Methanol biotransformation toward high-level production of fatty acid derivatives by engineering the industrial yeast Pichia pastoris

Abstract

Methanol-based biorefinery is a promising strategy to achieve carbon neutrality goals by linking CO₂ capture and solar energy storage. As a typical methylotroph, Pichia pastoris shows great potential in methanol biotransformation. However, challenges still remain in engineering methanol metabolism for chemical overproduction. Here, we present the global rewiring of the central metabolism for efficient production of free fatty acids (FFAs; 23.4 g/L) from methanol, with an enhanced supply of precursors and cofactors, as well as decreased accumulation of formaldehyde. Finally, metabolic transforming of the fatty acid cell factory enabled overproduction of fatty alcohols (2.0 g/L) from methanol. This study demonstrated that global metabolic rewiring released the great potential of P. pastoris for methanol biotransformation toward chemical overproduction.

Keywords: biofuels; metabolic engineering; methylotrophic yeast; oleochemicals; synthetic biotechnology.

Fig. 1.

The concept of methanol-based biomanufacturing from renewable resources. Methanol can be produced economically and in a large scale from renewable resources and by using green energy, representing an ideal feedstock for future biomanufacturing, and will contribute to the carbon neutrality goal by capturing CO₂.

Fig. 2.

Blocking FFA activation and consumption in P. pastoris. (A) Schematic view of the interruption of fatty acids consumption. (B) FFA titers of engineered strains in shake flasks after 96 h of cultivation in 20 g/L glucose medium or 120 h of cultivation in 20 g/L methanol medium at 220 rpm and 30 °C. The histogram shows the FFA titers, and the rhombus scatter points show the biomass of engineered strains. (C) FFA profiles of the strain PC103 in methanol- and glucose medium. Error bars correspond to the SD of the mean ± SD (n = 3, corresponding to three biological replicates).

Fig. 3.

Metabolic rewiring for FFA production in P. pastoris. (A) Schematic illustration of rewired central metabolism for improved FFA production from methanol. Overexpressed genes are shown in bright blue. The Mus musculus ATP:citrate lyase gene MmACL was integrated into the genomic PNSI-2 site; the Bifidobacterium breve phosphoketolase gene BbXFPK and Clostridium kluyveri phosphotransacetylase gene CkPTA were integrated into the genomic PNSIII-5 site. For NADPH regeneration, the S. cerevisiae NADP⁺-dependent isocitrate dehydrogenase gene ScIDP2 was integrated at the PNSI-4 site. Another copy of endogenous DAS2 was overexpressed at the PNSII-4 site to improve formaldehyde assimilation. (B) FFA production and cell density of strains in shake flasks after 120 h of cultivation at 220 rpm and 30 °C with 20 g/L methanol minimal medium. (C–F) Growth curves, methanol consumption, formaldehyde accumulation and intracellular ROS levels. Error bars represent SD of triplicate samples.

Fig. 4.

Fed-batch fermentation for FFA production. Fed-batch fermentation of PC124H in shake flasks (A) or 1 L bioreactor (C). PC124H is a prototrophic strain with complementation of the HIS4 marker in PC124. Time courses of fatty acid titers (in orange), growth curves (in gray) and methanol consumption (in blue) are shown. Fatty acids production at the end of fermentation are also shown as filled orange circles. FFA profile of the strain PC124H in shake flasks (B) and bioreactors (D). Due to the precipitation, ethyl acetate was used for the dissolution of all fatty acids. Error bars correspond to the SD of the mean (n = 3 for shake flask fed-batch, corresponding to three biological replicates; n = 2 for bioreactor fed-batch, corresponding to two independent biological samples).

Fig. 5.

Metabolic transforming for fatty alcohol production. (A) Schematic illustration of metabolic transforming of an FFAs-overproducing strain for overproduction of fatty alcohols. (B) Comparison of FFA reduction (CAR) and fatty acyl-CoA reduction (FaCoAR) pathways for fatty alcohol production from methanol. (C) Comparing fatty alcohol production between the wild-type and metabolic rewired strain. One copy of the FaCoAR gene was expressed in PC111B (generating strain PC170) and PC124 (generating strain PC172 by simultaneously reintroducing FAA1). (D) Optimization of fatty acyl-CoA reduction pathway. The engineered strains were cultivated in shake flasks containing 20 g/L methanol for 120 h at 220 rpm and 30 °C. (E) Fed-batch fermentation of the prototrophic strain PC174H. Time courses of fatty alcohol titers (deep red), growth curve (gray-purple) and consumption of methanol (blue) are shown. (F) The composition (Left) and distribution (Right) of fatty alcohols from fed-batch fermentation. Error bars correspond to the SD of the mean (n = 3, corresponding to three biological replicates).