The adaptive landscape of wildtype and glycosylation-deficient populations of the industrial yeast Pichia pastoris

Abstract

Background: The effects of long-term environmental adaptation and the implications of major cellular malfunctions are still poorly understood for non-model but biotechnologically relevant species. In this study we performed a large-scale laboratory evolution experiment with 48 populations of the yeast Pichia pastoris in order to establish a general adaptive landscape upon long-term selection in several glucose-based growth environments. As a model for a cellular malfunction the implications of OCH1 mannosyltransferase knockout-mediated glycosylation-deficiency were analyzed.

Results: In-depth growth profiling of evolved populations revealed several instances of genotype-dependent growth trade-off/cross-benefit correlations in non-evolutionary growth conditions. On the genome level a high degree of mutational convergence was observed among independent populations. Environment-dependent mutational hotspots were related to osmotic stress-, Rim - and cAMP signaling pathways. In agreement with the observed growth phenotypes, our data also suggest diverging compensatory mutations in glycosylation-deficient populations. High osmolarity glycerol (HOG) pathway loss-of-functions mutations, including genes such as SSK2 and SSK4, represented a major adaptive strategy during environmental adaptation. However, genotype-specific HOG-related mutations were predominantly observed in opposing environmental conditions. Surprisingly, such mutations emerged during salt stress adaptation in OCH1 knockout populations and led to growth trade-offs in non-adaptive conditions that were distinct from wildtype HOG-mutants. Further environment-dependent mutations were identified for a hitherto uncharacterized species-specific Gal4-like transcriptional regulator involved in environmental sensing.

Conclusion: We show that metabolic constraints such as glycosylation-deficiency can contribute to evolution on the molecular level, even in non-diverging growth environments. Our dataset suggests universal adaptive mechanisms involving cellular stress response and cAMP/PKA signaling but also the existence of highly species-specific strategies involving unique transcriptional regulators, improving our biological understanding of distinct Ascomycetes species.

Keywords: Experimental evolution; Glucose; OCH1; Pichia Pastoris; Salt stress.

Fig. 1

Experimental setup. a Experimental setup to analyze consequences of environmental adaptation in P. pastoris wildtype and mutant (∆OCH1) populations. Wildtype populations and glycosylation-deficient populations were propagated for 500 generations by serial transfers and subsequently analyzed in terms of growth rates and competitive fitness on the population and single clone level, followed by next generation sequencing (NGS) of selected clones. b Yeast protein N-glycosylation pathway. The Och1 mannosyltransferase mediates the transfer of a mannose moiety to the precursor N-glycan and thereby initiates outer chain elongation and hypermannosylation in yeasts. The prevention of this step by OCH1 deletion results in several growth defects. Ost – Oligosaccharyltransferases, Mns1 – Mannosidase 1, Och1 – outer chain elongation factor 1, MnTs – Mannosyltransferases

Fig. 2

Correlation of growth rates and competitive fitness of evolved heterogeneous P. pastoris populations in adaptive conditions. Growth rate (x-axis) and competitive fitness (y-axis) relative to the ancestral strains. a P. pastoris X-33, (b) BG10 and (c) and BG10 ∆OCH1. Circles represent individual evolved populations adapted to YPD (yellow), YPDN (red), BMD (light blue) and BMDN (dark blue) growth conditions

Fig. 3

Growth rates of heterogeneous P. pastoris populations in non-evolutionary conditions. Growth rates for each genotype background are grouped based on the adaptive growth conditions (a) YPD, (b) YPDN, (c) BMD and (d) BMDN. Colored bars represent the average growth rates of all populations of each condition, whereas dots (green – eGFP populations, red – DsRED population) represent the growth rates of the individual populations. Growth rates: yellow – YPD, red – YPDN, light blue – BMD, dark blue – BMDN; important cross-benefit and trade-off correlations (either similar or contrasting) are highlighted in boxes and marked with arrows

Fig. 4

Mutations identified in evolved P. pastoris populations. a Overlap of mutational targets in the X-33 and BG10 wildtype and the BG10 ∆OCH1 populations. b Number of single and recurrent mutations grouped by growth environment. c Environment-dependent mutations of genes and their associated functional modules. Only recurrently affected modules are shown

Fig. 5

Osmotic stress-related adaptive mutations. a High osmolarity glycerol (HOG) MAPK signaling pathway (b) Mutations of HOG pathway related genes in the evolved P. pastoris clones isolated from independent populations. Mutations are grouped by their appearance in strain background and growth condition. For each growth condition the affected genes are shown. The four clones for each condition are represented by squares. The occurrence of a particular mutation is indicated by color (yellow to red for the presence of a mutation in each individual clone). A blank square indicates the absence of a mutation. Non-HOG-related mutations (ACS1/FLC2) in ∆OCH1 clones are highlighted by a dashed box. (c) Fitness of clones with a single HOG-related or ACS1/FLC2-related mutation (a-d, as described in Tables S13-S15). Values represent averages +/− standard deviation of n = 4; paired Student’s T-test values * p ≤ 0.05, ** p ≤ 0.01

Fig. 6

cAMP/PKA signaling mutations in P. pastoris clones adapted to minimal growth medium (BMD). a Occurrence of mutations grouped by strain background. The occurrence of a particular mutation is indicated by color (yellow to red for the presence of a mutation in each individual clone). A blank square indicates the absence of a mutation. b Enzymatic steps catalyzed by the mutated genes IRA1 and PDE2. c Fitness effect of a single IRA1 mutation in an evolved OCH1 clone (clone ∆OCH1 BMD G2b Tables S13-S15). Values represent averages +/− standard deviation; n = 4; paired Student’s T-test values * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001

Fig. 7

Effect of PAS-chr3_0669 overexpression in the ancestral strains. The effect of overexpression of the GAL4-like gene in comparison to an empty vector strain is shown for all ancestral strains in (a) YPD and YPDN rich media (b) BMD and BMDN minimal media (c) rich and minimal methanol growth media, YPM and BMM and (d) rich glycerol growth media, YPG and YPGN. Values represent averages +/− standard deviation; n = 4; paired Student’s T-test values * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001