Indigo is a commercially important blue pigment used to dye cotton and wool. It was originally extracted from plants but is now synthesized chemically. Microbial synthesis of indigo could provide an efficient and economical process. Genes from indigo-producing plants were inserted into bacteria and yeast, allowing these microorganisms to produce indigo through fermentation. Current research is focused on optimizing indigo yields through these microbial synthesis methods.

1. Microbial
synthesis
of
Indigo

2. Indigo
• A commercially important blue pigment that is used to dye both cotton and wool.
• Isolated from plant but is currently synthesized chemically.
• At present, approximately 13×106 kg of indigo, worth more than $250 million, is
produced every year.
• Ability to produced indigo from bacteria opens the possibility of developing an
efficient and economical commercial microbial process for its production.

3. History
• Until the late 19th century, indigo was extracted from plants such as the Indian
bean plant Indigofera tinctoria.
• It was first synthesized chemically in 1877, after which indigo cultivation
collapsed.
• Today indigo is made chemically, starting with aniline, but the current chemical
process is environmentally hazardous.
• That’s why in the 1990s the biotech companies Amgen and Genencor developed
methods for making the dye by fermentation of microorganisms genetically
modified to contain genes from indigo-producing plants.

4. Biosynthesis of Indigo
• A number of bacteria, most notably Pseudomonas species have the ability to use
a variety of organic compound such as naphthalene, toluene, xylene etc.
• The genes encoding the enzymes for these degradation of organic compounds
are located on large naturally occurring plasmids.
• Plasmid NAH7 has two separate and distinct operons that allow pseudomonas to
grow on naphthalene as the sole carbon source.
• As a first step, NAH7 was digested and ligated with Hind III in plasmid PBR322 and
introduced in E.coli.
• E. coli contains the enzyme tryptophanase, E. coli, the cells have the ability to
synthesize indigo from tryptophan.

5. Colonies of the bacterium Escherichia coli, genetically modified to produce the dye
indigo (blue).

6. Steps of microbial indigo productio
• Conversion of tryptophan in the growth medium to indole by the enzyme
tryptophanase, which is produced by the E. coli host cell.
• Oxidation of indole to cis-indole-2,3-dihydrodiol by naphthalene dioxygenase, which
is encoded by the DNA that was cloned from the NAH7 plasmid
• Spontaneous elimination of water
• Air oxidation to form indigo

8. Steps of Microbial Indigo
production(cont.)
• Thus, the combination of enzymes from two different pathways and two different
organisms resulted in the synthesis of an unexpected compound, the dye indigo.
• In addition, introduction of the gene for the enzyme xylene oxidase, which is encoded
in the TOL plasmid, can convert tryptophan to indoxyl, which then spontaneously
oxidizes to indigo.

11. Commercial Importance of Indigo
• Used to make hair dyes.
• Used for textile dyeing and printing.
• Indigo is as a dye for cotton yarn.
• Production of denim cloth for blue jeans.
• Small amounts are used for dyeing wool and silk.
• Indigo carmine or indigo, is an indigo derivative which is also used as a colorant.
• It is also used as a food colorant.