Biotechnological production of amino acids and derivatives: current status and prospects
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- Leuchtenberger, W., Huthmacher, K. & Drauz, K. Appl Microbiol Biotechnol (2005) 69: 1. doi:10.1007/s00253-005-0155-y
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For almost 50 years now, biotechnological production processes have been used for industrial production of amino acids. Market development has been particularly dynamic for the flavor-enhancer glutamate and the animal feed amino acids l-lysine, l-threonine, and l-tryptophan, which are produced by fermentation processes using high-performance strains of Corynebacterium glutamicum and Escherichia coli from sugar sources such as molasses, sucrose, or glucose. But the market for amino acids in synthesis is also becoming increasingly important, with annual growth rates of 5–7%. The use of enzymes and whole cell biocatalysts has proven particularly valuable in production of both proteinogenic and nonproteinogenic l-amino acids, d-amino acids, and enantiomerically pure amino acid derivatives, which are of great interest as building blocks for active ingredients that are applied as pharmaceuticals, cosmetics, and agricultural products. Nutrition and health will continue to be the driving forces for exploiting the potential of microorganisms, and possibly also of suitable plants, to arrive at even more efficient processes for amino acid production.
As the building blocks of life, amino acids have long played an important role in both human and animal nutrition and health maintenance (Bercovici and Fuller 1995). On account of its functionality and the special features arising from chirality, this class of compounds is biochemically extremely important and of great interest for the chemical industry (Leuchtenberger 1996). Of the 20 standard protein amino acids, the 9 essential amino acids-l-valine, l-leucine, l-isoleucine, l-lysine, l-threonine, l-methionine, l-histidine, l-phenylalanine, and l-tryptophan occupy a key position in that they are not synthesized in animals and humans but must be ingested with feed or food.
The remaining proteinogenic amino acids are required in the pharmaceutical and cosmetics industries and are also ideal raw materials for synthesis of chiral active ingredients, which in turn find application in such sectors as pharmaceuticals, cosmetics, and agriculture. According to a study by the Business Communication Company (Brown 2005), the amino acid market for synthesis applications is growing at an annual rate of 7% and is expected to reach a volume of US $1 billion in the year 2009, of which the share of amino acids for peptide sweeteners alone is expected to be more than US $400 million.
Microbial amino acid production
The rapid development of the amino acid market since the 1980s is due in no small part to major successes in cost-effective production and isolation of amino acid products. Of the four production methods for amino acids—extraction, synthesis, fermentation, and enzymatic catalysis—it is particularly the last two biotechnological processes, with their economic and ecological advantages, that are responsible for this spectacular growth.
Extraction of amino acids from protein hydrolysate as a method of obtaining l-amino acids is now of only limited importance; although still relevant for production of l-serine, l-proline, l-hydroxy-proline, and l-tyrosine, for example, it is not suitable for large-scale production of amino acids. The extraction method for obtaining l-glutamate was superseded nearly 50 years ago by fermentation, following a sharp increase in demand for the flavor-enhancer MSG. The discovery of the soil bacterium, Corynebacterium glutamicum, which is capable of producing l-glutamic acid with high productivity from sugar, paved the way for the success of the fermentation technique in amino acid production (Kinoshita et al. 1957). It was advantageous here that the wild strain could be used on an industrial scale under optimized fermentation conditions for mass production of glutamate. Glutamate biosynthesis and methods for improving production strains have been investigated in depth (Kimura 2003). The fermentation process is in principle very simple: a fermentation tank is charged under sterile conditions with a culture medium containing a suitable carbon source, such as sugar cane syrup, as well as the required nitrogen, sulfur, and phosphorus sources, and some trace elements. A culture of the production strain prepared in a prefermenter is added to the fermentation tank and stirred under specified conditions (temperature, pH, aeration). The l-glutamic acid released by the microorganism into the fermentation solution is then obtained by crystallization in the recovery section of the fermentation plant. MSG (1.5 million tons) is currently produced each year by this method, making l-glutamic acid the number one amino acid in terms of production capacity and demand (Ajinomoto 2003).
The fermentation method of production is also well established for the amino acids l-threonine (Debabov 2003) and l-tryptophan (Ikeda and Katsumata 1999), which are important as the second and third limiting amino acids in growing pigs. In this case, recombinant strains of Escherichia coli have proven to be particularly productive. World requirements for 2005 have been projected at 70,000 tons for l-threonine and 3,000 tons for l-tryptophan (Ajinomoto 2004).
Selected amino acid-producing strains (Ikeda 2003)
Estimated yield (g/100 g sucrose)
C. glutamicum B-6
E. coli KY 10935
C. glutamicum KY9218/pIK9960
E. coli MWPWJ304/pMW16
Brevibacterium flavum AJ12429
C. glutamicum F81/pCH99
E. coli H-8461
Methylobacterium sp. MN43
C. glutamicum VR 3
One exception is the sulfur-containing amino acid methionine, which, as first limiting amino acid in poultry, is of particular importance. Methionine has been manufactured synthetically, i.e., as the racemate (dl-methionine), from the starting materials acrolein, hydrocyanic acid, methyl mercaptan, and ammonia, and marketed as a feed additive for more than 50 years. The fact that the D-form, not found in nature, is enzymatically converted into the nutritive L-form in the animal organism by means of an oxidase and transaminase allows direct use of the synthetic racemic mixture. For other amino acids such as lysine and threonine, there is no comparable enzyme system for conversion of the D-form, so that for these amino acids, it is necessary to produce the pure L-form. Despite the experience gained from lysine and threonine fermentation, attempts to develop a cost-effective production of l-methionine by the fermentation pathway have so far proved unsuccessful.
Enzymatic production of proteinogenic amino acids
For l-cysteine, which previously was produced mainly by electrochemical reduction of l-cystine obtained from protein hydrolysis, an industrially used enzymatic process exists in which the thiazoline derivative dl-2-amino-2-thiazoline-4-carboxylic acid (ATC) is converted with the help of three enzymes (l-ATC hydrolase, S-carbamoyl-l-cysteine hydrolase, and ATC racemase) from Pseudomonas thiazolinophilum (Pae et al. 1992). However, one may expect that modern methods of strain development will lead to the establishment of fermentation technology in l-cysteine production.
Enzymatic production of nonproteinogenic amino acids
Enzymatic production of amino acid derivatives
Outlook and prospects
Biotechnological production of amino acids today serves a market with strong prospects of growth. In the foreground are the fermentation processes, which are now widely established in the production of proteinogenic amino acids. The potential that will be leveraged in the future by modern methods and new findings in system biology will further stimulate and strengthen microbial amino acid production. (Wendisch et al. 2005).
Enzyme catalysis will remain the preferred production method for nonproteinogenic amino acids and amino acid derivatives. Modern methods such as directed evolution will allow development of customized, highly selective, and stable enzymes and whole cell biocatalysts, as well as efficient and ecologically sustainable production of the required products.
Both the fermentation and enzyme-based production methods play a central role in any discussion of the future of white biotechnology and could show the way to sustainable production of active ingredients, fine chemicals, and even certain bulk products (Sijbesma and Schepens 2004). It would be extremely interesting, for example, to assess the feasibility of implementing, in addition to the established chemical processes, a biorefinery concept based on renewable raw materials. “Green” biotechnology could also be used here to obtain products of interest from plants. As an example, transgenic plants with increased methionine (Aragao et al. 1999) and lysine content (Alvarez et al. 1998) have already been developed, but do not show commercial competitiveness at the present time.