Presentation Information
[1Chemi-13]Bioproduction of 3-Hydroxypropionic Acid from glucose: Oxaloacetate Pathway Engineering in Komagataella pastoris.
○Johannes Malherbe Malherbe1, Kim M Trollope1, Johann F Gorgens1, Heinrich Volschenck1 (1. Stellenbosch University (South Africa))
Keywords:
3-hydroxypropionic acid,Komagataella pastoris,Oxaloacetate pathway,Yeast engineering,Sugarcane biorefineries
The yeast Komagataella pastoris, widely used as a recombinant protein producer, has only recently been explored as a microbial cell factory for 3-hydroxypropionic acid (3-HP), primarily from glycerol or methanol via malonyl CoA and β-alanine pathways with reported titers up to 37 g/L and 48 g/L, respectively. No K. pastoris based 3 HP technologies currently target sugar based bioprocesses or biorefineries, and alternative 3-HP biosynthetic pathways remain largely unexplored in yeast.
The oxaloacetate pathway, which is thermodynamically favorable for 3-HP production, has so far only been implemented in Saccharomyces cerevisiae. Flux balance analysis predicted a maximum 3-HP yield of 0.99 g/g glucose for the oxaloacetate pathway in K. pastoris, exceeding those of the malonyl CoA (0.868 g/g) and β-alanine (0.947 g/g) routes. To evaluate this, we constitutively expressed benzoylformate decarboxylase and 3-hydroxyisobutyrate dehydrogenase from Pseudomonas putida to implement the oxaloacetate pathway in K. pastoris. Following extensive screening, a transformant was identified that produced 3.5 g/L 3-HP within 72 hrs, and media optimization increased the titer to 5 g/L from 20 g/L of glucose in 24 deep well cultivations. The strain was then cultivated in 1 L fed batch bioreactors with 40 g/L glucose and a constant 400 g/L glucose feed for 48 hrs. This yielded 50.6 g/L 3 HP, corresponding to 0.25 g/g glucose and a productivity of 0.77 g/L/h. To our knowledge, this is the first report of 3-HP production via the oxaloacetate pathway in K. pastoris and achieves among the highest reported titers, rates and yields for this pathway in a microbial chassis. The performance is comparable to 3-HP processes in S. cerevisiae using other pathways, while requiring fewer strain-engineering steps. These results establish K. pastoris as a promising cell factory for 3 HP production from glucose and other sugar-derived substrates and highlight the value of extensive transformant screening for identifying superior chassis strains.
The oxaloacetate pathway, which is thermodynamically favorable for 3-HP production, has so far only been implemented in Saccharomyces cerevisiae. Flux balance analysis predicted a maximum 3-HP yield of 0.99 g/g glucose for the oxaloacetate pathway in K. pastoris, exceeding those of the malonyl CoA (0.868 g/g) and β-alanine (0.947 g/g) routes. To evaluate this, we constitutively expressed benzoylformate decarboxylase and 3-hydroxyisobutyrate dehydrogenase from Pseudomonas putida to implement the oxaloacetate pathway in K. pastoris. Following extensive screening, a transformant was identified that produced 3.5 g/L 3-HP within 72 hrs, and media optimization increased the titer to 5 g/L from 20 g/L of glucose in 24 deep well cultivations. The strain was then cultivated in 1 L fed batch bioreactors with 40 g/L glucose and a constant 400 g/L glucose feed for 48 hrs. This yielded 50.6 g/L 3 HP, corresponding to 0.25 g/g glucose and a productivity of 0.77 g/L/h. To our knowledge, this is the first report of 3-HP production via the oxaloacetate pathway in K. pastoris and achieves among the highest reported titers, rates and yields for this pathway in a microbial chassis. The performance is comparable to 3-HP processes in S. cerevisiae using other pathways, while requiring fewer strain-engineering steps. These results establish K. pastoris as a promising cell factory for 3 HP production from glucose and other sugar-derived substrates and highlight the value of extensive transformant screening for identifying superior chassis strains.
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