Thermococcus sp. KS-1 PPIase as a fusion partner improving soluble production of aromatic amino acid decarboxylase

Takashi Koyanagi,Ayumi Hara,Yuji Habara,Hiromichi Minami,Takane Katayama,Akira Nakagawa,Norihiko Misawa,Kanako Kobayashi

doi:10.1186/s13568-021-01340-3

Takashi Koyanagi, Ayumi Hara + Show 6 more

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https://doi.org/10.1186/s13568-021-01340-3

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Abstract

Peptidyl-prolyl cis-trans isomerase (PPIase, EC 5.2.1.8) catalyzes the racemization reaction of proline residues on a polypeptide chain. This enzyme is also known to function as a molecular chaperon to stabilize protein conformation during the folding process. In this study, we noted FK506 binding protein (FKBP)-type PPIase from a hyperthemophilic archaeon Thermococcus sp. strain KS-1 (PPIase KS−1) to improve the solubility of Pseudomonas putida aromatic amino acid decarboxylase (AADC) that is an indispensable enzyme for fermentative production of plant isoquinoline alkaloids. AADC fused N-terminally with the PPIase KS−1 (PPIase KS−1-AADC), which was synthesized utilizing Escherichia coli host, showed improved solubility and, consequently, the cell-free extract from the recombinant strain exhibited 2.6- to 3.4-fold elevated AADC activity than that from the control strain that expressed the AADC gene without PPIase KS−1. On the other hand, its thermostability was slightly decreased by fusing PPIase KS−1. The recombinant E. coli cells expressing the PPIase KS−1-AADC gene produced dopamine and phenylethylamine from L-dopa and phenylalanine by two- and threefold faster, respectively, as compared with the control strain. We further demonstrated that the efficacy of PPIase KS−1-AADC in solubility and activity enhancement was a little but obviously higher than that of AADC fused N-terminally with NusA protein, which has been assumed to be the most effective protein solubilizer. These results suggest that PPIase KS−1 can be used as one of the best choices for producing heterologous proteins as active forms in E. coli.

Highlights

Peptidyl-prolyl cis-trans isomerase (PPIase, EC 5.2.1.8) is an enzyme that catalyzes the racemization reaction of amino acid proline (Fischer and Schmid 1999)
Overproduction of amino acid decarboxylase (AADC) and comparison of solubility among the expression systems E. coli BL21(DE3) carrying pAADC, pNusA-AADC, or pPPIase KS−1-AADC were cultivated in LB and the expression of target genes from the T7 promoter were induced by IPTG
These results indicated that PPIase KS−1 greatly improved the solubility of AADC, and the effectiveness of PPIase KS−1 was comparable to the NusA system, which is regarded as one of the most efficient protein solubilizer generally used among the E. coli overexpression constructs

Summary

Introduction

Peptidyl-prolyl cis-trans isomerase (PPIase, EC 5.2.1.8) is an enzyme that catalyzes the racemization reaction of amino acid proline (Fischer and Schmid 1999). In case of Escherichia coli, various types of protein stabilizer have been developed, e.g., DnaK/DnaJ/ GrpE and GroEL/ES (molecular chaperons co-expressed or fused with a target protein) (Bhandari and Houry 2009; Kyratsous et al 2009), thioredoxin DsbA and DsbC (cysteine-bonds improvers fused to the N-terminus of a target protein) (Collins-Racie et al 1995; Nozach et al 2013), and N utilization substance A (NusA, high-performance protein solubilizer fused to the N-terminus of a target protein) (Davis et al 1999) These systems can generally contribute to successful protein overproduction in the E. coli cells, but still have the probability that fails in the folding of soluble proteins case-dependently. It is desirable to retain the multiple options for functional expression systems as many as possible

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