Metabolic engineering of Komagataella phaffii for enhanced 3-hydroxypropionic acid (3-HP) production from methanol

Abstract

Background: Bioconversion of methanol derived from CO₂ reduction into value-added chemicals provides a unique approach for mitigating global warming and reducing fossil fuels dependence. Production of 3-hydroxypropionic acid (3-HP), a key building block for the development of biobased products such as acrylates and 1,3-propanediol, has been successfully achieved using methanol as the sole carbon and energy source in the methylotrophic yeast Komagataella phaffii (syn. Pichia pastoris). However, challenges remain in meeting commercially relevant concentrations, yields and productivities of 3-HP, prompting further strain optimization. In the present study, we have combined metabolic engineering strategies aiming at increasing metabolic precursors supply and redirecting carbon flux towards 3-HP production.

Results: A combinatorial metabolic engineering strategy targeting precursors supply and 3-HP export was applied to the original 3-HP producing K. phaffii strain harboring the synthetic β-alanine pathway and a mutated NADP-dependent formate dehydrogenase from Pseudomonas sp. 101 (PseFDH(V9)). To do so, several genes encoding enzymes catalyzing reactions immediately upstream of the β-alanine pathway were overexpressed to enhance precursors availability. However, only the overexpression of the pyruvate carboxylase PYC2 gene significantly increased the 3-HP yield on biomass (Y_P/X) in small-scale cultivations. Co-overexpression of PYC2 and the lactate permeases ESBP6 and JEN1 genes led to a 55% improvement in 3-HP titer and product yield in methanol deep-well plate cultures compared to the reference strain, mostly due to Esbp6 activity, proving its effectiveness as a 3-HP transporter. Deletion of the native formate dehydrogenase gene FDH1 did not increase methanol flux entering the assimilatory pathway. Instead, knockout strains showed severe growth defects due to toxic intermediates accumulation. Co-expression of the PseFDH(V9) encoding gene in these strains failed to compensate for the loss of the native FDH. The strain combining PYC2, ESBP6, and JEN1 overexpression was further tested in fed-batch cultures at pH 5, achieving a 3-HP concentration of 27.0 g l^{- 1}, with a product yield of 0.19 g g^{- 1}, and a volumetric productivity of 0.56 g l^{- 1} h^{- 1} for the methanol feeding phase of the cultivations. These results represent a 42% increase in final concentration and over 20% improvement in volumetric productivity compared to the original 3-HP-producing strain. Furthermore, bioreactor-scale cultivations at pH 3.5 revealed increased robustness of the strains overexpressing monocarboxylate transporters.

Conclusions: Our results point out the potential of lactate transporters to efficiently drive 3-HP export in K. phaffii, leading to higher titers, yields, and productivities, even at lower pH conditions.

Keywords: Komagataella phaffii; Pichia pastoris; 3-hydroxypropionic acid; Lactate transporters; Metabolic engineering; Methanol; ß-alanine pathway.

Fig. 1

Schematic representation of methanol metabolism, 3-HP production via the β-alanine pathway, and carboxylic acids transport mechanisms reported in yeast. The pathway of 3-HP synthesis is shaded in green; and the methanol dissimilatory pathway is shaded in red. Heterologous enzymes are indicated with colored gears. While uncharged 3-HP (HOCH₂CH₂COOH) can passively diffuse across the plasma membrane, its anionic form (HOCH₂CH₂COO⁻) is hypothesized to require active transport via ABC transporters or permeases (i.e., Jen1 symporter). Dissociation of 3-HP in the cytosol releases protons (H⁺), which are mainly extruded by H⁺-ATPases, maintaining pH balance and electrochemical potential across the membrane (Z∆pH) [70]. FDH1, NAD-dependent formate dehydrogenase; PseFDH(V9), mutated NADP-dependent formate dehydrogenase; PYC2, pyruvate carboxylase isoform 2; AAT2, aspartate aminotransferase 2; SpeAspDH, NAD- or NADP-dependent aspartate dehydrogenase; PAND, aspartate-1-decarboxylase; BAPAT, β-alanine-pyruvate aminotransferase; YDFG, 3-hydroxypropionate dehydrogenase

Fig. 2

Average global product yields calculated for the strains constructed in this study, Y_P/S and Y_P/X. The salmon-colored bars show the average 3-HP yield on methanol (g_3-HP g_MeOH^-1), while light blue was used for bars representing the average 3-HP yield on biomass (g_3-HP OD₆₀₀ unit^-1). The error bars show the standard deviation. Significant differences between two groups are shown in the graph. Statistical analysis was conducted using a two-tailed unpaired Student’s t-test (*p < 0.05, **p < 0.01, ***p < 0.001)

Fig. 3

Fed-batch phase profiles from the bioreactor-scale experiments with PpCβ21-P and PpCβ21-PEJY strains at pH 5. Concentrations of dry cell weight, 3-HP, and residual methanol are represented in the left-side y-axis. The total amount of methanol added, normalized by the actual volume of the reactor at every time, is represented using the right-side y-axis. Time axis corresponds to the exponential methanol feeding phase of the cultivations (preprogrammed at a µ = 0.03 h^− 1). The cultivation profile shown for each strain corresponds to the average result of two independent cultivations. The error bars denote the standard deviation for the duplicate

Fig. 4

Fed-batch phase profiles from the bioreactor-scale experiments with PpCβ21-P and PpCβ21-PEJY strains at pH 3.5. Concentrations of dry cell weight, 3-HP, and residual methanol are represented in the left-side y-axis. The total amount of methanol added is represented using the right-side y-axis. Time axis corresponds to the exponential methanol feeding phase of the cultivations (preprogrammed at a µ = 0.03 h^− 1). Data represent a single cultivation replicate for each strain. The error bars denote the standard deviation of triplicate measurements for each sample