Investigating the overproduction of aromatic amino acids and derived phenylpropanoids in cyanobacteria
Publikation: Bog/antologi/afhandling/rapport › Ph.d.-afhandling › Forskning
Cyanobacteria, as photosynthetic microorganisms, are promising candidates for sustainable production of high value compounds due to their ability to thrive on minimal nutrients, CO2, and sunlight (Part I). Despite their critical role in climate regulation and oxygen production, the full potential of cyanobacteria remains largely untapped. This thesis delves into the field of metabolic engineering of Synechocystis sp. PCC 6803 (hereafter Synechocystis), a unicellular cyanobacteria and model species. The aim of this thesis is to explore the use of Synechocystis for biotechnological applications, with a primary focus on the production of aromatic amino acids and derived compounds such as phenylpropanoids and flavonoids. These secondary metabolites, typically found in plants in small quantities, are of high value in the food, pharmaceutical and cosmetic industries.
In Part II, the review by Bolay et al. (2024), we provide an updated overview of the current knowledge of gene and protein regulation in cyanobacteria, by sigma factors, transcriptional regulators, and small regulatory proteins. Furthermore, we discuss the potential of manipulating these regulatory elements for more favorable phenotypes suitable for synthetic biology applications. Engineering transcriptional elements and other regulatory proteins in cyanobacteria can alter the entire cellular metabolism, redirecting carbon fluxes towards synthesis of the products of interest. Some studies have already demonstrated how altering expression of sigma factors and transcription factors can enhance stress tolerance and increase CO2 fixation. Combining these regulatory modifications, could lead to more robust strains with higher productivity.
In Part III, we aimed for a comprehensive characterization of a Synechocystis strain, AT-3, genetically engineered to overproduce aromatic amino acids. Overproduction of tyrosine and phenylalanine causes a visibly altered phenotype through changes in pigment composition, carbon storage and growth rates. By integrating both untargeted proteomics and metabolomics through a genome scale model, we were able to map underlying metabolic changes in AT-3. Several sigma factors were significantly upregulated, which are known to cause cell-wide stress responses. Overall, Part III sheds light on the intricate interplay between amino acid biosynthesis and cellular homeostasis and it implications for the biotechnological production of aromatic compounds in cyanobacteria.
In order to better to understand the potential negative growth effects of aromatic compounds on cyanobacteria, we performed a toxicity assay in Part IV. Exogenous supplementation of phenylalanine and tyrosine caused significant growth inhibition in wildtype Synechocystis, likely caused by intracellular feedback regulation. This prompted the investigation of potential protein-metabolite interactions, using Proteome Integral Solubility Alteration (PISA), a technique novel to cyanobacteria. Tyrosine and phenylalanine were incubated in vitro with the extracted proteome of Synechocystis, confirming some known interactions and revealing several previously unknown interactions, including a potential transporter. The binding domain of this transporter demonstrated stronger interactions with phenylalanine, which could explain why we see more phenylalanine in the media in Part III. Several protein-metabolite interactions were confirmed by in silico molecular docking, providing insights about the active sites and substrate binding of several potential target enzymes.
Expanding upon aromatic amino acid biosynthesis, Part V explores the production of phenylpropanoids in Synechocystis. Knockout of an endogenous laccase was confirmed to be beneficial for the production of p-coumaric acid, the phenylpropanoid derived from the deamination of tyrosine. Furthermore, several heterologous enzymes were expressed in Synechocystis in an attempt to produce caffeic acid. Although the expected product, caffeic acid was not detected in any samples, the substrate p-coumaric acid appears to be metabolized. Untargeted LCMS/MS analysis was employed in an attempt to identify these compounds. Several new phenolic compounds were found, as well as the endogenous production of the flavonoid, homoeriodictyol.
Although cyanobacteria are extremely complex and fascinating, we still have much to learn, to exploit their tremendous potential in biotechnology. They contain the reducing power and cofactors necessary to produce complex plant metabolites without the need for sugar or arable land. By unraveling metabolic pathways and developing innovative genetic engineering strategies, cyanobacteria can emerge as heterologous production platforms, contributing to a greener and more sustainable future.
In Part II, the review by Bolay et al. (2024), we provide an updated overview of the current knowledge of gene and protein regulation in cyanobacteria, by sigma factors, transcriptional regulators, and small regulatory proteins. Furthermore, we discuss the potential of manipulating these regulatory elements for more favorable phenotypes suitable for synthetic biology applications. Engineering transcriptional elements and other regulatory proteins in cyanobacteria can alter the entire cellular metabolism, redirecting carbon fluxes towards synthesis of the products of interest. Some studies have already demonstrated how altering expression of sigma factors and transcription factors can enhance stress tolerance and increase CO2 fixation. Combining these regulatory modifications, could lead to more robust strains with higher productivity.
In Part III, we aimed for a comprehensive characterization of a Synechocystis strain, AT-3, genetically engineered to overproduce aromatic amino acids. Overproduction of tyrosine and phenylalanine causes a visibly altered phenotype through changes in pigment composition, carbon storage and growth rates. By integrating both untargeted proteomics and metabolomics through a genome scale model, we were able to map underlying metabolic changes in AT-3. Several sigma factors were significantly upregulated, which are known to cause cell-wide stress responses. Overall, Part III sheds light on the intricate interplay between amino acid biosynthesis and cellular homeostasis and it implications for the biotechnological production of aromatic compounds in cyanobacteria.
In order to better to understand the potential negative growth effects of aromatic compounds on cyanobacteria, we performed a toxicity assay in Part IV. Exogenous supplementation of phenylalanine and tyrosine caused significant growth inhibition in wildtype Synechocystis, likely caused by intracellular feedback regulation. This prompted the investigation of potential protein-metabolite interactions, using Proteome Integral Solubility Alteration (PISA), a technique novel to cyanobacteria. Tyrosine and phenylalanine were incubated in vitro with the extracted proteome of Synechocystis, confirming some known interactions and revealing several previously unknown interactions, including a potential transporter. The binding domain of this transporter demonstrated stronger interactions with phenylalanine, which could explain why we see more phenylalanine in the media in Part III. Several protein-metabolite interactions were confirmed by in silico molecular docking, providing insights about the active sites and substrate binding of several potential target enzymes.
Expanding upon aromatic amino acid biosynthesis, Part V explores the production of phenylpropanoids in Synechocystis. Knockout of an endogenous laccase was confirmed to be beneficial for the production of p-coumaric acid, the phenylpropanoid derived from the deamination of tyrosine. Furthermore, several heterologous enzymes were expressed in Synechocystis in an attempt to produce caffeic acid. Although the expected product, caffeic acid was not detected in any samples, the substrate p-coumaric acid appears to be metabolized. Untargeted LCMS/MS analysis was employed in an attempt to identify these compounds. Several new phenolic compounds were found, as well as the endogenous production of the flavonoid, homoeriodictyol.
Although cyanobacteria are extremely complex and fascinating, we still have much to learn, to exploit their tremendous potential in biotechnology. They contain the reducing power and cofactors necessary to produce complex plant metabolites without the need for sugar or arable land. By unraveling metabolic pathways and developing innovative genetic engineering strategies, cyanobacteria can emerge as heterologous production platforms, contributing to a greener and more sustainable future.
Originalsprog | Engelsk |
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Forlag | Department of Food Science, Faculty of Science, University of Copenhagen |
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Antal sider | 181 |
Status | Udgivet - 2024 |
ID: 399178625