Novel tools for high-throughput genetic engineering, selection, and screening for improved Biotin production in E. coli cell factories
(2023) KBTM01 20231Biotechnology (MSc)
Biotechnology (M.Sc.Eng.)
- Abstract
- Biotin is an essential co-factor in various metabolic processes and is crucial for the growth and maintenance of living organisms. The market need for biotin is multifaceted and driven by its importance in human health, beauty and cosmetics applications, pharmaceuticals, animal nutrition, and other industries. Current methods of industrial biotin production are unsustainable and generate environmentally hazardous waste, fueling the demand for new, greener strategies. The production of biotin through microbial fermentation has thus become an attractive alternative. Biotin biosynthesis is a complex and energy-intensive process that requires several enzymatic reactions, and biotin titers in microbial production have not reached economically... (More)
- Biotin is an essential co-factor in various metabolic processes and is crucial for the growth and maintenance of living organisms. The market need for biotin is multifaceted and driven by its importance in human health, beauty and cosmetics applications, pharmaceuticals, animal nutrition, and other industries. Current methods of industrial biotin production are unsustainable and generate environmentally hazardous waste, fueling the demand for new, greener strategies. The production of biotin through microbial fermentation has thus become an attractive alternative. Biotin biosynthesis is a complex and energy-intensive process that requires several enzymatic reactions, and biotin titers in microbial production have not reached economically viable levels. Biotin Synthase (BioB), the enzyme that catalyzes the last step of this process in E. coli, the conversion of dethiobiotin to biotin, has been identified as the bottleneck for this pathway. Overexpression of the enzyme generates oxidative stress and inhibits growth, though the exact mechanism has not yet been elucidated. This thesis employed a multiplexed engineering approach via high-throughput genetic engineering, selection, and screening methods to explore whether creating E. coli strains more resistant to oxidative stress would lead to higher biotin production capabilities. Using the expression of a DNA methylase to introduce controlled genetic mutations in Biosyntia's proprietary biotin-producing E. coli strains and selecting strains with improved resistance to oxidative stress yielded a selection of strains with increased resistance, which were screened for their biotin production. The improved resistance to oxidative stress and BioB induction in the strains did not lead to higher biotin production levels. (Less)
- Popular Abstract
- The global need for biotin in the food, feed, and cosmetics industries is increasing steadily. Because current production methods use petrochemicals as precursors for synthesis, they are unsustainable and generate environmental waste. This has fueled the demand for alternative, more sustainable strategies. Biotin production via microbial fermentation could be a greener alternative but has not reached economically viable levels and cannot compete with current production methods. E. coli can naturally produce biotin but does so in minute amounts. This thesis aims to improve biotin production levels in biotin-producing E. coli strains to contribute towards a greener, more sustainable process.
A bottleneck in the biosynthetic pathway, which... (More) - The global need for biotin in the food, feed, and cosmetics industries is increasing steadily. Because current production methods use petrochemicals as precursors for synthesis, they are unsustainable and generate environmental waste. This has fueled the demand for alternative, more sustainable strategies. Biotin production via microbial fermentation could be a greener alternative but has not reached economically viable levels and cannot compete with current production methods. E. coli can naturally produce biotin but does so in minute amounts. This thesis aims to improve biotin production levels in biotin-producing E. coli strains to contribute towards a greener, more sustainable process.
A bottleneck in the biosynthetic pathway, which has limited production levels, has been identified in previous research: the enzyme BioB, which catalyzes the last step of biotin synthesis. This enzyme creates oxidative stress in the cells and thereby inhibits cell growth. It was hypothesized that creating strains more resistant to oxidative stress and higher levels of intracellular BioB could lead to higher biotin production capabilities. By generating a controlled level of genetic diversity or introducing new genes into the strains and then selecting those with higher oxidative stress resistance, this thesis aims to find a strain that can produce higher levels of biotin. By nature, the randomness of the mutagenesis process generates a high number of strains that need to be tested for their biotin production. In this thesis, the number of strains that need to be tested is systematically reduced by selecting strains with a higher oxidative stress resistance by exposing them to various stressor concentrations. Only the fittest, most resistant strains survive this and are subsequently tested for their biotin-production capabilities.
The work revealed that strains with higher resistance to oxidative stress and BioB production did not produce higher levels of biotin, indicating other limiting factors in the biosynthetic pathway. However, further investigations are needed to draw definitive conclusions, as not all the data generated during this project is reliable due to measurement issues. (Less)
Please use this url to cite or link to this publication:
http://lup.lub.lu.se/student-papers/record/9139321
- author
- Vellguth, Wibke LU
- supervisor
-
- Nélida Leiva LU
- organization
- course
- KBTM01 20231
- year
- 2023
- type
- H2 - Master's Degree (Two Years)
- subject
- keywords
- biotin, BioB, biotin synthase, oxidative stress, E. coli, biotechnology
- language
- English
- id
- 9139321
- date added to LUP
- 2023-10-02 13:17:35
- date last changed
- 2023-10-02 13:17:35
@misc{9139321,
abstract = {{Biotin is an essential co-factor in various metabolic processes and is crucial for the growth and maintenance of living organisms. The market need for biotin is multifaceted and driven by its importance in human health, beauty and cosmetics applications, pharmaceuticals, animal nutrition, and other industries. Current methods of industrial biotin production are unsustainable and generate environmentally hazardous waste, fueling the demand for new, greener strategies. The production of biotin through microbial fermentation has thus become an attractive alternative. Biotin biosynthesis is a complex and energy-intensive process that requires several enzymatic reactions, and biotin titers in microbial production have not reached economically viable levels. Biotin Synthase (BioB), the enzyme that catalyzes the last step of this process in E. coli, the conversion of dethiobiotin to biotin, has been identified as the bottleneck for this pathway. Overexpression of the enzyme generates oxidative stress and inhibits growth, though the exact mechanism has not yet been elucidated. This thesis employed a multiplexed engineering approach via high-throughput genetic engineering, selection, and screening methods to explore whether creating E. coli strains more resistant to oxidative stress would lead to higher biotin production capabilities. Using the expression of a DNA methylase to introduce controlled genetic mutations in Biosyntia's proprietary biotin-producing E. coli strains and selecting strains with improved resistance to oxidative stress yielded a selection of strains with increased resistance, which were screened for their biotin production. The improved resistance to oxidative stress and BioB induction in the strains did not lead to higher biotin production levels.}},
author = {{Vellguth, Wibke}},
language = {{eng}},
note = {{Student Paper}},
title = {{Novel tools for high-throughput genetic engineering, selection, and screening for improved Biotin production in E. coli cell factories}},
year = {{2023}},
}