Screening non-conventional yeasts for acid tolerance and engineering Pichia occidentalis for production of muconic acid

Abstract

Saccharomyces cerevisiae is a workhorse of industrial biotechnology owing to the organism's prominence in alcohol fermentation and the suite of sophisticated genetic tools available to manipulate its metabolism. However, S. cerevisiae is not suited to overproduce many bulk bioproducts, as toxicity constrains production at high titers. Here, we employ a high-throughput assay to screen 108 publicly accessible yeast strains for tolerance to 20 g L^-1 adipic acid (AA), a nylon precursor. We identify 15 tolerant yeasts and select Pichia occidentalis for production of cis,cis-muconic acid (CCM), the precursor to AA. By developing a genome editing toolkit for P. occidentalis, we demonstrate fed-batch production of CCM with a maximum titer (38.8 g L^-1), yield (0.134 g g^-1 glucose) and productivity (0.511 g L^-1 h^-1) that surpasses all metrics achieved using S. cerevisiae. This work brings us closer to the industrial bioproduction of AA and underscores the importance of host selection in bioprocessing.

Fig. 1. Growth of 83 candidate yeast strains in YPD supplemented with citric and adipic acids.

a Growth of yeast strains in YPD compared to YPD supplemented with 0.1 M citric acid (YPD_cit). b Growth of yeast strains in YPD compared to YPD supplemented with 0.15 M adipic acid (YPD_AA). Growth was assessed by measuring area under the curve (AUC) of growth curves. Each data point represents a single replicate of n = 3 independent biological replicates for each yeast strain. Dashed lines show average AUCs for S. cerevisiae CEN.PK113-7D in each growth condition. Source data are provided as a Source Data file.

Fig. 2. Growth of 10 candidate Pichia strains in various dicarboxylic acids.

a Maximum growth rates of candidate strains in 3× YNB supplemented with succinic, glutaric, or adipic acids (0.15 M each). All dicarboxylic-acid-containing media were pH adjusted to 2.8 after the addition of 0.15 M of the respective dicarboxylic acid. Acidic medium is 3× YNB medium with the pH adjusted to 2.8 and neutral medium is 3× YNB medium without pH adjustment. Error bars represent the mean ± s.d. of n = 3 independent biological samples. b Representative growth curves of S. cerevisiae CEN.PK and P. occidentalis strain Y-7552 in 3× YNB supplemented with 0.15 M (22 g L⁻¹) adipic acid. Growth was assessed by measuring OD₆₀₀ in arbitrary units (arb. units). Three independent biological replicates are overlaid for each growth condition. Source data are provided as a Source Data file.

Fig. 3. Xylose utilization, antibiotic susceptibility, plasmid transformation, and CRISPR-Cas9 editing of adipic acid tolerant Pichia.

a Xylose utilization, antibiotic susceptibility and transformation efficiency of adipic acid tolerant Pichia spp. Strains are sorted by adipic acid tolerance (AUC in YPD containing 20 g L⁻¹ adipic acid) and xylose utilization and susceptibility to four common yeast antibiotics is shown. Transformation efficiency using a plasmid conferring Hyg^R is provided rounded to the nearest order of magnitude. Transformants of P. kudriavzevii 2658 NCYC and P. membranifaciens NCYC55 were not detected (ND). Repeating plasmid transformation and antibiotic susceptibility assays routinely yielded similar results. b ADE2 deletion strategy utilized in adipic acid tolerant Pichia. Large homology regions (~800 bp) were designed flanking the ADE2 coding sequence. c Structure of ADE2 pCas CRISPR-Cas9 vectors containing precloned ade2Δ donor cassettes. Donor cassettes were precloned into the BglII site of pCas vectors. d Gap repair strategy of pCas vector assembly in adipic acid tolerant Pichia. pCas vectors harboring precloned ade2Δ donor cassettes were linearized by digestion with BsaI or NotI and used to transform Pichia species along with an overlapping ADE2 gRNA PCR product. e Deletion of ADE2 yields pink colonies in adipic acid tolerant Pichia. Transformants were plated onto YPD agar plates containing hygromycin without adenine supplementation. A representative YPD agar plate containing P. occidentalis Y-7552 is shown. f Screening ADE2 deletion in adipic acid tolerant Pichia. A pCas-Hyg-CEN6ARS4 vector possessing an ADE2 gRNA and repair donor was transferred to P. kluyveri and three strains of P. occidentalis. The ADE2 locus was screened for deletion in randomly selected Hyg^R transformants prior to color development. Source data are provided as a Source Data file.

Fig. 4. Characterization of P. occidentalis promoters and terminators.

a OD-normalized fluorescence intensity of P. occidentalis Y-7552 strains expressing common GFP gene variants. GFP-encoding variants were expressed using P_TDH3 and T_RPL3 from P. occidentalis Y-7552. GFP-expressing colonies are shown with UV exposure. b OD-normalized fluorescence intensity of mNeonGreen expressed from 12 P. occidentalis gene promoters with the RPL3 terminator. c OD-normalized fluorescence intensity of mNeonGreen expressed from the GPM1 promoter and 12 P. occidentalis gene terminators. Error bars represent the mean ± s.d. of n = 3 independent biological samples. All GFP constructs were integrated into the FCY1 locus of P. occidentalis. Source data are provided as a Source Data file.

Fig. 5. Engineering P. occidentalis Y-7552 for efficient synthesis of CCM.

a Heterologous pathway for synthesis of CCM from endogenous 3-dehydroshikimate (DHS). The native yeast shikimate pathway is shown in blue, while the heterologous CCM synthesis pathway is shaded and shown in red. b Titers of CCM pathway intermediates in culture supernatants of successive engineered P. occidentalis production strains. Metabolite titers are depicted in g L⁻¹ for PCA, CAT, and CCM. Asterisks (*) denote a significant increase or decrease in metabolite titer relative to the respective parent strain (P < 0.05). Statistical differences between parent and derivative strains were tested using two-tailed Student’s t-test. Error bars represent the mean ± s.d. of n = 3 independent biological samples. c Cultivation of a CCM-producing P. occidentalis strain (LP635) in a fed-batch fermentor using a mineral medium at pH 6.0. Growth of biomass (OD₆₀₀) and accumulation of CCM pathway metabolites in the culture medium were measured during cultivation. OD₆₀₀ is reported in arbitrary units (arb. units). CAT catechol, CCM cis,cis-muconic acid, DAHP 3-deoxy-d-arabinoheptulosonate 7-phosphate, DHQ 3-dehydroquinate, DHS 3-dehydroshikimate, E4P erythrose 4-phosphate, FMN flavin mononucleotide, PCA protocatechuic acid, PEP phosphoenolpyruvate, prFMN prenylated flavin mononucleotide. Source data are provided as a Source Data file.