Design of fully synthetic signal peptide library and its use for enhanced secretory production of recombinant proteins in Corynebacterium glutamicum

Abstract

Background: Corynebacterium glutamicum is an attractive host for secretory production of recombinant proteins, including high-value industrial enzymes and therapeutic proteins. The choice of an appropriate signaling peptide is crucial for efficient protein secretion. However, due to the limited availability of signal peptides in C. glutamicum, establishing an optimal secretion system is challenging.

Result: We constructed a signal peptide library for the isolation of target-specific signal peptides and developed a highly efficient secretory production system in C. glutamicum. Based on the sequence information of the signal peptides of the general secretion-dependent pathway in C. glutamicum, a synthetic signal peptide library was designed, and validated with three protein models. First, we examined endoxylanase (XynA) and one potential signal peptide (C1) was successfully isolated by library screening on xylan-containing agar plates. With this C1 signal peptide, secretory production of XynA as high as 3.2 g/L could be achieved with high purity (> 80%). Next, the signal peptide for ⍺-amylase (AmyA) was screened on a starch-containing agar plate. The production titer of the isolated signal peptide (HS06) reached 1.48 g/L which was 2-fold higher than that of the well-known Cg1514 signal peptide. Finally, we isolated the signal peptide for the M18 single-chain variable fragment (scFv). As an enzyme-independent screening tool, we developed a fluorescence-dependent screening tool using Fluorescence-Activating and Absorption-Shifting Tag (FAST) fusion, and successfully isolated the optimal signal peptide (18F11) for M18 scFv. With 18F11, secretory production as high as 228 mg/L was achieved, which was 3.4-fold higher than previous results.

Conclusions: By screening a fully synthetic signal peptide library, we achieved improved production of target proteins compared to previous results using well-known signal peptides. Our synthetic library provides a useful resource for the development of an optimal secretion system for various recombinant proteins in C. glutamicum, and we believe this bacterium to be a more promising workhorse for the bioindustry.

Keywords: Corynebacterium glutamicum; Library screening; Sec-dependent pathway; Secretion; Synthetic signal peptide.

Fig. 1

Sequence analysis of signal peptides and library construction (A) WebLogo depicting frequency of amino acid changes within the signal peptides of C. glutamicum. (B) Overall scheme of the synthetic signal peptide library construction. Nucleotide symbols: V represents G, C or A; M represents A or C; B represents G, T, or C; Y represents C or T; and N represents A, T, G or C. The entire synthetic signal peptide library was generated via PCR using degenerate primers without a template and subsequently cloned into pHCMS, yielding pHCP-SP-L

Fig. 2

Isolation of signal peptide for endoxylanase (XynA). (A) Screening of pHCP-SP-XynA library on xylan-agar plate. The red arrows indicate halo-forming colonies. (B) SDS-PAGE analysis of culture supernatant from C. glutamicum ATCC13032 in BHI medium. Lane M, protein size marker; lane 1, negative control (pHCP-XynA); lane 2, pHCP-C1-XynA; lane 3, pHCP-G1-XynA; lane 4, pHCP-G8-XynA; lane 5, pHCP-Cg1514-XynA; lane 6, pHCP-Cg2052-XynA; lane 7, pHCP-PorB-XynA. (C) Amino acid sequences of synthetic signal peptides (C1, G1, and G8). N-terminal amino acid sequences of XynA are indicated in grey shading. (D) SDS-PAGE analysis of the culture supernatant from C. glutamicum SP002 in modified CGXII medium. Lane M; protein size marker; lane 1, negative control (pHCP-XynA); lanes 2 and 3, pHCP-C1-XynA; lanes 4 and 5, pHCP-G1-XynA. All culture supernatants are concentrated thirty times and 10 µL was loaded on each well

Fig. 3

Fed-batch cultivation of C. glutamicum SP002 harboring pHCP-C1-XynA. (A) Time profiles of cell density (OD₆₀₀), dry cell weight (DCW, g/L), glucose conc. (g/L) and XynA conc. in culture medium (g/L). Symbols: ◆, cell density; ◼, glucose conc; ⬤, dry cell weight; ▲, XynA conc. (B) SDS-PAGE analysis of culture supernatants. Lane M represents protein size makers, and each lane represent protein fraction in each sampling time. All culture supernatants are not concentrated and 1 uL loaded on each well. Arrowhead indicates the band of XynA

Fig. 4

Isolation of signal peptide for ⍺-amylase (AmyA). (A) Screening of pHCP-SP-AmyA library on starch-agar plate. (B) SDS-PAGE analysis of culture supernatant of isolated colonies. Lane M, protein size marker; lanes 1 to 7, HS01, HS02, HS03, HS04, HS05, HS06 and HS07. All culture supernatants are concentrated thirty times and 10 µL was loaded on each well. Arrowhead indicates the band of AmyA. (C) Amino acid sequences of synthetic signal peptide of HS06. N-terminal three amino acid sequences of AmyA are indicated in grey shading

Fig. 5

Fed-batch cultivation of C. glutamicum SP002 harboring HS06. (A) Time profiles of cell density (OD₆₀₀), glucose conc. (g/L) and AmyA conc. in culture medium. (g/L). Symbols: ◆, cell density; ◼, glucose conc.; ▲, AmyA conc. (B) SDS-PAGE analysis of culture supernatants. Lane M represents protein size makers, and each lane represent protein fraction in each sampling time. On each well, 2 µL of culture supernatants were loaded. Arrowhead indicates the band of AmyA

Fig. 6

FAST-based screening methods for signal peptide library. (A) Overall scheme of FAST-based screening methods using 96-well plates. (B) Validation of FAST-based screening methods with various XynA expression systems. SDS-PAGE analysis of XynA expression systems with FAST. Lane M, protein size marker; lane 1, negative control (pHCP-XynA); lane 2, pHCP-C1-XynA-FAST; lane 3, pHCP-Cg2052-XynA-FAST; lane 4, pHCP-Cg1514-XynA-FAST; lane 5, pHCP-PorB-XynA-FAST. All culture supernatants were concentrated ten times and 10 µL was loaded on each well. Arrowhead indicates the band of XynA (C) Analysis of fluorescent intensity of XynA secretion systems with FAST

Fig. 7

Isolation of signal peptide from the library for M18 secretion. (A) SDS-PAGE analysis of isolated candidates with FAST. M; protein size marker, 1; pHCP-PorB-M18-FAST, 2; pHCP-18F11-M18-FAST, 3; pHCP-20G1-M18-FAST, 4; pHCP-24G1-M18-FAST. (B) SDS-PAGE analysis of isolated candidate (18F11) without FAST. M; protein size marker, 1; pHCP-PorB-M18, 2; pHCP-18F11-M18. (C) Western blot analysis of isolated candidate (18F11) without FAST. M; protein size marker, 1; pHCP-PorB-M18, 2; pHCP-18F11-M18. All culture supernatants are concentrated thirty times and 10 µL was loaded on each well. (D) Amino acid sequences of synthetic signal peptide of 18F11. N-terminal amino acid sequences of M18 scFv are indicated in grey shading

Fig. 8

Fed-batch cultivation of C. glutamicum SP002 harboring 18F11. (A) Time profiles of cell density (OD₆₀₀), glucose conc. (g/L) and M18 scFv conc. in culture medium (g/L). Symbols: ◆, cell density (OD₆₀₀); ◼, glucose conc.; ▲, M18 scFv conc. (B) SDS-PAGE analysis of culture supernatants. Lane M represents protein size makers, and each lane represent protein fraction in each sampling time. On each well, 10 µL of culture supernatants were loaded. Arrowhead indicates the band of M18 scFv