Isolation and characterization of a novel cyanophycin synthetase from a deep-sea sediment metagenomic library

Abstract

Cyanophycin is non-ribosomally synthesized protein-like copolymer. Synthesis of cyanophycin is catalyzed by cyanophycin synthetase (CphA). In this study, a novel cyanophycin synthetase CphA49 belonging to NOR5 clade of Gammaproteobacteria was identified with primer-based screening from a deep-sea sediment metagenomic library. The cphA49 gene contained an open reading frame of 2,637 bp and encoded a protein with a predicted molecular mass of 100 kDa. A recombinant CphA49 was obtained by the functional expression of cphA49 in Escherichia coli BL21 (DE3). The biochemical properties of the purified CphA49 were determined. The optimum pH and temperature of the recombinant CphA49 were 9.0 and 40 °C, respectively. The enzyme was stable at temperatures below 40 °C. The recombinant CphA49 exhibited strict primer dependency and broad substrate specificities. Cyanophycin catalyzed by CphA49 exhibited homogenous molecular mass. The amino acid composition of cyanophycin was determined and constitutes arginine, aspartic acid, and lysine.

Microbiome Dynamics in a Large Artificial Seawater Aquarium

“…All three MAGs contained genes for CphA and CphB. Orthologs are found in most cyanobacteria as well as in a small but diverse group of heterotrophic bacteria ( 15 , 29 , 30 ). However, none of the publicly available Kordiimonas species genome sequences contain cyanophycin-related genes.…”

Cyanophycin and its biosynthesis: not hot but very cool

“…To realize its full commercial potential, cyanophycin must be produced in large amounts and at low cost. The most promising approach is in vivo production, and many studies have been performed with different native 8,38,48,50,91,92 and heterologous 25,26,87,[93][94][95][96][97][98][99][100] hosts, with various CphA1 enzymes 15,16,91,96,[100][101][102][103][104][105][106] and growth conditions. An excellent review by Frommeyer, Wiefel and Steinbüchel summarizes this eld comprehensively.…”

Recombinant Multi‐l‐Arginyl‐Poly‐l‐Aspartate (Cyanophycin) as an Emerging Biomaterial

“…Numerous aforementioned host strains have been used for MAPA preparation; however, considering the availability of expression vector, the easiness of cultivation and the productivity, E. coli has been widely adopted as a host strain for the routine preparation of MAPA. [2,17,18,24,25,27,29,30,33,[35][36][37][38][41][42][43] The E. coli BL21(DE3) series of protein expression hosts include several variants, such as BL21 Star(DE3), BL21-CodonPlus(DE3), ClearColi BL21(DE3), each of which can serve different purposes for MAPA preparation. The expression of cphA1 gene is generally put under the control of an inducible promoter, for example the T7lac promoter.…”

“…Swain et al, 2023 [40] CphA BP-1 E. coli BL21(DE3) pET-21a(+) LB iMAPA (yield not specified) Arai & Kino 2008 [17] CphA Wiefel et al, 2019 [39] CphA 49 E. coli BL21 (DE3) pET-28a(+) LB sMAPA 16%, 1100 mg L −1 [3.5 mol% Lys] 35 kDa Du et al 2013 [42] CphA 49 E. coli BL21 (DE3) pET-28a(+) M R+ 10 mM Asp + 9 mM Arg +1 mM Lys sMAPA 1720 mg L −1 [3.5 mol% Lys]…”