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Functional expression in Escherichia coli of the tyrosine-inducible tyrosine ammonia-lyase enzyme from yeast Trichosporon cutaneum for production of p-hydroxycinnamic acid

Biochemical and Engineering Sciences, DuPont CR&D, Wilmington, DE 19880, United States
Enzyme and Microbial Technology (Impact Factor: 2.32). 09/2007; 41(4):413-422. DOI: 10.1016/j.enzmictec.2007.03.013

ABSTRACT

Deamination of aromatic amino acids phenylalanine or tyrosine is accomplished by the phenylalanine/tyrosine ammonia-lyase (PAL/TAL) enzyme. TAL enzyme is of interest since deamination of tyrosine produces p-hydroxycinnamic acid (pHCA), which has potential for a variety of applications. Among nine microorganisms tested for their ability to produce tyrosine-inducible TAL activity, the yeast, Trichosporon cutaneum showed the highest TAL catalytic activity and the lowest PAL/TAL catalytic efficiency ratio (0.8). The enzyme was purified to near homogeneity and its kinetics studied. The native enzyme appears to be a homo-tetramer with a calculated MW of 294 kDa, subunit MW of 73.5 kDa, and a pI of 5.8. When phenylalanine was used as substrate, the Vmax, Kcat and Km were ∼4.0 ± 0.2 μg/min/mg purified enzyme), 588 ± 29 per min and 4.9 ± 0.9 mM, respectively. However, when tyrosine served as the substrate the Vmax and Kcat were 0.59 ± 0.02 μg/min/mg purified enzyme), and 86.7 ± 29 per min, and substrate binding was apparently cooperative (nH ∼ 2.6 ± 0.4), with S0.5 ∼ 0.6 mM. This is the first reported positive cooperativity for a TAL enzyme. Based on the NH2-terminal and partial internal peptide sequences, the cDNA encoding the enzyme was cloned. Sequence analysis of TcTAL showed 56–62% similarity to other fungal PAL/TAL enzymes. High-level expression (∼30% of total soluble protein, based on SDS-PAGE analysis) of the cDNA in Escherichia coli was achieved using the arabinose inducible araB promoter. The recombinant enzyme possessed both PAL and TAL activities, as evident from the presence of both pHCA and CA in the culture medium.

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Available from: Todd Vannelli, Jun 11, 2014
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    • "Thus, the importance of glycosylation in PAL function needs to be further studied. Although genes encoding PAL have been cloned and sequenced from both plant and microbial sources (Faulkner et al., 1994; Minami et al., 1989; Vannelli et al., 2007a) and over-expressed as active enzymes in yeast, E. coli and insect cell cultures (Langer et al., 1997; McKegney et al., 1996; Vannelli et al., 2007b), the yields of recombinant enzymes obtained were still low. In terms of large-scale production of PAL for industrial and medical uses, expression levels in E. coli need to be improved substantially. "
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    ABSTRACT: Export Date: 18 October 2014
    Full-text · Article · Jan 2014 · Critical Reviews in Biotechnology
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    • "Thus, the importance of glycosylation in PAL function needs to be further studied. Although genes encoding PAL have been cloned and sequenced from both plant and microbial sources (Faulkner et al., 1994; Minami et al., 1989; Vannelli et al., 2007a) and over-expressed as active enzymes in yeast, E. coli and insect cell cultures (Langer et al., 1997; McKegney et al., 1996; Vannelli et al., 2007b), the yields of recombinant enzymes obtained were still low. In terms of large-scale production of PAL for industrial and medical uses, expression levels in E. coli need to be improved substantially. "
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    ABSTRACT: Abstract Phenylalanine ammonia lyase (PAL) catalyzes the nonoxidative deamination of L-phenylalanine to form trans-cinnamic acid and a free ammonium ion. It plays a major role in the catabolism of L-phenylalanine. The presence of PAL has been reported in diverse plants, some fungi, Streptomyces and few Cyanobacteria. In the past two decades, PAL has gained considerable significance in several clinical, industrial and biotechnological applications. Since its discovery, much knowledge has been gathered with reference to the enzyme's importance in phenyl propanoid pathway of plants. In contrast, there is little knowledge about microbial PAL. Furthermore, the commercial source of the enzyme has been mainly obtained from the fungi. This study focuses on the recent advances on the physiological role of microbial PAL and the improvements of PAL biotechnological production both from our laboratory and many others as well as the latest advances on the new applications of microbial PAL.
    Full-text · Article · May 2013 · Critical Reviews in Biotechnology
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    • "The necessary addition of p-coumarate to the culture medium has the consequence that the TAL or PAL activities remained low in the bacterium. As a result, much of the ongoing efforts are dedicated to increasing these activities in bacteria, for example, by aiming to develop novel TAL or PAL enzymes [71, 72]. Two more recent works constructed the complete resveratrol biosynthetic pathway in S. cerevisiae to produce resveratrol from phenylalanine. "
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    ABSTRACT: Microbes constitute important platforms for the biosynthesis of numerous molecules of pharmaceutical interest such as antitumor, anticancer, antiviral, antihypertensive, antiparasitic, antioxidant, immunological agents, and antibiotics as well as hormones, belonging to various chemical families, for instance, terpenoids, alkaloids, polyphenols, polyketides, amines, and proteins. Engineering microbial factories offers rich opportunities for the production of natural products that are too complex for cost-effective chemical synthesis and whose extraction from their originating plants needs the use of many solvents. Recent progresses that have been made since the millennium beginning with metabolic engineering of microorganisms for the biosynthesis of natural products of pharmaceutical significance will be reviewed.
    Full-text · Article · Apr 2013
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