Chitin Biodegradation by Lytic Polysaccharide Monooxygenases from Streptomyces coelicolor In Vitro and In Vivo

Abstract

Lytic polysaccharide monooxygenases (LPMOs) have the potential to improve recalcitrant polysaccharide hydrolysis by the oxidizing cleavage of glycosidic bond. Streptomyces species are major chitin decomposers in soil ecological environments and encode multiple lpmo genes. In this study, we demonstrated that transcription of the lpmo gene, Sclpmo10G, in the Streptomyces coelicolor A3(2) (ScA3(2)) strain is strongly induced by chitin. The ScLPMO10G protein was further expressed in Escherichia coli and characterized in vitro. The ScLPMO10G protein showed oxidation activity towards chitin. Chitinase synergy experiments demonstrated that the addition of ScLPMO10G resulted in a substantial in vitro increase in the reducing sugar levels. Moreover, in vivo the LPMO-overexpressing strain ScΔLPMO10G(+) showed stronger chitin-degrading ability than the wild-type, leading to a 2.97-fold increase in reducing sugar level following chitin degradation. The total chitinase activity of ScΔLPMO10G(+) was 1.5-fold higher than that of ScA3(2). In summary, ScLPMO10G may play a role in chitin biodegradation in S. coelicolor, which could have potential applications in biorefineries.

Keywords: Streptomyces coelicolor A3(2); chitin biodegradation; expression; lytic polysaccharide monooxygenases; transcription.

Figure 1

Time course of relative transcript level of Sclpmos in the presence of 0.1% chitin. Relative expression level of Sclpmos cultured with glucose as control. All experiments were performed in triplicate and standard deviation analysis was conducted (SD, n = 3). Asterisks indicate significant differences (p < 0.01).

Figure 2

Activity of ScLPMO10G on chitin. (A) Chitin degradation products by ScLPMO10G activity identified using MALDI TOF MS analysis. DP indicates the degree of polymerisation of oxidized oligosaccharides. Possible products in these clusters are the sodium adducts of the lactone (m/z 851.18, 1054.20, 1257.30), the sodium adducts of the aldonic acid (m/z 869.19, 1072.25, 1275.32), and the sodium adduct of the aldonic acid sodium salt (m/z 891.17, 1094.22, 1297.29). 100% relative intensity represents 1.9 × 10⁴ arbitrary units (a.u.) for full spectra. (B) Degradation of 10.0 mg/mL chitin by 0.2 μM chitinases in the presence (solid circle) or absence (hollow square) of 2 μM ScLPMO10G, with ascorbic acid (1.0 mM) used as the electron donor. All experiments were performed in triplicate and standard deviation analysis was conducted (SD, n = 3).

Figure 3

Influence of pH on enzymatic activity (A) and stability (B) of ScLPMO10G against 2,6-DMP. For (A,B), the activity at pH 8 and initial activity were set at 100%, respectively. Influence of temperature on enzymatic activity (C) and stability (D) of ScLPMO10G against 2,6-DMP. For (C,D), the activity at 30 °C and initial activity were set at 100%, respectively. All experiments were performed in triplicate and standard deviation was analysed (SD, n = 3).

Figure 4

Chitin degradation by wild-type and the ScΔLPMO10G(+) mutant strains. (A) The reducing sugar concentration of the culture supernatant during the incubation of wild-type Streptomyces coelicolor A3(2) (ScA3(2)) and its mutant strain ScΔLPMO10G(+). The strains were cultivated on MM medium using chitin as the sole carbon source at 28 °C. (B) The total chitinase activity of ScA3(2) and its mutant strain ScΔLPMO10G(+). (C) Chitin degradation by extracellular enzyme produced by the wild type ScA3(2) and its mutant strain ScΔLPMO10G(+). All experiments were performed in triplicate and standard deviation was analysed (SD, n = 3).