Enzyme engineering and evolution for improvement of methanol utilization in yeast

Background.

Komagataella phaffii is a yeast species of growing biotechnological interest in part due to its ability to utilize methanol to generate both biomass (assimilation) and energy (dissimilation). The first reaction in the methanol utilization pathway in K. phaffii is carried out by the alcohol oxidase enzymes AOX1 and AOX2, which catalyze the oxidation of methanol to formaldehyde in the peroxisome (Cregg et al. 1989). During assimilation, the formaldehyde then enters the xylulose monophosphate cycle resulting in the production of glyceraldehyde-3-phosphate which may be incorporated into biomass (Rußmayer et al. 2015). Alternatively, the formaldehyde may be dissimilated via a 4-step cytosolic pathway where it is further oxidized to carbon dioxide, with the energy being captured by the production of two molecules of NADH.

The evolved pathway in yeast, while rapid, is energetically wasteful. During the alcohol oxidase-catalyzed reaction, electrons are transferred from methanol to oxygen, producing hydrogen peroxide and generating heat. This reaction could instead be performed by an alcohol dehydrogenase enzyme, as is seen in some thermophilic bacterial species, which would allow for the transfer of electrons from methanol to NAD+. The resulting production of an additional NADH molecule per molecule of methanol consumed would constitute a dramatic increase in efficiency for methanol utilization in yeasts. Previous work in the Mattanovich group in this area revealed that there is a native enzyme in K. phaffii capable of carrying out this reaction. The enzyme identified, Adh2, is a cytosolic, Zn-dependent medium-chain alcohol dehydrogenase with roles in NAD+ recycling and ethanol metabolism (Karaoglan et al. 2016). It was demonstrated that Adh2 can support the oxidation of methanol and its incorporation of into biomass; however, a robust growth phenotype is yet to be achieved (Zavec et al. 2021).

Aims and methods.

The aim of this project is to improve the energetic efficiency of methanol utilization in yeast for applications in biotechnology. The initial focus of the project will be on the native Adh2 enzyme in K. phaffii. The protein will be isolated to determine key biochemical characteristics and high throughput directed evolution will used to increase the catalytic efficiency with methanol as a substrate. As a part of the ACIB Lighthouse Project, an emphasis will be placed on applying the resulting findings for improvement of engineered autotrophic strains of K. phaffii being developed within the group (Gassler et al., 2020). These efforts will then be brought together to generate a yeast strain with improved methanol utilization that can be optimized for production of industrially relevant compounds.

Cregg JM, Madden KR, Barringer KJ, Thill GP, Stillman CA. 1989. Functional characterization of the two alcohol oxidase genes from the yeast Pichia pastoris. Mol Cell Biol. 9:1316-1323
Gassler T, Sauer M, Gasser B, Egermeier M, Troyer C, Causon T, Hann S, Mattanovich D, Steiger MG. 2020. The industrial yeast Pichia pastoris is converted from a heterotroph into an autotroph capable of growth on CO2. Nat Biotechnol. 38:210-216.
Karaoglan M, Karaoglan FE, Inan M. 2016. Functional analysis of alcohol dehydrogenase (ADH) genes in Pichia pastoris. Biotechnol Lett. 38:463-499
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
Zavec D, Troyer C, Maresch D, Altmann F, Hann S, Gasser B, Mattanovich D. 2021. Beyond alcohol oxidase: The methylotrophic yeast Komagataella phaffii utilizes methanol also with its native alcohol dehydrogenase Adh2. FEMS Yeast Res. 21. doi: 10.1093/femsyr/foab009