Engineering synthetic pathways for adipic acid biosynthesis
Doctoral thesis, 2021

Utilization of petroleum in consumer product manufacturing is causing irreversible environmental damage. Its impact on land, sea, and air calls for the development of more sustainable technologies based on the use of renewable materials such as lignocellulosic biomass and its conversion into platform chemicals. Engineering microorganisms to produce chemicals is an important undertaking to address such issues and bio-based production of adipic acid especially has gained recent attention. In the present thesis I assess the in vivo and in silico action of enzymes involved in microbial production of adipic acid from simple sugar molecules. The aim of this work was to comprehensively map out the metabolic pathways leading to adipic acid biosynthesis and to investigate the enzymatic components of the L-lysine pathway, the reverse β-oxidation pathway, and cis,cis-muconic acid reduction.
Investigation of theoretical and in silico aspects in the deamination step in the L-lysine pathway revealed deamination of L-lysine was determined to be chemically difficult to occur. Removal of the β-amino group from β-D-lysine was deemed more feasible than the α-amino group from L-lysine, and an alternative route via β-D-lysine deamination was suggested. Homology modeling and molecular docking studies shed light on the substrate binding mechanisms of enzymes responsible for the reduction of the intermediates in the L-lysine pathway. Potential mechanism and feasibility of α,β-reduction were explained in terms of substrate interaction in the enzyme-binding pockets. Corynebacterium glutamicum was chosen as the host chassis for achieving adipic acid synthesis via reverse β-oxidation. Stepwise construction of a five-step synthetic pathway demonstrated functionality of each step in C. glutamicum. Biosynthesized and secreted 3-hydroxyadipate was detected in the cultivation broth using GC/MS. Weak trans-2-hexenedioic acid and adipic acid signals was observed using LC/MS after concentrating the cultivation broth. Dehydration of 3-hydroxyadipyl-CoA was identified as a potential bottleneck hindering this pathway. While implementing the reverse β-oxidation pathway, a new pathway involving cis,cis-muconic acid and 3-oxoadipic acid was observed and experimented on. The modified strategy for bio-conversion of benzoic acid to cis,cis-muconic acid was successful and molecular docking studies were carried out to better understand how oxidoreductases might reduce cis,cis-muconic acid.
Taking multiple approaches to generate adipic acid revealed different challenges in each pathway. One approach led to biosynthesis of adipic acid. Further investigation will allow multiple options for bio-based adipic acid production for better sustainability.

in silico docking

lignocellulose

L-lysine

GC/MS

cell factory

pathway engineering

cultivation

adipic acid

analytical chemistry

Corynebacterium glutamicum

muconic acid

benzoic acid

metabolic engineering

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Opponent: Stephan Noack, Forschungszentrum Jülich GmbH, Germany

Author

Jae Ho Shin

Chalmers, Biology and Biological Engineering, Industrial Biotechnology

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 requires robust and efficient microorganisms capable of converting sugars into suitable chemical building blocks. More types of bio-based chemicals mean more types of bio-based products. To achieve this, more microorganisms equipped with the machinery to synthesize more types of starting chemicals should be developed. One of the most desired chemicals for such bio-based transition is adipic acid. Adipic acid is used in the production of nylon, which in turn has wide applicability in the manufacturing of shoes, textiles, automobile parts, belts, and many others. Adipic acid is rarely found in nature and several attempts to commercially produce it viabio-based methods have encountered limited 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 the biosynthesis of adipic acid to better understand this process. One of the strategies I explored resulted in a miniscule amount ofadipic acid, yet could not be identified with certainty with the instrumentation at hand. Exploiting the full potential of each strategy may require further studies.

A novel strategy for cell factory design applied to adipic acid production - combining synthetic pathway and electrofermentation

Swedish Research Council (VR) (2016-03344), 2017-01-01 -- 2021-12-31.

Upgrading of renewable domestic raw materials to value-added bulk and fine chemicals for a biobased economy: technology development, systems integration and environmental impact assessment (BioBuF)

Region Västra Götaland (RUN612-0806-13), 2013-11-01 -- 2018-10-31.

Formas (213-2013-78), 2013-06-17 -- 2018-12-31.

Driving Forces

Sustainable development

Subject Categories

Biochemistry and Molecular Biology

Renewable Bioenergy Research

Other Biological Topics

Microbiology

Infrastructure

Chalmers Infrastructure for Mass spectrometry

C3SE (Chalmers Centre for Computational Science and Engineering)

Roots

Basic sciences

Areas of Advance

Life Science Engineering (2010-2018)

ISBN

978-91-7905-419-9

Doktorsavhandlingar vid Chalmers tekniska högskola. Ny serie: 4886

Publisher

Chalmers University of Technology

Zoom

Online

Opponent: Stephan Noack, Forschungszentrum Jülich GmbH, Germany

More information

Latest update

8/30/2021