Synthetic Pathway engineering toward Biosynthesis of Adipic Acid
Doktorsavhandling, 2021

Utilization of petroleum in consumer product manufacturing has caused irreversible and detrimental environmental damages globally. Recent awareness of apparent impact of such pollutions on land, sea, and air urgently prompts development of technologies to produce consumer goods independently of petroleum and sustainably. Motivation of such development points towards use of renewable materials such as lignocellulosic biomass and to further raises a necessity to develop technologies to convert biomass into platform monomers. The thesis addresses the in vivo and in silico assessment of various enzymes involved in different metabolic pathways for microbial production of adipic acid from simple sugar molecules.

The aim of the work presented in this thesis was to map out the comprehensive metabolic pathways leading to adipic acid biosynthesis as well as the corresponding enzymatic components. Reduction and deamination parts in one of the theoretical pathways based on L-lysine metabolism were assessed. Molecular docking studies accompanied with molecular dynamics studies as well as interaction energy studies shed light on to possible binding mechanisms for Old yellow enzyme class enzymes. Potential mechanism and feasibility of α,β-reduction of L-lysine pathway intermediate compounds were explained in terms of substrate interaction in the enzyme binding pockets. Deamination reaction of L-lysine was determined to be less likely to occur. D-β-lysine was chosen to be the better intermediate for α, β-elimination. Removal of β-amino group from β-lysine is deemed to be more feasible if an enzyme is additionally developed to accommodate the unnatural substrate.

In terms of reversed β-oxidation pathway and implementation, Corynebacterium glutamicum was chosen as the host chassis for extending the synthetic pathway toward adipic acid. Stepwise construction of 5-step synthetic pathway demonstrated functionality of the condensation and the first reduction steps. Non-decarboxylative condensation of acetyl-CoA and succinyl-CoA was demonstrated in C. glutamicum in vivo for the first time. Reduction of 3-oxoadipyl-CoA was performed in C. glutamicum for the first time as well. Biosynthesis of 3-hydroxyadipate as well as secretion was detected in the cultivation broth using GC/MS methodology. Potential bottleneck hindering the pathway was identified to be the reduction step of 3-hydroxyadipyl-CoA. In order to alleviate the problem, expression optimization and choosing an alternative enzyme is suggested. Along the course of implementing the reversed β-oxidation pathway, a new pathway was observed and experimented on. A new strategy for reaching adipic acid from 3-oxoadipate via cis,cis-muconic acid was  pursued. Bio-conversion of benzoic acid to cis,cis-muconic acid was successful and molecular docking studies were carried out further improve knowledge regarding the currently refuted knowledge regarding enoate reductases for their ability to reduce cis,cis-muconic acid.

lignocellulose

analytical chemistry

pathway engineering

GC/MS

benzoic acid

cultivation

cell factory

L-lysine

adipic acid

muconic acid

metabolic engineering

in silico docking

Corynebacterium glutamicum

Zoom
Opponent: Stephan Noack, Forschungszentrum Jülich GmbH, Germany

Författare

Jae Ho Shin

Chalmers, Biologi och bioteknik, Industriell bioteknik

Biobased adipic acid – The challenge of developing the production host

Biotechnology Advances,; Vol. 36(2018)p. 2248-2263

Artikel i vetenskaplig tidskrift

Shin J.H., Andersen A.J.C., Achterberg P., Olsson L. Production of 3-hydroxyadipic acid byreverse β-oxidationpathway in Corynebacterium glutamicum

Shin, J.H., Breard, C., Olsson, L. Assessment of Bioconversion for benzoic acid to adipic acid by engineered Corynebacterium glutamicum

Enzymes Involved in Adipic Acid Biosynthesis
Bio-based plastic bags, plastic bottles, shoes, utensils, teabags, coffee capsules, and many other products have been available for some time. Producing such bio-based goods require robust and efficient microorganisms for converting sugars into starting chemicals. More types of bio-based chemicals mean more types of bio-based products. To enable production of more bio-based products, we need more types of microorganisms equipped with capability to biosynthesize more types of starting chemicals. One the most sought chemicals for such bio-based transition is called adipic acid. Adipic acid is used in production of nylon which is in turn used in production in shoes, textiles, automobile parts, belts, and many others. Adipic acid is rarely found chemical in nature. Many attempts have been made to commercially produce adipic acid from bio-based methods with no tangible success. There are multiple metabolic and process strategies to produce adipic acid from sugars and biodiesel waste. Each strategy uses a different set of enzymes for sequentially converting sugar into adipic acid. In this thesis I explored different enzymes involved in biosynthesis of adipic acid to better understand this process. One of the strategies I explored resulted in miniscule amount of compound that resembles adipic acid. Understanding of full potential in each strategy may require further examination.

En ny strategi för design av cellfabriker tillämpad för adipinsyraproduktion - kombination av introduktion av en syntetisk metabolsk väg och elektrofermentering

Vetenskapsrådet (VR), 2017-01-01 -- 2021-12-31.

Drivkrafter

Hållbar utveckling

Ämneskategorier

Biokemi och molekylärbiologi

Förnyelsebar bioenergi

Annan biologi

Mikrobiologi

Infrastruktur

Chalmers infrastruktur för masspektrometri

C3SE (Chalmers Centre for Computational Science and Engineering)

Fundament

Grundläggande vetenskaper

Styrkeområden

Livsvetenskaper och teknik (2010-2018)

ISBN

978-91-7905-419-9

Doktorsavhandlingar vid Chalmers tekniska högskola. Ny serie: 4886

Utgivare

Chalmers tekniska högskola

Zoom

Opponent: Stephan Noack, Forschungszentrum Jülich GmbH, Germany

Mer information

Senast uppdaterat

2021-01-05