Whole-cell biocatalyst of recombinant tyrosine ammonia lyase with fusion protein and integrative chaperone in Escherichia coli for high-level p-Coumaric acid production

Abstract

Backgroundp-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. MethodsThree 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 findingsOptimal 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 μM 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.