Role of Dissimilative Pathway of Komagataella phaffii (Pichia pastoris): Formaldehyde Toxicity and Energy Metabolism

Abstract

Komagataella phaffii (aka Pichia pastoris) is a yeast able to grow in methanol as the sole carbon and energy source. This substrate is converted into formaldehyde, a toxic intermediary that can either be assimilated to biomass or dissimilated to CO₂ through the enzymes formaldehyde dehydrogenase (FLD) and formate dehydrogenase, also producing energy in the form of NADH. The dissimilative pathway has been described as an energy producing and a detoxifying route, but conclusive evidence has not been provided for this. In order to elucidate this theory, we generated mutants lacking the FLD activity (Δfld1) and used flux analysis to evaluate the metabolic impact of this disrupted pathway. Unexpectedly, we found that the specific growth rate of the Δfld1 strain was only slightly lower (92%) than the control. In contrast, the sensitivity to formaldehyde pulses (up to 8mM) was significantly higher in the Δfld1 mutant strain and was associated with a higher maintenance energy. In addition, the intracellular flux estimation revealed a high metabolic flexibility of K. phaffii in response to the disrupted pathway. Our results suggest that the role of the dissimilative pathway is mainly to protect the cells from the harmful effect of formaldehyde, as they were able to compensate for the energy provided from this pathway when disrupted.

Keywords: Komagataella phaffii; Pichia pastoris; dissimilative pathway; formaldehyde dehydrogenase; methanol.

Figure 1

Main steps of the methanol utilisation (Mut) pathway in K. phaffii. Relevant enzymes involved are shown in red boxes. (Adapted from [10]). AOX: alcohol oxidase; CAT: catalase; DAS: dihydroxyacetone synthase; FLD: formaldehyde dehydrogenase; FGH: S-formylglutathione hydrolase; FDH; formate dehydrogenase; GS(H): glutathione; Pmp20: peroxisomal glutathione peroxidase; GLR: glutathione reductase.

Figure 2

Specific growth rate (µ) of control and Δfld1 strains grown on a defined medium using either methanol or glycerol 3 g/L as the only carbon source. * Significant difference evaluated by an unpaired t-test * p < 0.01. Cell growth profiles are shown in Supplementary Figures S2 and S3.

Figure 3

(A) Example of biomass growth kinetics (shown as lnX) of control strain with glycerol (triangles) or methanol (circles) as the carbon source. The growth behaviour before (blue symbols) and after (red symbols) a formaldehyde pulse (5 mM) performed after 4 h of inoculation (red arrow) is shown. The experimental error in biomass measurements was lower than 3% (not shown). (B) The effect of formaldehyde pulses (2, 5 and 8 mM) on the cell growth kinetics (slope of semi log plot) of control (blue) and Δfld1 (orange) strains using glycerol as the only carbon source. (C) The compared effect of formaldehyde pulses (2, 5 and 8 mM) on control (blue triangles) and Δfld1 (red triangles) strains using methanol as the only carbon source. Model adjustment Equation (5) is also shown with continuous lines for each strain. SEM < 5% (not shown).

Figure 4

Correlation between the specific consumption rate of methanol (q_S) and the specific growth rate (µ) for control (blue) and Δfld1 (red) stains. The apparent maintenance coefficient m′_S was estimated from Equation (6). Control: blue open circle; Δfld1 strain: red open circle.

Figure 5

Simplified map of the metabolic flux distribution of methanol uptake in K. phaffii (blue columns: control strain; red columns: Δfld1 strain). The list of the metabolic reactions is provided in Supplementary File S1. Results are shown as percentage of the methanol uptake flux with no pulse (0) and after 5 or 8 mM formaldehyde pulse (rows). AOX: alcohol oxidase; DP: dissimilative pathway; AP: assimilative pathway; GNG: gluconeogenesis; GLC: glycolysis (from G3P onwards); PDH: pyruvate dehydrogenase; APP: anaplerotic pathways; G3P: glycerdaldyde-3-phosphate; G6P: glucose- 6-phosphate; Pyr: pyruvate; Ac-CoA: actyl coenzime A; α-KG: α -ketoglutarate; Succ-CoA: succinyl conezyme A; Mal: malate; OA: oxaloacetate.

Figure 6

(A) Carbon flux distribution from pyruvate (Pyr) to the TCAc through the anaplerotic pathway or pyruvate dehydrogenase (B) Net flux of ATP equivalents flux (in mmol/g_DCW·h) produced (positive values) or consumed (negative values) in control and Δfld1 strains. Mut: methanol utilisation pathway; Glyc+TCAc: glycolysis plus TCAc; PPP: pentose phosphate pathway.