Breeding Of Pyrroloquinoline Quinone Producing Strain

Pyrroloquinoline quinone (PQQ) was discovered as a new kind of coenzyme for oxidoreductase in the 1960s. The distinct structure of the compound make it differ from the normal cofactor NAD、NADP and FM、FAD, and have broad pharmaceutical prospect. This study focused on the yield of PQQ. The object was to isolate high-yielding PQQ producing strains and optimize the fermentation system. Besides, traditional random mutagenesis with the screening techniques was also studied.In this paper, the concentrations of PQQ were measured by using a high-performance liquid chromatograph and a column (100 by 21 mm,5 μm) on Thermo Hypersil GOLD, and the column temperature at 35 ℃. The solvent used was water-acetonitrile (95:5, vol/vol) containing 1‰ TFA. The flow rate was 0.5 mL/min. Detections were done with UV spectrophotometric detector at 254 nm and 330 nm.A large number of methanol-utilizing bacteria that can produce extracellular pyrroloquinoline quinone were screened by using methanol as the sole carbon and energy sources. A highest-yield strain FJNU-6 was isolated and identified as Hyphomicrobium denitrificans based on morphology, physiological and biochemical property together with 16S rDNA sequence analysis. We named it H.denitrificans FJNU-6. The concentrations of PQQ were measured by HPLC and the yield of PQQ in H. denitrificans FJNU-6 could reach up to 10.8356 mg/L without any optimization or genetic techniques after 60 h.Filter design and central composite design were applied to optimize the medium. The optimal medium for H. denitrificans FJNU-6 was composed of:MeOH 10 g/L, (NH4)2SO4 3 g/L, MgCl2 2.8 g/L, KH2PO42.8 g/L, Na2HPO49 g/L, CaCl2 0.035 g/L, ZnSO40.021 g/L, FeSO4 0.009 g/L, mineral water 0.6 mL/L, vitamin solution 1.4 mL/L. The optimal culture conditions as follows:the initial pH was not controlled, temperature 30℃, medium volume 30 mL/250 mL, rotational speed 100 rpm, culture time 88 h. Under these conditions, the production of pyrroloquinoline quinone increased to 87.0179 mg/L, with an increase of 3.54 fold compared to the original medium components.UV mutagenesis and high methanol were used to screen high-yielding strains of PQQ. Meanwhile, H. denitrificans FJNU-6 was taken as the original strain, treated with UV mutation and methanol domestication. Finally we have got a mutant which has high methanol tolerance and PQQ production, named H4-189. The methanol tolerance for mutant H4-189 was increased to 80 g/L, and which was 20 g/L for the original strain. H4-189 produced 158.5314 mg/L PQQ, respectively, which were 82.77% higher than the original strain. The Genetic stability experiment showed that H4-189 could be serially passaged 9 times and maintained stable solvents production.The composite kinetic equation for cell growth, PQQ formation and methanol consumption was proposed according to the Logistic and Luedeking-Piret equations. Model the average relative error is only 5.425%, better reflect the mutant H4-189 fermentation kinetics characteristics of the process. Under the conditions of shake-flask, the effects of different adding concentration of methanol on PQQ production were studied. The result showed that using multi-addiing method accessed to the larger of PQQ production, while the adding concentration of method was 0.3%, PQQ production can be up to 326.5542 mg/L, increased by 110.59%.