Recombinant protein production and streptomycetes

Anné, Jozef; Maldonado, Barbara; Impe, Jan Van; Mellaert, Lieve Van; Bernaerts, Kristel

doi:10.1016/j.jbiotec.2011.06.028

Cited by 93 publications

(79 citation statements)

References 103 publications

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“…Bacteria of the genus Streptomyces are the major producers of industrially relevant compounds, such as antibiotics, enzymes, antiviral and even anticancer drugs (Demain & Sanchez, 2009). Their ability to express and secrete heterologous proteins in a properly folded and biologically active state makes them even more attractive for biotechnological applications (Anné et al, 2012).…”

Section: Introductionmentioning

confidence: 99%

Synthetic riboswitches for the conditional control of gene expression in Streptomyces coelicolor

2013

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We have demonstrated the portability of theophylline-dependent synthetic riboswitches for the conditional control of gene expression in Streptomyces coelicolor. The riboswitches mediate dose-dependent, up to 260-fold activation of reporter gene expression. Riboswitch regulation is a simple method requiring a sequence of only~85 nt to be inserted between a transcriptional start site and the start codon; no additional auxiliary factors are necessary. The promoters galP2, ermEp1 and SF14 worked well in concert with the riboswitches. They allowed theophyllinedependent expression of not only the heterologous b-glucuronidase reporter gene but also dagA, an endogenous agarase gene. The successful combination of all tested promoters with the riboswitches underlines the orthogonality of riboswitch regulation. We anticipate that any additional natural or synthetic promoters can be combined with the riboswitch.

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Section: Introductionmentioning

confidence: 99%

Synthetic riboswitches for the conditional control of gene expression in Streptomyces coelicolor

2013

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“…The competence of streptomycetes to directly secrete enzymes into the culture broth makes them attractive hosts for the heterologous production of commercially valuable enzymes (26,27). However, the pellets suffer from poor efficiency in oxygen and nutrient uptake and thus compromise the overall efficiency of the fermentation process.…”

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confidence: 99%

Heterogeneity in the Histidine-brace Copper Coordination Sphere in Auxiliary Activity Family 10 (AA10) Lytic Polysaccharide Monooxygenases

Chaplin

Wilson

Hough

et al. 2016

Journal of Biological Chemistry

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Copper-dependent lytic polysaccharide monooxygenases (LPMOs) are enzymes that oxidatively deconstruct polysaccharides. The active site copper in LPMOs is coordinated by a histidine-brace. This utilizes the amino group and side chain of the N-terminal His residue with the side chain of a second His residue to create a T-shaped arrangement of nitrogen ligands. We report a structural, kinetic, and thermodynamic appraisal of copper binding to the histidine-brace in an auxiliary activity family 10 (AA10) LPMO from Streptomyces lividans (SliLPMO10E). Unexpectedly, we discovered the existence of two apo-SliLPMO10E species in solution that can each bind copper at a single site with distinct kinetic and thermodynamic (exothermic and endothermic) properties. The experimental EPR spectrum of copper-bound SliLPMO10E requires the simulation of two different line shapes, implying two different copper-bound species, indicative of three and two nitrogen ligands coordinating the copper. Amino group coordination was probed through the creation of an N-terminal extension variant (SliLPMO10E-Ext). The kinetics and thermodynamics of copper binding to SliLPMO10E-Ext are in accord with copper binding to one of the apo-forms in the wild-type protein, suggesting that amino group coordination is absent in the two-nitrogen coordinate form of SliLPMO10E. Copper binding to SliLPMO10B was also investigated, and again it revealed the presence of two apo-forms with kinetics and stoichiometry of copper binding identical to that of SliLPMO10E. Our findings highlight that heterogeneity exists in the active site copper coordination sphere of LPMOs that may have implications for the mechanism of loading copper in the cell.Lytic polysaccharide monooxygenases (LPMOs) 3 are enzymes that utilize copper as their functional active site metal and act to enhance the depolymerization of recalcitrant polysaccharides in nature such as cellulose and chitin (1, 2). Identification and characterization of members of this cuproenzyme family from fungal and bacterial species have gathered pace in recent years due in part to their promising uses in biorefinery applications of biomass to derive second generation biofuels (2, 3). LPMOs are classed into auxiliary activity (AA) families in the CAZy (carbohydrate active enzyme) database with four AA families, AA9, AA10, AA11, and AA13 identified so far (4). AA9 (formerly GH61) and AA10 (formerly CBM33) families have been the most extensively studied, with AA9 members having activity for cellulose, hemicellulose, and cellodextrin substrates (5-8) and AA10 members having activity with either cellulose or chitin substrates (9, 10). The AA11 and AA13 families are the most recently discovered, and so far have been determined to have activity with chitin and starch substrates, respectively (11, 12). All LPMOs coordinate a single copper ion by three nitrogen ligands to from the active site. Two of these are provided from the amino group (Nt) and side chain of the N-terminal His with the third from a second His side chain to giv...

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“…During the last years, considerable efforts were made in order to maximize the yield and quality of the proteins produced in heterologous systems [17]. This is even more critical in the case of peptides or proteins that are potentially toxic for the producing organism.…”

Section: Discussionmentioning

confidence: 99%

“…Additionally, when these strategies are employed, the cytoplasmic accumulation of the expressed proteins in the form of inclusion bodies is frequently observed [16], inducing activity inhibition and rendering the purification process difficult. Consequently, this is reflected in low yield values [17].…”

Section: Introductionmentioning

confidence: 99%

Streptomyces as Overexpression System for Heterologous Production of an Antimicrobial Peptide

Roldán-Tapia

Anné

Reyes³

et al. 2017

PPL

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Defense mechanisms of plants against phytopathogens include cationic antimicrobial peptides (CAPs). The broad-spectrum activity of these peptides has been evaluated previously against different phytopathogens. Their lack of toxicity for plants and animals is a promising feature for pest control. Although, some attempts have been made previously in order to increase their heterologous expressions, the employed strategies have so far proven to be ineffective. Low production yield and elevated costs are the obstacles to overcome. In this study, a strategy for CAP overexpression is presented based on the construction of an expression cassette for Streptomyces lividans TK24. This system contains elements that allow the increase of the efficiency of the peptide's expression. The main elements included in this cassette are the sequences of the promoter and signal peptide from a subtilisin inhibitor gene of Streptomyces venezuelae. This allows the efficient secretion of the peptide to the growth medium, thereby simplifying its recovery and avoiding its toxic effect on the producing organism. The results obtained demonstrate the system's efficiency by achieving a peptide concentration of 11.61 mg/ml. This represents at least a 10-fold increase compared to previously established strategies.

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Recombinant protein production and streptomycetes

Cited by 93 publications

References 103 publications

Synthetic riboswitches for the conditional control of gene expression in Streptomyces coelicolor

Synthetic riboswitches for the conditional control of gene expression in Streptomyces coelicolor

Heterogeneity in the Histidine-brace Copper Coordination Sphere in Auxiliary Activity Family 10 (AA10) Lytic Polysaccharide Monooxygenases

Streptomyces as Overexpression System for Heterologous Production of an Antimicrobial Peptide

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