Active tyrosine phenol-lyase aggregates induced by terminally attached functional peptides in Escherichia coli

Abstract

The formation of inclusion bodies (IBs) without enzyme activity in bacterial research is generally undesirable. Researchers have attempted to recovery the enzyme activities of IBs, which are commonly known as active IBs. Tyrosine phenol-lyase (TPL) is an important enzyme that can convert pyruvate and phenol into 3,4-dihydroxyphenyl-l-alanine (L-DOPA) and IBs of TPL can commonly occur. To induce the correct folding and recover the enzyme activity of the IBs, peptides, such as ELK16, DKL6, L6KD, ELP10, ELP20, L6K2, EAK16, 18A, and GFIL16, were fused to the carboxyl terminus of TPL. The results showed that aggregate particles of TPL-DKL6, TPL-ELP10, TPL-EAK16, TPL-18A, and TPL-GFIL16 improved the enzyme activity by 40.9%, 50.7%, 48.9%, 86.6%, and 97.9%, respectively. The peptides TPL-DKL6, TPL-EAK16, TPL-18A, and TPL-GFIL16 displayed significantly improved thermostability compared with TPL. L-DOPA titer of TPL-ELP10, TPL-EAK16, TPL-18A, and TPL-GFIL16, with cells reaching 37.8 g/L, 53.8 g/L, 37.5 g/L, and 29.1 g/L, had an improvement of 111%, 201%, 109%, and 63%, respectively. A higher activity and L-DOPA titer of the TPL-EAK16 could be valuable for its industrial application to biosynthesize L-DOPA.

Catalytically Active Inclusion Bodies─Benchmarking and Application in Flow Chemistry

“…Among the more widely used tags for inducing CatIB formation, ,− ,− ,,− we chose 18AWT, L6KD, GFIL8, and TdoT, which were genetically fused to the 5′ or 3′ end of the genes encoding red fluorescent protein mCherry from Discosoma striata, alcohol dehydrogenase from Ralstonia sp. (RADH), and lipase A from Bacillus subtilis (BsLA) as target proteins, thereby generating fusion proteins with either N- or C-terminally fused tag (Figure ).…”

Combining Protein Engineering and Pathway Engineering to Achieve Green Biosynthesis of l-Hypaphorine in Escherichia coli from Glucose

“…In addition, we found that all of the test methyltransferases showed low soluble expression levels in E. coli BL21­(DE3) (Figure b), and this may also lead to low l -HYP yield, even no yield . In order to increase the soluble expression level of MsE and to increase the l -HYP production, the expression conditions and the expression patterns were optimized focusing on inducible temperature, molecular chaperone, and fusion tag.…”

“…33 In addition, we found that all of the test methyltransferases showed low soluble expression levels in E. coli BL21(DE3) (Figure 2b), and this may also lead to low L-HYP yield, even no yield. 34 In order to increase the soluble expression level of MsE and to increase the L-HYP production, the expression conditions and the expression patterns were optimized focusing on inducible temperature, molecular chaperone, and fusion tag. 30 °C was selected as the best inducible temperature despite not significantly increasing the soluble MsE level (Figure 3a) because cell growth at 30 °C was almost consistent with that at 37 °C (Figure 3b).…”

Emerging Solutions for in Vivo Biocatalyst Immobilization: Tailor-Made Catalysts for Industrial Biocatalysis

“…Particularly noteworthy is the successful implementation of a two-step cascade reaction, which was realized using CatIBs with colocalized PfBAL and RADH, where PfBAL converted benzaldehyde and acetaldehyde to ( R )-2-hydroxy-1-phenylpropanone, which was further converted by RADH to benzyl alcohol and (1 R , 2 R )-1-phenylpropane-1,2-diol, a precursor of the calcium channel blocker diltiazem . Recently, the tetrameric, PLP-dependent tyrosine phenol-lyase, which can be employed to produce enantiomerically pure α-deuterated ( S )-amino acids, such as the dopamine precursor l -dihydroxyphenylalanine, has been immobilized using C-terminal fusions of nine different artificial peptide tags . Notably, two of those constructs (bearing GFIL16 and 18AWT tags) yielded CatIBs with improved thermostability and half-lives, reaching 87–98% of the activity detected in the supernatant of the soluble enzyme, respectively.…”

“…60 Recently, the tetrameric, PLP-dependent tyrosine phenol-lyase, which can be employed to produce enantiomerically pure α-deuterated (S)-amino acids, such as the dopamine precursor Ldihydroxyphenylalanine, has been immobilized using Cterminal fusions of nine different artificial peptide tags. 66 Notably, two of those constructs (bearing GFIL16 and 18AWT tags) yielded CatIBs with improved thermostability and half-lives, reaching 87−98% of the activity detected in the supernatant of the soluble enzyme, respectively. To the best of our knowledge, the most complex oligomeric POI that was immobilized with the CatIB strategy is the PLP-dependent, decameric L-lysine decarboxylase from E. coli, using TdoT and 3HAMP as tags.…”