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Design and application of an in vivo reporter assay for phenylalanine ammonia-lyase

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Abstract

Phenylalanine ammonia-lyase (PAL) is an important enzyme that links primary metabolism to secondary metabolism. Its efficiency is often a critical factor that affects the overall flux of a related metabolic pathway, the titer of the final products, and the efficacy of PAL-based therapies. Thus, PAL is a common target for metabolic engineering, and it is of significant interest to screen efficient PALs for industrial and medical applications. In this study, a novel and efficient visible reporter assay for screening of PAL efficiency in Escherichia coli was established based on a plant type III polyketide biosynthetic pathway. The candidate PALs were co-expressed with a 4-coumarate:CoA ligase 4CL1 from Arabidopsis thaliana and curcuminoid synthase (CUS) from Oryza sativa in E. coli BL21(DE3) to form a dicinnamoylmethane biosynthetic pathway. Taking advantage of the yellow color of the product, a microplate-based assay was designed to measure the titer of dicinnamoylmethane, which was validated by HPLC analysis. The different titers of the product reflect the overall performance (expression level and enzymatic activity) of the individual PALs in E. coli. Using this system, we have screened three PALs (PAL1, PAL3, and PAL4) from Trifolium pratense, among which PAL1 showed the best performance in E. coli. The engineered E. coli strain containing PAL1, 4CL1, and CUS led to the production of dicinnamoylmethane at a high level of 0.36 g/l. Supplement of 2-fluoro-phenylalanine yielded two fluorinated dicinnamoylmethane derivatives, 6,6′-difluoro-dicinnamoylmethane and 6-fluoro-dicinnamoylmethane, of which the latter is a new curcuminoid.

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Acknowledgments

We thank Dr. Mike Sullivan (USDA), Dr. Claudia Schmidt-Dannert (University of Minnesota), and Dr. Nobutaka Funa (University of Shizuoka) for providing the corresponding plasmids. This research was supported by Award W81XWH-11-1-0458 from the Congressionally Directed Medical Research Programs of the Department of Defense, USA.

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  1. Department of Biological Engineering, Utah State University, 4105 Old Main Hill, Logan, UT, 84322-4105, USA

    Siyuan Wang, Shuwei Zhang, Tong Zhou, Jia Zeng & Jixun Zhan

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  1. Siyuan Wang
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  2. Shuwei Zhang
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  3. Tong Zhou
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  4. Jia Zeng
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  5. Jixun Zhan
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Correspondence to Jixun Zhan.

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Wang, S., Zhang, S., Zhou, T. et al. Design and application of an in vivo reporter assay for phenylalanine ammonia-lyase. Appl Microbiol Biotechnol 97, 7877–7885 (2013). https://doi.org/10.1007/s00253-013-5122-4

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  • DOI: https://doi.org/10.1007/s00253-013-5122-4

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