Background: Methanol, synthesized from CO₂, is a potentially sustainable one-carbon (C1) resource for biomanufacturing. The use of methanol as a feedstock to produce single cell protein (SCP) has been investigated for decades as an alternative to alleviate the high global demand for animal-derived proteins. The methylotrophic yeast Pichia pastoris is an ideal host for methanol-based SCP synthesis due to its natural methanol assimilation ability. However, improving methanol utilization, tolerance to higher temperature, and the protein content of P. pastoris are also current challenges, which are of great significance to the economical industrial application using methanol as a feedstock for SCP production. Results: In the present work, adaptive laboratory evolution (ALE) has been employed to overcome the low methanol utilization efficiency and intolerance to a higher temperature of 33 °C in P. pastoris, associated with reduced carbon loss due to the lessened detoxification of intracellular formaldehyde through the dissimilation pathway and cell wall rearrangement to temperature stress resistance following long-term evolution as revealed by transcriptomic and phenotypic analysis. By strengthening nitrogen metabolism and impairing cell wall synthesis, metabolic engineering further increased protein content. Finally, the engineered strain via multi-strategy produced high levels of SCP from methanol in a pilot-scale fed-batch culture at 33 °C with a biomass of 63.37 g DCW/L, methanol conversion rate of 0.43 g DCW/g, and protein content of 0.506 g/g DCW. SCP obtained from P. pastoris contains a higher percentage of protein compared to conventional foods like soy, fish, meat, whole milk, and is a source of essential amino acids, including methionine, lysine, and branched-chain amino acids (BCAAs: valine, isoleucine, leucine). Conclusions: This study clarified the unique mechanism of P. pastoris for efficient methanol utilization, higher temperature resistance, and high protein synthesis, providing a P. pastoris cell factory for SCP production with environmental, economic, and nutritional benefits.

Lignocellulosic biomass is an abundant renewable feedstock, but its complex structure of lignocellulose poses barriers to its enzymatic hydrolysis and fermentation. Fungi possess diverse lignocellulolytic enzyme systems that synergistically deconstruct lignocellulose into soluble sugars for fermentation. This review elucidates recent advances in understanding the molecular mechanisms underpinning fungal degradation of lignocellulose. We analyze major enzyme classes tailored by fungi to depolymerize cellulose, hemicellulose, and lignin. Highlighted are the concerted actions and intimate partnerships between these biomass‐degrading enzymes. Current challenges impeding large‐scale implementation of enzymatic hydrolysis are discussed, along with emerging biotechnological opportunities. Advanced pretreatments, high‐throughput enzyme engineering platforms, and machine learning or artificial intelligence‐guided lignocellulolytic enzyme cocktail optimization represent promising ways to improve hydrolytic efficiencies. Elucidating the coordinated interplay and regulation of fungal lignocellulolytic machinery can facilitate optimization of fungal biotechnology platforms. Harnessing the efficiency of fungal biomass deconstruction promises to enhance the development of biorefinery processes for sustainable bioenergy.

Four cellobiohydrolase I (CBHI) glycoforms, namely, CBHI-A, CBHI-B, CBHI-C, and CBHI-D, were purified from the cultured broth of Penicillium decumbens JU-A10. All glycoforms had the same amino acid sequence but displayed different characteristics and biological functions. The effects of the N-glycans of the glycoforms on CBH activity were analyzed using mass spectrum data. Longer N-glycan chains at the Asn-137 of CBHI increased CBH activity. After the N-glycans were removed using site-directed mutagenesis and homologous expression in P. decumbens, the specific CBH activity of the recombinant CBHI without N-glycosylation increased by 65% compared with the wild-type CBHI with the highest specific activity. However, the activity was not stable. Only the N-glycosylation at Asn-137 can improve CBH activity by 40%. rCBHI with N-glycosylation only at Asn-470 exhibited no enzymatic activity. CBH activity was affected whether or not the protein was glycosylated, together with the N-glycosylation site and N-glycan structure. N-Glycosylation not only affects CBH activity but may also bring a new feature to a nonhydrolytic CBHI glycoform (CBHI-A). By supplementing CBHI-A to different commercial cellulase preparations, the glucose yield of lignocellulose hydrolysis increased by >20%. After treatment with a low dose (5 mg/g substrate) of CBHI-A at 50 °C for 7 days, the hydrogen-bond intensity and crystalline degree of cotton fibers decreased by 17 and 34%, respectively. These results may provide new guidelines for cellulase engineering.

Algal biomass, known as a potential feedstock for biofuel production, has cell wall structures that differ from terrestrial biomass. The existing methods for processing algae are limited to conventional pretreatments for terrestrial biomass. In this study, we investigated a novel hydroxyl radical-aided approach for pretreating different types of algal biomass. In this process, hydroxyl radicals formed by a Fenton system were employed in combination with heating to alter the crystalline structure and hydrogen bonds of cellulose in the algal biomass. FeSO4 and H2O2 at low concentrations were employed to initiate the formation of hydroxyl radicals. This method releases trapped polysaccharides in algal cell walls and converts them into fermentable sugars. The effects of temperature, time, and hydroxyl radical concentration were analyzed. The optimal pretreatment condition [100 °C, 30 min, and 5.3 mM H2O2 (determined FeSO4 concentration of 11.9 mM)] was identified using a central composite design. Complete (100 %) carbohydrate recovery was achieved with some algal biomass without formation of inhibitors such as hydroxymethylfurfural and furfural as by-products. Both microalgal and macroalgal biomasses showed higher enzymatic digestibility of cellulose conversion (>80 %) after the milder pretreatment condition. Hydroxyl radical-aided thermal pretreatment was used as a novel method to convert the carbohydrates in the algal cell wall into simple sugars. Overall, this method increased the amount of glucose released from the algal biomass. Overall, enhanced algal biomass digestibility was demonstrated with the proposed pretreatment process. The new pretreatment requires low concentration of chemical solvents and milder temperature conditions, which can prevent the toxic and corrosive effects that typically result from conventional pretreatments. Our data showed that the advantages of the new pretreatment include higher carbohydrate recovery, no inhibitor production, and lower energy consumption. The new pretreatment development mimicking natural system could be useful for biochemical conversion of algal biomass to fuels and chemicals.

A new β-glucosidase (Cel3B) was first isolated from cellulytic fungi, designated as PpCel3B. Although PpCel3B was classified to GH family 3 based on the homology sequence, PpCel3B had different biological functions in cellulose degradation and signaling molecules production. PpCel3B was constitutive and could form multiple soluble lignocellulose inducers for cellulase and hemicellulase synthesis via high tranglycosylation activity and new enzymatic activity. Moreover, PpCel3B showed apparent synergism with cellulases by removing several inhibitors. Supplementing low doses of PpCel3B (52 μg/g substrate) increased saccharification efficiency of cellulase produced by Trichoderma reesei and Penicillium piceum by 15% and 35%, respectively on delignified corn stover. PpCel3B had important application in boosting cellulase yield and efficiency.

Single-cell protein (SCP) production in the methylotrophic yeast Pichia pastoris has the potential to achieve a sustainable protein supply. However, improving the methanol fermentation efficiency and reducing carbon loss has been a long-standing challenge with far-reaching scientific and practical implications. Here, comparative transcriptomics revealed that PAS_0305, a gene directly associated with cell wall thickness under methanol stress, can be used as a target for unlocking cell wall sensors. Intracellular trehalose accumulation confirmed that cell wall sensors were activated after knocking out PAS_0305, which resulted in increased cell wall permeability. Genome-wide signal perturbations were transduced through the HOG module and the CWI pathway, which was confirmed to connected by Pbs2-Mkk. As a consequence of CWI pathway activation, ΔPAS_0305 elicited a rescue response of cell wall remodeling by increasing the β-1,3-glucan content and decreasing the chitin/mannose content. Remarkably, perturbations in global stress signals led to a fine-tuning of the metabolic network of ΔPAS_0305, resulting in a superior phenotype with highest crude protein and methanol conversion rate of 67.21% and 0.46 gDCW/g. Further genome-scale metabolic models were constructed to validate the experimental results, confirming that unlocking cell wall sensors resulted in maximized flux from methanol towards SCP and effectively addressing the issue of carbon loss in methanol fermentation. This work sheds new light on the potential of manipulating cellular signaling pathways to optimize metabolic networks and achieve exceptional phenotypic characteristics, providing new strategies for constructing versatile cell factories in P. pastoris.