Komagataella phaffii is a prominent methylotrophic yeast widely used in industrial biotechnology for heterologous protein production, chemical synthesis, and C1 feedstock utilization. However, its industrial application is limited by thermal stress and methanol toxicity, while the relationship between heat adaptation and methanol tolerance remains poorly understood. Here, we obtained a thermotolerant mutant ATH through ARTP mutagenesis followed by adaptive laboratory evolution (ALE) under heat stress (37 °C). ATH tolerated heat and methanol robustly under individual stress conditions, achieving 3-fold and 8-fold higher cell densities than GS115 at 37 °C or in 5% (v/v) methanol, respectively, yet failed under the combined stress of 37 °C and 5% methanol. Using three proteins (GFP, OPH, and ChiA) as reporters, we found that ATH showed higher heterologous expression than GS115 at 33 °C. Physiologically, ATH displayed enhanced cell wall/membrane integrity, reduced ROS accumulation and lower lipid peroxidation (>30% reduction in MDA), and accelerated formaldehyde clearance. Transcriptomics identified 561 shared differentially expressed genes, with coordinated upregulation of heat shock proteins (HSP70/40/90 families; Hsp104 and Hsp70-SSA2), antioxidant enzymes (catalase, peroxiredoxin), and cell wall/membrane remodeling genes, alongside downregulation of amino acid and secondary metabolite biosynthesis. Whole-genome resequencing further distinguished ATH from GS115 by only 19 variants (9 SNPs and 10 InDels), highlighting a compact genetic basis for the evolved phenotype. This work provides a K. phaffii chassis with improved individual heat and methanol tolerance and elucidates the physiological, transcriptional, and genomic mechanism underlying heat–methanol cross-adaptation.
Proton exchange membranes (PEMs) serve as essential elements in direct methanol fuel cells (DMFCs). Although sulfonated poly(ether ether ketone) (SPEEK) has shown competitive proton conductivity at high sulfonation levels, the elevated sulfonation inevitably induces excessive swelling. In this study, chitosan (CS) was modified to synthesize phosphorylated chitosan (PCS), which served as a dual-functional ionic crosslinker for SPEEK to fabricate SPEEK/PCS composite PEMs. Owing to the in-situ ionic crosslinking between the -NH2 groups of PCS and the -SO3H groups of SPEEK, the SPEEK/PCS-6 composite membrane achieves a tensile strength of 67.7 MPa and elongation at break of 203.3% in the hydrated state. Simultaneously, the phosphate groups of PCS provide extra proton-conducting pathways, yielding a high proton conductivity of 148.3 mS cm- 1 at 80 degrees C. Moreover, assembled DMFC achieves a low methanol crossover current density of 136 mA cm- 2 and high power density of 160.3 mW cm- 2, demonstrating promising potential for DMFCs.
Dillon-type Boolean functions are trace polynomial functions from 1F22n to 1F2, with all the exponents being multiples of 2n-1, often referred to as Dillon-type exponents. This paper investigates a class of bent functions within this family associated with trace rational functions. Specifically, we introduce a novel infinite family of trace rational functions and generalize a key bentness criterion established by Li et al. (2013) [22] for binary Dillon-type functions. Using our family of functions as structural components, we explicitly construct and characterize three distinct classes of bent functions, each derived via a different bridge function with a small number of variables. These characterizations are given either explicitly or in terms of the well-known binary Kloosterman sums. The choice of bridge functions proves to be crucial. We show that when the bridge function is the product of three variables, the resulting construction degenerates, yielding no new bent functions. Remarkably, our first two subclasses of Boolean bent functions can be naturally extended to vectorial bent functions. To the best of our knowledge, these constitute the first explicitly characterized classes of vectorial bent functions within the partial spread class. Moreover, our theoretical analysis and experimental results reveal that these constructions yield new bent functions that are not Extended-Affine equivalent to any previously known class of monomial functions. (c) 2026 Elsevier B.V. All rights are reserved, including those for text and data mining, AI training, and similar technologies.
Aromatic amines are one kind of crucial raw materials used to manufacture fine chemicals, biofuel, fuel additives, resins, polymers, pharmaceuticals and other biobased materials, which can be obtained by aminating lignin-valorized aromatic alcohols and aldehydes. In this study, a sustainable biosynthetic route for producing vanillylamine and related aromatic amines from lignin-derived alcohols was developed. First, the galactose oxidase gene from Fusarium NRRL 2903 was introduced into E. coli to construct recombinant strain GO for entirely oxidizing 100 mM vanillyl alcohol into vanillin in ethyl acetate:Triton X-114:water = 5:5:90 (vol/vol/ vol) containing Cu2+ (1 mM) (25 degrees C, pH 7.5). In addition, the CV w-transaminase gene from Chromobacterium violaceum was acquired to construct recombinant E. coli CV for efficiently converting vanillin-derived vanillin into vanillylamine through transamination (35 degrees C, pH 7.5) with amine donor L-alanine (L-alanine/vanillin molar ratio 10:1), achieving the mass yield of 1.0 g vanillylamine per g vanillyl alcohol. This established tandem biocatalysis process for converting vanillyl alcohol was an efficient and ecofriendly way for synthesizing vanillylamine, and lignin-derived aromatic aldehydes could be transformed into organic amines, rendering the high-value valorization of biomass and its derivatives. In addition, this study has expanded the efficient oxidation transformation of furfuryl alcohol and aromatic alcohol compounds (anisyl alcohol, cinnamyl alcohol, benzyl alcohol, veratryl alcohol, and syringic alcohol) that can be produced from lignocellulose, supplemented the substrate pedigree of the one-pot two-enzyme method, and provided a new idea for the high-value transformation of lignocellulose-derived compounds.
Komagataella phaffii is a prominent methylotrophic yeast widely used in industrial biotechnology for heterologous protein production, chemical synthesis, and C1 feedstock utilization. However, its industrial application is limited by thermal stress and methanol toxicity, while the relationship between heat adaptation and methanol tolerance remains poorly understood. Here, we obtained a thermotolerant mutant ATH through ARTP mutagenesis followed by adaptive laboratory evolution (ALE) under heat stress (37 °C). ATH tolerated heat and methanol robustly under individual stress conditions, achieving 3-fold and 8-fold higher cell densities than GS115 at 37 °C or in 5% (v/v) methanol, respectively, yet failed under the combined stress of 37 °C and 5% methanol. Using three proteins (GFP, OPH, and ChiA) as reporters, we found that ATH showed higher heterologous expression than GS115 at 33 °C. Physiologically, ATH displayed enhanced cell wall/membrane integrity, reduced ROS accumulation and lower lipid peroxidation (>30% reduction in MDA), and accelerated formaldehyde clearance. Transcriptomics identified 561 shared differentially expressed genes, with coordinated upregulation of heat shock proteins (HSP70/40/90 families; Hsp104 and Hsp70-SSA2), antioxidant enzymes (catalase, peroxiredoxin), and cell wall/membrane remodeling genes, alongside downregulation of amino acid and secondary metabolite biosynthesis. Whole-genome resequencing further distinguished ATH from GS115 by only 19 variants (9 SNPs and 10 InDels), highlighting a compact genetic basis for the evolved phenotype. This work provides a K. phaffii chassis with improved individual heat and methanol tolerance and elucidates the physiological, transcriptional, and genomic mechanism underlying heat–methanol cross-adaptation.
