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Engineering of phosphoserine aminotransferase and new metabolic pathways for microbial production of 1,3-propanediol and 1,2,4-butanetriol from sugar
Citation Link: https://doi.org/10.15480/882.2666
Publikationstyp
Doctoral Thesis
Publikationsdatum
2020
Sprache
English
Author
Advisor
Referee
Title Granting Institution
Technische Universität Hamburg
Place of Title Granting Institution
Hamburg
Examination Date
2020-01-17
TORE-URI
Citation
Dr. Hut (2020)
Publisher
Dr. Hut
1,3-Propanediol (1,3-PDO) is an important chemical compound with lots of applications in the fields of polymers, cosmetics, food and pharmaceutical. The significant advances of metabolic engineering in the past thirty years have made it possible to develop efficient industrial strains to synthesize 1,3-PDO from different resources. A milestone for microbial production of 1,3-PDO is the DuPont Tate & Lyle process from sugar via glycerol. To circumvent the use of expensive vitamin B12 in the ‘glucose-glycerol-PDO’ process, a completely new homoserine-derived 1,3-PDO pathway was developed by Chen et al. (2015) in our lab. In this pathway, 1,3-PDO is produced from L-homoserine via three heterologous enzymatic reactions. The bottleneck step is the first deamination step of L-homoserine to 4-hydroxy-2-oxobutanoic acid (HOBA).
In this work, a phosphoserine aminotransferase (SerC) from E. coli was investigated and engineered to achieve the crucial deamination of L-homoserine. To alter the substrate specificity of SerC from L-phosphoserine to L-homoserine, a computation-based rational method was firstly implemented. Key residues responsible for the substrate binding specificity were identified by calculating the binding free energy based on molecular dynamics simulations and this was followed by in silico site-directed saturation mutagenesis. After three rounds of screening, a few candidates were selected and experimentally verified. The specific activity of the best mutant, SerC(R42W-R77W), was improved by 4.2-fold towards L-homoserine in comparison to the wild type, while its activity towards the natural substrate L-phosphoserine was decreased by 43-fold.
However, the improvement of SerC is still not satisfactory. Considering the limitations of rational design, other screening strategies have also been developed in order to achieve better mutants of SerC. Apart from the method of site-directed mutagenesis, random mutagenesis and semi-rational design method were also used to generate larger and more diverse libraries of mutants. Afterwards, three different strategies were employed to screen the mutant libraries: a) generation and use of glutamate-dependent auxotrophic strain; b) GDH-coupled photometric detection; and c) mercaptopyruvate sulfurtransferase (MPST)-coupled colorimetric screening. Finally, a better mutant SerC(R42W-R77W-R329P) was identified. This mutant showed an increased activity towards L-homoserine by 5.5-fold compared to the wild type and its activity towards L-phosphoserine was completely deactivated. With 3 mM L-homoserine as the substrate, the Km of SerC(R42W-R77W-R329P) was decreased by 68-fold compared to that of the wild type.
To examine the performance of the improved SerC, the complete “homoserine to 1,3-PDO” pathway was constructed by combining SerC with pyruvate decarboxylase (PDC) and alcohol dehydrogenase (YqhD) and introduced into E. coli. To enhance the L-homoserine supply, a homoserine-producing strain was constructed by overexpressing aspartate kinase III (LysC) and homoserine dehydrogenase (MetL) with a medium-copy plasmid. In addition, the genes ldhA and adhE encoding lactate dehydrogenase and aldehyde/alcohol dehydrogenase (AdhE2), respectively, were also removed from the E. coli genome to increase NADH supplementation and decrease byproduct formation under oxygen-limited conditions. Finally, the engineered recombinant E. coli was tested for 1,3-PDO production in both shake flask and fed-batch fermentation. The mutant strain, S147, was able to produce 3.03 g/L 1,3-PDO after 62 h of fermentation, which is 13-fold higher than the wild type strain (S144, 0.24 g/L).
Given the largely improved performance of the engineered SerC for 1,3-PDO production, a new pathway for 1,2,4-butanetriol (BT) biosynthesis, was also constructed from L-homoserine. BT is best-known as a precursor for the production of 1,2,4-butanetriol trinitrate (BTTN), which is of interest as propellant and energetic plasticizer. The new BT pathway involves five consecutive enzymatic reactions catalyzed by four heterologous enzymes, including an engineered SerC, a lactate dehydrogenase, a 4-hydroxybutyrate CoA-transferase and an aldehyde/alcohol dehydrogenase. Implementation of this new pathway in an engineered E. coli resulted in a production of up to 19.6 5.9 mg/L BT in fed-batch fermentations, which is much higher than that (120 ng/L) reported in literature for the production route from L-malate.
In summary, different strategies were employed to develop SerC with an increased activity towards L-homoserine and mutants with improved performance were successfully identified. By integrating these SerC mutants into the homoserine-derived 1,3-PDO pathway and the new BT pathway, their production can be dramatically increased. These studies also provide a basis for developing potential microbial processes for other homoserine-derived biosynthetic pathways.
In this work, a phosphoserine aminotransferase (SerC) from E. coli was investigated and engineered to achieve the crucial deamination of L-homoserine. To alter the substrate specificity of SerC from L-phosphoserine to L-homoserine, a computation-based rational method was firstly implemented. Key residues responsible for the substrate binding specificity were identified by calculating the binding free energy based on molecular dynamics simulations and this was followed by in silico site-directed saturation mutagenesis. After three rounds of screening, a few candidates were selected and experimentally verified. The specific activity of the best mutant, SerC(R42W-R77W), was improved by 4.2-fold towards L-homoserine in comparison to the wild type, while its activity towards the natural substrate L-phosphoserine was decreased by 43-fold.
However, the improvement of SerC is still not satisfactory. Considering the limitations of rational design, other screening strategies have also been developed in order to achieve better mutants of SerC. Apart from the method of site-directed mutagenesis, random mutagenesis and semi-rational design method were also used to generate larger and more diverse libraries of mutants. Afterwards, three different strategies were employed to screen the mutant libraries: a) generation and use of glutamate-dependent auxotrophic strain; b) GDH-coupled photometric detection; and c) mercaptopyruvate sulfurtransferase (MPST)-coupled colorimetric screening. Finally, a better mutant SerC(R42W-R77W-R329P) was identified. This mutant showed an increased activity towards L-homoserine by 5.5-fold compared to the wild type and its activity towards L-phosphoserine was completely deactivated. With 3 mM L-homoserine as the substrate, the Km of SerC(R42W-R77W-R329P) was decreased by 68-fold compared to that of the wild type.
To examine the performance of the improved SerC, the complete “homoserine to 1,3-PDO” pathway was constructed by combining SerC with pyruvate decarboxylase (PDC) and alcohol dehydrogenase (YqhD) and introduced into E. coli. To enhance the L-homoserine supply, a homoserine-producing strain was constructed by overexpressing aspartate kinase III (LysC) and homoserine dehydrogenase (MetL) with a medium-copy plasmid. In addition, the genes ldhA and adhE encoding lactate dehydrogenase and aldehyde/alcohol dehydrogenase (AdhE2), respectively, were also removed from the E. coli genome to increase NADH supplementation and decrease byproduct formation under oxygen-limited conditions. Finally, the engineered recombinant E. coli was tested for 1,3-PDO production in both shake flask and fed-batch fermentation. The mutant strain, S147, was able to produce 3.03 g/L 1,3-PDO after 62 h of fermentation, which is 13-fold higher than the wild type strain (S144, 0.24 g/L).
Given the largely improved performance of the engineered SerC for 1,3-PDO production, a new pathway for 1,2,4-butanetriol (BT) biosynthesis, was also constructed from L-homoserine. BT is best-known as a precursor for the production of 1,2,4-butanetriol trinitrate (BTTN), which is of interest as propellant and energetic plasticizer. The new BT pathway involves five consecutive enzymatic reactions catalyzed by four heterologous enzymes, including an engineered SerC, a lactate dehydrogenase, a 4-hydroxybutyrate CoA-transferase and an aldehyde/alcohol dehydrogenase. Implementation of this new pathway in an engineered E. coli resulted in a production of up to 19.6 5.9 mg/L BT in fed-batch fermentations, which is much higher than that (120 ng/L) reported in literature for the production route from L-malate.
In summary, different strategies were employed to develop SerC with an increased activity towards L-homoserine and mutants with improved performance were successfully identified. By integrating these SerC mutants into the homoserine-derived 1,3-PDO pathway and the new BT pathway, their production can be dramatically increased. These studies also provide a basis for developing potential microbial processes for other homoserine-derived biosynthetic pathways.
DDC Class
600: Technik
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