Development of a Potential Protein Display Platform in Corynebacterium glutamicum Using Mycolic Acid Layer Protein, NCgl1337, as an Anchoring Motif

Abstract

In the cell surface display, the choice of host cell and anchoring motif are the most crucial for the efficient display of passenger proteins. Corynebacterium glutamicum has mycolic acid layer in outer membrane and the use of protein in the mycolic acid layer as an anchoring motif can provide a potential platform for surface display in C. glutamicum. All 19 mycolic acid layer proteins of C. glutamicum are analyzed, and two proteins, NCgl0535 and NCgl1337, which have a signal peptide and predicted O-mycoloylation site, are selected as anchoring motifs candidates. Among them, NCgl1337, which shows better expression with higher display efficiency, is chosen as a potential anchoring motif. Two forms of the NCgl1337 anchoring motif, a full-length (1-324 amino acids) and a short-length (1-50 amino acids) containing only signal peptide and O-mycoloylation site, are constructed and their abilities for surface display are examined using two protein models, endoxylanase from Streptomyces coelicolor and α-amylase from Streptococcus bovis. For both model proteins, the short-length NCgl1337 anchoring motif exhibits higher yield of protein display on the surface of C. glutamicum than the full-length NCgl1337. Finally, with C. glutamicum displaying α-amylase, a batch fermentation is performed for the production of l-lysine from starch degradation, and a production of l-lysine as high as 10.8 ± 0.92 g L was achieved after 18 h of culture.

Recombinant Protein Expression System in Corynebacterium glutamicum and Its Application

“…Recent reports of Choi et al suggested that the proteins from 19 known mycolic acid layers in the extracellular membrane of C. glutamicum can be used as anchoring motifs in surface display systems (Choi et al, 2018 ). The α-amylase of S. bovis was screened using a portion of NCgl1337 as an anchoring motif; this portion has a signal peptide and a predicted O-mycoloylation site.…”

Application of Corynebacterium glutamicum engineering display system in three generations of biorefinery

“…Mycoloylated proteins are special proteins that exist on the mycolic acid layer of the C. glutamicum outer membrane and are fixed on mycolic acids through O-acylation covalent modification, which occurs on serine residues [ 54 ]. The currently known mycoloylated proteins that can be used as anchor motifs are NCgl1337, ion-selective channel protein NCgl0933 (porin B, PorB), NCgl0932 (porin C, PorC), and PorH(NCgl number of PorH have not been assigned [ 55 ]) [ 39 , 40 ]. PorH combines with PorA to form a cation-selective channel called the PorHA channel.…”

“…2 ). In addition, mycolic acid layer proteins PorC, NCgl1337, and membrane protein Msc were used as anchoring motifs to display α-amylase, and these recombinant strains produced glutamic acid or L-lysine from starch successfully [ 39 , 41 , 44 ] (Table 1 ).…”

Genome-Scale Mining of Novel Anchor Proteins of Corynebacterium glutamicum

“…In order to display proteins on the surface of C. glutamicum, several anchoring motifs have been used. At present, the display system of C. glutamicum only has three types of anchor proteins: foreign protein (PgsA), mycoloylated proteins [NCgl1337, NCgl0933 (PorB), NCgl0932 (PorC), PorH (NCgl number of PorH have not been assigned)], and membrane protein (NCgl1221, mechanosensitive channel, MscCG) (Tateno et al, 2007b(Tateno et al, , 2009Choi et al, 2018;Inui and Toyoda, 2020). These recombinant strains with anchor proteins have been modified to display carbohydrate-active enzymes (CAZy), such as amylase (Tateno et al, 2007a), glucanase (Ryu and Karim, 2011), glucosidase (Muñoz-Gutiérrez et al, 2012), cellulase complex (Kim et al, 2014) etc., which enable the engineered strains to degrade and utilize cheap biomass.…”

“…In recent years, the screening of the anchoring proteins of C. glutamicum has been based mainly on partial screening rather than direct genome-wide screening of the characteristics of anchor proteins-for example, the process of screening Ncgl1337 as the anchor protein was to identify and analyze the mycolic acid layer protein by SignalP and tied-mixture hidden Markov models (TMHMM) and then display the reporter for further analysis and verification (Choi et al, 2018). The process of screening porin anchor proteins was similar to that (Tateno et al, 2009;Choi et al, 2018).…”