Microbial Production of Indigo

While synthetic indigo has enjoyed a virtual monopoly for nearly a century, an environmentally friendly microbial production of indigo is under development. The microbial production of indigo has been known since the 1920s. Indigo production with hydrocarbon degrading bacteria expressing mono-oxygenases or dioxygenases has also been investigated in search of a possible alternative for the chemical synthesis of indigo. In 2002 Berry et al. and his companions developed a fermentation process where indigo was produced from glucose with recombinant Escherichia coli that had been modified with Pseudomonas putida genes. However, the method produced also indirubin, which gave an undesirable red hue to the dyeing.

Several bacteria, most notably Pseudomonas species, can use a variety of organic compounds such as naphthalene, toluene, xylene and phenol as their sole carbon source. In many instances, the genes encoding the enzymes for the degradation of these organic compounds are located on large, naturally occurring plasmids. For example, pseudomonads that contain NAH7 plasmid could grow on naphthalene as a sole carbon source. The clone bank was then introduced into E. coli cells. During the characterization of one of the transformants that could convert naphthalene to salicylic acid, it was observed that when the growth medium contained tryptophan, it turned blue. A thorough analysis of the blue color revealed that the transformed E. coli cells were synthesizing the dye indigo. This synthesis is achieved in four steps:

• 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 NAH7 plasmid.
• Spontaneous elimination of water.
• Air oxidation to form indigo.

In addition, introduction of the gene for enzyme xylene oxidase, which is encoded in the TOL plasmid, can convert tryptophan to indoxyl, which then spontaneously oxidizes to indigo. In pathway A, the naphthalene dioxygenase is derived from the NAH plasmid. In pathway B, the xylene oxidase is from the TOL plasmid. E. coli transformants that synthesize indigo contain either pathway A or B. The conditions for large scale growth of an E. coli strain capable of synthesizing indigo, including temperature, pH and the amount of tryptophan that must be added to the medium to give maximum yields, are being tested.

Although this system has not yet been commercialized, a microbial process for the synthesis of indigo might include a bioreactor in which the recombinant E. coli is chemically immobilized to a solid matrix (e.g. cellulose or silica gel). The unit could be run continuously by adding tryptophan to one end and removing indigo at the other. Genencor International, of Rochester, New York, is experimenting on a process to produce indigo using biotechnology. However, at this stage the technology is expensive and production costs might be prohibitive.

The research and development efforts made in the field of microbial synthesis of indigo from 1927 onwards has been critically reviewed. The highlights of this critical review indicated that biosynthesis of indigo could be divided into three periods: biosynthesis by wild microbes, whole cell catalysis by engineering bacteria and biotransformation regulated by metabolic engineering. Most aromatic degrading microbes and their relevant enzymes possess the ability to convert indole to indigo.

New technologies such as directed evolution, metagenome and two-phase reaction systems could facilitate in-depth investigations of the enzyme resources, and they will play a crucial role in indigo biosynthesis research. Meanwhile, hydroxyl-indoles and indigo derivatives produced in the process are promising pharmaceutical and chemical precursors with great research interest. However, the transformation interactions between intermediates and by-products are still unclear. Besides, low indigo yield and efficiency with high cost have hampered practical production. Therefore, it is essential to combine the molecular biology and metabolic engineering technologies to investigate the mechanisms and industrial application of indigo biosynthesis in the future.