Metabolic engineering of the fungal D-galacturonate pathway: Dissertation

Joosu Kuivanen

Research output: ThesisDissertation

Abstract

Industrial biotechnology is one of the enabling technologies for biorefineries. In addition to biofuels, several platform and fine chemicals can be produced from biomass taking advantage of metabolic pathways in the cell. However, genetic engineering is often needed to redirect the cellular metabolism towards a product of interest. In this thesis, one of these metabolic pathways catabolizing constituents of pectin - the catabolic D-galacturonate pathway in filamentous fungi- was engineered and redirected to desired end products. Biotechnological production of L-galactonic acid, a potential platform chemical, was demonstrated in this thesis for first time. The production was obtained in Aspergillus niger and Hypocrea jecorina (Trichoderma reesei) by deleting the second gene, encoding a dehydratase, from the fungal D-galacturonate pathway. In addition to the production from pure D-galacturonic acid, a consolidated bioprocess from citrus processing waste, a pectin-rich biomass was investigated. L-galactonic acid can be lactonised and further oxidised to L-ascorbic acid (vitamin C) via chemical or biochemical routes. In this thesis, an A. niger strain was engineered for direct conversion of D-galacturonic acid to L-ascorbic acid by introducing two plant genes: L-galactono-1,4-lactone lactonase and dehydrogenase. The resulting strains were capable of L-ascorbic acid production from pure D-galacturonic acid or citrus processing waste. In addition to lactonization, two other biochemical reactions towards L-galactonic acid are known: dehydration, which is the second reaction in the fungal Dgalacturonate pathway, and oxidation to D-tagaturonic acid, which occurs in the catabolic L-galactonic acid pathway in bacteria. Both of these biochemical reactions and responsible enzymes from A. niger and Escherichia coli were investigated more detailed. As a result, the substrate specifities for four dehydratases from A. niger were determined and the bacterial L-galactonate-5-dehydrogenase was characterised and applied in a colorimetric assay for Lgalactonic and L-gulonic acids. Pectin-rich biomass has potential as a raw material for the production of renewable chemicals. This thesis presents new ways to utilise this residual biomass by using industrial biotechnology. In addition, the thesis broadens basic understanding of the fungal catabolic D-galacturonate pathway and how it can be engineered for production of useful chemicals.
Original languageEnglish
QualificationDoctor Degree
Awarding Institution
  • Aalto University
Supervisors/Advisors
  • Frey, Alexander D., Supervisor, External person
  • Richard, Peter, Advisor
Award date23 Oct 2015
Place of PublicationEspoo
Publisher
Print ISBNs978-951-38-8340-9
Electronic ISBNs978-951-38-8341-6
Publication statusPublished - 2015
MoE publication typeG5 Doctoral dissertation (article)

Fingerprint

Metabolic engineering
Ascorbic Acid
Biomass
Hydro-Lyases
Biotechnology
Oxidoreductases
Genetic engineering
Gene encoding
Biofuels
Aspergillus
Processing
Fungi
Dehydration
Metabolism
Escherichia coli
Assays
Bacteria
Raw materials
Genes
Oxidation

Keywords

  • filamentous fungi
  • Aspergillus niger
  • pectin
  • D-galacturonic acid
  • Lgalactonic acid
  • L-ascorbic acid
  • metabolic engineering

Cite this

Kuivanen, J. (2015). Metabolic engineering of the fungal D-galacturonate pathway: Dissertation. Espoo: VTT Technical Research Centre of Finland.
Kuivanen, Joosu. / Metabolic engineering of the fungal D-galacturonate pathway : Dissertation. Espoo : VTT Technical Research Centre of Finland, 2015. 122 p.
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abstract = "Industrial biotechnology is one of the enabling technologies for biorefineries. In addition to biofuels, several platform and fine chemicals can be produced from biomass taking advantage of metabolic pathways in the cell. However, genetic engineering is often needed to redirect the cellular metabolism towards a product of interest. In this thesis, one of these metabolic pathways catabolizing constituents of pectin - the catabolic D-galacturonate pathway in filamentous fungi- was engineered and redirected to desired end products. Biotechnological production of L-galactonic acid, a potential platform chemical, was demonstrated in this thesis for first time. The production was obtained in Aspergillus niger and Hypocrea jecorina (Trichoderma reesei) by deleting the second gene, encoding a dehydratase, from the fungal D-galacturonate pathway. In addition to the production from pure D-galacturonic acid, a consolidated bioprocess from citrus processing waste, a pectin-rich biomass was investigated. L-galactonic acid can be lactonised and further oxidised to L-ascorbic acid (vitamin C) via chemical or biochemical routes. In this thesis, an A. niger strain was engineered for direct conversion of D-galacturonic acid to L-ascorbic acid by introducing two plant genes: L-galactono-1,4-lactone lactonase and dehydrogenase. The resulting strains were capable of L-ascorbic acid production from pure D-galacturonic acid or citrus processing waste. In addition to lactonization, two other biochemical reactions towards L-galactonic acid are known: dehydration, which is the second reaction in the fungal Dgalacturonate pathway, and oxidation to D-tagaturonic acid, which occurs in the catabolic L-galactonic acid pathway in bacteria. Both of these biochemical reactions and responsible enzymes from A. niger and Escherichia coli were investigated more detailed. As a result, the substrate specifities for four dehydratases from A. niger were determined and the bacterial L-galactonate-5-dehydrogenase was characterised and applied in a colorimetric assay for Lgalactonic and L-gulonic acids. Pectin-rich biomass has potential as a raw material for the production of renewable chemicals. This thesis presents new ways to utilise this residual biomass by using industrial biotechnology. In addition, the thesis broadens basic understanding of the fungal catabolic D-galacturonate pathway and how it can be engineered for production of useful chemicals.",
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author = "Joosu Kuivanen",
year = "2015",
language = "English",
isbn = "978-951-38-8340-9",
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publisher = "VTT Technical Research Centre of Finland",
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Kuivanen, J 2015, 'Metabolic engineering of the fungal D-galacturonate pathway: Dissertation', Doctor Degree, Aalto University, Espoo.

Metabolic engineering of the fungal D-galacturonate pathway : Dissertation. / Kuivanen, Joosu.

Espoo : VTT Technical Research Centre of Finland, 2015. 122 p.

Research output: ThesisDissertation

TY - THES

T1 - Metabolic engineering of the fungal D-galacturonate pathway

T2 - Dissertation

AU - Kuivanen, Joosu

PY - 2015

Y1 - 2015

N2 - Industrial biotechnology is one of the enabling technologies for biorefineries. In addition to biofuels, several platform and fine chemicals can be produced from biomass taking advantage of metabolic pathways in the cell. However, genetic engineering is often needed to redirect the cellular metabolism towards a product of interest. In this thesis, one of these metabolic pathways catabolizing constituents of pectin - the catabolic D-galacturonate pathway in filamentous fungi- was engineered and redirected to desired end products. Biotechnological production of L-galactonic acid, a potential platform chemical, was demonstrated in this thesis for first time. The production was obtained in Aspergillus niger and Hypocrea jecorina (Trichoderma reesei) by deleting the second gene, encoding a dehydratase, from the fungal D-galacturonate pathway. In addition to the production from pure D-galacturonic acid, a consolidated bioprocess from citrus processing waste, a pectin-rich biomass was investigated. L-galactonic acid can be lactonised and further oxidised to L-ascorbic acid (vitamin C) via chemical or biochemical routes. In this thesis, an A. niger strain was engineered for direct conversion of D-galacturonic acid to L-ascorbic acid by introducing two plant genes: L-galactono-1,4-lactone lactonase and dehydrogenase. The resulting strains were capable of L-ascorbic acid production from pure D-galacturonic acid or citrus processing waste. In addition to lactonization, two other biochemical reactions towards L-galactonic acid are known: dehydration, which is the second reaction in the fungal Dgalacturonate pathway, and oxidation to D-tagaturonic acid, which occurs in the catabolic L-galactonic acid pathway in bacteria. Both of these biochemical reactions and responsible enzymes from A. niger and Escherichia coli were investigated more detailed. As a result, the substrate specifities for four dehydratases from A. niger were determined and the bacterial L-galactonate-5-dehydrogenase was characterised and applied in a colorimetric assay for Lgalactonic and L-gulonic acids. Pectin-rich biomass has potential as a raw material for the production of renewable chemicals. This thesis presents new ways to utilise this residual biomass by using industrial biotechnology. In addition, the thesis broadens basic understanding of the fungal catabolic D-galacturonate pathway and how it can be engineered for production of useful chemicals.

AB - Industrial biotechnology is one of the enabling technologies for biorefineries. In addition to biofuels, several platform and fine chemicals can be produced from biomass taking advantage of metabolic pathways in the cell. However, genetic engineering is often needed to redirect the cellular metabolism towards a product of interest. In this thesis, one of these metabolic pathways catabolizing constituents of pectin - the catabolic D-galacturonate pathway in filamentous fungi- was engineered and redirected to desired end products. Biotechnological production of L-galactonic acid, a potential platform chemical, was demonstrated in this thesis for first time. The production was obtained in Aspergillus niger and Hypocrea jecorina (Trichoderma reesei) by deleting the second gene, encoding a dehydratase, from the fungal D-galacturonate pathway. In addition to the production from pure D-galacturonic acid, a consolidated bioprocess from citrus processing waste, a pectin-rich biomass was investigated. L-galactonic acid can be lactonised and further oxidised to L-ascorbic acid (vitamin C) via chemical or biochemical routes. In this thesis, an A. niger strain was engineered for direct conversion of D-galacturonic acid to L-ascorbic acid by introducing two plant genes: L-galactono-1,4-lactone lactonase and dehydrogenase. The resulting strains were capable of L-ascorbic acid production from pure D-galacturonic acid or citrus processing waste. In addition to lactonization, two other biochemical reactions towards L-galactonic acid are known: dehydration, which is the second reaction in the fungal Dgalacturonate pathway, and oxidation to D-tagaturonic acid, which occurs in the catabolic L-galactonic acid pathway in bacteria. Both of these biochemical reactions and responsible enzymes from A. niger and Escherichia coli were investigated more detailed. As a result, the substrate specifities for four dehydratases from A. niger were determined and the bacterial L-galactonate-5-dehydrogenase was characterised and applied in a colorimetric assay for Lgalactonic and L-gulonic acids. Pectin-rich biomass has potential as a raw material for the production of renewable chemicals. This thesis presents new ways to utilise this residual biomass by using industrial biotechnology. In addition, the thesis broadens basic understanding of the fungal catabolic D-galacturonate pathway and how it can be engineered for production of useful chemicals.

KW - filamentous fungi

KW - Aspergillus niger

KW - pectin

KW - D-galacturonic acid

KW - Lgalactonic acid

KW - L-ascorbic acid

KW - metabolic engineering

M3 - Dissertation

SN - 978-951-38-8340-9

T3 - VTT Science

PB - VTT Technical Research Centre of Finland

CY - Espoo

ER -

Kuivanen J. Metabolic engineering of the fungal D-galacturonate pathway: Dissertation. Espoo: VTT Technical Research Centre of Finland, 2015. 122 p.