Protein engineering of cinnamate 4 -hydroxylase for production of non-natural phenylpropanoids

Hao Chen, Purdue University

Abstract

Phenylpropanoids are a diverse class of natural compounds derived mainly from plants. Besides their important physiological roles in plants, some phenylpropanoids such as flavonoids are valuable compounds for human health. The general goal of this study is to produce non-natural phenylpropanoids through recombinant Saccharomyces cerevisiae. The first objective was to optimize and characterize the heterologous expression of cinnamate 4-hydroxylase (C4H), a key enzyme in plant phenylpropanoid pathway. A single stage procedure combining both cell growth and induction was developed to simplify cell culture and enzyme expression. The induction time and medium were optimized, and a 63% increase in specific whole cell activity was achieved. A high throughput screening method was developed based on the spectrophotometric detection of whole cell C4H activity in 96-well microtiter plates. This method could be generalized to the screening of membrane-bound cytochrome P450s. Thirty-four substrate analogues were screened against C4H, thirteen of which were found to be reactive. The structures of nine products were characterized and two of them are novel compounds. The second objective was to alter the selectivity of C4H through protein engineering. Ten active site residues of C4H were identified through analysis of homology models, and their functional significance was assessed with site-directed and saturation mutagenesis. The subsequent mutants were screened with both natural and non-natural substrates. Most of the mutants showed altered preference of substrates. The mutant V305A completely lost activity towards all tested substrates. A replacement of F488 with all standard amino acids resulted in a mutant library with 88% of mutants remained cinnamate activity. The third objective was to synthesize novel flavonoids by feeding non-natural substrates to the engineered yeasts. Six cinnamate analogues were fed to the recombinant S. cerevisiae containing C4H, 4CL, and CHS. Preliminary data indicated that the feeding of 3-methyl, 3-fluoro, 3-amino, 2-bromo, and 2-cyno substituted cinnamate analogues could lead to the production of non-natural flavonoids, and all of the major products are novel compounds. Substrate analogue screening also indicated broad activity of the engineered yeast coexpressing 4CL and CHS: ten tested analogues could be metabolized sequentially by the engineered pathway.

Degree

Ph.D.

Advisors

Morgan, Purdue University.

Subject Area

Chemical engineering

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