초록
<P><B>Abstract</B><P>Background<P>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 <I>Pichia pastoris</I> 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 <I>P. pastoris</I> are also current challenges, which are of great significance to the economical industrial application using methanol as a feedstock for SCP production.</P></P><P>Results<P>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 <I>P. pastoris</I>, 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 <I>P. pastoris</I> 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).</P></P><P>Conclusions<P>This study clarified the unique mechanism of <I>P. pastoris</I> for efficient methanol utilization, higher temperature resistance, and high protein synthesis, providing a <I>P. pastoris</I> cell factory for SCP production with environmental, economic, and nutritional benefits.</P></P></P>