期刊
JOURNAL OF THE TAIWAN INSTITUTE OF CHEMICAL ENGINEERS
卷 128, 期 -, 页码 64-72出版社
ELSEVIER
DOI: 10.1016/j.jtice.2021.08.038
关键词
p-Coumaric acid; Tyrosine ammonia lyase; Whole-cell biocatalyst; GroELS; Fusion protein
This study successfully established a whole-cell biocatalyst with recombinant tyrosine ammonia lyase, which exhibited high activity, stability, and robustness for one-step pCA biosynthesis.
Background: p-Coumaric acid (pCA) is an essential bioactive compound for nutraceutical and pharmaceutical purposes; however, high-level pCA biosynthesis via in vivo is challenging due to the complexity of pathways and genetic designs. Hence, to produce pCA in a one-step reaction, it is crucial to establish whole-cell (WC) biocatalyst of recombinant tyrosine ammonia lyase (EC 4.3.1.23) with high activity, stability, and robustness properties. Methods: Three different tyrosine ammonia lyase (TAL) genes were cloned and overexpressed under T7 promoter in Escherichia coli BL21(DE3). First, a copy GroELS was integrated in E. coli chromosome using a phage attack approach while rare codons and fusion TrxA-tag were cloned in a plasmid with the best TAL (Rhodotorula toluroides; RtTAL) for pCA production. Further optimization was conducted to the effect of temperature, medium, inducer, and metal ions, as well as biotransformation parameters. Significant findings: Optimal pCA biotransformation was first explored using recombinant RtTAL in a low-cost medium with glycerol and lactose. Integrating a single copy of GroELS in E. coli chromosome and inserting the TrxA-tag, p32a-RtTAL/BD::7G, exhibited markedly elevated specific enzymatic activity of 16.14 mM mg-1 min-1 and conversion up to 94.8%. Moreover, RtTAL showed a 50% increase in the presence Mn2+ metal ions, while the whole-cell biocatalyst converted tyrosine to high-level of pCA up to 2.1 g/L by summation of 5-repeated batch reaction. (c) 2021 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
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