretion system, and the nature of the hydrolysis products must be such that they can be taken up and metabolized by the yeast. The engineered strains, particularly when co-cultivated, display robust growth and fermentation of GX, and represent a significant step forward towards a sustainable and cost-effective bioprocessing of GX-rich biomass. They also provide valuable insights for future strain and process development targets.

Metabolic engineering of Caldicellulosiruptor bescii for hydrogen production.

Hydrogen is an alternative fuel for transportation vehicles because it is clean, sustainable, and highly flammable. However, the production of hydrogen from lignocellulosic biomass by microorganisms presents challenges. This microbial process involves multiple complex steps, including thermal, chemical, and mechanical treatment of biomass to remove hemicellulose and lignin, as well as enzymatic hydrolysis to solubilize the plant cell walls. These steps not only incur costs but also result in the production of toxic hydrolysates, which inhibit microbial growth. A hyper-thermophilic bacterium of Caldicellulosiruptor bescii can produce hydrogen by decomposing and fermenting plant biomass without the need for conventional pretreatment. It is considered as a consolidated bioprocessing (CBP) microorganism. This review summarizes the basic scientific knowledge and hydrogen-producing capacity of C. bescii. Its genetic system and metabolic engineering strategies to improve hydrogen production are also discussed. KEY POINTS: • Hydrogen is an alternative and eco-friendly fuel. • Caldicellulosiruptor bescii produces hydrogen with a high yield in nature. • Metabolic engineering can make C. bescii to improve hydrogen production.

Enhanced ethanol production from paper sludge waste under high-solids conditions with industrial and cellulase-producing strains of Saccharomyces cerevisiae.

Reported ethanol titres from hydrolysis-fermentation of the degraded fibres in paper sludge (PS) waste, generally obtained under fed-batch submerged conditions, can be improved through fermentation processes at high solids loadings, as demonstrated in the present study with two industrial PS wastes at enzyme dosages appropriate for solids loadings up to 40% (w/w). The industrial yeast,Saccharomyces cerevisiaestrain Ethanol Red®, was compared to two genetically engineeredS. cerevisiaestrains, namely Cellusec® 1.0 and Cellusec® 2.0, capable of xylose utilisation, and xylose utilisation and cellulase production, respectively. High-solids batch fermentations were conducted in 3 L horizontal rotating reactors and ethanol titres of 100.8 and 73.3 g/L were obtained for virgin pulp and corrugated recycle PS, respectively, at 40% (w/w) solids loading using Ethanol Red®. Xylose utilisation by Cellusec® 1.0 improved ethanol titres by up to 10.3%, while exogenous cellulolytic enzyme requirements were reduced by up to 50% using cellulase-producing Cellusec® 2.0.

Unveiling malic acid biorefinery: Comprehensive insights into feedstocks, microbial strains, and metabolic pathways.

The over-reliance on fossil fuels and resultant environmental issues necessitate sustainable alternatives. Microbial fermentation of biomass for malic acid production offers a viable, eco-friendly solution, enhancing resource efficiency and minimizing ecological damage. This review covers three core aspects of malic acid biorefining: feedstocks, microbial strains, and metabolic pathways. It emphasizes the significance of utilizing biomass sugars, including the co-fermentation of different sugar types to improve feedstock efficiency. The review discusses microbial strains for malic acid fermentation, addressing challenges related to by-products from biomass breakdown and strategies for overcoming them. It delves into the crucial pathways and enzymes for malic acid production, outlining methods to optimize its metabolism, focusing on enzyme regulation, energy balance, and yield enhancement. These insights contribute to advancing the field of consolidated bioprocessing in malic acid biorefining.

Effective application of immobilized second generation industrial Saccharomyces cerevisiae strain on consolidated bioprocessing.

Integrated bioprocessing strategies can facilitate ethanol production from both cellulose and hemicellulose fractions of lignocellulosic biomass. Consolidated bioprocessing (CBP) is an approach that combines enzyme production, biomass hydrolysis and sugar fermentation in a single step. However, technologies that propose the use of microorganisms together with solid biomass present the difficulty of the recovery and reuse of the biocatalyst, which can be overcome by cell immobilization. In this regard, this work applied immobilized cells of AC14 yeast, a recombinant yeast that secretes 7 hydrolytic enzymes, in the CBP process in a successful proof-of-concept for the enzyme access to the substrate polymers. The most appropriate cell load for CBP under the conditions studied with immobilized cells was selected among three optical densities (OD) 10, 55 and 100. These experiments were performed with free cells to ensure that the results were not biased by mass limitations effects. OD 10 achieved 100% of the sugar consumption and the higher specific production of enzymes, being selected for further studies. Diffusional effects were observed with immobilized cells under static conditions. However, mass transfer limitations were mitigated under agitation, with an 18.5% increase in substrate consumption rate (from 2.7 to 3.5 g/L/h), reaching the same substrate uptake rates as free cells. In addition, immobilized cells achieved 100% hydrolysis and consumption of all substrates offered within only 12 h. Overall, this is the first report of a successful application of immobilized yeast cells in CBP processes for bioethanol production, a promising technology that can be extended to other biorefinery bioproducts.

Engineering natural isolates of Saccharomyces cerevisiae for consolidated bioprocessing of cellulosic feedstocks.

Saccharomyces cerevisiae has gained much attention as a potential host for cellulosic bioethanol production using consolidated bioprocessing (CBP) methodologies, due to its high-ethanol-producing titres, heterologous protein production capabilities, and tolerance to various industry-relevant stresses. Since the secretion levels of heterologous proteins are generally low in domesticated strains of S. cerevisiae, natural isolates may offer a more diverse genetic background for improved heterologous protein secretion, while also displaying greater robustness to process stresses. In this study, the potential of natural and industrial S. cerevisiae strains to secrete a core set of cellulases (CBH1, CBH2, EG2, and BGL1), encoded by genes integrated using CRISPR/Cas9 tools, was evaluated. High levels of heterologous protein production were associated with a reduced maximal growth rate and with slight changes in overall strain robustness, compared to the parental strains. The natural isolate derivatives YI13_BECC and YI59_BECC displayed superior secretion capacity for the heterologous cellulases at high incubation temperature and in the presence of acetic acid, respectively, compared to the reference industrial strain MH1000_BECC. These strains also exhibited multi-tolerance to several fermentation-associated and secretion stresses. Cultivation of the strains on crystalline cellulose in oxygen-limited conditions yielded ethanol concentrations in the range of 4-4.5 g/L, representing 35-40% of the theoretical maximum ethanol yield after 120 h, without the addition of exogenous enzymes. This study therefore highlights the potential of these natural isolates to be used as chassis organisms in CBP bioethanol production. KEY POINTS: • Process-related fermentation stresses influence heterologous protein production. • Transformants produced up to 4.5 g/L ethanol, ~ 40% of the theoretical yield in CBP. • CRISPR/Cas9 was feasible for integrating genes in natural S. cerevisiae isolates.

Additional glucoamylase genes increase ethanol productivity on rice and potato waste streams by a recombinant amylolytic yeast.

The implementation of consolidated bioprocessing for converting starch to ethanol relies on a robust yeast that produces enough amylases for rapid starch hydrolysis. Furthermore, using low-cost substrates will assist with competitive ethanol prices and support a bioeconomy, especially in developing countries. This paper addresses both challenges with the expression of additional glucoamylase gene copies in an efficient amylolytic strain (Saccharomyces cerevisiae ER T12) derived from the industrial yeast, Ethanol Red™. Recombinant ER T12 was used as a host to increase ethanol productivity during raw starch fermentation; the ER T12.7 variant, selected from various transformants, displayed enhanced raw starch conversion and a 36% higher ethanol concentration than the parental strain after 120 h. Unripe rice, rice bran, potato waste and potato peels were evaluated as alternative starchy substrates to test ER T12.7's fermenting ability. ER T12.7 produced high ethanol yields at significantly improved ethanol productivity, key criteria for its industrial application.

Consolidated bioprocessing for bioethanol production by metabolically engineered cellulolytic fungus Myceliophthora thermophila.

Using cellulosic ethanol as fuel is one way to help achieve the world's decarbonization goals. However, the economics of the present technology are unfavorable, especially the cost of cellulose degradation. Here, we reprogram the thermophilic cellulosic fungus Myceliophthora thermophila to directly ferment cellulose into ethanol by mimicking the aerobic ethanol fermentation of yeast (the Crabtree effect), including optimizing the synthetic pathway, enhancing the glycolytic rate, inhibiting mitochondrial NADH shuttles, and knocking out ethanol consumption pathway. The final engineered strain produced 52.8 g/L ethanol directly from cellulose, and 39.8 g/L from corncob, without the need for any added cellulase, while the starting strain produced almost no ethanol. We also demonstrate that as the ethanol fermentation by engineered M. thermophila increases, the composition and expression of cellulases that facilitate the degradation of cellulose, especially cellobiohydrolases, changes. The simplified production process and significantly increased ethanol yield indicate that the fungal consolidated bioprocessing technology that we develop here (one-step, one-strain ethanol production) is promising for fueling sustainable carbon-neutral biomanufacturing in the future.

Rewiring metabolism of Clostridium thermocellum for consolidated bioprocessing of lignocellulosic biomass poplar to produce short-chain esters.

Consolidated bioprocessing (CBP) of lignocellulosic biomass uses cellulolytic microorganisms to enable enzyme production, saccharification, and fermentation to produce biofuels, biochemicals, and biomaterials in a single step. However, understanding and redirecting metabolisms of these microorganisms compatible with CBP are limited. Here, a cellulolytic thermophile Clostridium thermocellum was engineered and demonstrated to be compatible with CBP integrated with a Co-solvent Enhanced Lignocellulosic Fractionation (CELF) pretreatment for conversion of hardwood poplar into short-chain esters with industrial use as solvents, flavors, fragrances, and biofuels. The recombinant C. thermocellum engineered with deletion of carbohydrate esterases and stable overexpression of alcohol acetyltransferases improved ester production without compromised deacetylation activities. These esterases were discovered to exhibit promiscuous thioesterase activities and their deletion enhanced ester production by rerouting the electron and carbon metabolism. Ester production was further improved up to 80-fold and ester composition could be modulated by deleting lactate biosynthesis and using poplar with different pretreatment severity.

Effect of itaconic acid production on Neurospora crassa in consolidated bioprocessing of cellulose.

A system for itaconic acid synthesis from cellulose by Neurospora crassa was established, resulting in the highest yield of itaconic acid was 354.08 + 35.99 mg/L. Meanwhile, cellulase activity increased significantly, without any strain modifications for improved cellulase production. Multi-omics analyses showed that itaconic acid synthesis reduced energy production, leading to decreases in trehalose, cell wall, fatty acids synthesis and downregulations in MAPK signaling pathway, cell cycle and meiosis. More importantly, the low-energy environment enhanced the energy-efficient cellobionic acid/gluconic acid pathway, and the cellulase composition also changed significantly, manifested as the up-regulation of LPMOs and the down-regulation of ß-glucosidases. Enhancing LPMOs-cellobionic acid/gluconic acid system has the potential to reduce energy consumption of the consolidated bioprocessing. These findings offer an overview of resource allocations by N. crassa in response to itaconic acid synthesis and highlight a series of intriguing connections between itaconic acid synthesis and cellulase synthesis in consolidated bioprocessing.

Regulation of lignocellulose degradation in microorganisms.

Microbial strategies for biomass deconstruction involve an incredible repertoire of enzymatic, structural, and regulatory proteins. From carbohydrate active enzymes to cellulosomes, bacteria, yeast, and filamentous fungi adapt their functional machinery to grow from alternative carbon sources such as lignocellulose and survive starvation. In that context, microbes must be able to sense, bind, degrade, and utilize lignin, cellulose, and hemicelluloses. Nature has developed specialized protein modules, RNA structures, and regulatory systems operating at a genomic, transcription, and translation level. This review briefly summarizes the main regulatory pathways involved in lignocellulose microbial degradation, including carbon catabolite repression; anti-sigma factors; regulatory RNA elements such as small RNAs, antisense RNA, RNA-binding proteins, and selective RNA processing and stabilization; and transcriptional regulators and unfolded protein response. Interplay with global regulators controlling pH response and nitrogen utilization is also revised.

Supplementation of recombinant cellulases with LPMOs and CDHs improves consolidated bioprocessing of cellulose.

The increased demand for energy has sparked a global search for renewable energy sources that could partly replace fossil fuel resources and help mitigate climate change. Cellulosic biomass is an ideal feedstock for renewable bioethanol production, but the process is not currently economically feasible due to the high cost of pretreatment and enzyme cocktails to release fermentable sugars. Lytic polysaccharide monooxygenases (LPMOs) and cellobiose dehydrogenases (CDHs) are auxiliary enzymes that can enhance cellulose hydrolysis. In this study, four LPMO and two CDH genes were subcloned and expressed in the Saccharomyces cerevisiae Y294 laboratory strain. SDS-PAGE analysis confirmed the extracellular production of the LPMOs and CDHs in the laboratory S. cerevisiae Y294 strain. A rudimentary cellulase cocktail (cellobiohydrolase 1 and 2, endoglucanase and ß-glucosidase) was expressed in the commercial CelluX™ 4 strain and extracellular production of the individual cellulases was confirmed by SDS-PAGE analysis. In vitro cooperation of the CDHs and LPMOs with the rudimentary cellulases produced by strain CelluX™ 4[F4-1] was demonstrated on Whatman filter paper. The significant levels of soluble sugars released from this crystalline cellulose substrate indicated that these auxiliary enzymes could be important components of the CBP yeast cellulolytic system.