Vanillin in Novel Plants Project

Vanillin is one of the major compounds in the vanilla flavor. A phenolic aldehyde, vanillin is also used in the pharmaceutical industry, beverages, as well as a fragrant compound in different products. The demand of Vanillin exceeded the natural production, so annually most vanillin is produced via chemical synthesis. We intend to produce vanillin in different plants as a natural alternative for vanillin production.

Introduction

Whether it’s a child’s first ice cream cone or the secret ingredient of a five star chef, everyone loves vanilla flavoring. Europeans first became aware of vanilla around 1520 and ever since then countless products featuring the beloved aromatic have been created. Vanillin, one of the major compounds in the vanilla flavor, is often produced by chemical synthesis due to the scarcity and high cost of natural vanilla extract. Hydroxycinnamoyl-CoA hydratase-lysase, more commonly known as HCHL, is an enzyme that converts feruloyl-CoA to vanillin and acetyl-CoA. HCHL is naturally produced by Pseudomonas putida Kt2440, a gram-negative rod-shaped saprotrophic soil bacterium. As plants naturally possess large pools of phenolic compounds used in lignin biosynthesis, we hope to be able to express HCHL within plants to convert feruoyl-CoA to vanillin.

Background

In order to express this enzyme within plants, HCHL must be cloned from Pseudomonas putida and modified for codon optimization in plants. This modification will involve the use of the Kozak sequence, which through experimental observation has been recognized as playing a major role in translational initiation. Additionally, a double stop codon along with the CaMV 35s promoter and Nos terminator will be utilized.

Method

In order to transform plants we first obtained cultures of Pseudomonas putida kt2440. We then PCR amplified the genomic DNA in order to amplify the HCHL enzyme. The 35s promoter and NOS terminator were amplified from p cambia p1201 vector. The 35s promoter was amplified with 2 sticky ends, at the beginning of the sequence with an EcorI restriction site and at the end with Xba1. The HCHL gene was amplified with a Xba1 restriction site at the beginning and a SpeI and the end. The NOS terminator with and SpeI restriction site at the beginning and BamH1 at the end. The pcambia p1201 and p1281z vectors were double digested with EcorI and BamHi, 35s promoter, HCHL enzyme and NOS terminator were digested and ligated together. Finally, the construct was ligated to the vector and then transformed into bacteria.

Further Study

Additional research is required in order to troubleshoot why transformation failed. The next steps will include stabilizing the construct in the pcambia vectors and transformed agrobacterium. Later the transformed agrobacterium, with the desired plasmid, will be used to transforms plants. The plant tissue then should be analyzed to determine if the desired products are present.

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