Project Inspiration and Description

Methylotrophic yeasts have the potential to convert single carbon compounds such as greenhouse gases into organic compounds of greater value. Currently, there exists engineered Pichia pastoris, a type of methylotrophic yeast, that is capable of converting methanol into medical compounds such as insulin. However, in Pichia pastoris, the metabolism of methanol is highly specific and results in significant oxygen consumption and heat generation, which have limited its industrial applications.

We aim to maximize the methanol conversion rate and lower oxygen consumption and heat generation in Pichia pastoris metabolism. This may be achieved via 1. Refining the metabolic pathways of Pichia pastoris so that it may consume methanol and other carbon sources (such as glycerine) at the same time and 2. Converting metabolic byproducts (formic acid) back to methanol so that methanol may be utilized to the greatest extent.

In Pichia pastoris, there are three transcription factors for the AOX1 gene that encodes aldehyde oxidase (the protein that allows it to metabolize methanol), respectively Prm1, Mit1, and Mxr1. These transcription factors are inhibited in the presence of other carbon sources such as glucose and glycerine.

original metabolic pathway of Pichia pastoris [1]

We plan to re-engineer the metabolic pathways of Pichia pastoris to allow it to simultaneously consume methanol and glycerine, hence maximizing the methanol conversion rate while lowering oxygen consumption and heat generation. This may be achieved by regulating the expression level of Prm1 and Mit1 by interchanging their promoter sequences (Pprm1 and Pmit1).

Mit1 is relatively cytotoxic and Pprm1 is a relatively strong promoter, but the promoter can be inhibited by Mit1. Meanwhile, Pmit1 is a relatively weak promoter, but it can be enhanced by Prm1, which is not very cytotoxic. Therefore, this combination of Pprm1-Mit1 and Pmit1-Prm1 may allow for the moderate overexpression of AOX1 transcription factors Mit1 and Prm1, which in turn should facilitate the expression of aldehyde oxidase despite the presence of glycerine.

We also plan to knock out the gene that facilitates glycerine inhibition to further allow the yeast to metabolize methanol despite the presence of glycerine.