Microbial production of 3-hydroxypropionic acid and poly(3-hydroxypropionate): Investigation of Lactobacillus reuteri propanediol utilization pathway enzymes
Author
Summary, in English
Concerns regarding environmental issues such as greenhouse gas emissions and
climate change have led to a shift within the research community and chemical and
energy industry sectors for finding sustainable routes for producing fuels and
chemicals from renewable resources, thereby minimizing our dependence on
petroleum. The C3-chemical 3-hydroxypropionic acid has been identifed as a top
candidate for the biobased chemical industry. This platform chemical is a β-hydroxy
acid containing two functional groups (hydroxyl and carboxyl) enabling its
conversion into value-added chemicals such as 1,3-propanediol, acrolein, malonic
acid, acrylamide and acrylic acid, which can be used in resins, coatings, paints,
adhesives, lubricants, and in the textile industry as anti-static agent. Polymerized 3-
HP, poly(3-hydroxypropionate) (poly(3-HP)), is a biodegradable and stable polymer
which, besides its potential role as a biomaterial, can be degraded to 3-HP monomer.
In recent years, a dramatic increase in the interest for microbial production of 3-HP
and poly(3-HP) has been observed. Metabolic engineering and recombinant
expression of various enzymatic pathways in a number of bacterial strains have been
suggested and implemented, with mainly renewable glucose and glycerol as substrates.
This thesis presents a novel pathway called the propanediol utilization pathway
present in Lactobacillus reuteri that catalyzes dehydration of glycerol to 3-
hydroxypropionaldehyde (3-HPA) and further to 3-HP by a series of reactions
catalyzed by propionaldehyde dehydrogenase (PduP), phosphotransacylase (PduL)
and propionate kinase (PduW). Through structural modeling and kinetic
characterization of PduP, its 3-HPA consuming ability was confirmed and catalytic
mechanism proposed. PduP, PduL and PduW-mediated conversion of 3-HPA to 3-
HP was confirmed through their recombinant expression in Escherichia coli. 3-HPA
produced from glycerol by L. reuteri was used as a substrate for conversion to 3-HP
by the recombinant E. coli. A yield of 1 mol/mol was reached with a titer of 12 mM
3-HP. Depletion of the cofactor NAD+ required for the catalysis of 3-HP to 3-HPCoA,
was deemed responsible for the low titer. Regeneration of NAD+, used up in
PduP catalyzed reaction, was achieved by recombinant expression of NADH oxidase
(Nox) from L. reuteri in E. coli expressing PduP, PduL and PduW. The final 3-HP
titer by this recombinant strain was at least twice that of E. coli carrying solely PduP,
PduL and PduW.
For the production of poly(3-HP), PduL and PduW in the recombinant strain were
replaced by polyhydroxyalkanoate synthase of Chromobacterium sp. that converts 3-
HP-CoA to poly(3-HP). A poly(3-HP) content of up to 40% (w/w) cell dry weight
was reached in an efficient and cheap process requring no additivies or expensive
cofactors.
climate change have led to a shift within the research community and chemical and
energy industry sectors for finding sustainable routes for producing fuels and
chemicals from renewable resources, thereby minimizing our dependence on
petroleum. The C3-chemical 3-hydroxypropionic acid has been identifed as a top
candidate for the biobased chemical industry. This platform chemical is a β-hydroxy
acid containing two functional groups (hydroxyl and carboxyl) enabling its
conversion into value-added chemicals such as 1,3-propanediol, acrolein, malonic
acid, acrylamide and acrylic acid, which can be used in resins, coatings, paints,
adhesives, lubricants, and in the textile industry as anti-static agent. Polymerized 3-
HP, poly(3-hydroxypropionate) (poly(3-HP)), is a biodegradable and stable polymer
which, besides its potential role as a biomaterial, can be degraded to 3-HP monomer.
In recent years, a dramatic increase in the interest for microbial production of 3-HP
and poly(3-HP) has been observed. Metabolic engineering and recombinant
expression of various enzymatic pathways in a number of bacterial strains have been
suggested and implemented, with mainly renewable glucose and glycerol as substrates.
This thesis presents a novel pathway called the propanediol utilization pathway
present in Lactobacillus reuteri that catalyzes dehydration of glycerol to 3-
hydroxypropionaldehyde (3-HPA) and further to 3-HP by a series of reactions
catalyzed by propionaldehyde dehydrogenase (PduP), phosphotransacylase (PduL)
and propionate kinase (PduW). Through structural modeling and kinetic
characterization of PduP, its 3-HPA consuming ability was confirmed and catalytic
mechanism proposed. PduP, PduL and PduW-mediated conversion of 3-HPA to 3-
HP was confirmed through their recombinant expression in Escherichia coli. 3-HPA
produced from glycerol by L. reuteri was used as a substrate for conversion to 3-HP
by the recombinant E. coli. A yield of 1 mol/mol was reached with a titer of 12 mM
3-HP. Depletion of the cofactor NAD+ required for the catalysis of 3-HP to 3-HPCoA,
was deemed responsible for the low titer. Regeneration of NAD+, used up in
PduP catalyzed reaction, was achieved by recombinant expression of NADH oxidase
(Nox) from L. reuteri in E. coli expressing PduP, PduL and PduW. The final 3-HP
titer by this recombinant strain was at least twice that of E. coli carrying solely PduP,
PduL and PduW.
For the production of poly(3-HP), PduL and PduW in the recombinant strain were
replaced by polyhydroxyalkanoate synthase of Chromobacterium sp. that converts 3-
HP-CoA to poly(3-HP). A poly(3-HP) content of up to 40% (w/w) cell dry weight
was reached in an efficient and cheap process requring no additivies or expensive
cofactors.
Department/s
Publishing year
2015
Language
English
Full text
Document type
Dissertation
Publisher
Division of Biotechnology, Lund University
Topic
- Industrial Biotechnology
Keywords
- Platform chemicals
- biopolymers
- 3-hydroxypropionic acid
- 3-hydroxypropionaldehyde
- glycerol
- poly(3-hydroxypropionate)
- polyhydroxyalkanoates
- Escherichia coli
- Lactobacillus reuteri
- propanediol utilization pathway
- propionaldehyde dehydrogenase
- biotransformation
- cofactor regeneration
- NADH oxidase
Status
Published
Supervisor
- Rajni Hatti-Kaul
- Javier Linares-Pastén
- Shukun Yu
ISBN/ISSN/Other
- ISBN: 978-91-7623-274-3
Defence date
30 April 2015
Defence time
10:00
Defence place
Lecture hall B, Kemicentrum, Getingevägen 60, Lund University, Faculty of Engineering LTH, Lund
Opponent
- An-Ping Zeng (Professor)