Respiratory reoxidation of NADH is a key contributor to high oxygen requirements of oxygen-limited cultures of Ogataea parapolymorpha

Abstract

While thermotolerance is an attractive trait for yeasts used in industrial ethanol production, oxygen requirements of known thermotolerant species are incompatible with process requirements. Analysis of oxygen-sufficient and oxygen-limited chemostat cultures of the facultatively fermentative, thermotolerant species Ogataea parapolymorpha showed its minimum oxygen requirements to be an order of magnitude larger than those reported for the thermotolerant yeast Kluyveromyces marxianus. High oxygen requirements of O. parapolymorpha coincided with a near absence of glycerol, a key NADH/NAD+ redox-cofactor-balancing product in many other yeasts, in oxygen-limited cultures. Genome analysis indicated absence of orthologs of the Saccharomyces cerevisiae glycerol-3-phosphate-phosphatase genes GPP1 and GPP2. Co-feeding of acetoin, whose conversion to 2,3-butanediol enables reoxidation of cytosolic NADH, supported a 2.5-fold increase of the biomass concentration in oxygen-limited cultures. An O. parapolymorpha strain in which key genes involved in mitochondrial reoxidation of NADH were inactivated did produce glycerol, but transcriptome analysis did not reveal a clear candidate for a responsible phosphatase. Expression of S. cerevisiae GPD2, which encodes NAD+-dependent glycerol-3-phosphate dehydrogenase, and GPP1 supported increased glycerol production by oxygen-limited chemostat cultures of O. parapolymorpha. These results identify dependence on respiration for NADH reoxidation as a key contributor to unexpectedly high oxygen requirements of O. parapolymorpha.

Keywords: Ogataea parapolymorpha; Custers effect; anaerobic growth; genome sequence; glycerol metabolism; thermotolerance.

Figure 1.

(A) Reactions and proteins involved in glycerol metabolism in S. cerevisiae. Gpd1 is mainly located in peroxisomes and Gpd2 in the cytosol and in mitochondria (Valadi et al. 2004). Red arrows represent the glycerol-3-phosphate shuttle, the dashed arrow linking DHAP and DHA indicates the hypothetical formation, in non-Saccharomyces yeasts, of glycerol via DHAP phosphatase and NAD(P)H-dependent DHA reductase (blue arrow; Klein et al. 2017). (B) Occurrence of orthologs of S. cerevisiae structural genes encoding glycerol-3P dehydrogenase (Gpd2), glycerol-3P phosphatase (Gpp1), and FAD-dependent mitochondrial glycerol-3P dehydrogenase (Gut2) in Ogataea sp., Brettanomyces (syn. Dekkera) bruxellensis, K. marxianus, and S. cerevisiae. Black and white squares indicate presence and absence, respectively, of orthologs, based on homology searches of whole-genome translated sequences with S. cerevisiae S288c sequences as queries. Species are mapped to the phylogenetic tree of Saccharomycotina yeasts (Shen et al. 2020).

Figure 2.

Genome-wide transcriptional responses of O. parapolymorpha (opar), K. marxianus (kmar), and S. cerevisiae (scer) to oxygen limitation. Aerobic (regime 1, 21 × 10⁴ ppm O₂ in inlet gas, and 7.5 g l^–1 glucose in feed medium) and oxygen-limited (regime 2, 840 ppm O₂ in inlet gas; 20 g l^–1 glucose in feed medium) chemostat cultures were grown at D = 0.1 h^–1 and 30°C. Data for K. marxianus and S. cerevisiae were obtained from a previous study (Dekker et al. 2021). (A) Gene-set enrichment analysis showing GO-terms overrepresented among genes showing a transcriptional response to oxygen limitation (regime 2 versus regime 1) in at least two of the three yeast species. Distinct directionalities calculated with Piano (Väremo et al. 2013) are indicated as distinct-directional down (pdddn), mixed-directional down (pmddn), nondirectional (pnd), mixed-directional up (pmdup), and distinct-directional up (pddup). Hierarchical clustering was based on degree of overrepresentation. Data on all enriched GO-terms for biological processes are shown in Figures S2–S4 (Supporting Information). (B) Log-fold changes (regime 2 versus regime 1) of orthologs in the three yeasts, (C) Orthologs showing higher transcript levels in oxygen-limited cultures of all three yeasts. (D) Orthologs showing lower transcript levels in oxygen-limited cultures of all three yeasts.

Figure 3.

Transcriptional regulation of specific pathways and genes in O. parapolymorpha, K. marxianus, and S. cerevisiae subjected to different aeration regimes. (A) Chemostat cultures were grown on glucose at D = 0.1 h^–1 and 30°C. Regime 1 (aerobic): 21 × 10⁴ ppm O₂ in inlet gas, 7.5 g l^–1 glucose in feed medium); Regime 2 (oxygen limitation): 840 ppm O₂ in inlet gas; 20 g l^–1 glucose in feed; and Regime 3 (extreme oxygen limitation): < 0.5 ppm O₂ medium 20 g l^–1 glucose in feed). Ogataea parapolymorpha washed out under regime 3. Data for K. marxianus and S. cerevisiae were obtained from a previous study (Dekker et al. 2021). In comparisons of transcript levels Regime 1 was used as the reference. (B) Single biochemical reactions are represented by arrows, lumped reactions by dashed arrows; some metabolites and cofactors are omitted to facilitate visualization. Respiratory complexes are indicated by Roman numerals. The O. parapolymorpha genome encodes all subunits of Complex I (Riley et al. 2016), which is absent in S. cerevisiae. Colored boxes indicate upregulation (blue-green) or downregulation (brown), color intensities indicate log 2 fold-change (log FC, capped to a maximum value of 5). Enzymes are indicated as S. cerevisiae orthologs; absence of orthologs in the other yeasts is indicated by grey dots. Abbreviations: G6P, glucose-6-phosphate; F6P, fructose-6-phosphate; F1,6P, fructose-1,6-bisphosphate; DHAP, dihydroxyacetone phosphate; G3P, glycerol-3-phosphate; GAP, glyceraldehyde-3-phosphate; IM, inner mitochondrial membrane; and OM, outer mitochondrial membrane.

Figure 4.

Impact of expression of ScGPP1 and/or ScGPD2 in O. parapolymorpha CBS11895 on glycerol production in oxygen-limited chemostat cultures. Biomass-specific conversion rates (q) were measured in oxygen-limited chemostat cultures of O. parapolymorpha strains (D = 0.1 h^–1; 840 ppm O₂ in inlet gas; and 20 g l^–1 glucose in feed medium). Data on anaerobic and oxygen-limited chemostat cultures of S. cerevisiae CEN.PK113-7D were derived from (Dekker et al. 2021). Symbols: white circles, S. cerevisiae CEN.PK113-7D; blue circles, O. parapolymorpha CBS11895; blue boxes IMX2119 O. parapolymorpha (ScGPP1), blue triangles up O. parapolymorpha IMX2587 (ScGPD2), and blue triangles down O. parapolymorpha IMX2588 (ScGPP1 ScGPD2). Data are represented as mean ± standard deviation of data obtained from independent chemostat cultures of each strain. (A) Biomass-specific glycerol production rates versus biomass-specific oxygen consumption rates. The dashed line depicts a stoichiometric relationship between glycerol production and oxygen consumption in S. cerevisiae cultures, based on the assumption that, for NADH reoxidation, consumption of one mol O₂ corresponds to production of 2 moles of glycerol (Weusthuis et al. 1994). (B) Biomass yields on oxygen versus biomass-specific rates of glycerol production.