Microbial production of 3-hydroxypropionic acid and poly(3-hydroxypropionate): Investigation of Lactobacillus reuteri propanediol utilization pathway enzymes

Abstract

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.

Abstract (Swedish)

Popular Abstract in English

Since the onset of the 20th century, human society has been using non-renewable

resources, mainly oil, for the production of fuels and chemicals that are now an

integral part of our everyday life. In the last few decades however, concerns regarding

environmental effects, geopolitical issues and the eventual depletion of oil have led to

us re-evaluating our dependency on this resource. Current production of a majority of

everday chemicals is based on refining petroleum to a small number of other

molecules, also known as platform chemicals, which can then be converted to a much

larger number of chemicals through various processes.

In our efforts to move from a fossil to biobased economy in which renewable

resources like sugars or glycerol, obtained through plants, trees, grasses, and/or as

residues/wastes of agro-/forestry based industries, will constitute the feedstock for

industry, sustainable technologies for processing of the biomass and its components in

an environmentally-friendly manner need to be develooped. Biotechnological

production of platform chemicals from the biomass feedstocks is mainly based on

replacing traditional chemical reactors with microorganisms. Microorganisms can be

considered small reactors as they contain mechanisms for the conversion of a large

variety of natural as well as synthetic molecules to others. These mechanisms are

driven by enzymes which are in turn encoded in their genomes. As microorganisms

are present in pretty much every type of environment imaginable on the planet, the

number of naturally occurring reactions is very high. Some microorganisms are

known to produce biodegradable plastics as a protection mechanism in harsh

conditions. Microbial processes for the production of chemicals and materials are

based in water and require no organic solvents. They can be performed in lower

temperatures and are very specific. As petroleum is cheap however, there is no

economic incentive to shift to these greener processes. Thus, it is important to

develop microbial methods for the production of these chemicals from cheap

resources and in high concentrations and yields.

This thesis deals mainly with the production of the platform chemical 3-

hydroxypropionic acid (3-HP) and its polymer poly(3-hydroxypropionate). These

products are not available commercially. 3-HP is of great promise as it can be further

converted into wide array of chemicals, e.g. resins, coatings, lubricants and in the

textile industry as anti-static agent. Poly(3-HP) is a biodegradable polymer that can

replace certain fossil-based polymers in different applications.

3-HP is produced in smaller amounts by certain microorganisms that grow slowly

and/or are expensive to cultivate. Therefore, a copy and paste-strategy has been

implemented for moving some of the reactions to well-known organisms that are

easier and cheaper to cultivate. Lactobacillus reuteri, a probiotic bacteria, contains a

mechanism for the production of 3-hydroxypropionic acid from glycerol, which is

currently produced in large amounts as a byproduct from the production of bio-diesel

from several plant oils. By copying relevant genes and transferring them to the

cheaply cultivated bacterium Escherichia coli, it was proven that 3-HP could be

produced with a high yield from glycerol. Such a strategy does require the

understanding of the metabolic system in the bacteria in order to avoid any

interference and to incorporate strategies for improving the formation of the product

in a selective and clean manner. Such strategies were implemented in the present

work, thereby increasing the final concentration of 3-HP. Some key enzymes in these

processes were studied further in order to gain a better understanding of their

function and structure.

It the same manner, a relevant gene from Lactobacillus reuteri and a gene from

another bacteria, Chromobacterium sp. known to produce a bioplastic, were copied

and pasted in Escherichia coli, resulting in a strain with the capacity to produce

poly(3-hydroxypropionate) in a cheap and efficient manner.

• author: Sabet Azad, Ramin
• supervisor: Rajni Hatti-Kaul; Javier Linares-Pastén; Shukun Yu
• opponent: Professor Zeng, An-Ping, Hamburg University of Technology, Germany
• organization: Biotechnology
• publishing date: 2015
• type: Thesis
• publication status: published
• subject: 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
• pages: 132 pages
• defense location: Lecture hall B, Kemicentrum, Getingevägen 60, Lund University, Faculty of Engineering LTH, Lund
• defense date: 2015-04-30 10:00:00
• ISBN: 978-91-7623-274-3
• language: English
• LU publication?: yes
• id: 80f1f3bd-73a5-4688-a6eb-ea78166599ce (old id 5265772)
• date added to LUP: 2016-04-04 12:23:08
• date last changed: 2018-11-21 21:10:39