Research Article: Improving extracellular production of Serratia marcescens lytic polysaccharide monooxygenase CBP21 and Aeromonas veronii B565 chitinase Chi92 in Escherichia coli and their synergism

Date Published: September 7, 2017

Publisher: Springer Berlin Heidelberg

Author(s): Yalin Yang, Juan Li, Xuewei Liu, Xingliang Pan, Junxiu Hou, Chao Ran, Zhigang Zhou.

http://doi.org/10.1186/s13568-017-0470-6

Abstract

Lytic polysaccharide monooxygenases (LPMOs) can oxidize recalcitrant polysaccharides and boost the conversion of the second most abundant polysaccharide chitin by chitinase. In this study, we aimed to achieve the efficient extracellular production of Serratia marcescens LPMO CBP21 and Aeromonas veronii B565 chitinase Chi92 by Escherichia coli. Twelve signal peptides reported with high secretion efficiency were screened to assess the extracellular production efficiency of CBP21 and Chi92, with glycine used as a medium supplement. The results showed that PelB was the most productive signal peptide for the extracellular production of CBP21 and Chi92 in E. coli. Furthermore, CBP21 facilitated the degradation of the three chitin substrates (colloidal chitin, β-chitin, and α-chitin) by Chi92. This study will be valuable for the industrial production and application of the two enzymes for chitin degradation.

Partial Text

Chitin, a water insoluble poly-β-1,4-N-acetylglucosamine, is the second most abundant natural polysaccharide and is widely distributed in organisms such as fungi, arthropods and nematodes. Chitooligosaccharides (COS), the degraded products of chitin, have attracted increasing interest because of their physicochemical properties and potential food and pharmaceutical applications (Zou et al. 2016).

Signal peptides showed large variations in secretion efficiencies, depending on the nature of the secreted protein. A suitable signal peptide for one protein may not be efficient for another protein (Low et al. 2013). A similar outcome was obtained in the present study, all 12 selected signal peptides increased the production level of Chi92 in varying degrees, whereas only four signal peptides, including PelB, CBHI, SacB and XCs, increased the production level of CBP21. Without glycine addition, StII, LMSEA, LSEA-mut and XCs can efficiently improve the release of intracellular Chi92 to culture media, while only PelB can promote the release of CBP21 to culture media with high efficiency. The cleavage efficiency of the signal peptide affects protein secretion levels (Low et al. 2013). Interleukin-2 production in E. coli periplasm was increased drastically by a single amino acid substitution in OmpA cleavage site (Robbens et al. 2006). Substitution of the Staphylococcus staphilococcic enterotoxin A leader sequence of Val–Asn–Gly with a conserved E. coli signal peptidase recognition sequence of Ala–Ser–Ala significantly increased enterotoxin A secretion to the periplasm (Manuvera et al. 2010). The modified signal peptide (OmpASIL2 and LSEA-mut) and their respective controls (WompA and LMSEA) reported previously (Manuvera et al. 2010; Robbens et al. 2006) were also used for the secretion of CBP21 or Chi92, but the reported improvements in efficiency were not observed in CBP21 or Chi92, which is consistent with the variation in secretion efficiency of signal peptides for different proteins. The effects of glycine on the extracellular production of various signal peptides are also different. Glycine addition can significantly improve the extracellular production of Chi92 fused with PelB, WOmpA, OmpASIL2, gIII or LMSEA, but can’t improve the extracellular production of StII- and CBHI-fused Chi92. This may be due to the differences in the ability of the different signal peptides to transport protein to periplasm.

 

Source:

http://doi.org/10.1186/s13568-017-0470-6

 

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