Inhibition of coenzyme Qs accumulation in engineered escherichia coli by high concentration of farnesyl diphosphate

Coenzyme Q[10] [CoQ[10]] is an isoprenoid component used widely in nutraceutical industries. Farnesyl diphosphate synthase [FPPS] is a responsible enzyme for biosynthesis of farnesyl diphosphate [FPP], a key precursor for CoQs production. This research involved investigating the effect of FPPS over-expression on CoQs production in engineered CoQ[10]-producing Escherichia coli [E. coli]. Two CoQ[10]-producing strains, as referred to E. coli Ba and E. coli Br, were transformed by the encoding gene for FPPS [ispA] under the control of either the trc or P[BAD] promoters. Over-expression of ispA under the control of P[BAD] promoter led to a relative increase in CoQ[10] production only in recombinant E. coli Br although induction by arabinose resulted in partial reduction of CoQ[10] production in both recombinant E. coli Ba and E. coli Br strains. Over-expression of ispA under the control of stronger trc promoter, however, led to a severe decrease in CoQ[10] production in both recombinant E. coli Ba and E. coli Br strains, as reflected by reductions from 629 +/- 40 to 30 +/- 13 and 564 +/- 28 to 80 +/- 14 micro g/g Dried Cell Weight [DCW], respectively. The results showed high level of FPP reduces endogenous CoQ8 production as well and that CoQs are produced in a complimentary manner, as the increase in production of one decreases the production of the other. The reduction in CoQ[10] production can be a result of Dds inhibition by high FPP concentration. Therefore, more effort is needed to verify the role of intermediate metabolite concentration and to optimize production of CoQ[10]

Production and characterization of monoclonal antibodies against human prostate specific antigen

Prostate Specific Antigen [PSA] is an important laboratory marker for diagnosis of prostatic cancer. Thus, development of diagnostic tools specific for PSA plays an important role in screening, monitoring and early diagnosis of prostate cancer. In this paper, the production and characterization of a panel of murine monoclonal antibodies [mAbs] against PSA have been presented. Balb/c mice were immunized with PSA, which was purified from seminal plasma. Splenocytes of hyperimmunized mice were extracted and fused with Sp2/0 cells. By adding selective HAT medium, hybridoma cells were established and positive clones were selected by ELISA after four times of cloning. The isotypes of produced mAbs were determined by ELISA and then purified from ascitic fluids using Hi-Trap protein G column. The reactivities of the mAbs were examined with the purified PSA and seminal plasma by ELISA and western blot techniques. Furthermore, the reactivities of the mAbs were assessed in Prostate Cancer [PC alpha], Benign Prostatic Hyperplasia [BPH] and brain cancer tissues by Immunohistochemistry [IHC]. Five anti-PSA mAbs [clones: 2G2-B2, 2F9-F4, 2D6-E8, IgG1/K] and clones [2C8-E9, 2G3-E2, IgG2 alpha/K] were produced and characterized. All mAbs, except 2F9-F4 detected the expression of PSA in PCa and BPH tissues and none of them reacted with PSA in brain cancer tissue in IHC. Besides, all mAbs could detect a protein band around 33 kD alpha in human seminal plasma in western blot. These mAbs can specifically recognize PSA and may serve as a component of PSA diagnostic kit in various biological fluids

Effect of concomitant lycopene biosynthesis on CoQ[10] accumulation in transformed Escherichia coli strains

CoQ[10] and lycopene are isoprenoid compounds with nutraceutical and pharmaceutical benefits. In this study, the effect of concomitant lycopene biosynthesis on CoQ[10] accumulation in transformed Escherichia coli DH5 alpha was studied. A lycopene production pathway including geranyl diphosphate synthase [crtE], phytoene synthase [crtB], and phytoene desaturase [crtI] from Erwinia herbicola was constructed in two CoQ[10]-producing E. coli strains. E. coli Ba and E. coli Br containing dds orthologs encoding for decaprenyl diphosphate synthase [Dds], respectively from Agrobacterium tumefaciens and Rhodobacter sphaeroides were transformed by the lycopene pathway resulting in E. coli Ba-lyc and E. coli Br-lyc. The lycopene pathway in E. coli Br-lyc interestingly resulted in a significant increase in CoQ[10] production from 564 +/- 28 to 989 +/- 22 micro g /g DCW. To confirm that the improvement of CoQ[10] production in E. coli Br-lyc was due to lycopene biosynthesis and not just geranylgeranyl diphosphate formation in the lycopene pathway, crtE was only introduced into E. coli Ba and E. coli Br strains. Surprisingly, crtE expression had adverse effects on CoQ[10] production in both strains. The results shed light on the Dds-catalyzed reaction as a bottleneck controlled by precursors; and the efficiency of a parallel lycopene pathway to streamline the flow of metabolites