Codon-Optimized Rhodotorula glutinis PAL Expressed in Escherichia coli With Enhanced Activities

Phenylalanine ammonia lyase (PAL; E.C.4.3.1.5), which catalyses the biotransformation of L-phenylalanine to trans-cinnamic acid and ammonia, was first described in 1961 by Koukol and Conn. Since its discovery, much knowledge has been gathered with reference to the enzyme's catabolic role in microorganisms and its importance in the phenyl propanoid pathway of plants. The 3-dimensional structure of the enzyme has been characterized using X-ray crystallography. This has led to a greater understanding of the mechanism of PAL-catalyzed reactions, including the discovery of a recently described cofactor, 3,5-dihydro-5-methyldiene-4H-imidazol-4-one. In the past 3 decades, PAL has gained considerable significance in several clinical, industrial, and biotechnological applications. The reversal of the normal physiological reaction can be effectively employed in the production of optically pure L-phenylalanine, which is a precursor of the noncalorific sweetener aspartame (L-phenylalanyl-L-aspartyl methyl ester). The enzyme's natural ability to break down L-phenylalanine makes PAL a reliable treatment for the genetic condition phenylketonuria. In this mini-review, we discuss prominent details relating to the physiological role of PAL, the mechanism of catalysis, methods of determination and purification, enzyme kinetics, and enzyme activity in nonaqueous media. Two topics of current study on PAL, molecular biology and crystal structure, are also discussed.

The expression of heterologous proteins in microorganisms using genetic recombination is still the high point in the development and exploitation of modern biotechnology. People can produce bioactive proteins from relatively cheap culture medium instead of expensive extraction. Host cell systems for the expression of heterologous genes are generally prokaryotic or eukaryotic systems, both of which have inherent advantages and drawbacks. An optimal expression system can be selected only if the productivity, bioactivity, purpose, and physicochemical characteristics of the interest protein are taken into consideration, together with the cost, convenience and safety of the system itself. Here, we concisely review the most frequently used prokaryotic, yeast, insect and mammalian expression systems, as well as expression in eukaryote individuals. The merits and demerits of these systems are discussed.

Background Due to the degeneracy of the genetic code, most amino acids can be encoded by multiple synonymous codons. Synonymous codons naturally occur with different frequencies in different organisms. The choice of codons may affect protein expression, structure, and function. Recombinant gene technologies commonly take advantage of the former effect by implementing a technique termed codon optimization, in which codons are replaced with synonymous ones in order to increase protein expression. This technique relies on the accurate knowledge of codon usage frequencies. Accurately quantifying codon usage bias for different organisms is useful not only for codon optimization, but also for evolutionary and translation studies: phylogenetic relations of organisms, and host-pathogen co-evolution relationships, may be explored through their codon usage similarities. Furthermore, codon usage has been shown to affect protein structure and function through interfering with translation kinetics, and cotranslational protein folding. Results Despite the obvious need for accurate codon usage tables, currently available resources are either limited in scope, encompassing only organisms from specific domains of life, or greatly outdated. Taking advantage of the exponential growth of GenBank and the creation of NCBI’s RefSeq database, we have developed a new database, the High-performance Integrated Virtual Environment-Codon Usage Tables (HIVE-CUTs), to present and analyse codon usage tables for every organism with publicly available sequencing data. Compared to existing databases, this new database is more comprehensive, addresses concerns that limited the accuracy of earlier databases, and provides several new functionalities, such as the ability to view and compare codon usage between individual organisms and across taxonomical clades, through graphical representation or through commonly used indices. In addition, it is being routinely updated to keep up with the continuous flow of new data in GenBank and RefSeq. Conclusion Given the impact of codon usage bias on recombinant gene technologies, this database will facilitate effective development and review of recombinant drug products and will be instrumental in a wide area of biological research. The database is available at hive.biochemistry.gwu.edu/review/codon. Electronic supplementary material The online version of this article (doi:10.1186/s12859-017-1793-7) contains supplementary material, which is available to authorized users.