Engineering Yarrowia lipolytica for methanol utilization

Abstract

Abstract
The utilization of one-carbon compounds, such as methanol, as a feedstock for production of high value compounds is a fast developing area within cell factory engineering. Methanol is a cheap and easily stored compound, which can be produced from CO2 and renewable energy. Engineering microbial cell factories for utilization of methanol could therefore contribute to decreasing the amounts of abundant CO2 and thereby contribute to a more sustainable world. In this project, the non-conventional oleaginous yeast Yarrowia lipolytica was engineered for utilization of methanol as a substrate. Methanol is a toxic compound which can be oxidized into the even more toxic formaldehyde. Therefore, prior to the introduction of heterologous genes, the methanol and formaldehyde toxicity was determined on Strain A. The cells were determined to tolerate methanol to a level up to X M and formaldehyde to a level of Y mM. From the cultivation data the optimal methanol concentration for cultivations in this project was determined to be Z%. The first step of assimilation of methanol into the core metabolism was to introduce heterologous alcohol dehydrogenases which would oxidase the methanol into formaldehyde. For this, three candidates were tested, Enzyme A, Enzyme B and Enzyme C. The evaluation of the strain containing these genes revealed that Enzyme B and Enzyme C were not functioning well in Y. lipolytica and they were therefore discarded in favour of Enzyme A. Next, Pathway A and Pathway B were integrated separately into the strain carrying the Enzyme A genes. The Pathway A expressing strain showcased the highest efficiency in formaldehyde assimilation as well as in methanol consumption. To sum up, we successfully engineered Y. lipolytica to assimilate methanol. Further evaluation and optimization will be needed on the strain to ensure that the carbon provided by the methanol is contributing to a production of high value compounds.

Popular Abstract

Engineering yeast cells to consume methanol could reduce CO2 emissions
The handling of CO2 emissions is one of the big challenges in today’s society. An excessive use of fossil fuels over several decades have led to an imbalance of the carbon cycle, which has led to an abundance of CO2 in the atmosphere and an increased global warming. Because of this, almost all sectors of industry today are making considerate efforts towards decreasing their CO2 emissions. Electric cars, windmills and increasing alternatives for meat are all examples of strategies developed for reducing the carbon footprint. However, the question still stands, what are we going to do with all the abundant CO2 that already exist in the atmosphere? What if there was a way to utilize this CO2 for a profitable production of compounds and in the process contribute to a better world? Microorganisms might be the answer! In this project, I tried to introduce edits in the genome of the yeast Yarrowia lipolytica to promote consumption of methanol for production of high value compounds.

Microorganisms, in form of yeast, have been utilized for centuries in the form of bread baking and beer brewing. However, it hasn’t been until recently that the true potential of these microorganisms have been discovered. Today, thanks to scientific advances, we are able to engineer these microorganisms and use them in so-called microbial factories to produce a variety of products used in everyday life, such as pharmaceuticals and biofuels. In order for these microbes to be happy and produce these chemicals, they need some kind of food, normally in forms of sugars such as glucose. This provides energy as well as carbon, which is an essential building block for the production of the high value products. This in itself is already a favourable reaction that could however be made even more favourable if the cells were engineered to be fed CO2 instead. One problem however, is that it is very difficult to capture and use CO2 as food for the cells. An alternative could instead be to feed the cells methanol, as there are already methods in place to convert CO2 into methanol.

When first hearing this you might think, hang on, isn’t methanol bad for you? Almost everyone in their teenage years have heard stories about people turning blind when drinking moonshine that was made with methanol instead of ethanol. And yes, it is bad for you, and it is also bad for the cells if they try to consume too much of it. Therefore, before making changes to the genome, I had to determine how much methanol the yeast Y. lipolytica could consume before dying. This was necessary to find the optimal methanol concentrations that would be sufficient to feed the cells but not be so high that it became toxic.

The problem is however, that the Y. lipolytica cells are smart and it knows that methanol is not good for it. Therefore, I also had to teach the cells to consume the methanol. This is done with the so-called CRISPR-Cas9 system, which basically is a molecular pair of scissors that can cut in the genome and paste information into the cells on how to consume methanol. By pasting several batches of information into the genome the cells were able to consume the methanol. However, the cells was still not entirely convinced with the methanol and still needed glucose as well to grow and be happy. The cells therefore has to be provided with additional edits to the genome. By doing this, Y. lipolytica could be used as a cell factory relying solely on methanol as a feedstock that can produce profitable compounds and simultaneously contribute to a more sustainable world.

• author: Olofsson-Dolk, Mattis
• supervisor: Marie-Francoise Gorwa-Grauslund
• organization: Applied Microbiology
• course: KMBM05 20201
• year: 2020
• type: H2 - Master's Degree (Two Years)
• subject: Technology and Engineering
• keywords: Biochemistry, methanol, Yarrowia lipolytica, engineering, applied microbiology, teknisk mikrobiologi
• language: English
• id: 9027084
• date added to LUP: 2020-09-16 13:09:33
• date last changed: 2020-09-16 13:09:33