Large-scale production of a thermostable Rhodothermus marinus cellulase by heterologous secretion from Streptomyces lividans

Abstract

Background: The gene encoding a thermostable cellulase of family 12 was previously isolated from a Rhodothermus marinus through functional screening. CelA is a protein of 260 aminoacyl residues with a 28-residue amino-terminal signal peptide. Mature CelA was poorly synthesized in some Escherichia coli strains and not at all in others. Here we present an alternative approach for its heterologous production as a secreted polypeptide in Streptomyces.

Results: CelA was successfully over-expressed as a secreted polypeptide in Streptomyces lividans TK24. To this end, CelA was fused C-terminally to the secretory signal peptide of the subtilisin inhibitor protein (Sianidis et al. in J Biotechnol. 121: 498-507, 2006) from Streptomyces venezuelae and a new cloning strategy developed. Optimal growth media and conditions that stall biomass production promote excessive CelA secretion. Under optimal growth conditions in nutrient broth medium, significant amounts of mature CelA (50-90 mg/L or 100-120 mg/g of dry cell weight) are secreted in the spent growth media after 7 days. A protocol to rapidly purify CelA to homogeneity from culture supernatants was developed and specific anti-sera raised against it. Biophysical, biochemical and immmuno-detection analyses indicate that the enzyme is intact, stable and fully functional. CelA is the most thermostable heterologous polypeptide shown to be secreted from S. lividans.

Conclusion: This study further validates and extends the use of the S. lividans platform for production of heterologous enzymes of industrial importance and extends it to active thermostable enzymes. This study contributes to developing a platform for poly-omics analysis of protein secretion in S. lividans.

Keywords: Cellulase; Protein secretion biotechnology; Protein translocase; Secretion; Signal peptide; Streptomyces lividans.

Fig. 1

Heterologous synthesis of CelA in E. coli and synthesis/secretion in S. lividans. a CelA_His6 was synthesized in BL21 (DE3) cells. Expression of CelA_His6 carried on a pET vector was induced by 0.2 mM IPTG for 3 h at 30 °C. b Synthesis in and secretion of CelA from S. lividans. Polypeptides (1–8 µg/lane) from culture supernatants (~ 0.1 mL) that is equivalent to 0.2 mg of dry cell mass. The samples from cells grown for 2 days in the indicated media (see “Methods”) were harvested by precipitation with TCA (25%), analyzed by SDS-PAGE and stained with Coomassie blue. Lane 1, molecular weight markers: β-galactosidase (116 kDa), bovine serum albumin (66.2 kDa), ovalbumin (45 kDa), lactate dehydrogenase (35 kDa), restriction endonuclease Bsp98I (25 kDa), β-lactoglobulin (18.4 kDa), lysozyme (14.4 kDa). Pha, phage medium; NB, nutrient broth; NB_2X, nutrient broth double strength; MM, minimal medium; C₅ and C₁₅, casamino acids (5 or 15 g/L); TSB, tryptic soy broth; Ben, Bennet medium

Fig. 2

Time course of CelA secretion and the effect of additional carbon source. a Polypeptides (0.1–5 µg/lane) from culture supernatants (0.9–16 µL/lane) that is equivalent to (0.08 mg of dry cell mass) from cells grown for the indicated times in the indicated media were analyzed by SDS-PAGE and silver-stained. Lane 1, MW markers as in Fig. 1b. Secreted CelA (filled arrow) is indicated. b The growth curves of S. lividans TK24 carrying pIJ486 expressing SP^Vsi-CelA in the indicated media expressed as values of dry cell weight. c The dry cell weight in gram per liter produced by S. lividans TK24 with pIJ486 expressing SP^Vsi-CelA in the different media (as in Fig. 1b) at the indicated growth phases. Ben, Bennet medium. d The amount of CelA secreted (mg) correlated to a gram of dry cell mass by S. lividans TK24 with pIJ486 expressing SP^Vsi-CelA in the different media (as in Fig. 1b) for the indicated time related to its growth curves in the same media. e, f The amount of CelA secreted (mg) correlated to a gram of dry cell mass (e) or 1L of culture (f) produced by S. lividans TK24 with pIJ486 expressing SP^Vsi-CelA in the different media at late exponential, early stationary and late stationary phases from d difference in CelA secretion was compared using an unpaired t test without assumption of equal variance (see “Methods”; NS, non-significant difference). Error bars represent standard error of the mean (SEM). n = 3

Fig. 3

Time course of S. lividans TK24 with pIJ486-vsi-celA and S. lividans TK24 with pIJ486 (as a wild type) growth in NB medium. a The growth curves of S. lividans TK24 carrying empty pIJ486 or pIJ486 expressing SP^Vsi-CelA in the nutrient broth expressed as values of dry cell weight. b Polypeptides (1.2–6 µg/lane) from culture supernatants (~ 7 µL/lane; equivalent to 0.08 mg of dry cell weight) from cells grown for the indicated times in NB were analyzed by SDS-PAGE and silver-stained. Lanes 1 and 12, MW markers as in Fig. 1b. Secreted CelA (filled arrow) is indicated

Fig. 4

Large-scale purification of R. marinus CelA expressed and secreted in S. lividans culture supernatants. Protein samples (15 µg/lane) from the various purification steps (see “Methods”) were analyzed as in Fig. 2a and were stained with Coomassie blue. Lane 1: mass markers as in Fig. 1. Lane 2: culture supernatants from S. lividans TK24 with pIJ486 expressing SP^Vsi-CelA grown in NB for 48 h; lane 3: the culture supernatant after concentration with rotary evaporator; lane 4: The 35–55% ammonium sulfate fraction; lane 5: the elution fraction with the highest CelA activity from Q-Sepharose; lane 6: the fraction from lane 5 after heating at 80 °C for 4 h and centrifugal removal of precipitates

Fig. 5

Physical and functional characterization of S. lividans-secreted CelA. a Size exclusion chromatography of recombinant purified CelA. Arrows indicate migration positions for 35 kDa (CesAB/EspA) and 27 kDa (CesAB) [39]. b Circular dichroism spectrometry. 5 µM or protein in buffer (5 mM MOPS pH 7.5, 5 mM NaCl, 1 mM DTT) 20 °C was analyzed using a 190–260 nm wavelength scan. c Thermal denaturation curves monitored by circular dichroism. Purified CelA (5 µM) in buffer (5 mM MOPS pH 7.5, 5 mM NaCl, 1 mM DTT) was exposed to gradual temperature rise and changes in ellipticity were monitored at 213 nm, as described [22]. d Biochemical activity of S. lividans-secreted CelA. Cellulase activity by CelA (20 µg/mL) was determined by hydrolysis of carboxymethyl cellulose (CMC) (see “Methods”). The activity of CelA secreted and purified from S. lividans was compared to CelA_His6 produced in E. coli and to a commercial preparation from A. niger of 24 U/mg total protein estimated to contain ~ 15 µg of unknown cellulases. n = 3; values represent mean ± SD