Abstract
BackgroundBacterial secretory proteins often require the formation of disulphide bonds outside the cell to acquire an active conformation. Thiol-disulphide oxidoreductases are enzymes that catalyse the formation of disulphide bonds. The bacterium Streptomyces lividans is a well-known host for the efficient secretion of overproduced homologous and heterologous secretory proteins of industrial application. Therefore, the correct conformation of these extracellular proteins is of great importance when engineering that overproduction.ResultsWe have identified four acting thiol-disulphide oxidoreductases (TDORs) in S. lividans TK21, mutants in all TDOR candidates affect the secretion and activity of the Sec-dependent alpha-amylase, which contains several disulphide bonds, but the effect was more drastic in the case of the Sli-DsbA deficient strain. Thus, the four TDOR are required to obtain active alpha-amylase. Additionally, only mutations in Sli-DsbA and Sli-DsbB affect the secretion and activity of the Tat-dependent agarase, which does not form a disulphide bond, when it is overproduced. This suggests a possible role of the oxidised Sli-DsbA as a chaperone in the production of active agarase.ConclusionsEnzymes involved in the production of the extracellular mature active proteins are not fully characterised yet in Streptomyces lividans. Our results suggest that the role of thiol-disulphide oxidoreductases must be considered when engineering Streptomyces strains for the overproduction of homologous or heterologous secretory proteins of industrial application, irrespective of their secretion route, in order to obtain active, correctly folded proteins.
Highlights
Bacterial secretory proteins often require the formation of disulphide bonds outside the cell to acquire an active conformation
Identification of the thiol‐disulphide oxidoreductases in S. lividans TK21 Using the Bacillus subtilis BdbD sequence to search for homologous proteins, four possible thiol-disulphide oxidoreductases were identified in the S. lividans TK24 genome [13] namely SLIV_08650, SLIV_27370, SLIV_27535, and SLIV_28015 containing 25%, 25%, 26% and 26% of identical residues and 45%, 41%, 45%, 43% of equivalent similar amino acids, respectively
Based on the four possible candidates identified in S. lividans TK24, oligonucleotides containing possible regulatory region and the coding sequences of the different genes encoding the potential oxidoreductases were designed and used to amplify the corresponding genes from the Streptomyces lividans TK21 genome (Oxidoreductases genes, Additional file 1)
Summary
Bacterial secretory proteins often require the formation of disulphide bonds outside the cell to acquire an active conformation. The action of an enzyme that oxidises substrate cysteines is required for the formation of disulphide bonds in the secreted proteins and, to maintain this oxidised state, that enzyme needs to be subsequently re-oxidised by other oxidase, which must transfer the accepted electrons to the last electron acceptor, usually the electron transport components [2, 3]. In the gram-negative bacteria E. coli five thiol-disulphide oxidoreductases have been described to be involved in disulphide bond formation in the secreted proteins. Gullón et al Microb Cell Fact (2019) 18:126 catalyses disulphide bond formation in secreted proteins immediately after their translocation into the periplasm, and it is maintained in its oxidised active state by the transmembrane protein DsbB. DsbA, DsbC and DsbG display a chaperone activity [7,8,9]
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