Methanol, as an alternative to sugar, is a renewable one-carbon source that can be obtained from CO2 or methane through chemical catalysis, photocatalysis or electrocatalysis (Goeppert et al., 2014). Methanol-based bioconversion emerges as a sustainable bio-manufacturing process with nearly zero CO2 emissions (Li et al., 2020), offering potential solutions to the land-food crisis (Zhou et al., 2018) and contributing to achieving global carbon neutrality (Liu et al., 2021). While methylotrophic bacteria, including natural microorganisms like Eukaryotic methylotrophic yeast, Bacillus methanolicus, and Methylobacterium extorquens (Chen and Lan, 2020), as well as synthetic microorganisms such as Escherichia coli (Chen et al., 2020, Keller et al., 2022), Corynebacterium glutamate (Tuyishime et al., 2018, Wang et al., 2020) and Saccharomyces cerevisiae (Dai et al., 2017, Zhan et al., 2023), demonstrate the capability for methanol bioconversion, the limited diversity of methylotrophs species and their low methanol utilization efficiency significantly constrain the vast application potential of methanol-based biomanufacturing (Espinosa et al., 2020).

Komagataella phaffii (K. phaffii), formerly known as Pichia pastoris, emerges as an ideal host for methanol biotransformation due to its inherent methanol utilization pathway and highly regulated expression system (Vogl et al., 2016). Presently, it has been widely used in the manufacture of chemical products such as fatty acids (Cai et al., 2022), terpenoids (Zuo et al., 2022), and biofuels (Pena et al., 2018). However, only a few studies achieved the synthesis of target products that used methanol as the only carbon source (Guo et al., 2021). In addition, the yield of target products from methanol carbon sources was much lower than that from glucose. For instance, when methanol and glucose were used as carbon sources, the malic acid yield of K. phaffii was 2.7 g/L and 8.7 g/L, respectively (Gonzalez et al., 2018), which might be caused by insufficient understanding of methanol metabolism (Hou et al., 2022). Methanol assimilation pathways in nature mainly include the serine cycle and xylulose monophosphate pathway (XuMP), the latter occurs in methylotrophic yeasts and is used to assimilate methanol to produce glyceraldehyde 3-phosphate (G3P). Approximately one-third of G3P enters glycolysis/gluconeogenesis and pentose phosphate pathways for the generation of substances and energy (Russmayer et al., 2015). The methanol dissimilation pathway (MDIP) provides energy for cells by the oxidation of formaldehyde (Jiang et al., 2021). The XuMP in K. phaffii mainly occurs in peroxisome (van der Klei et al., 2006), which can help cells alleviate the toxic effect of formaldehyde (Hou et al., 2022). Notably, the biomass composition of protein in methanol carbon sources (50 %) is about 10 % higher than that in glucose or glycerol (Tomas-Gamisans et al., 2018), suggesting an increased demand for protein to resist oxygen stress response. Although the relative stability of tricarboxylic acid (TCA) cycle indicated the similar energy produced by methanol and glucose for cell growth (Hou et al., 2022), the growth-related maintenance energy (GAME) in the cells using the methanol carbon source was increased by 1.4 times, while the non-growth-related maintenance energy (NGAME) was reduced by 84.3 % and 82.4 %, respectively (Tomas-Gamisans et al., 2018). This discrepancy might be caused by the inefficiency of the XuMP (Guo et al., 2023). This inherent cellular protective mechanism may lead to metabolic overload, in other words, most of the energy is wasted to compensate for maintaining the energy balance of methanol metabolism (Vogl et al., 2016).

This study aimed to construct a new alcohol-aldehyde conversion system to enhance methanol metabolism efficiency, alleviate metabolic overload, and mitigate the toxic effects of formaldehyde on cells. In this study, this new system was constructed and the synergistic promoting effect of histidine was validated. This is the first report that a new endogenous alcohol-aldehyde system cooperated with histidine to promote the assimilation efficiency of methanol in K. phaffii. Ultimately, this synergistic effect resulted in a remarkable 63 % increase in cell dry weight when utilizing methanol carbon source.