An effective combination of codon optimization, gene dosage, and process optimization for high-level production of fibrinolytic enzyme in Komagataella phaffii (Pichia pastoris).

BACKGROUND: 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). RESULTS: After 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. CONCLUSIONS: The 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.

Investigations in ultrasound-assisted anticoagulant production by marine Bacillus subtilis ZHX.

Anticoagulants are the main drugs for the prevention and treatment of thromboembolism. However, most of the present anticoagulants have shortcomings and novel anticoagulants are in great demand. Marine microorganisms are an important source of new drugs. Therefore, in this study, ultrasound was applied to enhance anticoagulant accumulation by marine Bacillus subtilis ZHX. Ultrasound parameters were optimized by single-factor experiments exploring the effects of ultrasound power, duration, duty cycle and the cell growth phases. The optimum conditions were exponential prophase (5 h) with 25 kHz frequency, 140 W power, and a 40% duty cycle for 5 min. The maximum anticoagulant activity (55.36 U/mL) was 1.73 times that of the control group, and the fermentation time was shortened by 3 h. Under optimal conditions, ultrasound increased the carbon utilization by Bacillus subtilis ZHX without significant changes in morphology, favoring cell growth and anticoagulant production. However, excessive ultrasound caused intracellular damage, which inhibited biomass accumulation, decreasing anticoagulant activity and even leading to cell rupture. This is the first report on the use of ultrasound to enhance anticoagulant production by Bacillus, and it provides useful information for scaling-up the process.

Production of poly-γ-glutamic acid by a thermotolerant glutamate-independent strain and comparative analysis of the glutamate dependent difference.

Poly-γ-glutamic acid (γ-PGA) is a promising microbial polymer with wide applications in industry, agriculture and medicine. In this study, a novel glutamate-independent γ-PGA producing strain with thermotolerant characteristics was isolated and identified as Bacillus subtilis GXG-5, then its product was also characterized. The fermentation process was optimized by single-factor tests, and results showed that high temperature (50 °C) was especially suitable for the γ-PGA production by GXG-5. The γ-PGA yield reached 19.50 ± 0.75 g/L with substrate conversion efficiency of 78% at 50 °C in 10 L fermentor. Comparison of GXG-5 and GXA-28 (glutamate-dependent strain) under respective optimal fermentation conditions, the γ-PGA yield of GXG-5 was 19.0% higher than that of GXA-28, and GXG-5 was also superior to GXA-28 in the availability of carbon sources and substrates. Furthermore, the glutamate dependent difference between GXA-28 and GXG-5 was analyzed by genomic sequencing, results indicated that genes related to the glutamate dependent difference mainly involved in carbohydrate transport and metabolism and amino acid metabolism, and 13 genes related to γ-PGA synthesis were mutated in GXG-5. This study provided a potential glutamate-independent strain to replace glutamate-dependent strain for γ-PGA production, and shared novel information for understanding the glutamate dependent difference at the genomic level.

Characterization of a novel beta-glucosidase-like activity from a soil metagenome.

We report the cloning of a novel beta-glucosidase-like gene by function-based screening of a metagenomic library from uncultured soil microorganisms. The gene was named bgllC and has an open reading frame of 1,443 base pairs. It encodes a 481 amino acid polypeptide with a predicted molecular mass of about 57.8 kDa. The deduced amino acid sequence did not show any homology with known beta-glucosidases. The putative beta-glucosidase gene was subcloned into the pETBlue-2 vector and overexpressed in E. coli Tuner (DE3) pLacI; the recombinant protein was purified to homogeneity. Functional characterization with a high performance liquid chromatography method demonstrated that the recombinant BgllC protein hydrolyzed D-glucosyl-beta-(l-4)-D-glucose to glucose. The maximum activity for BgllC protein occurred at pH 8.0 and 42 degrees C using p-nitrophenyl-beta-D-glucoside as the substrate. A CaCl(2) concentration of 1 mM was required for optimal activity. The putative beta-glucosidase had an apparent K(m) value of 0.19 mM, a V(max) value of 4.75 U/mg and a k (cat) value of 316.7/min under the optimal reaction conditions. The biochemical characterization of BgllC has enlarged our understanding of the novel enzymes that can be isolated from the soil metagenome.