The Study On Free Fatty Acids And Artemisinin Biosynthesis In Photosynthetic Cyanobacteria

Given that cyanobacteria can grow by photosynthesis and environmental CO₂,it has become host of choice to produce value-added products in a sustainable way.Thus,we extracted promises by genetic modification,applying state-of art synthetic biology and metabolic engineering techniques,so that this host could produce biodiesel component and anti-malarial drug.Previously,it has been reported that photosynthetic production of extracellular free fatty acids（FFAs）based biofuel in cyanobacteria is possible by expressing thioesterase（Tes A）which could hydrolyze fatty acyl-ACP,generate free fatty acid and subsequently excretion outside of cell into media.But,two major drawbacks of these genetically modified strains need to be addressed before scaling it up into commercial outcome;first,toxicity of free fatty acids,second,the diversity of carbon lengths of fatty acids that could mimic the fossil fuel.Toward this goal,we localized a leaderless thioesterase（Ac Tes A）,from Acinetobacter baylyi,using membrane scaffolding system on the cytosolic side of inner membrane of Synechocystis sp PCC6803.The engineered Synechocystis sp PCC 6803 strain consisting Ac Tes A on its membrane（m Ac T）was found to produce free extracellular fatty acid up to 171.9±13.22 mg/L compared with cytosol-expressed Ac Tes A and wild type（WT）strains which produce40.24±10.94mg/L and 1.904±0.158 mg/L respectively.About 78%of total FFAs were extracted from culture media which facilitated cells to reduce cellular toxicity caused by accumulated FFAs.The secretion of FFAs in engineered strain（m Ac T）was at average rate of 1mg L^-1h^-1,that two times higher than those reported in Synechocystis sp.PCC 6803.The mutant strains showed distinct FFAs profile than those previously reported.Higher amount of monounsaturated fatty acids（MUFAs）,about more than 60%of total secreted fatty acid,is C18:1 in mutant strains.MUFAs are considered one of the best precursors for biodiesel.We have also engineered Synechocystis to produce anti-malarial drug component of artemisinin,Artemisinic acid.Artemisinin（also known as qinghaosu）is a sesquiterpene lactone having a special epoxide bridge.This drug was found to have effect as not only anti-malarial but also as anti-cancer,anti-tumor and anti-pathogenic activity.Artemisinin is originally produced in Chinese harb Artemisinia annua,locally called sweet woodworm.After first isolation in 1972 by Noble Laureate（2015）Tu Youyou,it has been considered number one drug to combat against deadly malarial treatment.However,the lengthy cultivation time,low abundance and costly isolation procedure make it difficult for large scale production that make this drug so expensive which is beyond the capacity of malaria sufferer.Efforts have been made to get genetically modified A.annua plant but resulted either no significant improvement or unstable strain.Synthetic biology approaches have also been noticed to elevate production of this drug in yeast and E.coli but failed to get the final product Atremisinin.Rather,those engineered hosts can produce Artemisinic acid,the component that needs further chemical treatment to convert Artemisinin.In addition,those engineered hosts need glucose as carbon source that,again,is not cost effective as it needed to be.Knowing cyanobacteria are the evolutionary ancestor of plant,we then planned to produce Artemisinin in Synechocystis.Heterologous Artemisinin biosynthetic pathway was constructed by expressing ADS,CYP/CPR in Synechocystis that could convert intermediate of MEP pathway to Amorphadine,Artimisinic Acid and Artemisinin.By engineering the common MEP/DXP pathway in Synechosystis for sustainable production of artemisinin we developed the genetically engineered strain ACC,which expressed amorpha,4-11diene synthase（ADS）,cytochrome P450 monooxygenase（CYP71AV1）and cytochrome P450reductase（CPR）genes form plant A.annua.We found the strain expressing ADS could produce amorpha-4,11-dine（5mg/L）.Further expression of CYP/CPR could convert this amorphadine into Artemisinic acid（6.258 mg/gm DCW）,the product that was previously been produced in engineered E.coli and yeast,Saccharomyces cerevisiae.The final ACC strain showed the artemisinin content was 114μg/gm DCW.Moreover,we maintained modification of growth media（BG-11）of Synechocystis by adding nutrient such as glycerol,sodium acetate etc and found those modifications can change the product level.Our findings demonstrate that cyanobacteria as a possible“molecular firm”can provide the platform to produce artemisinin as like plant and the first example of artemisinin production in any microbial host other than plant source.Taken together,this dissertation described promising approaches that shed light on huge benefits from Cyanobacteria.It is thus convincible that further advancement and application of synthetic biology and metabolic engineering and their appropriate application on this host could bring some solution that the humanity is looking for long time.