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

Article (PDF Available)inEnzyme and Microbial Technology 41(4):413-422 · September 2007with73 Reads
DOI: 10.1016/j.enzmictec.2007.03.013
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
    • "What follows is a comparison of strain performance among those studies providing data that can be related to our study. Similar or higher CA titers to those observed for E. coli strain WPJAt with arabinose have been reported with S. lividans expressing the PAL from Streptomyces maritimus and employing culture medium containing tryptone and carbon sources such as xylose, glycerol or xylooligosa- charides [16]. However, these titers were reached after 6 to 8 days of culture time. "
    [Show abstract] [Hide abstract] ABSTRACT: Background The aromatic compounds cinnamic acid (CA) and p-hydroxycinnamic acid (pHCA) are used as flavoring agents as well as precursors of chemicals. These compounds are present in plants at low concentrations, therefore, complex purification processes are usually required to extract the product. An alternative production method for these aromatic acids is based on the use of microbial strains modified by metabolic engineering. These biotechnological processes are usually based on the use of simple sugars like glucose as a raw material. However, sustainable production processes should preferably be based on the use of waste material such as lignocellulosic hydrolysates.ResultsIn this study, E. coli strains with active (W3110) and inactive phosphoenolpyruvate:sugar phosphotransferase system (PTS) (VH33) were engineered for CA and pHCA production by transforming them with plasmids expressing genes encoding phenylalanine/tyrosine ammonia lyase (PAL/TAL) enzymes from Rhodotorula glutinis or Arabidopsis thaliana as well as genes aroG fbr and tktA, encoding a feedback inhibition resistant version of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase and transketolase, respectively. The generated strains were evaluated in cultures with glucose, xylose or arabinose, as well as a simulated lignocellulosic hydrolysate containing a mixture of these three sugars plus acetate. Production of CA was detected in strains expressing PAL/TAL from A. thaliana, whereas both CA and pHCA accumulated in strains expressing the enzyme from R. glutinis. These experiments identified arabinose and W3110 expressing PAL/TAL from A. thaliana, aroG fbr and tktA as the carbon source/strain combination resulting in the best CA specific productivity and titer. To improve pHCA production, a mutant with inactive pheA gene was generated, causing an 8-fold increase in the yield of this aromatic acid from the sugars in a simulated hydrolysate.Conclusions In this study the quantitative contribution of active or inactive PTS as well as expression of PAL/TAL from R. glutinis or A. thaliana were determined for production performance of CA and pHCA when growing on carbon sources derived from lignocellulosic hydrolysates. These data will be a useful resource in efforts towards the development of sustainable technologies for the production of aromatic acids.
    Full-text · Article · Jan 2015
    • "When both 20% glycerol and 400 mg/L thioglycolic acid were simultaneously present in the reaction mixture, conversion rate and overall productivity of L-phenylalanine were higher than that of the control. Recent studies showed that PAL could be used to produce trans-p-hydroxycinnamic acid (pHCA) from glucose via deamination of L-phenylalanine or tyrosine by PAL/TAL enzyme (Vannelli et al., 2007aVannelli et al., , 2007b). Vannelli et al. (2007a) first developed expression systems for the production of pHCA from glucose in both E. coli and S. cerevisiae using the PAL/TAL enzyme from Rh. glutinis. "
    [Show abstract] [Hide abstract] ABSTRACT: Export Date: 18 October 2014
    Full-text · Article · Jan 2014
    • "However, so far, the entire phenylpropanoid pathway has not been found in one microorganism, although the key enzymes in this pathway have been separately found in different microorganisms. For example, PAL has been found in Rhodotorula glutinis and Rhodosporidium toruloids (Vannelli et al. 2007a; Wu et al. 2009); TAL in R. glutinis , Trichosporon cutaneum , and Saccharothrix Vannelli et al. 2007b; Berner et al. 2006); and 4CL in Streptomyces coelicolor (Park et al. 2009; Kaneko et al. 2003). To date, only plant STS has been used in genetically modified resveratrol-producing microorganisms . "
    [Show abstract] [Hide abstract] ABSTRACT: To identify the substrates and enzymes related to resveratrol biosynthesis in Alternaria sp. MG1, different substrates were used to produce resveratrol, and their influence on resveratrol production was analyzed using high performance liquid chromatography (HPLC). Formation of resveratrol and related intermediates was identified using mass spectrum. During the biotransformation, activities of related enzymes, including phenylalanine ammonia-lyase (PAL), trans-cinnamate 4-hydroxylase (C4H), and 4-coumarate-CoA ligase (4CL), were analyzed and tracked. The reaction system contained 100 mL 0.2 mol/L phosphate buffer (pH 6.5), 120 g/L Alternaria sp. MG1 cells, 0.1 g/L MgSO4, and 0.2 g/L CaSO4 and different substrates according to the experimental design. The biotransformation was carried out for 21 h at 28 °C and 120 rpm. Resveratrol formation was identified when phenylalanine, tyrosine, cinnamic acid, and p-coumaric acid were separately used as the only substrate. Accumulation of cinnamic acid, p-coumaric acid, and resveratrol and the activities of PAL, C4H, and 4CL were identified and changed in different trends during transformation with phenylalanine as the only substrate. The addition of carbohydrates and the increase of phenylalanine concentration promoted resveratrol production and yielded the highest value (4.57 μg/L) when 2 g/L glucose, 1 g/L cyclodextrin, and phenylalanine (4.7 mmol/L) were used simultaneously.
    Full-text · Article · Sep 2013
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