Tri-valorization of methanol in a single bioreactor: co-production of enzyme, chemical, and single-cell protein using engineered Pichia pastoris (Komagataella phaffii)
The economic viability of methanol-based biomanufacturing, particularly with green methanol as feedstock, is often limited by the low value of single-product processes. Here, we developed an integrated co-production strategy in the methylotrophic yeast Pichia pastoris (Komagataella phaffii) for the simultaneous conversion of methanol into three products: the sweetener erythritol, the industrial biocatalyst β-mannanase, and single-cell protein (SCP) biomass. This new strategy explores the inherent spatial and functional separation between the ER-Golgi secretory pathway for enzyme production and the cytosolic pathway for chemical synthesis, thereby reducing interference between the two pathways. The engineered co-production strain achieved β-mannanase and erythritol titers comparable to those of the corresponding β-mannanase- and erythritol-producing reference strains in both shake-flask and fed-batch fermentor cultures. Furthermore, transcriptomic analysis revealed distinct regulatory responses related to enzyme production and erythritol synthesis, supporting the limited cross-pathway interference between the two pathways. In addition, we showed that ultrafiltration enabled efficient downstream separation of the small-molecule erythritol from the secreted β-mannanase, with recovery efficiencies exceeding 89%. These results demonstrate a feasible strategy for methanol valorization into multiple value-added products and expand the potential of methylotrophic yeasts for integrated biomanufacturing.
The high cost of commercial enzyme cocktails remains a major barrier for lignocellulosic (second-generation) bioethanol production. Simultaneous saccharification and fermentation (SSF) at elevated temperatures using enzyme-secreting yeast can reduce enzyme demand, but is constrained by the limited thermotolerance of industrial strains. In this study, thermotolerant isolates of an inhibitor-tolerant, xylose-utilizing, enzyme-secreting industrial Saccharomyces cerevisiae strain were generated using whole-genome transformation (WGT). Screening in mixed-sugar fermentations at 41 °C identified several improved isolates, of which one isolate, designated Cellusec®4.0, achieved an ethanol titer of 5.45%(v/v), representing an 86% increase compared to the parental strain. This was driven by near-complete utilization of glucose, xylose, and cellobiose. In SSF at 40 °C with sorghum pulp, Cellusec®4.0 reached 5.83%(v/v) ethanol, 24% higher than the parental strain. Fed-batch SSF of pretreated softwood demonstrated the benefit of elevated temperature, with Cellusec®4.0 achieving 4.36%(v/v) ethanol at 40 °C, 29% higher than at 35 °C. In addition, fed-batch SSF of alkali-pretreated sugarcane bagasse at 39 °C using an in-house produced enzyme cocktail resulted in ethanol titers of up to 8.0% (v/v) within 48 h, corresponding to an 83% yield. These results demonstrate that WGT is an effective strategy to introduce thermotolerance into industrial yeast while maintaining key traits. The improved thermotolerance of Cellusec® 4.0 enabled high-temperature SSF, thereby increasing ethanol titers. Combined with retained inhibitor tolerance, enzyme secretion, and mixed-sugar utilization, this supported efficient ethanol production across multiple lignocellulosic substrates under industrially relevant conditions.
Bart Thevelein, Mekonnen M Demeke, Stijn De Graeve et al.· Bioresource Technology· 0 citations
Developing sustainable single-cell protein (SCP) from non-food feedstocks offers a promising strategy to address the escalating global demand for sustainable nutrition. However, the lack of industrially robust platforms capable of cost-efficient multi-substrate assimilation remains a key bottleneck. Here, we discover a Cyberlindnera jadinii strain, CGMCC34730, which can efficiently utilize diverse non-food carbon sources (e.g., acetate, ethanol, and xylose) for SCP production. Metabolic analysis reveals the assimilation mechanisms for these substrates, notably identifying the reductive glycine pathway as central to formic acid (FA) utilization. Scaled-up production in a 5-L bioreactor demonstrates excellent performance, with ethanol- and acetate-driven cultures reaching protein contents of 68.60 and 67.34% and volumetric productivities of 8.91 and 7.90 t m–3 y–1, respectively. The resulting SCP surpasses soybean meal and approaches the quality of fish meal, exhibiting superior essential amino acid indices alongside elevated carbohydrate and B-vitamin contents. Furthermore, we validate a circular bioeconomy model by converting electrochemically synthesized 13C-labeled acetate into SCP. Techno-economic analysis confirms that acetate-based fermentation offers optimal cost-effectiveness for industrial deployment. In summary, this GRAS-certified platform establishes a highly efficient and economically viable route for the sustainable production of high-value SCP from non-food substrates.
Single-cell oils produced by oleaginous yeasts are frequently proposed as alternatives to plant and fossil-derived lipids because they can be synthesised in controlled bioreactors, can exploit diverse carbon streams and can be compositionally tailored through strain and process engineering. Yet the industrial sustainability of yeast oil is not determined by lipid content alone. It depends on the interaction among carbon-source quality, substrate conversion, lipid titre, yield and volumetric productivity, oxygen and nutrient demand, strain robustness, downstream recovery, product specification, co-product strategy and the assumptions used in techno-economic and life-cycle assessment. This critical narrative review evaluates these interacting dimensions, with emphasis on Yarrowia lipolytica, Rhodotorula toruloides, Lipomyces starkeyi and Cutaneotrichosporon oleaginosus. Literature was selected from accessible scholarly databases and citation networks for the period 2000 to 19 June 2026, while earlier foundational studies were retained where mechanistically necessary. The evidence supports a mature biochemical understanding of nitrogen-limitation-driven lipogenesis and shows that metabolic engineering can markedly increase storage-lipid formation in model conditions. Evidence is also substantial that several oleaginous yeasts can convert lignocellulosic hydrolysates, crude glycerol and other residual streams. The principal weakness is translational: high lipid fractions reported in defined media are often not accompanied by equally strong titre, productivity, tolerance, feedstock reproducibility, extraction performance or system-level environmental evidence. Techno-economic and life-cycle studies consistently identify fermentation productivity, inexpensive carbon supply, energy use and downstream processing as decisive variables, while residue removal and co-product allocation can alter environmental conclusions. The most defensible pathway to sustainable single-cell oil therefore combines robust mixed-substrate biocatalysts, high-cell-density process control, low-energy recovery and product-market matching, with techno-economic and life-cycle analysis embedded during process development rather than added after optimisation.
O. Ayano, C. O. Bamigboye· Asian Journal of Biotechnolo...· 0 citations
Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBH) is a biodegradable copolymer whose mechanical properties can be tuned by the 3-hydroxyhexanoate (3HHx) fraction. However, current industrial production largely relies on established hosts and plant oil-based feedstocks. Here, we developed Rhodobacter capsulatus SB1003 as a new PHBH-producing platform by focusing on the two key determinants of copolymer formation: polyhydroxyalkanoate (PHA) synthase substrate specificity and intracellular monomer supply. A PHA synthase with broad-substrate specificity was integrated into the native phaC locus to generate a heterologous phaC strain. All PHA production experiments were performed under anaerobic photoheterotrophic conditions in 8-mL screw-cap tubes containing 7.6 mL of medium and illuminated with continuous white light. During butyrate cultivation under these conditions, the engineered strain accumulated polymer up to 41.5% of cell dry weight and incorporated detectable 3HHx, whereas the wild type showed no 3HHx incorporation. To increase 3HHx-CoA availability from butyrate, we introduced C4-to-C6 precursor-supply modules involving β-ketothiolase (BktB)/β-ketoacyl-CoA reductase (PhaB) and crotonyl-CoA carboxylase/reductase (Ccr)/ethylmalonyl-CoA decarboxylase (Emd), but these modifications led to only marginal improvements in the 3HHx fraction. In contrast, supplying C6 or longer fatty acids under the same conditions markedly increased 3HHx incorporation; cultivation on hexanoate yielded PHBH containing 32.4 mol% 3HHx. Collectively, this study demonstrates PHBH biosynthesis in R. capsulatus and indicates that limited 3HHx-CoA supply rather than polymerization capacity is the primary bottleneck, providing a foundation for further pathway and host optimization toward flexible PHBH production from diverse substrates.
Kako Miura, Takayuki Shimizu, Tomohisa Hasunuma et al.· Journal of Bioscience and Bi...· 0 citations
The sustainable production of hydroxy fatty acids, such as 10-hydroxystearic acid (10-HSA), by biocatalysis is a promising alternative to petrochemical and castor oil-derived products. However, industrial implementation still requires an efficient and scalable process for biocatalyst production by fermentation, which precedes the biotransformation to 10-HSA. In general, industrial biocatalysis is commonly performed using whole cells, which are cheaper than purified enzymes. Still, the fermentation process itself is a major cost contributor requiring a high-yielding and scalable process. Here, we established and scaled a lactose-induced fed-batch process to produce an Escherichia coli BL21 (DE3) based whole-cell biocatalyst containing an oleate hydratase from Stenotrophomonas nitritireducens. The two-phased process employs an initial growth phase on glucose, followed by an induced feed phase using glycerol and lactose. When comparing two different growth rates during enzyme expression, a higher growth rate was found beneficial, resulting in a higher biomass concentration of 69.2 ± 0.5 g L-1 and a yield increase of 80 % while maintaining biocatalyst activity at >85 % conversion. This highlights the importance of process design variables, such as growth rate settings, for a high-yielding and economic process. Feasibility of the process was demonstrated by scaling into a 150 L bioreactor, achieving a biomass concentration of 60.6 ± 0.4 g L-1 with a yield of 0.44 gC,Biomass gC -1 and 91.5 ± 1.4 % conversion. Supplemented with an initial test of suitable unit operations for technical biomass separation, this work provides a fermentation route for a whole-cell oleate hydratase biocatalyst, paving the way for further scaling toward industrial 10-HSA production.
Rebekka Horstmann, Mario Beckers, J. Viell et al.· Biotechnology progress (Prin...· 0 citations
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