Materials and methods br Results br

Materials and methods
Results
Discussion Hydroxylation of proline in bacterial BQ-123 mg is one of the oxygen-requiring metabolic reactions, which needs efficient oxygen. Oxygen supply is always a rate-limiting step for tyrosinase production even though pure oxygen is purged in a bioreactor (37). Due to consumption of oxygen in the hydroxylation reaction, oxygen supply will be more important for P4H production by recombinant strains. According to previous studies 12, 13, VHb can bind oxygen and deliver it to the terminal respiratory oxidases, especially under oxygen-limited conditions. VHb binding oxygen may increase the possibility of oxygen transfer to hydroxylase. Many studies have reported that VHb expression improves production of valuable compounds 16, 17, 18, 24. In this study, we integrated the vgb gene into the chromosome of E. coli WD3-VGB (pTrc99a-p4h) to enhance cell growth and Hyp production. E. coli WD3-VGB (pTrc99a-p4h) produced more Hyp, reaching concentrations of 8.23 g/L in shaker flask culture and 14.4 g/L in bioreactor fermentation, which showed an increase of 1.9-fold and 1.73-fold, respectively, compared to cultures of E. coli WD3 (pTrc99a-p4h). Meanwhile, the maximum cell concentration showed increases of 37% and 106%, respectively, in shaker flask culture and bioreactor fermentation, for the strain expressing VHb compared to that of E. coli WD3 (pTrc99a-p4h). Our results showed that expression of VHb enhanced cell growth and Hyp production by recombinant E. coli with P4H. E. coli WD3 (pTrc99a-p4h) and E. coli WD3-VGB (pTrc99a-p4h) were constructed to lack activity of α-ketoglutarate dehydrogenase, which would hinder TCA cycle and thereby cell growth if no P4H was expressed to catalyze α-ketoglutarate to succinate. In this study, P4H was introduced to produce Hyp from proline, while simultaneously restoring TCA cycle, thus coupling l-proline hydroxylation and cell growth. Hyp was produced in the presence of glucose and proline by both E. coli WD3 (pTrc99a-p4h) and E. coli WD3-VGB (pTrc99a-p4h). The design approach used in this study enabled cell growth to be coupled with Hyp production for both strains. The strain expressing VHb improved cell growth as well as increasing Hyp concentration. In E. coli WD3 (pTrc99a-p4h) and E. coli WD3-VGB (pTrc99a-p4h), glucose added to medium was decomposed by the glycolysis pathway or the pentose phosphate pathway which is one of the main glucose metabolic pathways. Subsequently, the intermediate metabolite pyruvic acid produced by the glycolysis pathway entered into aerobic fermentation pathway, or into the anaerobic fermentation pathway. When glucose was added to fermentation of both E. coli WD3 (pTrc99a-p4h) and E. coli WD3-VGB (pTrc99a-p4h), the TCA cycle coupling l-proline hydroxylation and cell growth was the main pathway under aerobic conditions. VHb expression resulted in a higher growth rate and Hyp production rate, while the glucose consumption rate was not significantly affected by introduction of the vgb gene. The possible reason is that VHb expression enhances the dissolved oxygen level, weakening the anaerobic fermentation pathway and enhancing the aerobic fermentation pathway (38). However, during bioreactor fermentation, the specific growth rate and Hyp production rate of E. coli WD3 (pTrc99a-p4h) were 0.148 g cells/L/h and 0.027 g Hyp/g cells/h, while those of E. coli WD3-VGB (pTrc99a-p4h) were 0.201 g cells/L/h and 0.022 g Hyp/g cells/h, respectively. It therefore seems that VHb simply improved cell growth (but did not directly improve Hyp production) and consequently the increase in the concentration of catalytic cells led to the high Hyp titer in bioreactor fermentation. The possible reason is that VHb expression delivers oxygen to terminal oxygenases to produce a higher growth rate when 5% tryptone and 5% yeast extract are added to the fermentation medium.