Maximizing resveratrol production in engineered E. coli.

Last Wednesday (January 9, 2012), I started my first day of the second semester with my internship:)

Since Eun Ji was still on break, I worked with Namita. But because she was busy with writing a paper recently and didn't do much lab work, she went over another paper published by Koffas group with me. The paper is about ways and factors that help to maximize resveratrol production in engineered E.coli., and here's the link to the paper--> http://www.ncbi.nlm.nih.gov/pubmed/21441338

Resveratrol is a natural products that is suspected to be responsible for a decreased risk of heart disease and diabetes. It is usually found in red wine, bushberries, peanuts, cranberries, etc. However, all those plants carry only a extremely tiny amount of resveratrol. Thus, the lab examined ways to optimized the production of the molecule.

Factors examined:

• E. coli. strain
• malonyl-CoA availability
• STS, 4CL 
• gene expression
• cerulemin
• promoter system

First of all, the general metabolic pathway for resveratrol production is:

E.coli is the most common used bacteria because there are more documented information about this bacteria, many strands are non-pathogenic, and they act closer to human cells.

There are 2major enzymes involved: 4CL and STS. The amount of the enzymes affects the efficiency of resveratrol synthesis. The group also searched for a more active STS that would increase production rates and levels. They identified 7 new STS, yet further research still need to be done.

One reason why resveratrol is produced in such low amount is the lack of malonyl-CoA. Malonyl-CoA is an important precursor (added reactant) because it is a key molecule for fatty acid biosynthesis. Thus, the group added cerulemin to slow down the biosynthesis of fatty acid, so there will be extra CoA for the production of resveratrol. The group chose slow down instead of cutting the fatty acid pathway completely because fatty acid is essential for life (cell membrane, etc.), and eliminating it entirely may cause negative effects.

What I think more interesting is the expression construct. An expression construct, or expression vector, is usually a plasmid or virus designed for protein expression in cells (such as production of insulin in bacteria). You can also delete a gene in a cell. First, you would design a particular sequence that code for the protein you want the bacteria to synthesize for you. The ends of the designed sequence (gene 1) must match the ends of the target sequence you want to delete (gene 2).

Then, you add recombinase, a genetic recombination enzyme. Recombinase would then switch the two genes. To test whether the genes are switched, we add antibiotic resistant gene in the designed vector. If the result bacteria grow on ampicillin, then that means bacteria successfully intake the designed gene. Here is a nice video that explain the detailed process, but the first 20 sec is the main idea --> http://www.youtube.com/watch?v=d4PFp43brvI

In the end, the Koffas group maintained to achieve a production of resveratrol of 2.3 g/liter (1.4 g/liter without adding cerulenin), compared to  4 μg/g of dry peanuts and grapes and 2 mg/liter of red wines.

Reading this article is quite hard, but I'm glad that I understand the basic concepts of it. Namita also gave me another article to read in prepare of the experiment we will do next week:)