Abstract
BackgroundAs a main drug for diseased thrombus, some clinically used thrombolytic agents have various disadvantages, safer novel thrombolytic agents are of great demand. This study aimed to achieve high and efficient production of a fibrinolytic enzyme with superior enzymatic properties, by a combination strategy of codon optimization, gene dosage and process optimization in Komagataella phaffii (K. phaffii).ResultsAfter codon optimization, the fibase from a marine Bacillus subtilis was expressed and secreted in K. phaffii GS115. Recombinant strains harboring different copies of the fib gene (fib-nc) were successfully obtained via Geneticin (0.25–4 mg/ml) screening on minimal dextrose selection plates and assessment via real-time quantitative PCR. The respective levels of fibase produced by strains expressing fib-5.4c, fib-6c, fib-8c, fib-9c, and fib-12c were 4428, 5781, 7323, 7930, and 2472 U/ml. Levels increased as the copy number increased from 4 to 9, but decreased dramatically at copy number 12. After high cell density fermentation optimization, the highest fibase activity of the strain expressing fib-9c was 7930 U/ml in a shake flask and increased to 12,690 U/ml after 3 days of continuous culture in a 5-L fermenter, which is one of the highest levels of production reported. The recombinant fibase was maximally active at pH 9.0 and 45 °C, and was remarkably stable at pH levels ranging from 5 to 10 and temperatures up to 50 °C. As a metal-dependent serine protease, fibase did not cause hemolysis in vitro and preferentially degraded fibrin directly.ConclusionsThe combination of codon optimization, gene dosage, and process optimization described herein could be used for the expression of other therapeutic proteins difficult to express. The characteristics of the recombinant fibase suggest that it has potential applications for thrombosis prevention and therapy.
Highlights
As a main drug for diseased thrombus, some clinically used thrombolytic agents have various disadvantages, safer novel thrombolytic agents are of great demand
E. coli was cultivated in Luria-Bertani medium at 37 °C, and K. phaffii was cultured in buffered glycerol-complex medium (BMGY, 1% yeast extract, 2% peptone, 100 mM potassium phosphate buffer, 4 × 10− 5% biotin, 1.34% Yeast Nitrogen Base, 1% glycerol, pH 6.0) for growth, buffered methanol-complex medium (BMMY, 1% yeast extract, 2% peptone, 100 mM potassium phosphate buffer, 4 × 10− 5% biotin, 1.34% Yeast Nitrogen Base, 0.5% methanol, pH 6.0) for fibase induction, or yeast extract peptone dextrose (YPD) free of glucose (1% yeast extract and 2% peptone) for growth and induction
Recombinant fibase was secreted into the supernatant by K. phaffii GS115
Summary
As a main drug for diseased thrombus, some clinically used thrombolytic agents have various disadvantages, safer novel thrombolytic agents are of great demand. This study aimed to achieve high and efficient production of a fibrinolytic enzyme with superior enzymatic properties, by a combination strategy of codon optimization, gene dosage and process optimization in Komagataella phaffii (K. phaffii). As the main medical drug for diseased thrombus, thrombolytic agents such as urokinase, streptokinase, and tissue-type plasminogen activator have been widely used in clinical therapy. Many fibrinolytic enzymes (fibases) from animals such as snakes [3], earthworms [4, 5], plants [6], the fungus Cordyceps militaris [7] and microbes [8, 9] have been discovered and studied extensively. There are few reports on fibase produced by marine microorganisms
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