Significance Methanol-based biomanufacturing is considered a promising way to achieve carbon neutrality. However, efficient methanol biotransformation toward chemical production is challenging. To address this challenge, we enhanced the precursor supply and coenzyme regeneration in Pichia pastoris, combined with reinforcing methanol assimilation, which resulted in significant improvements in fatty acid production. Furthermore, we offered an effective approach for rapidly constructing versatile cell factories that share common precursors. This study represents an important step forward in the rewiring of P. pastoris as industrial microbial cell factories for chemical production.

Methanol biotransformation toward high-level production of fatty acid derivatives by engineering the industrial yeast Pichia pastoris

Synthesis of bioengineered heparin by recombinant yeast Pichia pastoris

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.

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

https://doi.org/10.1016/j.btre.2022.e00710

Heterologous production of chondroitin

High-level de novo biosynthesis of cordycepin by systems metabolic engineering in Yarrowia lipolytica.

Biosynthesis of non-animal chondroitin sulfate from methanol using genetically engineered Pichia pastoris

Chondroitin sulfate (CS) is a linear polysaccharide, which is widely used in medical, health care and other fields. Compared with the traditional animal tissue extraction method, microbial synthesis of CS has the advantages of controllability and easiness of scaling-up. In order to achieve an efficient synthesis of chondroitin sulfate A (CSA), we constructed a recombinant Pichia pastoris GS115 strain capable of synthesizing chondroitin (Ch) from glycerol by introducing the Ch synthase coding genes kfoC, kfoA and UDP-glucose dehydrogenase coding gene tuaD into the P. pastoris chromosome. The titer of Ch reached 2.6 g/L in fed-batch cultures upon optimizing the synthesis pathway of Ch. After further expressing the chondroitin-4-O-sulfotransferase (C4ST), we developed a one-pot biosynthesis system for CSA production by directly adding 3'-adenosine-5'-phosphoryl sulfate and C4ST into the high-pressure homogenized recombinant P. pastoris cells. Eventually, controllable synthesis of 0-40% CSA with different sulfation degrees were achieved by optimizing the catalytic conditions. The one-pot biosynthesis system constructed here is easy to operate and easy to scale up for industrial production of CSA. The idea of the present study may also facilitate the biosynthesis of other glycosaminoglycan, for instance, heparin.

Jingyu Sheng

Papers

Biosynthesis of non-animal chondroitin sulfate from methanol using genetically engineered Pichia pastoris

[One-pot synthesis of chondroitin sulfate A by engineered Pichia pastoris].