Lycopene is the red pigment that gives tomatoes their color. This pigment is also made by microbes. In fact, transferring a 3-enzyme pathway to E. coli can convert farnesyl diphosphate (FPP) to lycopene. For the laboratory portion of this assignment, you will characterize lycopene production in E. coli. The computational tools and databases presented today can also be used to enhance lycopene production in E. coli or even produce different colors by adding additional genes.
Part I: Test pAC-LYC plasmid
The pAC-LYC plasmid is available from Addgene, and contains three genes from Erwinia herbicola: CrtE, CrtI, and CrtB. This plasmid is provided by Addgene on an agar stab transformed into Top10 E. coli. That means that it can simply be streaked onto LB Agar with chloramphenicol to make lycopene-producing colonies that should be pink to red in color.
It worked!
Streaking pAC-LYC on LB agar plates
pAC-LYC plasmid from Addgene
bio
production
LAB TASK
EXPERIMENTAL HOMEWORK
Part II: Design strategies for increasing lycopene production
Using tools such as Biocyc and KEGG, select enzymes that can be added to (or knocked out of) your lycopene-producing E. coli to increase the amount of lycopene they produce. The pAtipiTrc plasmid available in Addgene represents one possible strategy, as it overexpresses a heterologous idi enzyme. Labs working with PCR and cloning may want to try replacing the idi gene in pAtipiTrc with alternative enzymes to see how they impact colony color.
A chassis (host strain) is the microorganism to be engineered. Chassis selection is guided by many factors:
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Suitability to target environment
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Metabolic capabilities
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Ease of genetic manipulation
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Tolerance of desired product
Escherichia coli is a well-understood bacterial host with tons of genetic tools, which makes it a great platform for designing complex genetic circuits and also used in industry (with caveats). Saccharomyces cerevisiae is a domesticated eukaryotic host (yeast), also with great genetic tools. Great platform for beer and industry.
A pathway is the biosynthetic route from a feedstock (for example, sugar) to the end product. This typically requires enzymes that are not native to the chassis strain, aka “heterologous” enzymes. Heterologous enzymes can also be used to replace chassis enzymes.
There are three ways to design a pathway: by combining existing pathways, engineer existing pathways and de novo pathway design (Prather & Martin, 2008). The first is the easiest, the latter the most difficult.
References
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Prather KLJ, Martin CH. De novo biosynthetic pathways: rational design of microbial chemical factories. Curr Opin Biotechnol. 2008 Oct;19(5):468–74.
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iGEM Cambridge, UK (2009) http://2009.igem.org/Team:Cambridge/Project/Carotenoids
Resources
Using the KEGG tool, I looked for the lycopene pathway in the carotenoid biosynthesis map and selected enzymes that could be added or knocked out to increase lycopene production in E. coli.
CLASS ASSIGNMENT
THEORETICAL HOMEWORK
Part III: Design strategies for converting lycopene to beta-carotene
Use Biocyc and/or KEGG to identify the enzyme that can convert the red pigment lycopene to the orange pigment beta-carotene. Plasmids that can be co-transformed with pAC-LYC that produce this enzyme can be found in Addgene. Labs working on miniprep and transformation can order these plasmids to observe switch the color of their E. coli from red to orange.
The enzyme that can convert the red pigment lycopene into the orange pigment beta carotene is the Lycopene-beta-cyclase (CrtL, CrtL-b, CrtY). This enzyme catalyses the biosynthesis of β-carotene. β-Carotene is an organic, strongly colored red-orange pigment abundant in plants and fruits. It is a member of the carotenes, which are terpenoids (isoprenoids), synthesized biochemically from eight isoprene units and thus having 40 carbons. Among the carotenes, β-carotene is distinguished by having beta-rings at both ends of the molecule. β-Carotene is biosynthesized from geranylgeranyl pyrophosphate.
Overview of the Carotenoid pathway
We choose the Carotenoid synthesis Pathway that can easily display different steps in the pathway by different colors.
If E.coli is incubating below 37℃, mRNAs cannot be translated by ribosomes, because temperature-sensitive RBS(ribosome biding site) forms the hairpin. A ribosome cannot bind to the RBS so that it cannot translate CrtY. As a result, the E.coli only can produce CrtE, CrtB and CrtI which convert colorless Farnesyl pyrophosphate to red Lycopene.
When the temperature is at 37℃ or higher, the hairpin is denatured so temperature-sensitive RBS is activated . CrtY is translated, which convert red Lycopene to orange beta-Carotene.
The common starting point for carotenoid synthesis is farnesyl pyrophosphate (FPP), which derives from two precursors, isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). In general, there are two pathways for synthesising IPP and DMAPP: the Mevalonate Pathway (starting with acetyl CoA) and the Non-mevalonate Pathway (starting with pyruvate and glyceradehyde-3-phosphate). While the Mevalonate Pathway is present in all higher eukaryotes, the Non-mevalonate Pathway is present in E. coli. Part of the carotenoid biosynthesis pathway is shown in the diagram. Some of the intermediates in the pathways are colored, e.g. lycopene (red), beta-carotene (orange), and zeaxanthin (yellow). The enzymes involved in the biosynthetic pathway are CrtE, CrtB, CrtI, CrtY, and CrtZ (iGEM Cambridge, 2009)
