Building Blue E. Coli: Engineering the Anthocyanin Biosynthetic Pathway
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Building blue E. coli: Engineering the anthocyanin biosynthetic pathway Leah Johnston1, Lucas Jarche1, Alexander Porter1, Julie Ryu1, Emma Price1, Emma Finlayson-Trick1, Khaled Aly1, Paul Bissonnette2, Eric Pace3, Dr. John Rohde1 1Department of Microbiology and Immunology, 2Department of Chemistry, 3Department of Agriculture Dalhousie University, Halifax, Canada, B3H 1X5 THE ANTHOCYANIN PATHWAY METHODS RESULTS 1kb (B) Method of Template DNA for each of the genes required for the production of the Anthocyanin biosynthetic Gene Size (bp) Function Epitope ladder (B) Acquisition of pathway in E. coli were acquired from the following sources outlined in Figure 1 (B). (A) Name Tag Template DNA 10 Phenylalanine Lyase involved in Amplified from A. (A) 2762 polyphenol S-Tag thaliana cDNA, Ammonia . synthesis. insert in Phenylalanine Ammonia Lyase and 4-Coumaroyl CoA Ligase were acquired as inserts in a 3.0. Lyase pACYCDuet-1 pACYCDuet-1 plasmid from Sinyuan Wang et. al (2005) at Utah State University. Cinnamate 4- 2.0 Phenylalanine from Utah State Hydroxylase-HA fusion protein was inserted into this plasmid at the end of the PAL coding region 1.5 Phenylalanine ammonia lyase University using restriction enzymes and transformed into BL21 CaCl2 competent E. coli with T7 RNA Cinnamate 4- Oxidoreductase Amplified from A. Cinnamic Acid 1518 involved in HA-Tag thaliana cDNA, Polymerase activity. A western blot with anti-HA antibody was used to detect the presence of the 1.0 Hydroxylase phenylalanine purchased from C4H fusion protein in the resultant cells. Cinnamate 4-hydroxylase metabolism. TAIR database P-Coumaric Acid 4-Coumaroyl Ligase involved in Insert in 2546 phenylpropanoid His-Tag pACYCDuet-1 Chalcone Synthase, Chalcone Isomerase and Flavanone 3-Hydroxylase were amplified from the CoA Ligase 0.5 4-coumaryl-coenzyme A ligase biosynthesis. from Utah State TAIR library with primers designed to create overlapping areas on each gene, as well as adding Coumaryl-CoA (+ University an epitope tag to each gene. These overlaps were used to stitch together a 3-gene operon using Chalcone Sy---nthase Gibson Assembly, 1188 involved in plant FLAG- amplified from A. Gibson Assembly. The three genes were cloned into a pET-302/Nt-His overexpression plasmid Malonyl – CoA) Synthase immunity and thaliana cDNA which was used to transform the BL21 E. coli cells. After induction the E. coli strains were flavonoid Tag Chalcone synthase biosynthesis. subjected to expression testing through induction with IPTG and confirmation using Western Figure 2 .Title?. (A) Flavonone 3’5’-hydroxylase (1.5kb) produced through Gibson assembly Blots targeting epitope tags built into the proteins. For a list of the tags associated with each gene Naringenin Chalcone Chalcone Isomerase Gibson Assembly, and cloned into a pGEM T-easy vector, henceforth referred to as pDAL_bb3 (B) Western blot 741 involved in HA-Tag amplified from A. Isomerase see Figure 1 (B) probed with an HA antibody to detect the presence of a C4H-HA fusion protein cloned into the Chalcone isomerase flavonoid thaliana cDNA biosynthesis. pACYCDuet-1 plasmid. The first 3 lanes are lysates from E. coli that do not contain this plasmid Naringenin Dihydroflavanol 4-Reductase and Anthocyanidin Synthase were amplified from A. thaliana cDNA (C) The final location of the nine genes of the delphinidin pathway after cloning. Plasmid Flavanone 3- Hydroxylase Gibson Assembly, 744 involved in Myc-Tag amplified from A. and cloned into pGEM T-Easy vectors. Flavanone 3’,5’ Hydroxylase was also synthesized using Flavonone 3-hydroxylase Hydroxylase pDAL_bb1 contains phenylalanine ammonia lyase (PAL), cinnamate 4-hydroxylase (C4H), and flavonoid thaliana cDNA Gibson Assembly and assembled into a pGEM T-Easy plasmid. Dihydrokaempferol biosynthesis. 4-courmaryl-coenzyme A ligase (4CL). Plasmid pDAL_bb2 contains chalcone synthase (CS), chalcone isomerase (CI), and flavonone 3-hydroxylase (F3H). Flavonoid 3’5’-hydroxylase Hydroxylase Gibson Assembly Flavonoid 3’5’-hydroxylase Flavanone 3’, 1518 involved in - from 5’ (F3’5’H), dihydroflavonol 4-reductase (D4R), and anthocyanidin synthase (AS) are contained on flavonoid oligonucleotides Dihydromyricetin Hydroxylase biosynthesis. individual plasmids pDAL_bb3, bb4, and bb5 respectively. Dihydroflavonol 4-reductase RESULTS Dihydroflavan Oxidoreductase Amplified from A. 1149 involved in FLAG- thaliana cDNA -ol 4- Leucodelphinidin flavonoid Reductase biosynthesis. Tag (B) Anthocyanidin synthase (A) Anthocyanidi- Oxidoreductase Amplified d from PAL CS 1071 involved in HA-Tag A. thaliana cDNA Delphinidin n Synthase flavonoid pDAL_bb3 pDAL_bb5 biosynthesis. pDAL_bb1 C4H pDAL_bb2 CI F3’5’H AS Figure 1. The anthocyanin (delphinidin) pathway and its constituent enzymes. (A) The pDAL_bb4 conversion of phenylalanine to the blue anthocyanin delphinidin occurs in nine steps pDAL_bb2 catalyzed by nine separate enzymes. (B) Table displaying basic information on each of the 4CL F3H nine genes in the delphinidin pathway. Also shown is the epitope tag which was cloned into D4R each gene through PCR primer design and the acquisition method for each gene. (C) Empty Vector Empty Vector CONCLUSIONS AND FUTURE DIRECTIONS INTRODUCTION • The nine genes from the delphinidin pathway were successfully Plant secondary metabolites provide a diverse source of economically important products cloned into various vectors. such as drugs, flavors, fragrances, dyes, pigments, pesticides and food additives. An anthocyanin is an example of a plant secondary metabolite, a class of water soluble, • A yellow colour was detected in E. coli transformants expressing pigmented compounds that are useful for their antioxidant properties. Large scale production three of these genes. of these secondary metabolites through the cultivation of plant sources can prove to be • We plan to engineer all nine genes into a transmissible vector to use uneconomical due to low crop yields, high operation costs and difficulties involved in isolating and purifying target compounds. Recently, there has been a shift to circumvent the blue phenotype for screening genes that affect the anthocyanin these problems by inserting foreign biosynthetic pathways into microbial workhorses such as Figure 3. The recombinant plasmid pDAL_bb2 containing chalcone pathway E. coli. Taking into account this methodology, it was our objective to synthesize the plant synthase, chalcone isomerase, and flavonone 3-hydroxylase. (A) BL21 E. secondary metabolite delphinidin, an anthocyanin, by expressing the genes involved in coli containing empty vector (left) versus Bl21 E. coli containing a pDAL_bb2 ACKNOWLEDGEMENTS delphinidin biosynthesis in E. coli. The inspiration for this project came from the pioneering work done by the various research groups who were successful in bioengineering and recombinant plasmid with the coding sequence for CS, CI, and F3H fusion expressing the lycopene biosynthetic pathway. proteins (right). (B) Western blot probed with HA, Myc, and Flag antibodies to Dr. Vasantha Rupasinghe • Dr. Christine Barnes • Dr. Sophia Stone • detect the presence of a three fusion proteins from a BL21 E. coli cell Daryl McNeilly • Dr. Jean Marshall • Dr. Richard Florizone • Dr. Melanie Successful implementation of the pathway into E. coli would have two important transformed with the pDAL_bb2 plasmid. (C) Yellow pigment present in the consequences. Firstly, it would provide a system for procuring useful biological products organic phase of lysed BL21 pDAL_bb2 transformants. Coombs • Tennille Crossman • Dean of Medicine, Dalhousie • without the limitations of large scale cultivation of plant-derived sources. Secondly, Department of Microbiology and Immunology • NSERC • DSU • Faculty pigmented anthocyanin compounds can be used as colorimetric tools in the analysis of other important genes in the anthocyanin biosynthetic pathway. Our approach consists of of Science constructing three sets of operons, each consisting of three sequential genes in the anthocyanin pathway, and expressing these genes concurrently in E. coli which would lead to the production of delphinidin and an easily identifiable blue phenotype. .