Advanced

An NADH-Coupled Biosensor for Engineering Redox Metabolism in Saccharomyces cerevisiae

Knudsen, Jan LU (2015)
Abstract (Swedish)
Popular Abstract in English

The work described in this thesis is in the field of industrial biotechnology, which can be defined as a branch of biotechnology aimed at sustainable production of chemicals, materials and fuels with the help of microorganisms. Microorganisms are very diverse and possess a wide variety of enzymes allowing them to transform inexpensive molecules into valuable ones, or toxic ones into non-toxic ones. However, most microorganisms are not naturally equipped to mass produce specific, valuable compounds. During recent decades, massive investments have therefore been made in designing and engineering organisms, thus transforming the cells into small factories, for large-scale production. A common... (More)
Popular Abstract in English

The work described in this thesis is in the field of industrial biotechnology, which can be defined as a branch of biotechnology aimed at sustainable production of chemicals, materials and fuels with the help of microorganisms. Microorganisms are very diverse and possess a wide variety of enzymes allowing them to transform inexpensive molecules into valuable ones, or toxic ones into non-toxic ones. However, most microorganisms are not naturally equipped to mass produce specific, valuable compounds. During recent decades, massive investments have therefore been made in designing and engineering organisms, thus transforming the cells into small factories, for large-scale production. A common obstacle is the production of side-products or by-products. These are produced naturally by the cell in response to the environmental conditions, and they lower the yields of the desired primary products. Therefore, if their production could be monitored or measured it would be easier to avoid them, increasing the primary yield and reducing the cost of the final product. In this work I have studied different process related cell properties of baker’s yeast, also known as Saccharomyces cerevisiae, one of the most widely established microbial production organisms. Some of its properties have been exploited for thousands of years, for example, in beer and bread making, but many more applications have been developed in the past century, such as the production of insulin and bioplastics. In my studies, I evaluated how yeast can be optimized to become a more efficient cellular factory for the production of various types of chemicals. In particular, I developed tools for monitoring the cells’ metabolic mode and their need to produce side-products. These efforts resulted in a novel sensor, enabling easier monitoring of the metabolic mode of the cells. This sensor also pointed to the potential of a well-known promoter for the production of user-defined metabolites, and showed promising for use in the search for new enzymes. Additionally I studied how the side-products can be replaced with more valuable chemicals, such as chiral alcohols and amines that can be used as active pharmaceutical ingredients. (Less)
Abstract
Baker’s yeast, Saccharomyces cerevisiae’s potential in industrial biotechnology for producing valuable products e.g. biofuels, bulk chemicals, bio-flavours and pharmaceuticals is established. In order to achieve efficient production, the intracellular balance of the NADH/NAD + redox couple must be monitored and maintained, as this influences the feasibility of over 100 reactions in the cell. My approach based on the inherent ability of S. cerevisiae to control cytosolic NADH/NAD+ homeostasis by regulating the transcription of the glycerol phosphate-3 dehydrogenase 2 (GPD2) gene. It is responsible for glycerol synthesis in the cell, via the concurrent oxidation of NADH. It is known that its promoter is activated when there is a strong need... (More)
Baker’s yeast, Saccharomyces cerevisiae’s potential in industrial biotechnology for producing valuable products e.g. biofuels, bulk chemicals, bio-flavours and pharmaceuticals is established. In order to achieve efficient production, the intracellular balance of the NADH/NAD + redox couple must be monitored and maintained, as this influences the feasibility of over 100 reactions in the cell. My approach based on the inherent ability of S. cerevisiae to control cytosolic NADH/NAD+ homeostasis by regulating the transcription of the glycerol phosphate-3 dehydrogenase 2 (GPD2) gene. It is responsible for glycerol synthesis in the cell, via the concurrent oxidation of NADH. It is known that its promoter is activated when there is a strong need for NADH oxidation. An induction different from that of the homologous gene GPD1, which is induced under osmotic stress. GFP levels were correlated to the need for GPD2 synthesis by cloning a green fluorescent protein (GFP) encoding gene in a plasmid downstream of GPD2p. It showed to be an efficient fluorescent biosensor for monitoring the redox state of the cell, enabling the distinction between strains with different abilities to reoxidize NADH. The sensor was applied to monitor the regulation of GPD2p under various growth conditions. It provided single-cell level information on the mode of metabolism enabling the identification of possible subpopulations. When no aeration was applied, GPD2p could be induced at a level twice that of the constitutively high TDH3 promoter. However, under conditions of high aeration and when the growth rate was maintained at 0.3 h -1 , the promoter was inactive. These findings, together with that a gpd1Δgpd2Δ strain could be cultivated as efficiently as a wild-type strain, demonstrates the possibility of using the gpd1Δgpd2Δ strain as a microbial production platform and the GPD2p as an inducible promoter for recombinant protein production. The technical and physiological boundaries of a gpd1Δpgpd2Δ yeast for FACS-based discovery of xylose or acetophenone reductases were investigated. The reduction of xylose resulted in lower GFP fluorescence, but the opposite was observed when acetophenone was the substrate. Acetophenone was inhibitory to cell growth above 10 mM, and caused significant cell flocculation, challenging FACS-based screening. The results suggest that biosensor-based screening must be performed under well-controlled and substrate-specific conditions, as

the substrate-specific responses may have strong and unforeseen effects on the output signal. The highly reducing potential of the gpd1Δgpd2Δ background strain was separately evaluated for driving NADH-dependent whole-cell bioconversion with a transaminase and a ketone reductase for the production of the chiral pharmaceutical building blocks (R)-1-phenylethylamine and (S)-1-phenylethanol. The gpd1Δgpd2Δ strain exhibited a 3-fold higher reduction rate and a 10-fold lower glucose requirement than the wild-type strain. The results provide detailed information on the need for GPD2 activity, and highlight the potential of the reducing environment of a gpd1Δgpd2Δ strain to replace the production of glycerol with that of more economically interesting compounds. (Less)
Please use this url to cite or link to this publication:
author
supervisor
opponent
  • Professor Larsson, Christer, Chalmers Tekniska Högskola
organization
publishing date
type
Thesis
publication status
published
subject
keywords
Yeast, redox balance, glycerol whole-cell biocatalysis, single-cell monitoring, FACS
categories
Higher Education
pages
175 pages
publisher
Lunds Tekniska Högskola
defense location
Lecture hall B, Kemicentrum, Getingevägen 60, Lund University, Faculty of Engineering LTH, Lund
defense date
2015-06-05 10:30
ISBN
978-91-7422-401-6
language
English
LU publication?
yes
id
0cc4feea-c998-4f57-9875-52d193b102c5 (old id 5368364)
date added to LUP
2015-05-12 07:36:30
date last changed
2016-09-19 08:45:07
@phdthesis{0cc4feea-c998-4f57-9875-52d193b102c5,
  abstract     = {Baker’s yeast, Saccharomyces cerevisiae’s potential in industrial biotechnology for producing valuable products e.g. biofuels, bulk chemicals, bio-flavours and pharmaceuticals is established. In order to achieve efficient production, the intracellular balance of the NADH/NAD + redox couple must be monitored and maintained, as this influences the feasibility of over 100 reactions in the cell. My approach based on the inherent ability of S. cerevisiae to control cytosolic NADH/NAD+ homeostasis by regulating the transcription of the glycerol phosphate-3 dehydrogenase 2 (GPD2) gene. It is responsible for glycerol synthesis in the cell, via the concurrent oxidation of NADH. It is known that its promoter is activated when there is a strong need for NADH oxidation. An induction different from that of the homologous gene GPD1, which is induced under osmotic stress. GFP levels were correlated to the need for GPD2 synthesis by cloning a green fluorescent protein (GFP) encoding gene in a plasmid downstream of GPD2p. It showed to be an efficient fluorescent biosensor for monitoring the redox state of the cell, enabling the distinction between strains with different abilities to reoxidize NADH. The sensor was applied to monitor the regulation of GPD2p under various growth conditions. It provided single-cell level information on the mode of metabolism enabling the identification of possible subpopulations. When no aeration was applied, GPD2p could be induced at a level twice that of the constitutively high TDH3 promoter. However, under conditions of high aeration and when the growth rate was maintained at 0.3 h -1 , the promoter was inactive. These findings, together with that a gpd1Δgpd2Δ strain could be cultivated as efficiently as a wild-type strain, demonstrates the possibility of using the gpd1Δgpd2Δ strain as a microbial production platform and the GPD2p as an inducible promoter for recombinant protein production. The technical and physiological boundaries of a gpd1Δpgpd2Δ yeast for FACS-based discovery of xylose or acetophenone reductases were investigated. The reduction of xylose resulted in lower GFP fluorescence, but the opposite was observed when acetophenone was the substrate. Acetophenone was inhibitory to cell growth above 10 mM, and caused significant cell flocculation, challenging FACS-based screening. The results suggest that biosensor-based screening must be performed under well-controlled and substrate-specific conditions, as<br/><br>
the substrate-specific responses may have strong and unforeseen effects on the output signal. The highly reducing potential of the gpd1Δgpd2Δ background strain was separately evaluated for driving NADH-dependent whole-cell bioconversion with a transaminase and a ketone reductase for the production of the chiral pharmaceutical building blocks (R)-1-phenylethylamine and (S)-1-phenylethanol. The gpd1Δgpd2Δ strain exhibited a 3-fold higher reduction rate and a 10-fold lower glucose requirement than the wild-type strain. The results provide detailed information on the need for GPD2 activity, and highlight the potential of the reducing environment of a gpd1Δgpd2Δ strain to replace the production of glycerol with that of more economically interesting compounds.},
  author       = {Knudsen, Jan},
  isbn         = {978-91-7422-401-6},
  keyword      = {Yeast,redox balance,glycerol whole-cell biocatalysis,single-cell monitoring,FACS},
  language     = {eng},
  pages        = {175},
  publisher    = {Lunds Tekniska Högskola},
  school       = {Lund University},
  title        = {An NADH-Coupled Biosensor for Engineering Redox Metabolism in Saccharomyces cerevisiae},
  year         = {2015},
}