Hossein Vahidi

Optimization of Fermentation Conditions for Recombinant Human Interferon Beta Production by Escherichia coli Using the Response Surface Methodology.

Background: The periplasmic overexpression of recombinant human interferon beta (rhIFN-β)-1b using a synthetic gene in Escherichia coli BL21 (DE3) was optimized in shake flasks using Response Surface Methodology (RSM) based on the Box-Behnken Design (BBD). Objectives: This study aimed to predict and develop the optimal fermentation conditions for periplasmic expression of rhIFN-β-1b in shake flasks whilst keeping the acetate excretion as the lowest amount and exploit the best results condition for rhIFN-β in a bench top bioreactor. Materials and Methods: The process variables studied were the concentration of glucose as carbon source, cell density prior the induction (OD 600 nm) and induction temperature. Ultimately, a three-factor three-level BBD was employed during the optimization process. The rhIFN-β production and the acetate excretion served as the evaluated responses. Results: The proposed optimum fermentation condition consisted of 7.81 g L-1 glucose, OD 600 nm prior induction 1.66 and induction temperature of 30.27°C. The model prediction of 0.267 g L-1 of rhIFN-β and 0.961 g L-1 of acetate at the optimum conditions was verified experimentally as 0.255 g L-1 and 0.981 g L-1 of acetate. This agreement between the predicted and observed values confirmed the precision of the applied method to predict the optimum conditions. Conclusions: It can be concluded that the RSM is an effective method for the optimization of recombinant protein expression using synthetic genes in E. coli.

Fungal transformation of androsta-1,4-diene-3, 17-dione by Aspergillus brasiliensis

BackgroundThe biotransformation of steroids by fungal biocatalysts has been recognized for many years. There are numerous fungi of the genus Aspergillu s which have been shown to transform different steroid substances. The possibility of using filamentous fungi Aspergillus brasiliensis cells in the biotransformation of androsta-1,4-diene-3,17-dione, was evaluated.MethodsThe fungal strain was inoculated into the transformation medium which supplemented with androstadienedione as a substrate and fermentation continued for 5 days. The metabolites were extracted and isolated by thin layer chromatography. The structures of these metabolites were elucidated using 1H-NMR, broadband decoupled 13C-NMR, EI Mass and IR spectroscopies.ResultsThe fermentation yielded one reduced product: 17β-hydroxyandrost-1,4-dien-3-one and two hydroxylated metabolites: 11α-hydroxyandrost-1,4-diene-3,17-dione and 12β-hydroxyandrost-1,4-diene-3,17-dione.ConclusionsThe results obtained in this study show that A. brasiliendsis could be considered as a biocatalyst for producing important derivatives from androstadienedione.

Baeyer-Villiger oxidation of progesterone by Aspergillus sojae PTCC 5196

HIGHLIGHTSProgesterone converted by the fungus Aspergillus sojae PTCC 5196 produced testololactone.The production of testololactone indicated fungal Baeyer‐Villiger monooxygenase (BVMO) activity.Substrate‐induced cultures have a decisive impact on the metabolism of progesterone.Progesterone, a C‐21 steroidal compound, induced 17&bgr;‐acetyl side chain cleavage.Androstenedione, testosterone, and DHEA, C‐19 steroidal substances, induced ring‐D oxidation. ABSTRACT Microbial transformations are capable of producing steroid substances difficult to synthesize by chemical methods. Strains belonging to the genus Aspergillus are effective facilitators of microbial biotransformations due to their enzymatic diversity. In this study, the biotransformation of progesterone by the fungus Aspergillus sojae (A. sojae) PTCC 5196 was examined. Analysis of the bioconversion process revealed that progesterone was converted to testololactone through a three‐step pathway (17&bgr;‐acetyl side chain cleavage, 17&bgr;‐hydroxyl oxidation, and oxygenative lactonization of 17‐ketone), indicating the presence of Baeyer‐Villiger monooxygenase (BVMO) activity in the fungal strain. GC analysis confirmed the production of testololactone with a yield of 99% in 24h. Faster testololactone production was induced in the presence of both C‐21 (progesterone) and C‐19 (androstenedione, testosterone, and dehydroepiandrosterone [DHEA]) steroid substances. Due to the high biotransformation rate observed in the present study, A. sojae may be a novel and promising candidate in the production of testololactone.

Effect of Acyl Homoserine Lactone on Recombinant Production of Human Insulin-like Growth Factor-1 in Batch Culture of Escherichia coli

BACKGROUND IGF-I as a human growth factor produced in Escherichia coli is a single, non-glycosylated, polypeptide chain containing 70 amino acids and having a molecular mass of 7.6 kDa. Up to now, E. coli expression system has been widely used as the host to produce rhIGF-1 with high yields. Acyl Homoserine Lactones (AHLs) are intercellular signaling molecules used in quorum sensing by Gram-negative bacteria. Quorum sensing is a cell density-dependent gene regulation process that allows bacterial cells to express specific genes only when signaling molecules reach the sufficient concentration. OBJECTIVE For the first time, this study focuses on the N-hexanoyl-L- Homoserine Lactone (HHL) activity on increasing the cell growth and rh-IGF-1concentration in batch culture of E. coli. METHOD The maximum production of rhIGF-I was previously optimized in 32y culture medium at 32°C with 0.05 mM IPTG as inducer and 10 g/l glucose concentration. Under this condition, different amounts of HHL (0.001 µg/ml, 1 µg/ml, and 100µg/ml) were evaluated as an inducer for IGF-1 production. RESULTS Generally, with increasing of HHL concentration, an increase in dry cell weight (2.45 mg/ml to 4.63 mg/ml) and IGF-I expression level (0.4 mg/ml to 0.77 mg/ml) was observed. CONCLUSION HHL or other types of AHLs can be considered as protein production inducer in bacterial expression systems through the quorum sensing pathways.