The Manufacture of L-Tryptophan using Microbial Fermentation

The manufacture of L-tryptophan, an essential amino acid, using microbial fermentation involves the utilization of microorganisms to produce and accumulate L-tryptophan through metabolic pathways and enzymatic reactions. It is a feedstock too for many other chemical manufacturing processes in the pharmaceutical industry.

The traditional methods for this amino-acid include chemical synthesis, direct fermentation bioconversion, and enzymatic conversion of precursors. Microbial fermentation entails the production of l-tryptophan from cheap and renewable carbon source such as sucrose or glucose (Sprenger, 2007)

Here is a broad review of that fermentation process:

Microorganism Selection

Several microorganisms can be used for the production of L-tryptophan, including bacteria and fungi. The bacterium Escherichia coli and the fungus Corynebacterium glutamicum are commonly employed due to their well-studied metabolic pathways and genetic manipulation capabilities. E.coli is preferred because it has a faster reproductive cycle and genetic systems that are more amenable to modification. It is also better understood and widely used to produce a range of fermentation products including amino-acids such as valine and tyrosine as well as organic acids like malic etc.

Genetic Engineering

Genetic manipulation plays a crucial role in optimizing L-tryptophan production. The selected microorganism’s genome is modified to enhance the activity of enzymes involved in the tryptophan biosynthesis pathway. This can be achieved by overexpressing key enzymes or introducing genes from other microorganisms with higher tryptophan production capabilities.

Substrate Selection

The choice of substrate for fermentation depends on the metabolic capabilities of the selected microorganism. Common carbon sources include glucose, glycerol, and other carbohydrates. Nitrogen sources such as ammonium salts or amino acids are also provided as nutrients for microbial growth and tryptophan production.

Optimization of Fermentation Conditions

Fed-batch fermentation is often employed for this type of amino-acid production and the manufacture of L-tryptophan is no exception. A typical example uses recombinant E.coli NT367 with glycerol as the key source of carbon (Trondle et al., 2018). The growth conditions are optimized to maximize L-tryptophan production. Factors such as temperature, pH, oxygen levels, and nutrient concentrations are controlled to create an environment suitable for microbial growth and efficient metabolic activity. These parameters are adjusted based on the specific requirements of the chosen microorganism. 

Metabolic Pathway Engineering

The biosynthesis of L-tryptophan involves a series of enzymatic reactions in the shikimate pathway and the tryptophan biosynthesis pathway. The shikimate pathway produces the precursor molecules necessary for tryptophan synthesis. Genetic engineering techniques are used to enhance the activity of enzymes in these pathways, allowing for increased metabolic flux towards L-tryptophan production.

Recovery and Purification

After fermentation, L-tryptophan needs to be recovered and purified from the fermentation broth. Various techniques such as filtration, extraction, chromatography, and crystallization can be used for isolation and purification. The choice of purification method depends on the scale of production and the desired purity of the final product.

The manufacture of L-tryptophan using microbial fermentation offers a sustainable and cost-effective approach for obtaining this important amino acid. Genetic engineering allows for the optimization of metabolic pathways and enzymatic reactions, leading to increased yields and productivity. The fermentation conditions and substrate selection are tailored to the specific requirements of the chosen microorganism. Purification steps are employed to isolate and purify L-tryptophan from the fermentation broth, ensuring the production of a high-quality final product.

References

Sprenger, G. A. (2007). From scratch to value: engineering Escherichia coli wild type cells to the production of L-phenylalanine and other fine chemicals derived from chorismate. Applied Microbiology and Biotechnology, 75, pp. 739-749.

Tröndle, J., Trachtmann, N., Sprenger, G. A., & Weuster‐Botz, D. (2018). Fed‐batch production of l‐tryptophan from glycerol using recombinant Escherichia coli. Biotechnology and Bioengineering, 115(12), pp. 2881-2892 (Article) .