Grafting the ribulose monophosphate cycle for methanol assimilation into Komagataella phaffii

Background.

Methanol assimilation has evolved several times in nature, both in bacteria and in yeasts. The key step in all pathways is the C-C bond formation of the single carbon substrate which happens usually at the oxidation state of formaldehyde. The acceptor molecule is either a pentose phosphate or tetrahydrofolate. The pentose phosphate pathways are cyclic to enable regeneration of the acceptor molecule (Cotton et al. 2020). In methylotrophic yeasts such as Komagataella phaffii (Pichia pastoris) formaldehyde reacts with xylulose 5-phosphate (Xu5P) by dihydroxyacetone synthase (DAS) in a transketolase reaction to dihydroxyacetone and glyceraldehyde 3-phosphate (G3P), and Xu5P is regenerated in three cycles per molecule of G3P. This pathway is confined to peroxisomes and utilizes an extra set of enzymes (similar to pentose phosphate pathway enzymes but methanol regulated and compartmentalized (Rußmayer et al. 2015). One bacterial pathway is based on ribulose 5-phosphate. Formaldehyde is assimilated by 3-hexulose-6-phosphate synthase (HPS), and utilization sets off by 6-phospho-3-hexuloisomerase (PHI) (Cotton et al. 2020). All other reactions of the ribulose monophosphate (RuMP) cycle (or at least some variants of it) are present in K. phaffii peroxisomes as well.

Aims and methods.

It is postulated that the natural yeast XuMP cycle can be converted to the RuMP cycle by a minimum of interventions, namely deletion of the two DAS genes, and overexpression of bacterial HPS and PHI genes. Activities of the heterologous enzymes will be determined. If growth on methanol cannot be achieved in these strains directly, adaptive laboratory evolution will be applied as recently demonstrated in Escherichia coli to functionalize a heterologous RuMP cycle (Keller et al. 2022). When growth is achieved further fine-tuning will be performed to create different variants of the RuMP cycle which are described being more energy efficient than the XuMP cycle (Cotton et al. 2020). 13C tracer analysis will be employed to verify assimilation of methanol via the postulated pathways, and growth experiments will be used to determine growth associated ATP demand of the different pathway variants. Non-stationary ¹³C flux analysis will be set up and used to determine metabolic flux distributions.

Collaboration partners within this project are Stephan Hann and Özge Ata.

Cotton CA, Claassens NJ, Benito-Vaquerizo S, Bar-Even A. 2020. Renewable methanol and formate as microbial feedstocks. Curr Opin Biotechnol. 62:168-180. doi: 10.1016/j.copbio.2019.10.002.
Keller P, Reiter MA, Kiefer P, Gassler T, Hemmerle L, Christen P, Noor E, Vorholt JA. 2022. Generation of an Escherichia coli strain growing on methanol via the ribulose monophosphate cycle. Nat Commun. 13(1):5243. doi: 10.1038/s41467-022-32744-9.
Rußmayer H, Buchetics M, Gruber C, Valli M, Grillitsch K, Modarres G, Guerrasio R, Klavins K, Neubauer S, Drexler H, Steiger M, Troyer C, Al Chalabi A, Krebiehl G, Sonntag D, Zellnig G, Daum G, Graf AB, Altmann F, Koellensperger G, Hann S, Sauer M, Mattanovich D, Gasser B. 2015. Systems-level organization of yeast methylotrophic lifestyle. BMC Biol. 13:80. doi: 10.1186/s12915-015-0186-5.