Metabolic engineering of yeast for improved whole-cell biocatalytic amine production
(2025) KMBM01 20251Applied Microbiology
Biotechnology
Biotechnology (MSc)
- Abstract
- Saccharomyces cerevisiae has emerged as a versatile platform for whole-cell biocatalysis, owing to its intrinsic regeneration of cofactors and amenability to genetic engineering. However, efficient reductive amination of aromatic carbonyls remains challenging due to competing redox pathways and product inhibition by pyruvate. Building on the redox-rewired TMB4133 strain (∆gpd1,2) that accumulates high levels of cytosolic NADH, we integrated the Chromobacterium violaceum ω-transaminase (Cv-ATA) via chromosomal YIp‐mediated insertion and co-expressed a Bacillus subtilis alanine dehydrogenase (BsAlaDH) using CRISPR–Cas9. Under fully aerobic and reduced-oxygen conditions in Delft medium supplemented with vanillin, the engineered strain... (More)
- Saccharomyces cerevisiae has emerged as a versatile platform for whole-cell biocatalysis, owing to its intrinsic regeneration of cofactors and amenability to genetic engineering. However, efficient reductive amination of aromatic carbonyls remains challenging due to competing redox pathways and product inhibition by pyruvate. Building on the redox-rewired TMB4133 strain (∆gpd1,2) that accumulates high levels of cytosolic NADH, we integrated the Chromobacterium violaceum ω-transaminase (Cv-ATA) via chromosomal YIp‐mediated insertion and co-expressed a Bacillus subtilis alanine dehydrogenase (BsAlaDH) using CRISPR–Cas9. Under fully aerobic and reduced-oxygen conditions in Delft medium supplemented with vanillin, the engineered strain achieved complete vanillin conversion within 20 h at high cell density, yielding up to 1.26 mM vanillylamine. Flow-cytometric analysis of a GPD2p-yEGFP3 reporter confirmed sustained NADH overabundance, suggesting a link between elevated redox co-substrate availability and enhanced transamination. In contrast, the control strain with intact GPD1 and GPD2 exhibited minimal vanillylamine formation (<0.4 mM). It also showed impaired ethanol assimilation, indicating that this strain could not be used as a suitable control. These results establish a robust, redox-self-sufficient yeast whole-cell catalyst for vanillin amination that can serve as a platform for further studies into the effect of redox balance on transamination. Future work will focus on anaerobic cultivations, multi-replicate validation, and fed-batch reactor implementation to further boost amine yields. (Less)
- Popular Abstract
- Many important compounds, like medicines and perfume ingredients, are often made from special types of chemicals called chiral amines. Making these in factories often requires harsh conditions and expensive additives. Our goal was to turn baker’s yeast into a tiny factory that can produce these amines on its own at around room temperature, needing to add nothing more than some sugar to grow. As a test, we used vanillin and made the yeast convert it to vanillylamine. Normally, yeast cells use helper molecules like NADH and PLP (active forms of vitamins B3 and B6, respectively) to power their chemistry, but many natural pathways compete for these helpers and slow down the desired reaction.
We started with a specially designed yeast strain... (More) - Many important compounds, like medicines and perfume ingredients, are often made from special types of chemicals called chiral amines. Making these in factories often requires harsh conditions and expensive additives. Our goal was to turn baker’s yeast into a tiny factory that can produce these amines on its own at around room temperature, needing to add nothing more than some sugar to grow. As a test, we used vanillin and made the yeast convert it to vanillylamine. Normally, yeast cells use helper molecules like NADH and PLP (active forms of vitamins B3 and B6, respectively) to power their chemistry, but many natural pathways compete for these helpers and slow down the desired reaction.
We started with a specially designed yeast strain called TMB4133. In this strain, two enzymes that normally mop up extra NADH by making glycerol were removed, so the cells end up with plenty of NADH available. Next, we gave these cells two new tools: a “transaminase” enzyme (from a plant-loving bacterium) that transforms vanillin to vanillylamine, and an “alanine dehydrogenase” enzyme (from another harmless bacterium) that recycles one of the helper molecules (NADH) and keeps side products in check. We used stable genetic methods, similar to molecular scissors and glue, to insert these new tools into the yeast DNA so they stay there permanently.
When we grew these engineered yeast cells in simple sugar medium with vanillin, they grew to high density, used up all their sugar, and completely converted vanillin into vanillylamine within 20 hours. In contrast, normal yeast barely made any vanillylamine in the same time. We also had a green fluorescent reporter that lights up when NADH levels rise, and saw bright signals in our engineered strain, proof that our “NADH boost” worked as intended.
Surprisingly, adding these extra enzymes didn’t slow the cells down; instead, they seemed healthier because the extra NADH had somewhere useful to go. The result is a yeast-based process that needs no added chemicals or costly helpers, just sugar and vanillin, and runs at room-friendly temperatures.
Looking ahead, we plan to run this process in larger, controlled fermenters where we can monitor the NADH signal in real time and feed sugar or air at just the right moments. We’ll also try this approach on other natural compounds to make a range of useful amines in a greener, simpler way. (Less)
Please use this url to cite or link to this publication:
http://lup.lub.lu.se/student-papers/record/9208646
- author
- Seibold, Mikael Ralph LU
- supervisor
- organization
- course
- KMBM01 20251
- year
- 2025
- type
- H2 - Master's Degree (Two Years)
- subject
- keywords
- S. cerevisiae, amination, whole-cell bioconversion, metabolic engineering, HPLC, vanillin, vanillylamine, transaminase, applied microbiology
- language
- English
- id
- 9208646
- date added to LUP
- 2025-10-07 13:21:01
- date last changed
- 2025-10-07 13:21:01
@misc{9208646,
abstract = {{Saccharomyces cerevisiae has emerged as a versatile platform for whole-cell biocatalysis, owing to its intrinsic regeneration of cofactors and amenability to genetic engineering. However, efficient reductive amination of aromatic carbonyls remains challenging due to competing redox pathways and product inhibition by pyruvate. Building on the redox-rewired TMB4133 strain (∆gpd1,2) that accumulates high levels of cytosolic NADH, we integrated the Chromobacterium violaceum ω-transaminase (Cv-ATA) via chromosomal YIp‐mediated insertion and co-expressed a Bacillus subtilis alanine dehydrogenase (BsAlaDH) using CRISPR–Cas9. Under fully aerobic and reduced-oxygen conditions in Delft medium supplemented with vanillin, the engineered strain achieved complete vanillin conversion within 20 h at high cell density, yielding up to 1.26 mM vanillylamine. Flow-cytometric analysis of a GPD2p-yEGFP3 reporter confirmed sustained NADH overabundance, suggesting a link between elevated redox co-substrate availability and enhanced transamination. In contrast, the control strain with intact GPD1 and GPD2 exhibited minimal vanillylamine formation (<0.4 mM). It also showed impaired ethanol assimilation, indicating that this strain could not be used as a suitable control. These results establish a robust, redox-self-sufficient yeast whole-cell catalyst for vanillin amination that can serve as a platform for further studies into the effect of redox balance on transamination. Future work will focus on anaerobic cultivations, multi-replicate validation, and fed-batch reactor implementation to further boost amine yields.}},
author = {{Seibold, Mikael Ralph}},
language = {{eng}},
note = {{Student Paper}},
title = {{Metabolic engineering of yeast for improved whole-cell biocatalytic amine production}},
year = {{2025}},
}