TY - BOOK

T1 - Synthetic Biology with Cytochromes P450 Using Photosynthetic Chassis

AU - Gnanasekaran, Thiyagarajan

PY - 2014

Y1 - 2014

N2 - Synthetic biology is a rapidly growing engineering discipline in biology. It aims at building novel biological systems that do not exist in nature by selecting the interchangeable standardized biological parts that are already available in the nature, and assembling them in a specific order. Today, this modern field of synthetic biology is completely dependent on the nature of the chassis - the host organisms - for its endeavor. Of all the chassis, photosynthetic organisms such as cyanobacteria and plants gains special attention due to the remarkable amount of sunlight that is striking the Earth’s atmosphere and anthropogenic carbon dioxide (CO2) increase in the atmosphere. Hence, tapping into photosynthesis for synthetic biology endeavor is very rational, and for future, it has a huge potential for the industrial production of fuels and high value bioactive compounds in a sustainable way. Most of these commercially important high value bioactive compounds are plant derived, and in plants, some of the key enzymes that catalyze the production of these compounds are cytochromesP450 (P450s). This thesis focuses on three subprojects in which we expressed plant metabolic pathways involving P450 enzymes in cyanobacteria and plant chloroplasts for the purpose of light driven synthesis of bioactive compounds by using synthetic biology approaches. As model pathways, in this thesis, the pathway involved in the synthesis of the cyanogenic glucoside dhurrin from Sorghum bicolor, and the pathway involved in the synthesis of diterpene resin acid (isopimaric acid) from the genus Picea were exploited.In Sorghum bicolor, the dhurrin pathway is localized in the endoplasmic reticulum,and it is comprised of two P450s (CYP79A1 and CYP71E1) and a glycosyl transferase UGT85B1. We chose the dhurrin pathway because every catalytic step in the dhurrin pathway is well characterized and is an ideal model pathway for the synthetic biology exploitation. In the first subproject, we successfully engineered the dhurrin pathway in the chloroplasts of tobacco, and demonstrated that all the three enzymes were successfully expressed and remained highly active. In addition, we also demonstrated that the P450s can be successfully localized in the thylakoids, and can be driven by using electrons derived directly from the photosynthetic electron transport chain via ferredoxin without the need for a P450 oxidoreductase (POR) that extracts electrons from NADPH. This proof of concept is to our knowledge the first of its kind where we show that the plant P450s can be driven by the electrons extracted from the photosynthetic electron transport chain in planta. Furthermore,we also analyzed the metabolite profile in our engineered lines, and the accumulation of dhurrin constituted ~0.25 % of leaf dry weight in addition to few defined glycosidic derivaties that constituted ~0.5 % of leaf dry weight.In the second subproject, the aim was to express the dhurrin pathway in thecyanobacterial chassis Synechocystis sp. PCC 6803. For this purpose we used a similar approach as the first subproject, and designed constructs to express all the three enzymes of the dhurrin pathway individually and also in combination in the cyanobacterial chassis. By successfully expressing and localizing the P450s in the thylakoids, we demonstrated the P450s can be light-driven both in vitro and in vivo. Furthermore we also demonstrated that in vivo, the intermediate oxime gets secreted out of the cells into the growth media making the extraction of these kinds of compounds relatively easier in downstream processes, which eliminates the need for breaking the cells. The P450s were successfully expressed and localized to the thylakoids, but expressing the UGT85B1 along with the P450s has not beenachieved yet.In the third subproject, the aim was to transiently engineer the diterpene resin acidIsopimaric acid pathway in the chloroplast of Nicotiana benthamiana. For this purpose we chose the coding region for ISO from Sitka spruce (Picea sitchensis), and the coding region for CYP720B4 from Norway spruce (Picea abies). Upon infiltrating the gene constructs, the GC-MS analysis on leaf extracts confirmed the successful production of both isopimaradiene and isopimaric acid in N. benthamiana. This demonstrated that using transient infiltration of Nicotiana benthamiana with Agrobacterium tumefaciens as a tool, the expression of heterologous metabolic pathways that are involved in terpenoid biosynthesis is indeed feasible. However, during this study, we encountered difficulties in targeting CYP720B4 to the thylakoids, and hence, new gene constructs designs for targeting the P450 to the thylakoids were proposed. In the future, the expression and localization of the new P450s gene constructs will be analyzed in greater detail to achieve light-driven synthesis of high value compounds such as terpenoids.

AB - Synthetic biology is a rapidly growing engineering discipline in biology. It aims at building novel biological systems that do not exist in nature by selecting the interchangeable standardized biological parts that are already available in the nature, and assembling them in a specific order. Today, this modern field of synthetic biology is completely dependent on the nature of the chassis - the host organisms - for its endeavor. Of all the chassis, photosynthetic organisms such as cyanobacteria and plants gains special attention due to the remarkable amount of sunlight that is striking the Earth’s atmosphere and anthropogenic carbon dioxide (CO2) increase in the atmosphere. Hence, tapping into photosynthesis for synthetic biology endeavor is very rational, and for future, it has a huge potential for the industrial production of fuels and high value bioactive compounds in a sustainable way. Most of these commercially important high value bioactive compounds are plant derived, and in plants, some of the key enzymes that catalyze the production of these compounds are cytochromesP450 (P450s). This thesis focuses on three subprojects in which we expressed plant metabolic pathways involving P450 enzymes in cyanobacteria and plant chloroplasts for the purpose of light driven synthesis of bioactive compounds by using synthetic biology approaches. As model pathways, in this thesis, the pathway involved in the synthesis of the cyanogenic glucoside dhurrin from Sorghum bicolor, and the pathway involved in the synthesis of diterpene resin acid (isopimaric acid) from the genus Picea were exploited.In Sorghum bicolor, the dhurrin pathway is localized in the endoplasmic reticulum,and it is comprised of two P450s (CYP79A1 and CYP71E1) and a glycosyl transferase UGT85B1. We chose the dhurrin pathway because every catalytic step in the dhurrin pathway is well characterized and is an ideal model pathway for the synthetic biology exploitation. In the first subproject, we successfully engineered the dhurrin pathway in the chloroplasts of tobacco, and demonstrated that all the three enzymes were successfully expressed and remained highly active. In addition, we also demonstrated that the P450s can be successfully localized in the thylakoids, and can be driven by using electrons derived directly from the photosynthetic electron transport chain via ferredoxin without the need for a P450 oxidoreductase (POR) that extracts electrons from NADPH. This proof of concept is to our knowledge the first of its kind where we show that the plant P450s can be driven by the electrons extracted from the photosynthetic electron transport chain in planta. Furthermore,we also analyzed the metabolite profile in our engineered lines, and the accumulation of dhurrin constituted ~0.25 % of leaf dry weight in addition to few defined glycosidic derivaties that constituted ~0.5 % of leaf dry weight.In the second subproject, the aim was to express the dhurrin pathway in thecyanobacterial chassis Synechocystis sp. PCC 6803. For this purpose we used a similar approach as the first subproject, and designed constructs to express all the three enzymes of the dhurrin pathway individually and also in combination in the cyanobacterial chassis. By successfully expressing and localizing the P450s in the thylakoids, we demonstrated the P450s can be light-driven both in vitro and in vivo. Furthermore we also demonstrated that in vivo, the intermediate oxime gets secreted out of the cells into the growth media making the extraction of these kinds of compounds relatively easier in downstream processes, which eliminates the need for breaking the cells. The P450s were successfully expressed and localized to the thylakoids, but expressing the UGT85B1 along with the P450s has not beenachieved yet.In the third subproject, the aim was to transiently engineer the diterpene resin acidIsopimaric acid pathway in the chloroplast of Nicotiana benthamiana. For this purpose we chose the coding region for ISO from Sitka spruce (Picea sitchensis), and the coding region for CYP720B4 from Norway spruce (Picea abies). Upon infiltrating the gene constructs, the GC-MS analysis on leaf extracts confirmed the successful production of both isopimaradiene and isopimaric acid in N. benthamiana. This demonstrated that using transient infiltration of Nicotiana benthamiana with Agrobacterium tumefaciens as a tool, the expression of heterologous metabolic pathways that are involved in terpenoid biosynthesis is indeed feasible. However, during this study, we encountered difficulties in targeting CYP720B4 to the thylakoids, and hence, new gene constructs designs for targeting the P450 to the thylakoids were proposed. In the future, the expression and localization of the new P450s gene constructs will be analyzed in greater detail to achieve light-driven synthesis of high value compounds such as terpenoids.

UR - https://soeg.kb.dk/permalink/45KBDK_KGL/fbp0ps/alma99122153182605763

M3 - Ph.D. thesis

BT - Synthetic Biology with Cytochromes P450 Using Photosynthetic Chassis

PB - Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen

ER -