Trends in Biotechnology
Volume 16, Issue 10, 1 October 1998, Pages 412-418
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Novel biosynthetic approaches to the production of unnatural amino acids using transaminases

https://doi.org/10.1016/S0167-7799(98)01240-2Get rights and content

Abstract

Transaminase enzymes are being increasingly applied to the large-scale synthesis of unnatural and nonproteinogenic amino acids. Typically displaying relaxed substrate specificity, rapid reaction rates and lacking the need for cofactor regeneration, they possess many characteristics that make them desirable as effective biocatalysts. By judiciously combining the transaminase reaction with additional enzymatic steps, this approach can be used very efficiently to prepare a broad range of d- and l-amino acids.

Section snippets

General properties of aminotransferases

l-α-Amino acid transaminases (LATs) are ubiquitous in nature, being involved (directly or indirectly) in the biosynthesis of most natural amino acids. d-Amino acid transaminases (DATs) have been identified in a number of bacterial species, most notably the Bacilli, in which they provide d-amino acids for peptidoglycan and secondary metabolite biosynthesis10, 11, 12, 13, 14. The reaction mechanism is well understood, with a pyridoxal 5′-phosphate cofactor shuttling between pyridoxal and

The problem of reaction equilibrium

Although both types of transaminases have appropriate characteristics to function as commercial biocatalysts, with rapid reaction rates, broad substrate specificity and no requirement for external cofactor regeneration, the frequently cited drawback is the equilibrium constant for the reaction. This is typically close to one, which can often compromise amino acid product yield, isolation and recovery. Earlier applications of LATs have addressed this issue by coupling two aminotransferases (Fig.

Supply of α-keto acids

Many α-keto-acid substrates are available by chemical synthesis, but these are often the most expensive component in the reaction. Fortunately, the high value of the final product often offsets the relatively high substrate costs. In the case of the DAT reaction, keto acids are also accessible by enzymatic methods (such as l-amino acid deaminases), which are able to generate keto-acid substrates from inexpensive l-amino acids. For example, the l-amino acid deaminase (l-AAD) from Proteus

Production of unnatural amino acids using l-aminotransferases

The most commonly employed LATs used for l-amino acid synthesis are the transaminases of Escherichia coli, most of which have been cloned and overexpressed. They are generally used in whole-cell or immobilized systems for industrial processes and include: aspartate aminotransferase (E.C. 2.6.1.1), branched-chain aminotransferase (E.C. 2.6.1.42) and tyrosine aminotransferase (E.C. 2.6.1.5). Below are details of each specific enzyme, primarily from E. coli, along with a description of the

Production of unnatural amino acids using bacterial d-aminotransferases

Unlike the LATs, very few DATs (E.C. 2.6.1.21) have been extensively studied. One of the best understood is that from Bacillus sp. YM1, whose gene has been cloned and sequenced[24]and whose crystal structure has been solved[25]. Recently, the dat genes from Staphylococcus haemolyticus[26], Bacillus licheniformis[27] and Bacillus sphaericus (I. G. Fotheringham et al., unpublished) have been cloned, sequenced and overexpressed in E. coli. These factors, plus their broad substrate specificity,

Other aminotransferases

The enzyme 4-aminobutyrate-2-ketoglutarate transaminase (E.C. 2.6.1.19) has also been used to prepare the herbicide l-phosphinothricin37, 38. Most recently, this enzyme has been coupled with aspartate aminotransferase to drive the equilibrium in a similar way to the reaction shown in Fig. 2[39].

A novel d-phenylglycine aminotransferase from Pseudomonas stutzeri ST-210 has recently been purified and characterized[40]. This transaminase is unusual in that it has a very narrow substrate range and

Conclusions and future perspectives

The processes above illustrate a number of biotransformations carried out by bacterial transaminases acting in concert with additional biocatalysts in recombinant strains. The breadth of application is due in part to the inherent versatility of transaminases, but also to the detailed characterization of transaminase-encoding genes from a variety of microorganisms. Homology studies have indicated a high degree of primary-sequence similarity between many of the LATs[41], and the recent cloning of

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