A Rewired NADPH-Dependent Redox Shuttle for Testing Peroxisomal Compartmentalization of Synthetic Metabolic Pathways in Komagataella phaffii

Abstract

The introduction of heterologous pathways into microbial cell compartments offers several potential advantages, including increasing enzyme concentrations and reducing competition with native pathways, making this approach attractive for producing complex metabolites like fatty acids and fatty alcohols. However, measuring subcellular concentrations of these metabolites remains technically challenging. Here, we explored 3-hydroxypropionic acid (3-HP), readily quantifiable and sharing the same precursors-acetyl-CoA, NADPH, and ATP-with the above-mentioned products, as a reporter metabolite for peroxisomal engineering in the yeast Komagataella phaffii. To this end, the malonyl-CoA reductase pathway for 3-HP production was targeted into the peroxisome of K. phaffii using the PTS1-tagging system, and further tested with different carbon sources. Thereafter, we used compartmentalized 3-HP production as a reporter system to showcase the impact of different strategies aimed at enhancing the peroxisomal NADPH pool. Co-overexpression of genes encoding a NADPH-dependent redox shuttle from Saccharomyces cerevisiae (IDP2/IDP3) significantly increased 3-HP yields across all substrates, whereas peroxisomal targeting of the S. cerevisiae NADH kinase Pos5 failed to improve 3-HP production. This study highlights the potential of using peroxisomal 3-HP production as a biosensor for evaluating peroxisomal acetyl-CoA and NAPDH availability by simply quantifying 3-HP, demonstrating its potential for peroxisome-based metabolic engineering in yeast.

Keywords: 3-hydroxypropionic acid; Komagataella phaffii; NADPH; Pichia pastoris; acetyl-CoA; metabolic engineering; peroxisome; redox shuttle.

Figure 1

Metabolic pathway engineering for peroxisomal 3-HP synthesis in K. phaffii from various carbon sources. Heterologous and endogenous genes are presented in purple and black, respectively. MCR-C and MCR-N, C- and N-terminal domains of malonyl-CoA reductase, respectively; ACC, acetyl-CoA carboxylase; POS5, NADH kinase; IDP2 and IDP3, NADP-dependent isocitrate dehydrogenases isoenzymes; AOX, alcohol oxidase; PDC, pyruvate decarboxylase; ALD, acetaldehyde dehydrogenase; ACS, acetyl-CoA synthetase; YAT1,2 and CAT2, carnitine acetyl-transferases; FAS1,2, fatty acid synthases; FAA2, acyl-CoA synthetase; ANT1, ATP transporter; PXA1,2, ABC transporter family D; FALD, formaldehyde; Xu5P, xylulose-5-phosphate; G3P, glyceraldehyde-3-phosphate; DHA, dihydroxyacetone; DHAP, dihydroxyacetone phosphate; G6P, glucose-6-phosphate; GYC, glyoxylate cycle; Ac-carn, acetylcarnitine; MSA, malonate semialdehyde; 3-HP, 3-hydroxypropionic acid; αKG, α-ketoglutarate; FFA, free fatty acids; LCFA, long-chain fatty acids.

Figure 2

Average final 3-HP concentration produced from oleic acid by each K. phaffii recombinant strain. Data are presented as the mean ± SD of biological triplicates. Statistically significant differences between two groups are shown in the graph. Statistical analysis was conducted using a two-tailed unpaired Student’s t-test (* p < 0.01, ** p < 0.001, *** p < 0.0001). Raw data available in Supplementary File S2.

Figure 3

Final 3-HP concentrations and overall product yields (Y_P/S) obtained from BMO, BMM, BMD, and BMG by platform strain PpMA and its derivative strains. Data are presented as the mean ± SD of biological triplicates. Statistically significant differences between PpMA and each derivative strain are indicated in the graph. Statistical analysis was conducted using a two-tailed unpaired Student’s t-test (* p < 0.05, ** p < 0.001, *** p < 0.0001, **** p < 0.00001, n.s. not significant). Raw data available in Supplementary File S2.

Figure 4

Final 3-HP concentrations and global product yields (Y_P/S) obtained from BMO, BMM, BMD, and BMG by each cytosolic K. phaffii strain. Data are presented as the mean ± SD of biological triplicates. The p-values resulting from two-tailed unpaired Student’s t-tests comparing two groups (PpHP4 vs. PpHP2 and PpHP6 vs. PpHP4) on each substrate are indicated. Raw data available in Supplementary File S2.