Characterization and enhanced extracellular overexpression of a new salt‐activated alginate lyase

Abstract

BACKGROUND Alginate lyases (EC 4.4.2.3/4.4.2.11) have been applied to produce alginate oligosaccharides, which have physiological advantages such as prebiotic and antidiabetic effects, and are of benefit in the food and pharmaceutical industries. Extracellular production of recombinant proteins in Escherichia coli presents advantages including simplified downstream processing and high productivity; however, the presence of certain signal peptides does not always ensure successful secretion, which make the extracellular production of alginate lyase in E. coli rarely reported but of great significance. RESULTS A PL7 family alginate lyase, Aly01, with its native signal peptide from Vibrio natriegens SK42.001, was identified, characterized, and extracellularly expressed in E. coli. The enzyme specifically released trisaccharide from alginate and was strictly NaCl activated. Green fluorescent protein (GFP) was fused with the Aly01 signal peptide and successfully secreted in E. coli to expand the feasibility of using this signal peptide to produce other heterologous proteins extracellularly. Through a synergistic strategy of utilizing Terrific Broth (TB) medium supplemented with 120 mmol L–1 glycine and 10 mmol L–1 calcium, the lag phase of protein secretion was reduced to 3 h from 12 h; meanwhile calcium remedied glycine‐related cell growth impairment, leading to further enhancement of overall enzyme productivity, reaching a maximum of 4.55 U mL−1. CONCLUSION A new salt‐activated alginate lyase, Aly01, was identified and characterized. E. coli employed its signal peptide and extracellularly expressed both Aly01 and a GFP, which indicated the signal peptide of Aly01 could be a powerful tool for extracellular production of other heterologous proteins in E. coli. © 2021 Society of Chemical Industry

High-Level Extracellular Production of a Trisaccharide-Producing Alginate Lyase AlyC7 in Escherichia coli and Its Agricultural Application

“…Unlike the fed-batch method required for P. pastoris, PelB-AlyC7 only necessitates glycine supplementation during fermentation. In another study, Meng et al reported the secretory expression of the alginate lyase Aly01 in E. coli, which shares 71.4% sequence similarity with AlyC7 and primarily yields trisaccharides along with small amounts of disaccharides and tetrasaccharides [22]. Aly01's extracellular production was carried out in TB medium supplemented with 1 mM IPTG, 120 mM glycine, and 10 mM CaCl 2 at 18 • C for 48 h, achieving an extracellular activity of 796.3 U/mL after unit conversion [22].…”

“…In another study, Meng et al reported the secretory expression of the alginate lyase Aly01 in E. coli, which shares 71.4% sequence similarity with AlyC7 and primarily yields trisaccharides along with small amounts of disaccharides and tetrasaccharides [22]. Aly01's extracellular production was carried out in TB medium supplemented with 1 mM IPTG, 120 mM glycine, and 10 mM CaCl 2 at 18 • C for 48 h, achieving an extracellular activity of 796.3 U/mL after unit conversion [22]. Compared with the E. coli strain constructed by Meng et al, strain PelB-AlyC7 also exhibited higher extracellular activity.…”

“…The considerable heterogeneity of AOS products results in instability and variability regarding their biological activity. Secondly, while numerous alginate lyases have been overexpressed in the cytoplasm of the engineered strains [22,23], e.g., Escherichia coli, secretory production of recombinant proteins is highly regarded in industry due to its advantages such as simplified downstream processing at a low cost, efficient productivity, high activity levels, and stability [22]. Nevertheless, targeting proteins in the extracellular space has been constrained by complex secretory mechanisms and the limited intrinsic secretory capacities of strains.…”

Preparation of Alginate Oligosaccharides from Laminaria japonica Biomass by a Novel Biofunctional Alginate Lyase with pH and Salt Tolerance

“…C42 was successfully expressed in E. coli [22]. The alginate lyase Aly01 of PL7 family from Vibrio albicans SK42.001 has been extracellularly expressed in E. coli [23]. Based on the simplicity of purification, the alginate lyase genes also have been expressed in a yeast host.…”