- Open Access
Development of a secretory expression system with high compatibility between expression elements and an optimized host for endoxylanase production in Corynebacterium glutamicum
© The Author(s) 2019
- Received: 26 February 2019
- Accepted: 3 April 2019
- Published: 17 April 2019
In terms of protein production, the internal environment of the host influences the activity of expression elements, thus affecting the expression level of the target protein. Native expression elements from a specific strain always function well in the original host. In the present study, to enhance the endoxylanase (XynA) production level in Corynebacterium glutamicum CGMCC1.15647 with its native expression elements, approaches to reduce host expression obstacles and to promote expression were evaluated.
We identified the signal peptide of CspB2 in C. glutamicum CGMCC1.15647 by MALDI-TOF and applied it along with its promoter for the production of endoxylanase (XynA) in this strain. The native cspB2 promoter and cspB2 signal peptide are superior to the well-used cspB1 promoter and cspA signal peptide for XynA expression in C. glutamicum CGMCC1.15647, and expression in this strain is superior to the expression in C. glutamicum ATCC13032. The highest XynA secretion efficiency level in deep 24-well plates level (2492.88 U/mL) was achieved by disruption of the cell wall protein CspB2 and the protease ClpS, chromosomal integration of xynA and coexisting plasmid expression, which increased expression 11.43- and 1.35-fold compared to that of chromosomal expression and pXMJ19-xynA-mediated expression in the original strain, respectively. In fed-batch cultivation, the highest XynA accumulation (1.77 g/L) was achieved in the culture supernatant after 44 h of cultivation.
Adaptation between the expression elements and the host is crucial for XynA production in C. glutamicum CGMCC1.15647. Strategies including host optimization, chromosomal integration, and coexistence of plasmids were useful for efficient protein production in C. glutamicum.
- Corynebacterium glutamicum
- Chromosomal expression
- Coexisting plasmids
In heterologous protein production, the successful production of recombinant proteins is mainly dependent on the interaction between the expression elements and the host. The ideal bacterial host provides a simplified and stable host environment for the function of synthetic biological circuits , such as high expression level of target protein, no inclusion body produced, no degradation of the target protein and easy for secretion. Efforts have been made to increase the protein production levels by optimizing the adaptation between the expression elements and the host in Escherichia coli , Bacillus subtilis , Saccharomyces cerevisiae  and Corynebacterium glutamicum . Due to the following several characteristics such as (1) no endotoxin production and generally regarded as safe status , (2) the ability to secrete properly folded protein into the culture, (3) lack of detectable extracellular protease, C. glutamicum is a potential host cell for the secretory production of heterologous proteins, important enzymes and pharmaceutical proteins [7–9]. However, the state-of-the-art of C. glutamicum protein production is in its infancy due to its limited genetic tools compared to those for the most-widely used host, E. coli . To improve the secretory production level of target proteins in C. glutamicum, endogenous expression elements had been explored for protein production in C. glutamicum [11, 12]. In addition to the manipulations based on expression elements, the development of a suitable host that is suitable for the expression elements is also important, including decreasing expression barriers and enhancing the factors that facilitate secretion. Deletion of the cell wall component protein CspB and the penicillin-binding protein resulted in high secretion efficiency of an antibody Fab . Protease deletion is one of the way to improve the stability of heterologous protein in host, which can improve the accumulation level of the target protein. For example, disruption of the five protease genes tppA, pepE, nptB, dppIV and dppV showed 34% higher bovine chymosin production than that in double-disruption of tppA and pepE ; the GFP fluorescence intensity was increased by 40.6% in a ClpC disrupted C. glutamicum compared to the intensity in wild type . In addition, recombinant protein can also be expressed from a single chromosomal site. Chromosomal expression of GFP and AprE could be 2.9-fold and 1.5-fold-increased compared to that of expression from the common integration site amyE by random knock-in in B. subtilis . These cases indicated that host optimization would be a useful method to improve protein production, and we believe these approaches could be attempted to improve recombinant protein expression in C. glutamicum.
Endoxylanase (XynA) cleaves the β-1,4-glycosidic bonds present in the main chain of xylan, which is the major constituent of hemicellulose. A better digestibility could be achieved when poultry were fed with the XynA-treaded cereal ; addition of XynA could improve the bread volume, reduce stickiness and increase the shelf life ; the XynA treated pulp showed superior properties [19, 20]. Reports investigating the secretory production of XynA and its utilization in S. cerevisiae and C. glutamicum were mainly focused on expression elements [21, 22], and the production level could be increased by improving the complementarity between the host and expression elements.
In the present study, we present an efficient system based on native expression elements for the secretory production of XynA in C. glutamicum CGMCCl.15647. In addition to studies on expression elements, the effect of host optimization on the production level of XynA was performed, including cell surface layer protein and protease disruption, integration of the xynA gene into the chromosome, and coexisting plasmid expression. Large-scale production of XynA was also performed using the optimized expression system in fed-batch cultivation.
Identification of the CspB2 protein of C. glutamicum CGMCC1.15647
To confirm that CspB2 could be expressed and secreted with its promoter and signal peptide at a higher level by plasmid expression than by chromosomal expression, the cspB2 gene with its signal peptide and promoter was cloned into the pXMJ19 vector and transformed into ΔcspB2, which is a CspB2-disrupted strain derived from C. glutamicum CGMCC1.15647. The proteins in the culture medium of the transformant were analyzed by SDS-PAGE after cultivation for 48 h. One major protein that matched the molecular weight of CspB2 appeared in C. glutamicum CGMCC1.15647 and ΔcspB2 harboring pXMJ19-cspB2 (Fig. 1b), while the CspB2-disrupted strain ΔcspB2 lacked this band in the SDS-PAGE gel; we confirmed that this major protein was CspB2 by MALDI-TOF analysis. Figure 1b also shows that the plasmid expression had a higher expression level of CspB2 than did the chromosomal expression in wild-type C. glutamicum CGMCC1.15647.
Secretory production of XynA in C. glutamicum
To verify the strength of this cspB2 promoter and signal peptide combination in other C. glutamicum subspecies, the pXMJ19-xynA plasmid was transformed into the C. glutamicum ATCC 13032 and the resulting strain Cg32+P19-X (C. glutamicum ATCC 13032 carrying pXMJ19-xynA) was used for XynA production. Under the same culture conditions, the XynA secretion level in strain Cg32+P19-X was 528.93 U/mL (Fig. 2), which is only 28.61% of the activity in Cg47+P19-X, and the XynA activity per OD600 was 35.99% of the activity in Cg47+P19-X (Fig. 2).
Effect of CspB2 and ClpS disruption on XynA secretory expression
Both single mutants of ΔcspB2+P19-X and ΔclpS+P19-X enhanced XynA secretion. To assess the effect of double deletion of cspB2 and clpS on the productivity of XynA, we evaluated the effect of ΔcspB2 and ΔclpS on C. glutamicum CGMCCl.15647. However, the secretion level of XynA was 2036.15 U/mL in ΔcspB2ΔclpS+P19-X (ΔcspB2ΔclpS carrying pXMJ19-xynA) (Fig. 3), indicating that the cspB2 and clpS double mutation had no effect on XynA secretion compared to that of the clpS disruption alone (Fig. 3), but the XynA activity per OD600 increased 10.5% and 29.3% compared to that of Cg47+P19-X and ΔclpS+P19-X, respectively (Fig. 3).
Effect of xynA chromosomal integration and coexistence of plasmids on XynA secretion
Generally, multiple plasmids with different replication origins can exist in one host. pXMJ19-xynA and pEC-XK99E-xynA have different antibiotic resistance genes and replicons, so they can coexist in a single C. glutamicum cell. To investigate the production of XynA by compatible plasmids in C. glutamicum, the plasmids pXMJ19-xynA and pEC-XK99E-xynA were cotransformed into ΔcspB2ΔclpSInX to obtain strain ΔcspB2ΔclpSInX+P19-X+pEC-X. This coexisting plasmid construct resulted in lower biomass due to the extra burden of the plasmids (Fig. 4); however, highest activity of XynA reached 2492.88 U/mL were present in ΔcspB2ΔclpSInX+P19-X+pEC-X (Fig. 4), and the XynA activity per OD600 was 67.4% greater than in Cg47+P19-X. Finally, the highest yield of XynA was 11.43- and 1.35-fold greater than that for the chromosomal integration expression strain ΔcspB2InX and the wild-type C. glutamicum CGMCCl.15647 harboring pXMJ19-xynA, respectively, demonstrating that the strategies performed above can be useful for the production of XynA in C. glutamicum CGMCCl.15647.
Enhanced production of XynA by fed-batch cultivation
The adaptation between the expression elements and the internal environment of the host is crucial for protein production in synthetic biology. Native expression elements would be preferable for function in the internal environment of a specific strain. Here, we identified the native cspB2 promoter and cspB2 signal peptide from C. glutamicum CGMCCl.15647 and applied it for XynA expression, and they were superior to the reported cspB1 promoter and cspA signal peptide elements . It seems that the expression level of the target protein is highly related to the suitability of the expression elements for host because there was a much lower XynA expression level in C. glutamicum ATCC 13032 than in C. glutamicum CGMCC1.15647 with the same expression elements under the same culture conditions.
In addition to plasmid manipulation, another important factor that may affect protein secretion is host optimization, including decreasing barriers that may affect expression and secretion and promoting factors that could facilitate expression and secretion. The XynA expression level could be further increased in C. glutamicum CGMCCl.15647 by disrupting the S-layer protein CspB2 and protease ClpS. In E. coli, ClpS participates in the recognition of the N-terminus of specific proteins during degradation; ClpS is not essential for degradation, but it enhances degradation [30, 31]. XynA from Streptomyces coelicolor A3(2) is a heterologous protein for C. glutamicum, and the regulatory system, including the protease, may manipulate this foreign protein.
Moreover, chromosomal integration and coexisting plasmid transformation were performed to facilitate the expression and secretion of XynA. Considering the suitability between expression elements and the host, we chose the native AH6 promoter and cspB2 signal peptide of C. glutamicum CGMCCl.15647 for xynA integration. Coexistence of pXMJ19-xynA and pEC-XK99E-xynA in the optimized ΔcspB2ΔclpSInX strain (ΔcspB2ΔclpSInX+P19-X+pEC-X) resulted in the highest XynA production, and this coexisting plasmid expression system functioned well because the XynA production level increased with time during Fed-batch cultivation, indicating that coexistence of plasmids would be a useful approach for protein production. Plasmid replication and antibiotic resistance gene expression in coexisting plasmid expression increased the metabolic burden on the host and led to a lower growth rate, and homologous recombination of the xynA genes is another increased danger due to the two used plasmids. However, higher expression levels of the target protein could be achieved despite the extra burden or danger. The approaches performed here for XynA production could be useful for improving heterologous protein production in C. glutamicum.
Bacterial strains and growth conditions
Bacterial strains and plasmids used in this study
Strain or plasmid
E. coli DH5α
C. glutamicum ATCC13032
C. glutamicum CGMCCl.15647
C. glutamicum CGMCCl.15647, cspB2 disruption
C. glutamicum CGMCCl.15647, clpS disruption
C. glutamicum CGMCCl.15647, cspB2 and clpS disruption
C. glutamicum CGMCCl.15647, cspB2 disruption, xynA integration
C. glutamicum CGMCCl.15647, cspB2 disruption, clpS disruption, xynA integration
C. glutamicum CGMCCl.15647 harboring pXMJ19
C. glutamicum CGMCCl.15647 harboring pXMJ19-xynA
C. glutamicum CGMCCl.15647 harboring pXMJ19-cspB1-cspA-xynA
C. glutamicum ATCC13032 harboring pXMJ19-xynA
ΔcspB2 harboring pXMJ19-xynA
ΔclpS harboring pXMJ19-xynA
ΔcspB2ΔclpS harboring pXMJ19-xynA
ΔcspB2InX harboring pXMJ19-xynA
ΔcspB2ΔclpSInX harboring pXMJ19-xynA
ΔcspB2ΔclpSInX harboring pXMJ19-xynA and pEC-XK99E-xynA
E. coli–C. glutamicum shuttle vector, Chlr
pXMJ19 derivative, PcspB2, cspB2 signal peptide, cspB2 gene
PcspB1, cspA signal peptide, xynA
pXMJ19 derivative, PcspB2, cspB2 signal peptide, xynA
E. coli–C. glutamicum shuttle vector, Kmr
pEC-XK99E derivative, PAH6, cspB2 signal peptide, xynA
SacB, suicide vector, Kmr
pK18mobSacB derivative, for cspB2 disruption
pK18mobSacB derivative, for xynA integration
List of primer oligonucleotide sequences used in this study
Construction of CspB2 and ClpS deletion mutants and XynA integration mutants of C. glutamicum
To analyze the effect of host mutation on XynA production, mutations of the cell surface layer protein CspB2 and the protease ClpS were constructed. The protease ClpS (encoded by clpS, GenBank: CP025533.1)-disrupted mutant ΔclpS was constructed based on homologous recombination in the lab strain. The other mutations were constructed by homologous recombination as described previously . The pK18mobSacB-cspB2 vector was transformed into C. glutamicum CGMCCl.15647, the resulting mutant was designated ΔcspB2 after single-crossover and double-crossover selection. Then, the cspB2 and clpS double mutation was obtained using the same method and designated as ΔcspB2ΔclpS. Then, pK18mobSacB-xynA for integration of xynA into the chromosome was transformed into ΔcspB2 and ΔcspB2ΔclpS, resulting in the desired xynA-integrated mutants ΔcspB2InX and ΔcspB2ΔclpSInX, respectively. The pXMJ19-xynA and pEC-XK99E-xynA plasmids were then transformed into the C. glutamicum mutants alone or together for XynA expression.
ΔcspB2ΔclpSInX harboring pXMJ19-xynA and pEC-XK99E-xynA was inoculated into 50 mL BHI as seed medium in a 500-mL flask. After cultivation at 30 °C for 12 h with shaking at 230 rpm, the seed culture (100 mL) was inoculated into 1 L fermentation medium (17.61 g maltose, 44.87 g brain heart infusion, 9 g tryptone, 0.6 g MgSO4, 1.39 g FeSO4·7H2O, 1 mg biotin, 1 mg riboflavin and 1 mg ascorbic acid per liter distilled H2O; pH 7.0) in a 5-L jar bioreactor (Applikon EZ-control, Netherlands), and 10 mL ethyl alcohol was added into the jar upon inoculation. The temperature, dissolved oxygen (DO) concentration and the pH were maintained at 30 °C, 35% (v/v) and 7.0, respectively. To prevent glucose starvation, a maltose solution (400 g/L) was added to the culture medium 12 h after inoculation, and 20 mL was added immediately after sampling. Cell growth was monitored by measuring the optical density at 600 nm (OD600) with a spectrophotometer.
Protein preparation and analysis
After cell cultivation in deep plates for 48 h, the culture supernatant was collected by centrifugation at 12,000g and 4 °C for 5 min for enzyme activity analysis and SDS-PAGE. To identify the secreted CspB2 protein, the protein band at approximately 50 kDa was excised from the SDS-PAGE gel for the culture supernatant of C. glutamicum CGMCCl.15647 and analyzed by MALDI-TOF at Sangon. The activity of XynA was assayed by using the XylX6 kit method (Megazyme, Ireland); the XylX6 assay is highly sensitive and reproducible . One unit of XynA activity is defined as the amount of enzyme required to release 20 nmol of 4-nitrophenol from the XylX6 substrate in 1 min under the defined assay conditions according to the instructions of the XylX6 kit. The protein samples were also analyzed by performing electrophoresis in a 12% (w/v) SDS-PAGE gel. The XynA concentration was measured by using the bicinchoninic acid (BCA) assay.
ZW and LXX designed and performed most experiments. ZW, LXX, YYK, LCL and BZH analyzed data. ZW and LXX mainly wrote the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
Consent for publication
Ethics approval and consent to participate
This work was supported by the National Natural Science Foundation of China (21808082, 21878124), the 111 Project (111-2-06), National first-class discipline program of Light Industry Technology and Engineering (LITE2018-24) and the Opening Project of the Key Laboratory of Industrial Biotechnology, Ministry of Education (KLIB-KF201802).
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