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

Abstract

BackgroundThe 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.ResultsCelA 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.ConclusionThis 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.Electronic supplementary materialThe online version of this article (10.1186/s12934-017-0847-x) contains supplementary material, which is available to authorized users.

Multi-Omics and Targeted Approaches to Determine the Role of Cellular Proteases in Streptomyces Protein Secretion

“…Moreover, upon loading of secretome material derived from the same volume of culture, TK24Δ ftsH was also found to be a profuse secretor of polypeptides (not shown). As seen in other studies ( Hamed et al, 2017 ; Tsolis et al, 2018 ), there appears to be a good correlation between suppressed growth and improved secretion as seen by comparison of the total secretome expressed per unit cell biomass ( Figure 3B ).…”

“…The stable activity of secreted mRFP underlines the suitability of S. lividans for heterologous protein production and is reminiscent of what has been observed with several other heterologous proteins ( Pozidis et al, 2001 ; Sianidis et al, 2006 ; Hamed et al, 2017 ). Perhaps the deletion of multiple secreted proteases could be more beneficial for heterologous protein production, but this can be seen now under new light.…”

“…In contrast, when growing in casamino acid-supplemented minimal media, abundance peaks were for most of the proteases at early log or stationary phase. This late phase expression is reminiscent of the secretion from TK24 of some heterologous proteins in various growth media, in which high levels of secretion were linked to the cells entering the stationary phase ( Pozidis et al, 2001 ; Hamed et al, 2017 ).…”

Comprehensive subcellular topologies of polypeptides in Streptomyces

“…In addition, Streptomyces strains have been used as cell factories for heterologous protein expression, including interleukin-6 (IL-6) [ 5 ], human mature form of interferon alpha 2 (hIFN-alpha 2) [ 6 ], mouse tumor necrosis factor alpha (mTNF-alpha) [ 7 ], Xeg glucoside hydrolase from Jonesia sp. [ 8 ] and a thermophilic cellulase from Rhodothermus [ 9 ]. There are two widely used model strains in this family: Streptomyces coelicolor A3(2), used for the production of antibiotics and secondary metabolites [ 10 ], and its close relative Streptomyces lividans , which is utilized for heterologous protein production [ 11 ].…”

Streptomycetes: Attractive Hosts for Recombinant Protein Production

“…Unfortunately, only few of these studies reported a comparison of protein expression yield between streptomycetes and other microbial hosts. Hamed et al (2017) succeed in producing 90 mg/L of a thermostable cellulase from the bacteroidetes Rhodotermus marinus using S. lividans TK24 as host; the same protein could not be produced in E. coli. Very recently, Šnajder et al (2019) reported the first and so far the only case of expression of an archaeal thermozyme (pernisine) in Streptomyces rimosus.…”