Production of D-lactic acid containing polyhydroxyalkanoate polymers in yeast Saccharomyces cerevisiae

Abstract

Polyhydroxyalkanoates (PHAs) provide biodegradable and bio-based alternatives to conventional plastics. Incorporation of 2-hydroxy acid monomers into polymer, in addition to 3-hydroxy acids, offers possibility to tailor the polymer properties. In this study, poly(D-lactic acid) (PDLA) and copolymer P(LA-3HB) were produced and characterized for the first time in the yeast Saccharomyces cerevisiae. Expression of engineered PHA synthase PhaC1437Ps6–19, propionyl-CoA transferase Pct540Cp, acetyl-CoA acetyltransferase PhaA, and acetoacetyl-CoA reductase PhaB1 resulted in accumulation of 3.6% P(LA-3HB) and expression of engineered enzymes PhaC1Pre and PctMe resulted in accumulation of 0.73% PDLA of the cell dry weight (CDW). According to NMR, P(LA-3HB) contained D-lactic acid repeating sequences. For reference, expression of PhaA, PhaB1, and PHA synthase PhaC1 resulted in accumulation 11% poly(hydroxybutyrate) (PHB) of the CDW. Weight average molecular weights of these polymers were comparable to similar polymers produced by bacterial strains, 24.6, 6.3, and 1 130 kDa for P(LA-3HB), PDLA, and PHB, respectively. The results suggest that yeast, as a robust and acid tolerant industrial production organism, could be suitable for production of 2-hydroxy acid containing PHAs from sugars or from 2-hydroxy acid containing raw materials. Moreover, the wide substrate specificity of PHA synthase enzymes employed increases the possibilities for modifying copolymer properties in yeast in the future.

Control of D-lactic acid content in P(LA-3HB) copolymer in the yeast Saccharomyces cerevisiae using a synthetic gene expression system

“…The endogenous D-lactate dehydrogenase gene ( DLD1 ) of the strain CEN.PK111-9A was deleted with simultaneous integration of codon optimized, stereospecific D-lactate dehydrogenase from L. mesenteroides ( ldhA ) ( Baek et al, 2016 ; Ylinen et al, 2021 ). Deletion was carried with CRISPR/Cas9 method using two gRNA plasmids B11839 and B11840 presented in Table 1 .…”

“…The yeast strains were grown in 50 ml of synthetic complete media supplemented with 20 g L −1 glucose and 0–10 mg L −1 doxycycline in three replicates for 24–72 h at 30 °C with 220 rpm shaking. The cell growth, pH, and metabolite production were analyzed daily as described previously ( Ylinen et al, 2021 ). The cell growth was followed as optical density (OD 600 ), or as CDW.…”

“…The polymers were analyzed as described previously ( Ylinen et al, 2021 ). The cells were washed with distilled water three times and lyophilized over night.…”

“…Production of the P(LA-3HB) copolymer has been studied earlier in vitro and in vivo in two bacterial strains, Escherichia coli and Corynebacterium glutamicum, as reviewed in ( Choi et al, 2020 ), and most recently in vivo by us in the yeast Saccharomyces cerevisiae ( Ylinen et al, 2021 ) . In previous studies polymers with D-lactic acid contents from 3 to 30 mol% ( Kadoya et al, 2015 ), 20–50 mol% ( Yang et al, 2010 ), 8–73 mol% ( Nduko et al, 2014 ), and from 55 to 86 mol% ( Jung et al, 2010 ), were obtained by controlling physiological parameters such as aeration and monomer feeding, and by expressing different PHA synthases.…”

“…Engineering of PHB and mcl-PHA production in other yeasts has resulted in accumulation of up to 25%, 30%, and 52% PHAs of CDW in yeasts Y. lipolytica, Pichia Pastoris , and Arxula adeninivorans, respectively ( Biernacki et al, 2017 ; Rigouin et al, 2019 ; Vijayasankaran et al, 2005 ). In our previous study we engineered the yeast S. cerevisiae for the production of PDLA, PHB, and their copolymer P(LA-3HB) ( Ylinen et al, 2021 ). We obtained D-lactic acid contents of 46–65% in the copolymer P(LA-3HB) by expressing a stereospecific D-lactate dehydrogenase gene ( ldhA ) from Leuconostoc mesenteroides from a constitutive pTDH3 promoter.…”