Combining Metabolic Engineering and Multiplexed Screening Methods for 3-Hydroxypropionic Acid Production in Pichia pastoris

Abstract

Production of 3-hydroxypropionic acid (3-HP) in Pichia pastoris (syn. Komagataella phaffii) via the malonyl-CoA pathway has been recently demonstrated using glycerol as a carbon source, but the reported metrics were not commercially relevant. The flux through the heterologous pathway from malonyl-CoA to 3-HP was hypothesized as the main bottleneck. In the present study, different metabolic engineering approaches have been combined to improve the productivity of the original 3-HP producing strains. To do so, an additional copy of the gene encoding for the potential rate-limiting step of the pathway, i.e., the C-terminal domain of the malonyl-CoA reductase, was introduced. In addition, a variant of the endogenous acetyl-CoA carboxylase (ACC1 ^S1132A ) was overexpressed with the aim to increase the delivery of malonyl-CoA. Furthermore, the genes encoding for the pyruvate decarboxylase, aldehyde dehydrogenase and acetyl-CoA synthase, respectively, were overexpressed to enhance conversion of pyruvate into cytosolic acetyl-CoA, and the main gene responsible for the production of the by-product D-arabitol was deleted. Three different screening conditions were used to classify the performance of the different strains: 24-deep-well plates batch cultures, small-scale cultures in falcon tubes using FeedBeads® (i.e., slow release of glycerol over time), and mini bioreactor batch cultures. The best two strains from the FeedBeads® screening, PpHP8 and PpHP18, were tested in bioreactor fed-batch cultures using a pre-fixed exponentially increasing feeding rate. The strain PpHP18 produced up to 37.05 g L^-1 of 3-HP at 0.712 g L^-1 h^-1 with a final product yield on glycerol of 0.194 Cmol^-1 in fed-batch cultures. Remarkably, PpHP18 did not rank among the 2-top producer strains in small scale batch cultivations in deep-well plates and mini bioreactors, highlighting the importance of multiplexed screening conditions for adequate assessment of metabolic engineering strategies. These results represent a 50% increase in the product yield and final concentration, as well as over 30% increase in volumetric productivity compared to the previously obtained metrics for P. pastoris. Overall, the combination of glycerol as carbon source and a metabolically engineered P. pastoris strain resulted in the highest 3-HP concentration and productivity reported so far in yeast.

Keywords: 3-hydroxypropionic acid; Pichia pastoris; acetyl-CoA; glycerol; malonyl-CoA; metabolic engineering.

FIGURE 1

Summary of metabolic engineering strategies for the optimisation of 3-HP production in P. pastoris. Strain engineering started from the 3-HP producing strain PpHP6 containing the malonyl-CoA to 3-HP pathway (Fina et al., 2021). Heterologous enzymes are depicted in yellow, endogenous enzymes being overexpressed are shown in green, and the enzymes catalyzing the reactions of genes being deleted are shown in red. The (2x) indicates that a second copy of the MCR-C_Ca gene was introduced. Enzyme abbreviations: Pdc1, pyruvate decarboxylase; Ald6 _Sc , aldehyde dehydrogenase from S. cerevisiae; ACS _Se ^L641P, acetyl-CoA synthase from S. enterica with the mutation L641P; Acc1 _Yl , acetyl-CoA carboxylase from Y. lipolytica; Acc1^S1132A, acetyl-CoA carboxylase from P. pastoris with the mutation S1132A; MCR-C _Ca , C-terminal domain of the malonyl-CoA reductase from C. aurantiacus; MCR-N _Ca , N-terminal domain of the malonyl-CoA reductase from C. aurantiacus; cPos5 _Sc , NADH kinase from S. cerevisiae relocated into the cytoplasm; ArDH, D-arabitol dehydrogenase.

FIGURE 2

3-HP production yield of the recombinant P. pastoris strains tested in deep-well plates. The genetic modifications performed to each strain are shown at the left side of the graph. The product yield in Cmol⁻¹ is shown at the right side of the graph.

FIGURE 3

Comparison of the most relevant 3-HP producing P. pastoris strains under three different screening conditions. (A) 3-HP production yield in deep-well plates (grey solid bars), FeedBeads® (blue dotted bars), and mini bioreactors (orange striped bars). (B) Growth rate (yellow vertically striped bars) and arabitol production yield (green horizontally striped bars) for each strain grown in mini bioreactors.

FIGURE 4

Profiles of fed-batch cultivations of the PpHP8 (A) and PpHP18 (B) strains. Concentration of glycerol, biomass, 3-HP, and D-arabitol are represented using the left-side y-axis. The total amount of glycerol added to the reactor, normalized by the actual volume of the reactor at every time is represented using the y-axis at the right side. The average result of two independent cultivations is shown. The error bars correspond to the standard deviation for the duplicate.