Study On Riboflavin Fermentation Optimization And Metabolomics Of Recombinant Bacillus Subtilis

Riboflavin fermentation process by recombinant Bacillus subtilis RH44 was optimized, and then the mechanisms of the significant factors enhancing riboflavin production were discussed according to enzyme activity assay and metabolic flux analysis. The metabolomics of B. subtilis RH44 was investigated primarily using LC-MS.A sequential optimization strategy, based on statistical experimental designs, was used to enhance the production of riboflavin. Plackett-Burman design was implemented to screen medium components that significantly influence riboflavin production. Among the fifteen variables tested, glucose, NaNO₃, K₂HPO₄, ZnSO₄, and MnCl₂ were identified as the most significant factors for riboflavin production. The optimal values of these five variables were determined by response surface methodology (RSM) based on the central composite design (CCD). This optimum medium led to a maximum riboflavin concentration of 6.65 g/L in shake flasks. The optimal culture parameters (i.e. pH, inoculum level, stirring speed, temperature and airflow rate) for maximum riboflavin production by batch cultivations in a 5 L fermentor were determined by using artificial neural networks (ANN) incorporating genetic algorithm (GA) and RSM. ANN-GA led to the maximum riboflavin production of 7.53 g/L, which was 8.6% higher than that by RSM.Three fed-batch modes, intermittent, constant rate and glucose-limited, were carried out to reduce overflow in this work. The maximum riboflavin titer was obtained in a glucose-limited fed-batch process by maintaining constant (5¹0 g/L) glucose concentration in the culture broth. The effects of pH on riboflavin production were investigated using a series of glucose-limited fed-batch fermentations. Although constant pH 6.8 was favorable for the cell formation, constant pH 7.2 resulted in the highest riboflavin production and also maximum specific activity of riboflavin synthase of B. subtilis RH44. Hence, a pH-shift strategy was developed to improve riboflavin production. The results showed that the maximum riboflavin concentration was increased by 13.3% when compared with the best results of constant pH. The results of pH influence on both by-product levels and by-product forming enzyme activities indicated that the optimum pH-shift strategy had the capability of inhibiting the accumulation of by-products to a certain extent. In addition, when pH was adjusted with NH4OH instead of NaOH, in the pH-shift strategy, further improvement (17.4 g/L in 48 h) was achieved in riboflavin production.The mechamisms of three significant factors enhancing riboflavin production were discussed further. The comparison of the consumption courses of NH₄⁺ and NO₃^–of B. subtilis 168 and B. subtilis RH44 showed that the utilization of NO₃^– in B. subtilis RH44 did not appear to be inhibited by NH₄⁺. The metabolic flux shifted from EMP to PP during the mid- and later phases when the optimum nitrogen sources were used. Mn²⁺ has an activation function to glucose-6-phosphate dehydrogenase (G6PDH), while an inhibition to the activity of phosphortransacetylase (PTA). Possibly, the effect of Zn²⁺ on riboflavin biosynthesis had a close relationship with GCHII. The functions of Mn²⁺ and Zn²⁺ to riboflavin production were further investigated using metabolic flux analysis.A scheme of metabolomics analysis by LC-MS on B. subtilis was developed. The metabolomic analysis was valid to distinguish various culture phases by principal components analysis of negative ions ESI-MS data. In addition, 16 metabolites were determined by qualification analysis based on m/z of metabolites as well as standard and MS-MS. The limited factors of riboflavin production by B. subtilis RH44 were discussed according to the comparison of metabolites of different nitrogen sources and different strains by metabolic profiling analysis.