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

Feiyan Xue,Lanqing Ma,Yue Yu,Zihui Liu,Yangjie Wu,Yuxin Jin,Mingfeng Yang

doi:10.3389/fbioe.2020.610506

Abstract

PAL (phenylalanine ammonia lyase) is important for secondary metabolite production in plants and microorganisms. There is broad interest in engineering PAL for its biocatalytic applications in industry, agriculture, and medicine. The production of quantities of high-activity enzymes has been explored by gene cloning and heterogeneous expression of the corresponding protein. Here, we cloned the cDNA of Rhodotorula glutinis PAL (RgPAL) and introduced codon optimization to improve protein expression in Escherichia coli and enzyme activities in vitro. The RgPAL gene was cloned by reverse transcription and named pal-wt. It had a full-length of 2,121 bp and encoded a 706-amino-acid protein. The pal-wt was inefficiently expressed in E. coli, even when the expression host and physical conditions were optimized. Therefore, codon optimization was used to obtain the corresponding gene sequence, named pal-opt, in order to encode the same amino acid for the RgPAL protein. The recombinant protein encoded by pal-opt, named PAL-opt, was successfully expressed in E. coli and then purified to detect its enzymatic activity in vitro. Consequently, 55.33 ± 0.88 mg/L of PAL-opt protein with a specific activity of 1,219 ± 147 U/mg and Km value of 609 μM for substrate L-phenylalanine was easily obtained. The enzyme protein also displayed tyrosine ammonia lyase (TAL)–specific activity of 80 ± 2 U/mg and Km value of 13.3 μM for substrate L-tyrosine. The bifunctional enzyme RgPAL/TAL (PAL-opt) and its easy expression advantage will provide an important basis for further applications.

Highlights

Phenylalanine ammonia lyase (PAL, EC 4.3.1.5) is the first enzyme of the general phenylpropanoid pathway catalyzing ammonia elimination from phenylalanine (Phe) to give trans-cinnamic acid, or tyrosine (Tyr) deamination to form p-coumaric acid (p-hydroxycinnamic acids), indicating its additional tyrosine ammonia lyase (TAL) activity (Figure 1) (Jun et al, 2018)
The strains used in this work included Rhodotorula glutinis CGMCC2258 and E. coli BL21 (DE3), Rosetta-Gami 2 (DE3), and DH5α
The recombinant strain E. coli BL21 (DE3) harboring the plasmid pET-30a or pET-30a-pal-wt was induced by the addition of Isopropyl β-D-Thiogalactoside (IPTG) to identify Rhodotorula glutinis PAL (RgPAL) expression

Summary

Introduction

Phenylalanine ammonia lyase (PAL, EC 4.3.1.5) is the first enzyme of the general phenylpropanoid pathway catalyzing ammonia elimination from phenylalanine (Phe) to give trans-cinnamic acid, or tyrosine (Tyr) deamination to form p-coumaric acid (p-hydroxycinnamic acids), indicating its additional tyrosine ammonia lyase (TAL) activity (Figure 1) (Jun et al, 2018). The cloning and heterogeneous expression of Rhodotorula PAL in recombinant Escherichia coli have typically been used (Cui et al, 2008; Jia et al, 2008; Babich et al, 2013; Zhu et al, 2013; Vargas-Tah et al, 2015; Rowles et al, 2016; Levy et al, 2018). This lowcost and easy method reported here for obtaining abundant recombinant RgPAL with high activity offers an effective and sustainable PAL production source

Methods

Results

Conclusion