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Protein engineering and synthetic pathways in Escherichia coli for effective production of 5-hydroxytryptophan and serotonin
Citation Link: https://doi.org/10.15480/882.1685
Publikationstyp
Doctoral Thesis
Date Issued
2018
Sprache
English
Author(s)
Advisor
Referee
Title Granting Institution
Technische Universität Hamburg
Place of Title Granting Institution
Hamburg
Examination Date
2018-02-23
Institut
TORE-DOI
Metabolic engineering has improved the production of tryptophan in E. coli during the last two decades, opening itself a plethora of opportunities for the production of tryptophan derivatives. 5-Hydroxytryptophan (5HTP) and serotonin are two important derivatives not only important for their pharmaceutical value but also because they could serve as a precursor of other molecules which includes sleep cycle regulator, anti-migraine medications, sedatives, anticonvulsants, antitumors, antimicrobials, antivirals, among others.
To the present, 5HTP is mainly obtained by extraction from the plant Griffonia simplicifolia and serotonin is produced by chemical synthesis. In both cases, the processes involve the use of organic solvents and high temperature during the procedure. Moreover, in the case of serotonin, its chemical synthesis starts from a complex molecule (5-benzyloxyindole). A simple biotechnological method for the production of these compounds is desired.
This dissertation presents the work done in the extension of the tryptophan metabolism for the production of 5HTP and serotonin. For this purpose, serotonin production via tryptamine and via 5HTP were compared and analyzed. In both cases, the hydroxylation step seemed to be the bottleneck due to the low activity of the enzymes when expressed in E. coli and the requirement of a cofactor, plus its regeneration pathway. The serotonin production pathway via 5HTP was chosen, and for this purpose, an aromatic amino acid hydroxylase from Cupriavidus taiwanensis (CtAAAH) was selected using an in silico structure-based approach. Several substrate-determining residues were predicted and selected using sequence, phylogenetic and functional divergence analyses. Whole cells analysis with the wild-type and variants were done to study the shift of the enzyme preference from phenylalanine to tryptophan. All the variants increased the tryptophan hydroxylation activity in detriment to phenylalanine. The best performer, CtAAAH-W192F, was transformed into a strain that had the tryptophanase A gene disrupted and carried a human tetrahydrobiopterin (BH4) regeneration pathway. The resulting strain was capable of synthesizing 2.5 mM 5HTP after 24 hours in medium supplied with tryptophan.
After this first rational design round, a second semi-rational approach was selected to improve the efficiency of the enzyme. A tryptophan intracellular concentration sensor was used to screen two independent libraries, and the variants found in the best performer of each library were combined to create CtAAAH-LC. This double mutant showed higher activity and reaction velocity than its predecessor. CtAAAH-LC was transformed into a tryptophan producer strain (S028), which had been previously modified by the addition of a pterin (a cofactor that is consumed during hydroxylation) regeneration pathway. In this case, 5HTP was synthesized from glucose.
Tryptophan decarboxylase (TDC) was incorporated in the 5HTP producer strain to produce serotonin from glucose. However, the serotonin production was low and undesired side reactions were identified. To circumvent this problem, a two-step system was constructed in which the 5HTP production and the serotonin conversion are separated.
In this work, results of the highest biotechnologically produced concentration of 5HTP reported so far are presented, for this purpose protein engineering was done in CtAAAH and a synthetic pathway was incorporated in E. coli. Afterwards, 5HTP was decarboxylated to produce serotonin in a second fermentation. This is the first report of serotonin production from glucose. Still, the process can be further optimized by combining the hydroxylation and decarboxylation reaction in one strain. TDC selectivity can be engineered to shift the preference toward 5HTP in detriment of tryptophan. In this case, the development of a novel biosensor sensitive to 5HTP is critical for selection.
To the present, 5HTP is mainly obtained by extraction from the plant Griffonia simplicifolia and serotonin is produced by chemical synthesis. In both cases, the processes involve the use of organic solvents and high temperature during the procedure. Moreover, in the case of serotonin, its chemical synthesis starts from a complex molecule (5-benzyloxyindole). A simple biotechnological method for the production of these compounds is desired.
This dissertation presents the work done in the extension of the tryptophan metabolism for the production of 5HTP and serotonin. For this purpose, serotonin production via tryptamine and via 5HTP were compared and analyzed. In both cases, the hydroxylation step seemed to be the bottleneck due to the low activity of the enzymes when expressed in E. coli and the requirement of a cofactor, plus its regeneration pathway. The serotonin production pathway via 5HTP was chosen, and for this purpose, an aromatic amino acid hydroxylase from Cupriavidus taiwanensis (CtAAAH) was selected using an in silico structure-based approach. Several substrate-determining residues were predicted and selected using sequence, phylogenetic and functional divergence analyses. Whole cells analysis with the wild-type and variants were done to study the shift of the enzyme preference from phenylalanine to tryptophan. All the variants increased the tryptophan hydroxylation activity in detriment to phenylalanine. The best performer, CtAAAH-W192F, was transformed into a strain that had the tryptophanase A gene disrupted and carried a human tetrahydrobiopterin (BH4) regeneration pathway. The resulting strain was capable of synthesizing 2.5 mM 5HTP after 24 hours in medium supplied with tryptophan.
After this first rational design round, a second semi-rational approach was selected to improve the efficiency of the enzyme. A tryptophan intracellular concentration sensor was used to screen two independent libraries, and the variants found in the best performer of each library were combined to create CtAAAH-LC. This double mutant showed higher activity and reaction velocity than its predecessor. CtAAAH-LC was transformed into a tryptophan producer strain (S028), which had been previously modified by the addition of a pterin (a cofactor that is consumed during hydroxylation) regeneration pathway. In this case, 5HTP was synthesized from glucose.
Tryptophan decarboxylase (TDC) was incorporated in the 5HTP producer strain to produce serotonin from glucose. However, the serotonin production was low and undesired side reactions were identified. To circumvent this problem, a two-step system was constructed in which the 5HTP production and the serotonin conversion are separated.
In this work, results of the highest biotechnologically produced concentration of 5HTP reported so far are presented, for this purpose protein engineering was done in CtAAAH and a synthetic pathway was incorporated in E. coli. Afterwards, 5HTP was decarboxylated to produce serotonin in a second fermentation. This is the first report of serotonin production from glucose. Still, the process can be further optimized by combining the hydroxylation and decarboxylation reaction in one strain. TDC selectivity can be engineered to shift the preference toward 5HTP in detriment of tryptophan. In this case, the development of a novel biosensor sensitive to 5HTP is critical for selection.
Subjects
protein engineering
metabolic engineering
5-hydroxytryptophan
synthetic biology
Serotonin
DDC Class
610: Medizin
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Name
Thesis Final 20180613.pdf
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11.23 MB
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