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. 2017 Jul 4;8(4):424-427.
doi: 10.1080/21655979.2016.1230572. Epub 2016 Oct 18.

N-terminal engineering of glutamyl-tRNA reductase with positive charge arginine to increase 5-aminolevulinic acid biosynthesis

Junli Zhang  1   2 , Huanjiao Weng  1 , Wenwen Ding  1 , Zhen Kang  1   3
Affiliations

N-terminal engineering of glutamyl-tRNA reductase with positive charge arginine to increase 5-aminolevulinic acid biosynthesis

Junli Zhang et al. Bioengineered. .

Abstract

Five-Aminolevulinic acid (ALA), the universal precursor of all tetrapyrroles, has various applications in medicine and agriculture industries. Glutamyl-tRNA reductase (GluTR) as the first key enzyme of C5 pathway is feedback regulated by heme, and its N-terminus plays a critical role on its stability control. Here, the GluTR N-terminus was engineered by inserting different numbers of positively charged lysine and arginine residues. The results confirmed that insertion of lysine or arginine residues (especially one arginine residue) behind Thr2 significantly increased the stability of GluTR. By co-expression of the GluTR variant R1 and the glutamate-1-semialdehyde aminotransferase, ALA production was improved 1.76-fold to 1220 mg/L. The GluTR variant R1 constructed here could be used for engineering the C5 pathway to enhance ALA and other products.

Keywords: Five-aminolevulinic acid; N-terminal engineering; escherichia coli; glutamyl-tRNA reductase; heme.

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Figures

Figure 1.
Figure 1.
The model complex of GluTR and GSA-AT. The V-shaped dimer protein is GluTR while the other dimer is GSA-AT.
Figure 2.
Figure 2.
The illustration of GluTR variants with insertion of different numbers of lysine residues (A) and arginine residues (B). WT represented the wild-type GluTR.
Figure 3.
Figure 3.
ALA production of the variants with inserting different lysine residues (A) and arginine residues (B).
Figure 4.
Figure 4.
Structural analysis of N-terminal domain of GluTR. (A) Wild-type GluTR; (B) the variant GluTR K2 with insertion of 2 lysine residues; (C) the variant GluTR R1 with insertion of one arginine residue. The green dotted lines indicate the hydrogen bonds.
Figure 5.
Figure 5.
SDS-PAGE analysis of the GluTR variants with insertion of arginine residues.
See this image and copyright information in PMC

Erratum for

  • Addendum to: Zhang J, Kang Z, Ding W, Chen J, Du G. Integrated optimization of the in vivo heme biosynthesis pathway and the in vitro iron concentration for 5-aminolevulinate production. Applied Biochemistry and Biotechnology 2016; 178(6):1252–1262.

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