Engineering of a Bacillus subtilis strain with adjustable levels of intracellular biotin for secretory production of functional streptavidin.
Streptavidin is a biotin-binding protein which has been widely used in many in vitro and in vivo applications. Because of the ease of protein recovery and availability of protease-deficient strains, the Bacillus subtilis expression-secretion system is an attractive system for streptavidin production. However, attempts to produce streptavidin using B. subtilis face the problem that cells overproducing large amounts of streptavidin suffer poor growth, presumably because of biotin deficiency. This problem cannot be solved by supplementing biotin to the culture medium, as this will saturate the biotin binding sites in streptavidin. We addressed this dilemma by engineering a B. subtilis strain (WB800BIO) which overproduces intracellular biotin. The strategy involves replacing the natural regulatory region of the B. subtilis chromosomal biotin biosynthetic operon (bioWAFDBIorf2) with an engineered one consisting of the B. subtilis groE promoter and gluconate operator. Biotin production in WB800BIO is induced by gluconate, and the level of biotin produced can be adjusted by varying the gluconate dosage. A level of gluconate was selected to allow enhanced intracellular production of biotin without getting it released into the culture medium. WB800BIO, when used as a host for streptavidin production, grows healthily in a biotin-limited medium and produces large amounts (35 to 50 mg/liter) of streptavidin, with over 80% of its biotin binding sites available for future applications.
Wu
Sau-Ching
SC
Department of Biological Sciences, Division of Molecular, Cellular and Microbial Biology, University of Calgary, 2500 University Drive NW, Calgary, Alberta T2N 1N4, Canada.
Wong
Sui-Lam
SL
D001412:Bacillus subtilis / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001710:Biotin / Q000235:genetics / Q000378:metabolism*; D018833:Chaperonins; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D005818:Genetic Engineering / Q000379:methods*; D005942:Gluconates / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D009875:Operator Regions, Genetic / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D019809:Streptavidin / Q000096:biosynthesis*
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D005942:Gluconates; C028715:GroE protein, E coli; D006360:Heat-Shock Proteins; D001710:Biotin; D019809:Streptavidin; D018833:Chaperonins; C030691:gluconic acid