Metabolic engineering of cyanobacteria for the production of aromatic compounds

Laura Maria Furelos Brey

Metabolic engineering of cyanobacteria for the production of aromatic compounds

Laura Maria Furelos Brey

Abstract

In light of the climate change challenge, the advantageous trait of using solar energy and carbon dioxide to produce organic molecules has granted cyanobacteria deserved interest as hosts for metabolic engineering. Importantly, the production of valuable chemicals in these organisms does not directly compete with terrestrial agricultural resources such as productive land and fresh water. Through collective efforts, a range of cyanobacterial strains have been engineered to produce a number of valuable organic compounds. However, the investigation of cyanobacteria as chassis for heterologous production of valuable aromatic amino acids and derived aromatic compounds is so far limited. Aromatic amino acids and derived phenylpropanoids are of high importance because they are used by the pharmaceutical, food, cosmetic, and agricultural industries as precursors of active ingredients. The aim of this thesis was to engineer the metabolism of Synechocystis sp. PCC 6803 to explore the sustainable production of valuable aromatic amino acids and their derived phenylpropanoids. The work is organized in three parts comprising an introductory chapter and two manuscripts. The introduction chapter reviews several aspects of the physiology of Synechocystis sp. PCC 6803 necessary to understand the present work. It also summarizes the current molecular tools used to engineer the metabolism of cyanobacteria, and lists several successful examples of production of valuable chemicals in these organisms. The first manuscript depicts the strategies we followed to achieve the successful overproduction of aromatic amino acids in Synechocystis sp. PCC 6803. Our results show that it is possible to overproduce phenylalanine and tyrosine with high efficiency sorely from supplied carbon dioxide and inorganic nutrients. Production titers reached 905 53mg=gDW (580 34mg=l) of phenylalanine and 64:0 3:7mg=gDW (41:0 2:3mg=l) of tyrosine after 10 days of photoautotropic growth. Estimations suggest that 56 % of the fixed carbon was deviated from biomass to be channeled to phenylalanine and tyrosine production. The second manuscript comprises the experimental data and results obtained for the heterologous production of phenylpropanoids from phenylalanine and tyrosine in Synechocystis sp. PCC 6803. Using a phenylalanine/tyrosine overproducer strain generated in the first manuscript, we tested the activity of several phenylpropanoid biosynthetic enzymes and achieved the successful production of 470 70mg=gDW (207mg=l) of p-coumaric acid, 267 31mg=gDW (114mg=l) of cinnamic acid and 47 14mg=gDW (12:6mg=l) of caffeic acid after 5 days of photoautotrophic growth; and 104 μg=l of L-DOPA after 3 days of photomixotropic growth. To our knowledge, this are the highest titers of these phenylpropanoids produced by any cyanobacteria strain thus far. The results presented in this thesis show it is possible to engineer cyanobacteria to overproduce aromatic amino acids with a high efficiency and that these compounds can be effectively channel to valuable phenylpropanoids. Although a more detailed molecular characterization would be needed to understand the physiological bases and additionally improve titers, these findings suggest cyanobacteria are competitive host for sustainable production of valuable aromatic compounds that must be explored furthe.

Originalsprog	Engelsk

Forlag	Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen
Status	Udgivet - 2019

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@phdthesis{983f94cb0cd34dbba402cc4e1558b4a8,

title = "Metabolic engineering of cyanobacteria for the production of aromatic compounds",

abstract = "In light of the climate change challenge, the advantageous trait of using solar energy and carbon dioxide to produce organic molecules has granted cyanobacteria deserved interest as hosts for metabolic engineering. Importantly, the production of valuable chemicals in these organisms does not directly compete with terrestrial agricultural resources such as productive land and fresh water. Through collective efforts, a range of cyanobacterial strains have been engineered to produce a number of valuable organic compounds. However, the investigation of cyanobacteria as chassis for heterologous production of valuable aromatic amino acids and derived aromatic compounds is so far limited. Aromatic amino acids and derived phenylpropanoids are of high importance because they are used by the pharmaceutical, food, cosmetic, and agricultural industries as precursors of active ingredients. The aim of this thesis was to engineer the metabolism of Synechocystis sp. PCC 6803 to explore the sustainable production of valuable aromatic amino acids and their derived phenylpropanoids. The work is organized in three parts comprising an introductory chapter and two manuscripts. The introduction chapter reviews several aspects of the physiology of Synechocystis sp. PCC 6803 necessary to understand the present work. It also summarizes the current molecular tools used to engineer the metabolism of cyanobacteria, and lists several successful examples of production of valuable chemicals in these organisms. The first manuscript depicts the strategies we followed to achieve the successful overproduction of aromatic amino acids in Synechocystis sp. PCC 6803. Our results show that it is possible to overproduce phenylalanine and tyrosine with high efficiency sorely from supplied carbon dioxide and inorganic nutrients. Production titers reached 905 53mg=gDW (580 34mg=l) of phenylalanine and 64:0 3:7mg=gDW (41:0 2:3mg=l) of tyrosine after 10 days of photoautotropic growth. Estimations suggest that 56 % of the fixed carbon was deviated from biomass to be channeled to phenylalanine and tyrosine production. The second manuscript comprises the experimental data and results obtained for the heterologous production of phenylpropanoids from phenylalanine and tyrosine in Synechocystis sp. PCC 6803. Using a phenylalanine/tyrosine overproducer strain generated in the first manuscript, we tested the activity of several phenylpropanoid biosynthetic enzymes and achieved the successful production of 470 70mg=gDW (207mg=l) of p-coumaric acid, 267 31mg=gDW (114mg=l) of cinnamic acid and 47 14mg=gDW (12:6mg=l) of caffeic acid after 5 days of photoautotrophic growth; and 104 μg=l of L-DOPA after 3 days of photomixotropic growth. To our knowledge, this are the highest titers of these phenylpropanoids produced by any cyanobacteria strain thus far. The results presented in this thesis show it is possible to engineer cyanobacteria to overproduce aromatic amino acids with a high efficiency and that these compounds can be effectively channel to valuable phenylpropanoids. Although a more detailed molecular characterization would be needed to understand the physiological bases and additionally improve titers, these findings suggest cyanobacteria are competitive host for sustainable production of valuable aromatic compounds that must be explored furthe.",

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year = "2019",

language = "English",

publisher = "Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen",

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AU - Furelos Brey, Laura Maria

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Y1 - 2019

N2 - In light of the climate change challenge, the advantageous trait of using solar energy and carbon dioxide to produce organic molecules has granted cyanobacteria deserved interest as hosts for metabolic engineering. Importantly, the production of valuable chemicals in these organisms does not directly compete with terrestrial agricultural resources such as productive land and fresh water. Through collective efforts, a range of cyanobacterial strains have been engineered to produce a number of valuable organic compounds. However, the investigation of cyanobacteria as chassis for heterologous production of valuable aromatic amino acids and derived aromatic compounds is so far limited. Aromatic amino acids and derived phenylpropanoids are of high importance because they are used by the pharmaceutical, food, cosmetic, and agricultural industries as precursors of active ingredients. The aim of this thesis was to engineer the metabolism of Synechocystis sp. PCC 6803 to explore the sustainable production of valuable aromatic amino acids and their derived phenylpropanoids. The work is organized in three parts comprising an introductory chapter and two manuscripts. The introduction chapter reviews several aspects of the physiology of Synechocystis sp. PCC 6803 necessary to understand the present work. It also summarizes the current molecular tools used to engineer the metabolism of cyanobacteria, and lists several successful examples of production of valuable chemicals in these organisms. The first manuscript depicts the strategies we followed to achieve the successful overproduction of aromatic amino acids in Synechocystis sp. PCC 6803. Our results show that it is possible to overproduce phenylalanine and tyrosine with high efficiency sorely from supplied carbon dioxide and inorganic nutrients. Production titers reached 905 53mg=gDW (580 34mg=l) of phenylalanine and 64:0 3:7mg=gDW (41:0 2:3mg=l) of tyrosine after 10 days of photoautotropic growth. Estimations suggest that 56 % of the fixed carbon was deviated from biomass to be channeled to phenylalanine and tyrosine production. The second manuscript comprises the experimental data and results obtained for the heterologous production of phenylpropanoids from phenylalanine and tyrosine in Synechocystis sp. PCC 6803. Using a phenylalanine/tyrosine overproducer strain generated in the first manuscript, we tested the activity of several phenylpropanoid biosynthetic enzymes and achieved the successful production of 470 70mg=gDW (207mg=l) of p-coumaric acid, 267 31mg=gDW (114mg=l) of cinnamic acid and 47 14mg=gDW (12:6mg=l) of caffeic acid after 5 days of photoautotrophic growth; and 104 μg=l of L-DOPA after 3 days of photomixotropic growth. To our knowledge, this are the highest titers of these phenylpropanoids produced by any cyanobacteria strain thus far. The results presented in this thesis show it is possible to engineer cyanobacteria to overproduce aromatic amino acids with a high efficiency and that these compounds can be effectively channel to valuable phenylpropanoids. Although a more detailed molecular characterization would be needed to understand the physiological bases and additionally improve titers, these findings suggest cyanobacteria are competitive host for sustainable production of valuable aromatic compounds that must be explored furthe.

AB - In light of the climate change challenge, the advantageous trait of using solar energy and carbon dioxide to produce organic molecules has granted cyanobacteria deserved interest as hosts for metabolic engineering. Importantly, the production of valuable chemicals in these organisms does not directly compete with terrestrial agricultural resources such as productive land and fresh water. Through collective efforts, a range of cyanobacterial strains have been engineered to produce a number of valuable organic compounds. However, the investigation of cyanobacteria as chassis for heterologous production of valuable aromatic amino acids and derived aromatic compounds is so far limited. Aromatic amino acids and derived phenylpropanoids are of high importance because they are used by the pharmaceutical, food, cosmetic, and agricultural industries as precursors of active ingredients. The aim of this thesis was to engineer the metabolism of Synechocystis sp. PCC 6803 to explore the sustainable production of valuable aromatic amino acids and their derived phenylpropanoids. The work is organized in three parts comprising an introductory chapter and two manuscripts. The introduction chapter reviews several aspects of the physiology of Synechocystis sp. PCC 6803 necessary to understand the present work. It also summarizes the current molecular tools used to engineer the metabolism of cyanobacteria, and lists several successful examples of production of valuable chemicals in these organisms. The first manuscript depicts the strategies we followed to achieve the successful overproduction of aromatic amino acids in Synechocystis sp. PCC 6803. Our results show that it is possible to overproduce phenylalanine and tyrosine with high efficiency sorely from supplied carbon dioxide and inorganic nutrients. Production titers reached 905 53mg=gDW (580 34mg=l) of phenylalanine and 64:0 3:7mg=gDW (41:0 2:3mg=l) of tyrosine after 10 days of photoautotropic growth. Estimations suggest that 56 % of the fixed carbon was deviated from biomass to be channeled to phenylalanine and tyrosine production. The second manuscript comprises the experimental data and results obtained for the heterologous production of phenylpropanoids from phenylalanine and tyrosine in Synechocystis sp. PCC 6803. Using a phenylalanine/tyrosine overproducer strain generated in the first manuscript, we tested the activity of several phenylpropanoid biosynthetic enzymes and achieved the successful production of 470 70mg=gDW (207mg=l) of p-coumaric acid, 267 31mg=gDW (114mg=l) of cinnamic acid and 47 14mg=gDW (12:6mg=l) of caffeic acid after 5 days of photoautotrophic growth; and 104 μg=l of L-DOPA after 3 days of photomixotropic growth. To our knowledge, this are the highest titers of these phenylpropanoids produced by any cyanobacteria strain thus far. The results presented in this thesis show it is possible to engineer cyanobacteria to overproduce aromatic amino acids with a high efficiency and that these compounds can be effectively channel to valuable phenylpropanoids. Although a more detailed molecular characterization would be needed to understand the physiological bases and additionally improve titers, these findings suggest cyanobacteria are competitive host for sustainable production of valuable aromatic compounds that must be explored furthe.

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BT - Metabolic engineering of cyanobacteria for the production of aromatic compounds

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