Metabolic engineering of methylotrophic Pichia pastoris for the production of β-alanine

β-Alanine (3-aminopropionic acid) is the only naturally occurring β-amino acid and an important precursor for the synthesis of a variety of nitrogen-containing chemicals. Fermentative production of β-alanine from renewable feedstocks such as glucose has attracted significant interest in recent years. Methanol has become an emerging and promising renewable feedstock for biomanufacturing as an alternative to glucose. In this work, we demonstrated the feasibility of β-alanine production from methanol using Pichia pastoris (Komagataella phaffii) as a methylotrophic cell factory. L-Aspartate-α-decarboxylases (ADCs) from different sources were screened and expressed in P. pastoris, followed by the optimization of aspartate decarboxylation by increasing the ADC copy number and C4 precursor supply via the overexpression of aspartate dehydrogenase. The production potential of the best strain was further evaluated in a 1-L fermenter, and a β-alanine titer of 5.6 g/L was obtained. To our best knowledge, this is the highest metabolite production titer ever reached in P. pastoris using methanol as the substrate.