MICR3003 Molecular Microbiology Lecture 10

Molecular Basis for Expression of Multigene Phenotypes In E.coli Carotenoid Biosynthetic Pathways

Dr J M Pemberton 2008

HeterologousExpression Of Rhodobacter Carotenoids In Paracoccus

Humminbird Feeding on nectar Produced by An Hibiscus Flower

Rhodobacter Colony

Red Ibis in South America

Cassowary in North Queensland

1.Biological Functions of Carotenoids

Biological Functions in Bacteria
They occur in both photosynthetic and non-photosynthetic bacteria In photosynthetic bacteria e.g. Rhodobacter they act as secondary light harvesting pigments and as photoprotective pigments.

In non-photosynthetic bacteria Staphylococcus aureus (gold color is a carotenoid) where they protect against photo-oxidative damage e.g. ultra violet (UV) damage.

Biological Functions in Eukaryotes Beta-carotene is the precursor of Vitamin A. The visual photopigment.

2. The first cloning and heterologous expression of carotenoid

genes - eukaryotic or prokaryotic.
This was the cloning and heterologous expression of the carotenoid gene cluster of the Rhodobacter(formerly rhodopseudomonas) sphaeroides in a range of Gram negative bacteria including Paracoccus denitrificans (Pemberton & Harding, 1986;1987.see below)

3. Initial Steps in Carotenoid Synthesis

4.Carotenoid Biosynthesis Pathway of Rhodobacter

crtE

crtE

crtB

crtB

PPSR THE MASTER REGULATOR OF BACTERIAL PHOTOSYNTHESIS

ppsR

crt

Rhodobacter Carotenoid(crt) Gene Cluster

ITS REGULATORY ACTIVITY IS SEEN FROM SPACE

GULF OF MEXICO SHOWING RED TIDE

Algal blooms, dinoflagellates (marine algae such as Gonyaulax) and bacteria, produce red carotenoids. A 20-30% is made up a the marine Rhodobacter Roseobacter. When bloom consumes all the oxygen, PPSR is inactivated and carotenoid synthesis is switched on.

5. Synthesis of the Plant Carotenoid Zeaxanthin

6. Synthesis of the Algal Carotenoid Astaxanthin

7. A Kaleidiscope Of Colors

J. M. Jez and J. P. Noel.2000.A kaleidoscope of carotenoids. Nature Biotechnology Vol 18p825

8. Carotenoid Synthesis in E.coli

COLOUR ME RED Tracey Smith 2000 Nature Structural Biology Vol7 no.7 p536

9. Combinatorial Synthesis of Carotenoids in E.coli K12

The cloning and expression of the carotenoid biosynthesis pathway from Erwinia in E.coli provided the the opportunity to use combinatorial synthesis to produce a wide variety of carotenoids. Combinatorial synthesis is the construction of novel biosynthetic pathways by substituting one enzyme for another, adding new enzymes or using accelerated evolution to produce pathway enzymes with altered enzymatic activities. These altered pathways produce different or novel carotenoids. The need to engineer such pathways is based on demands for complex, biologically active molecules for use in medicine, agriculture and industry

SO WHAT WAS THE FIRST GENETICALLY ENGINEERED ANIMAL?

This animal is one of the most popular pets worldwide

This animal is not a dog, but it is named after the dog

It was first genetically engineered over 80 years ago And its appearance and its appearance in these lectures is not co-incidental

The scheme to develop the first genetically engineered animal was developed by two Germans in 1921 Duncker a school teacher with a strong background in Mendelian genetics Reich an enthusiastic canary breeder who bred German Roller nightingale canaries

The German Roller canary is the original miners canary

female female male

German Roller Canary

Black Hooded Red Siskin

male

female

Fertile Red Canary/Siskin Hybrids

The red color of canaries is the red carotenoid canthaxanthin which is added to their food when they molt (lose) their feathers. The new feathers are stained red with the red canthaxanthin. A gene(s) from the red siskin enables the red canary to concentrate red carotenoid canthaxanthin rather than the normal yellow carotenoid zeaxanthinn in their new feathers.

A couple of my red roller canaries