Study of genomic stability of heterologous production at simulated industrial-scale cell concentration with Yarrowia lipolytica
(2019) KMBM01 20191Applied Microbiology
Biotechnology
- Abstract
- Despite the increasing level and number of techniques deployed to generate more robust, better-producing microbial cell factories for production of chemicals, the number of products that reach commercial feasibility is still very low. Many hurdles come along the way as it has been observed that strains designed in the laboratory do not perform as expected in industrial-scale bioreactors. For instance, the production of the desired chemical is often in direct conflict with cell growth, resulting in a selection pressure to disrupt the production and restore a higher growth rate. In the light of this issue, four different strains of Yarrowia lipolytica were engineered by site-specific or multi-copy integration to have multiple copies of... (More)
- Despite the increasing level and number of techniques deployed to generate more robust, better-producing microbial cell factories for production of chemicals, the number of products that reach commercial feasibility is still very low. Many hurdles come along the way as it has been observed that strains designed in the laboratory do not perform as expected in industrial-scale bioreactors. For instance, the production of the desired chemical is often in direct conflict with cell growth, resulting in a selection pressure to disrupt the production and restore a higher growth rate. In the light of this issue, four different strains of Yarrowia lipolytica were engineered by site-specific or multi-copy integration to have multiple copies of heterologous genes for the biosynthesis of a product of biotechnological interest; either β-carotene or fatty alcohols. These strains were subjected to adaptive laboratory evolution (ALE) until successive subculturing reached between 60-80 generations; resembling the amount of cells at industrial-scale conditions. Then, production levels were evaluated for both non-evolved and evolved strains. For β-carotene, production decreased around 90% for both site-specific and multi-copy strains. And for fatty alcohols, there was no significant decrease in the production levels. Then, to correlate the production levels with a change in growth behavior, μmax was measured for strains before and after evolution. β-carotene producers displayed a significantly higher growth rate after ALE (between 20-30%); which rendered these strains not suitable for industrial-scale production of β-carotene. Furthermore, no integration stability has proven to be remarkably more stable than the other regarding β-carotene production. Fatty alcohol producers did not show any significant change in growth after ALE. The lack of any observed difference in the fatty alcohol strains was determined to be the result of product degradation, which must be addressed before future work can proceed. However, the β-carotene producers are fit to be sent for whole-genome sequencing (WGS), which could help reveal the mechanism by which the production is disrupted. (Less)
- Popular Abstract
- In this project, it was shown that an industrial yeast was not able to sustain production at industrially-relevant volumes after continuous growth over time.
The global economy is controlled by petroleum nowadays. For decades, it has supplied all our needs by creating the chemical compounds necessary to manufacture the things we encounter in our everyday lives. However, our society is on the brink of collapse as we know it. Climate crisis is a reality and our actions are not enough to mitigate its consequences. One alternative to unsustainable oil is cell factories. Scientists have always wondered what’s inside microorganisms. Why do they live? What is their function in the planet? Are they beneficial/detrimental for us? One thing we... (More) - In this project, it was shown that an industrial yeast was not able to sustain production at industrially-relevant volumes after continuous growth over time.
The global economy is controlled by petroleum nowadays. For decades, it has supplied all our needs by creating the chemical compounds necessary to manufacture the things we encounter in our everyday lives. However, our society is on the brink of collapse as we know it. Climate crisis is a reality and our actions are not enough to mitigate its consequences. One alternative to unsustainable oil is cell factories. Scientists have always wondered what’s inside microorganisms. Why do they live? What is their function in the planet? Are they beneficial/detrimental for us? One thing we know for sure is that they can deliver chemical compounds that have industrial applicability as products of biotechnological interest. We also have the tools to modify them so that they become production machines; the so-called cell factories. Nevertheless, engineered microorganisms do not perform as expected in industrial settings, since they usually drop production in exchange for growing better. These bugs don’t like to be stressed to produce compounds, they just want to live happily and grow as fast as possible!
In my project, the yeast Yarrowia lipolytica was engineered to produce either β-carotene or fatty alcohols; both products of biotechnological interest, by introducing many copies of genes by two different strategies (to find out the most stable) that enable their production. These yeasts were left growing for several generations in lab-scale in plates the same way they would grow for an industrial setup in bioreactors. After measuring the production of the compounds of interest, both decreased and yeasts showed more production fitness (only for β-carotene). Moreover, both strategies were equally unstable. This means that this problem is hampering the industrial application of microorganisms and therefore to help us stop pumping oil out of the ground. Innovative ways to engineer microorganisms so that they stop disliking producing a lot of a compound of interest are needed in order to generate a bio-based economy, which would create new jobs and rethink the whole system of chemicals manufacturing; towards a more sustainable industry.
My work could be used to show how easy it is to lose production in an industrial strain, but more importantly, future work should focus on analyzing the whole genome to unravel the mechanism by which production is lost in exchange of growth. This would light up new inventions to overcome this problem. (Less)
Please use this url to cite or link to this publication:
http://lup.lub.lu.se/student-papers/record/8997195
- author
- Cantero Torrecilla, Manuel LU
- supervisor
- organization
- course
- KMBM01 20191
- year
- 2019
- type
- H2 - Master's Degree (Two Years)
- subject
- keywords
- biotechnology, metabolic engineering, synbio, yeast, yarrowia, oleaginous, fatty alcohols, beta-carotene, applied microbiology, teknisk mikrobiologi
- language
- English
- id
- 8997195
- date added to LUP
- 2019-10-29 14:28:45
- date last changed
- 2019-10-29 14:28:45
@misc{8997195,
abstract = {{Despite the increasing level and number of techniques deployed to generate more robust, better-producing microbial cell factories for production of chemicals, the number of products that reach commercial feasibility is still very low. Many hurdles come along the way as it has been observed that strains designed in the laboratory do not perform as expected in industrial-scale bioreactors. For instance, the production of the desired chemical is often in direct conflict with cell growth, resulting in a selection pressure to disrupt the production and restore a higher growth rate. In the light of this issue, four different strains of Yarrowia lipolytica were engineered by site-specific or multi-copy integration to have multiple copies of heterologous genes for the biosynthesis of a product of biotechnological interest; either β-carotene or fatty alcohols. These strains were subjected to adaptive laboratory evolution (ALE) until successive subculturing reached between 60-80 generations; resembling the amount of cells at industrial-scale conditions. Then, production levels were evaluated for both non-evolved and evolved strains. For β-carotene, production decreased around 90% for both site-specific and multi-copy strains. And for fatty alcohols, there was no significant decrease in the production levels. Then, to correlate the production levels with a change in growth behavior, μmax was measured for strains before and after evolution. β-carotene producers displayed a significantly higher growth rate after ALE (between 20-30%); which rendered these strains not suitable for industrial-scale production of β-carotene. Furthermore, no integration stability has proven to be remarkably more stable than the other regarding β-carotene production. Fatty alcohol producers did not show any significant change in growth after ALE. The lack of any observed difference in the fatty alcohol strains was determined to be the result of product degradation, which must be addressed before future work can proceed. However, the β-carotene producers are fit to be sent for whole-genome sequencing (WGS), which could help reveal the mechanism by which the production is disrupted.}},
author = {{Cantero Torrecilla, Manuel}},
language = {{eng}},
note = {{Student Paper}},
title = {{Study of genomic stability of heterologous production at simulated industrial-scale cell concentration with Yarrowia lipolytica}},
year = {{2019}},
}