Engineering yeast to produce bioplasticizers
July 31, 2024
.jpg "Sasha Yogiswara presenting her research at the VIB Seminar")
-written by Sasha Yogiswara, a PhD student at the VIB-KU Leuven Center for Microbiology
The word ‘plastic’ means pliable, easily shaped. But what makes the plastics in our lives so flexible? After all, the plastics we use are made of polymers – long chains of large molecules. How do we ensure that those linked chains are just ‘plastic’ enough for our , from pipes to roofs, to food packaging?
We have plasticizers to thank for that – chemicals that we mix with plastic polymers to make plastics flexible. One type of plasticizer is a compound made from isoamyl alcohol and phthalates. Isoamyl alcohol is currently made from petroleum, which renders it unsustainable.
So, how can we produce isoamyl alcohol more sustainably? The answer is yeast!
Yeast, specifically the species Saccharomyces cerevisiae, is a microorganism that we have used since the beginning of civilization to ferment bread and beer. Upon consuming sugars, yeast produces metabolites such as alcohols, acids, and other aroma compounds. Aside from ethanol, which comprises the largest portion of alcohol produced, yeast naturally also produces isoamyl alcohol, but in very low quantities (around 0.3% of the total alcohol).
Consider then the bioethanol industry, one of the most significant renewable energy transitions away from petroleum. The bioethanol industry uses yeast to produce roughly 100 billion liters annually. And if 0.3% of the total alcohol is isoamyl alcohol, we have been wasting 300 million liters of isoamyl alcohol that could have been used to make plasticizers. We been using this waste stream because it also contains other alcohols, such as isobutanol and propanol, which means we need extra energy and money for distillation.
So, what if the alcohol waste stream contains only isoamyl alcohol, without isobutanol or propanol? Distillation would no longer be needed, and plasticizer producers could directly obtain high-purity isoamyl alcohol from bioethanol producers.
How do we make this happen? Again, the answer is yeast! Or rather, an engineered yeast.
One project in Kevin Verstrepen’s lab at the VIB-KU Leuven Center for Microbiology aims to engineer bioethanol yeasts to produce solely isoamyl alcohol as an alcohol byproduct without compromising ethanol yields. We need two steps to achieve this. First, we screened for a robust S. cerevisiae strain that can produce ethanol (maybe even more than the current commercial bioethanol strain). Then, we modified several genes to optimize the isoamyl alcohol biosynthetic pathway.
Screening for a robust yeast
Industrial yeasts, although classified within the same species, Saccharomyces cerevisiae, have a large genomic and phenotypic diversity depending on where they are isolated from (check out this paper). No yeast strain serves as a one-size-fits-all solution for various industrial applications. For example, strains isolated from beer production may easily consume maltose, a disaccharide in beer wort, but strains isolated from a winery won’t. With this in mind, we decided not to start this project with a standard laboratory strain like many peers in the yeast scientific community.
In the Verstrepen lab, we have more than 1,000 S. cerevisiae strains isolated from different industries, such as beer, wine, bioethanol, bread, and spirits. Using this collection, we searched for strains that can grow in sugarcane molasses, a feedstock for bioethanol production. This experiment was done on a micro-liter scale, using plates containing hundreds of wells to allow fast screening. The top candidates were then tested in flasks for ethanol production yield. We found five strains performing better than the standard commercial bioethanol strain!
Engineering the isoamyl alcohol biosynthesis
The isoamyl alcohol biosynthetic pathway branches from the valine and leucine biosynthetic pathway (an essential amino acid in the yeast cell). Similar to other amino acid biosynthesis, the pathway is highly regulated by the amount of amino acid present in the cell. For example, leucine inhibits one of the enzymes responsible for producing itself, leu4, such that leucine levels in the cell are never too high or too low. The same applies to valine and its enzyme, ilv6. However, to produce high quantities of isoamyl alcohol, we need a high and constant activity of leu4 and ilv6 enzymes.
So, we introduced a single nucleotide change in the LEU4 and ILV6 genes that alters the inhibition site of leucine and valine – no more stop sign, in other words. This strategy resulted in five times more isoamyl alcohol produced, and an increase from 30% to 65% of purity within the waste alcohol stream. Also, ethanol production has even improved.
This project demonstrates the possibility of co-producing isoamyl alcohol with bioethanol. The advantage is that the engineered strain can be incorporated as such into the existing bioethanol infrastructure. Potentially, millions of liters of high-purity isoamyl alcohol could be procured directly from bioethanol producers with minimal post-processing by plasticizer producers.
The co-production strategy can also be useful for many biotechnological applications, especially commodity chemicals, where the full utilization of feedstocks can increase profitability (check out this paper). This strategy can come in many forms and can be as complicated as having two sequential tanks containing different microbial species where one feeds on the other’s byproducts. But it can also be as simple as one microbe and one tank, just like our ethanol-isoamyl alcohol idea.
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