Production of medium-chain, a, omega-bifunctional monomers from fatty acids and n-alkanes

Youri M. Nuland

Research output: Thesisinternal PhD, WU

Abstract

In chapter 1, we give an introduction to bifunctional monomers that play an important role in the chemical industry. Briefly, the conventional production processes of α,ω-dicarboxylic acids and α,ω-diols are discussed. Strategies for more sustainable alternatives for production of medium-chain bifunctional monomers are discussed. Monooxygenase-based processes seem promising, if the problem of poor diterminal oxidation capacities of monooxygenases is solved. Esterification could be a tool to solve this problem.

In chapter 2 we have investigated the ω-oxidation activities of E. coli expressing AlkBGT or AlkBGTL, with various esters having an alkyl chain >1. These strains were able to ω-oxidize ethyl, propyl and butyl esters of C6-C10 fatty acids. Using esters with a longer alkyl chain enhanced ω-oxidation activities for C6 and C7 fatty acids. The major products were ω-hydroxy fatty acid esters, but over oxidation to the aldehyde and carboxylic acid also occurred. AlkL improved whole-cell ω-oxidation activities for substrates with a logPo/w above 4.

Since the major products were ω-hydroxy fatty acid esters in chapter 2, we investigated further conversion of these compounds to mono-esterified dicarboxylic acids in chapter 3. Alcohol dehydrogenase AlkJ and aldehyde dehydrogenase AlkH were functionally expressed in E. coli. AlkJ is functional with 9-hydroxy ethyl nonanoate as substrate, AlkH is functional with 9-oxo methyl nonanoate. Expansion of the AlkBGTL system with AlkJ and AlkH yielded strain E. coli AlkBGTHJL. This strain accumulated mono-ethyl azelate exclusively from ethyl nonanoate. Adding the substrate dissolved in a carrier solvent increased final product titers.

Subsequently, we investigated if in vivo esterification could enhance the ω-oxidation of AlkB in chapter 4. E. coli expressing AlkBGTHJL can ω-oxidize octanoate and nonanoate, but not efficiently. When acyl-CoA ligase AlkK and acyltransferase AtfA or Eeb1 were also expressed, ω-oxidation was more efficient. Furthermore, complete oxidation to the carboxylic acid was much more efficient when also in vivo esterification was achieved. Also di-ethyl esters were produced, meaning that esterification occurred twice.

Since ω-oxidation of fatty acids was improved with in vivo esterification in chapter 4, we were interested to investigate whether this system could also work with n-alkanes in chapter 5. Mono-esters of dicarboxylic acids were produced from n-alkanes by E. coli expressing AlkBGTHJKL and either AtfA or Eeb1. Starting from n-alkanes would also allow production of alcohols if overoxidation could be prevented. Application of a different alcohol acyltransferase (Atf1), limited the overoxidation by AlkB. ω-Oxidation of the formed ester resulted in the production of ω-alcohols, which were again esterified by Atf1.

Chapter 6 is the general discussion of this thesis, which evaluates the combination of esterification and terminal oxidation. Suggestions for improvements of the biocatalytic pathway are provided and critical factors for experiments in bioreactors are identified.

Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Wageningen University
Supervisors/Advisors
  • Eggink, Gerrit, Promotor
  • Sanders, J.P.M., Promotor
  • Weusthuis, Ruud, Co-promotor
Award date20 Oct 2017
Place of PublicationWageningen
Publisher
Print ISBNs9789463436809
DOIs
Publication statusPublished - 2017

Fingerprint

Alkanes
Fatty Acids
Monomers
Oxidation
Esterification
Esters
Escherichia coli
Dicarboxylic Acids
Acyltransferases
Alcohols
Carboxylic Acids
Mixed Function Oxygenases
Substrates
Acyl Coenzyme A
Aldehyde Dehydrogenase
Alcohol Dehydrogenase
Ligases
Chemical industry
Bioreactors
Aldehydes

Cite this

Nuland, Youri M.. / Production of medium-chain, a, omega-bifunctional monomers from fatty acids and n-alkanes. Wageningen : Wageningen University, 2017. 161 p.
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abstract = "In chapter 1, we give an introduction to bifunctional monomers that play an important role in the chemical industry. Briefly, the conventional production processes of α,ω-dicarboxylic acids and α,ω-diols are discussed. Strategies for more sustainable alternatives for production of medium-chain bifunctional monomers are discussed. Monooxygenase-based processes seem promising, if the problem of poor diterminal oxidation capacities of monooxygenases is solved. Esterification could be a tool to solve this problem. In chapter 2 we have investigated the ω-oxidation activities of E. coli expressing AlkBGT or AlkBGTL, with various esters having an alkyl chain >1. These strains were able to ω-oxidize ethyl, propyl and butyl esters of C6-C10 fatty acids. Using esters with a longer alkyl chain enhanced ω-oxidation activities for C6 and C7 fatty acids. The major products were ω-hydroxy fatty acid esters, but over oxidation to the aldehyde and carboxylic acid also occurred. AlkL improved whole-cell ω-oxidation activities for substrates with a logPo/w above 4. Since the major products were ω-hydroxy fatty acid esters in chapter 2, we investigated further conversion of these compounds to mono-esterified dicarboxylic acids in chapter 3. Alcohol dehydrogenase AlkJ and aldehyde dehydrogenase AlkH were functionally expressed in E. coli. AlkJ is functional with 9-hydroxy ethyl nonanoate as substrate, AlkH is functional with 9-oxo methyl nonanoate. Expansion of the AlkBGTL system with AlkJ and AlkH yielded strain E. coli AlkBGTHJL. This strain accumulated mono-ethyl azelate exclusively from ethyl nonanoate. Adding the substrate dissolved in a carrier solvent increased final product titers. Subsequently, we investigated if in vivo esterification could enhance the ω-oxidation of AlkB in chapter 4. E. coli expressing AlkBGTHJL can ω-oxidize octanoate and nonanoate, but not efficiently. When acyl-CoA ligase AlkK and acyltransferase AtfA or Eeb1 were also expressed, ω-oxidation was more efficient. Furthermore, complete oxidation to the carboxylic acid was much more efficient when also in vivo esterification was achieved. Also di-ethyl esters were produced, meaning that esterification occurred twice. Since ω-oxidation of fatty acids was improved with in vivo esterification in chapter 4, we were interested to investigate whether this system could also work with n-alkanes in chapter 5. Mono-esters of dicarboxylic acids were produced from n-alkanes by E. coli expressing AlkBGTHJKL and either AtfA or Eeb1. Starting from n-alkanes would also allow production of alcohols if overoxidation could be prevented. Application of a different alcohol acyltransferase (Atf1), limited the overoxidation by AlkB. ω-Oxidation of the formed ester resulted in the production of ω-alcohols, which were again esterified by Atf1. Chapter 6 is the general discussion of this thesis, which evaluates the combination of esterification and terminal oxidation. Suggestions for improvements of the biocatalytic pathway are provided and critical factors for experiments in bioreactors are identified.",
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Production of medium-chain, a, omega-bifunctional monomers from fatty acids and n-alkanes. / Nuland, Youri M.

Wageningen : Wageningen University, 2017. 161 p.

Research output: Thesisinternal PhD, WU

TY - THES

T1 - Production of medium-chain, a, omega-bifunctional monomers from fatty acids and n-alkanes

AU - Nuland, Youri M.

N1 - WU thesis 6784 Includes bibliographical references. - With summary in English

PY - 2017

Y1 - 2017

N2 - In chapter 1, we give an introduction to bifunctional monomers that play an important role in the chemical industry. Briefly, the conventional production processes of α,ω-dicarboxylic acids and α,ω-diols are discussed. Strategies for more sustainable alternatives for production of medium-chain bifunctional monomers are discussed. Monooxygenase-based processes seem promising, if the problem of poor diterminal oxidation capacities of monooxygenases is solved. Esterification could be a tool to solve this problem. In chapter 2 we have investigated the ω-oxidation activities of E. coli expressing AlkBGT or AlkBGTL, with various esters having an alkyl chain >1. These strains were able to ω-oxidize ethyl, propyl and butyl esters of C6-C10 fatty acids. Using esters with a longer alkyl chain enhanced ω-oxidation activities for C6 and C7 fatty acids. The major products were ω-hydroxy fatty acid esters, but over oxidation to the aldehyde and carboxylic acid also occurred. AlkL improved whole-cell ω-oxidation activities for substrates with a logPo/w above 4. Since the major products were ω-hydroxy fatty acid esters in chapter 2, we investigated further conversion of these compounds to mono-esterified dicarboxylic acids in chapter 3. Alcohol dehydrogenase AlkJ and aldehyde dehydrogenase AlkH were functionally expressed in E. coli. AlkJ is functional with 9-hydroxy ethyl nonanoate as substrate, AlkH is functional with 9-oxo methyl nonanoate. Expansion of the AlkBGTL system with AlkJ and AlkH yielded strain E. coli AlkBGTHJL. This strain accumulated mono-ethyl azelate exclusively from ethyl nonanoate. Adding the substrate dissolved in a carrier solvent increased final product titers. Subsequently, we investigated if in vivo esterification could enhance the ω-oxidation of AlkB in chapter 4. E. coli expressing AlkBGTHJL can ω-oxidize octanoate and nonanoate, but not efficiently. When acyl-CoA ligase AlkK and acyltransferase AtfA or Eeb1 were also expressed, ω-oxidation was more efficient. Furthermore, complete oxidation to the carboxylic acid was much more efficient when also in vivo esterification was achieved. Also di-ethyl esters were produced, meaning that esterification occurred twice. Since ω-oxidation of fatty acids was improved with in vivo esterification in chapter 4, we were interested to investigate whether this system could also work with n-alkanes in chapter 5. Mono-esters of dicarboxylic acids were produced from n-alkanes by E. coli expressing AlkBGTHJKL and either AtfA or Eeb1. Starting from n-alkanes would also allow production of alcohols if overoxidation could be prevented. Application of a different alcohol acyltransferase (Atf1), limited the overoxidation by AlkB. ω-Oxidation of the formed ester resulted in the production of ω-alcohols, which were again esterified by Atf1. Chapter 6 is the general discussion of this thesis, which evaluates the combination of esterification and terminal oxidation. Suggestions for improvements of the biocatalytic pathway are provided and critical factors for experiments in bioreactors are identified.

AB - In chapter 1, we give an introduction to bifunctional monomers that play an important role in the chemical industry. Briefly, the conventional production processes of α,ω-dicarboxylic acids and α,ω-diols are discussed. Strategies for more sustainable alternatives for production of medium-chain bifunctional monomers are discussed. Monooxygenase-based processes seem promising, if the problem of poor diterminal oxidation capacities of monooxygenases is solved. Esterification could be a tool to solve this problem. In chapter 2 we have investigated the ω-oxidation activities of E. coli expressing AlkBGT or AlkBGTL, with various esters having an alkyl chain >1. These strains were able to ω-oxidize ethyl, propyl and butyl esters of C6-C10 fatty acids. Using esters with a longer alkyl chain enhanced ω-oxidation activities for C6 and C7 fatty acids. The major products were ω-hydroxy fatty acid esters, but over oxidation to the aldehyde and carboxylic acid also occurred. AlkL improved whole-cell ω-oxidation activities for substrates with a logPo/w above 4. Since the major products were ω-hydroxy fatty acid esters in chapter 2, we investigated further conversion of these compounds to mono-esterified dicarboxylic acids in chapter 3. Alcohol dehydrogenase AlkJ and aldehyde dehydrogenase AlkH were functionally expressed in E. coli. AlkJ is functional with 9-hydroxy ethyl nonanoate as substrate, AlkH is functional with 9-oxo methyl nonanoate. Expansion of the AlkBGTL system with AlkJ and AlkH yielded strain E. coli AlkBGTHJL. This strain accumulated mono-ethyl azelate exclusively from ethyl nonanoate. Adding the substrate dissolved in a carrier solvent increased final product titers. Subsequently, we investigated if in vivo esterification could enhance the ω-oxidation of AlkB in chapter 4. E. coli expressing AlkBGTHJL can ω-oxidize octanoate and nonanoate, but not efficiently. When acyl-CoA ligase AlkK and acyltransferase AtfA or Eeb1 were also expressed, ω-oxidation was more efficient. Furthermore, complete oxidation to the carboxylic acid was much more efficient when also in vivo esterification was achieved. Also di-ethyl esters were produced, meaning that esterification occurred twice. Since ω-oxidation of fatty acids was improved with in vivo esterification in chapter 4, we were interested to investigate whether this system could also work with n-alkanes in chapter 5. Mono-esters of dicarboxylic acids were produced from n-alkanes by E. coli expressing AlkBGTHJKL and either AtfA or Eeb1. Starting from n-alkanes would also allow production of alcohols if overoxidation could be prevented. Application of a different alcohol acyltransferase (Atf1), limited the overoxidation by AlkB. ω-Oxidation of the formed ester resulted in the production of ω-alcohols, which were again esterified by Atf1. Chapter 6 is the general discussion of this thesis, which evaluates the combination of esterification and terminal oxidation. Suggestions for improvements of the biocatalytic pathway are provided and critical factors for experiments in bioreactors are identified.

U2 - 10.18174/422088

DO - 10.18174/422088

M3 - internal PhD, WU

SN - 9789463436809

PB - Wageningen University

CY - Wageningen

ER -