Studies On Efficient Bioproductions Of Pyrroloquinoline Quinone And Poly (β-L-malic Acid)

Pyrroloquinoline quinone(PQQ)and polymalic acid(PMLA)are two kinds of important biochemicals,which are widely used in the fields of pharmacy,chemistry,agriculture,food and etc.In this thesis,the biosynthetic mechanism and new technology development for the efficient production of these two chemicals were carried out systematically from multiple perspectives.At first,the comparison of three PQQ determination methods was performed to find out their characteristics and scopes of application.Based on the high sensitivity of GDH method,an efficient method for the screening of PQQ producing strains from soil samples was established.One high-yield PQQ strain,Methylobacillus sp.zju323,which could use methanol as the sole carbon source,was isolated and stored in China Center for Type Cultured Collection(CCTCC),numbered as M2016079.The titer of PQQ was 25.8 mg/L in shaking flask.Medium optimization for PQQ production was further carried out using the Plackett-Burman method.By using response surface analysis combined with artificial neural network and genetic algorithm optimization,the key best medium components and their concentrations were determined as follows:CoCl2·6H2O 3.2 mg/L,PABA 418.7 μg/L,MgSO4·7H2O 1.5 g/L.For further improvement of PQQ yield,two-stage pH control fermentation(pH 6.8 from the beginning to 48 h,and then pH 5.8 to the end)was carried out by feeding methanol in earlier stage coupled with yeast extract in later stage.At last,the strain improvement by using atmospheric and room temperature plasma(ARTP)physical mutagenesis,as well as nitrosoguanidine(NTG)chemical mutagenesis,was carried out.The final highest PQQ concentration of 450 mg/L was obtained under the optimal conditions.By using PMLA producing strains Aurobasidium pullulans ZD-3d(CGMCC 4605)previously screened by our lab,one novel PMLA high-yielding strategy was developed.At first,the effects of the glucose and raw cassava starch hydrolysate on PMLA synthesis were studied.The result showed that raw cassava starch hydrolysate was more favorable to PMLA accumulation.And the fed batch fermentation coupled with cell separation by membrane filtration or centrifugation was studied.By using centrifugation cell recycle fermentation,the cell vitality was obviously improved and the recycle of fermentation process could last for 5 rounds.The concentration,productivity and yield of PMLA were 76.2～39.6 g/L,0.98-1.76 g L-1 h-1 and 0.78～0.86 g/g,respectively,which were all higher than those using the membrane filtration cell recycle fermentation.The proteomics analysis was also applied to study the Ca2+ effects on PMLA biosynthesis,458 and 726 differential proteins were found in Ca2+ group and the control,respectively.GO enrichment analysis on the differential proteins showed that there were 104 kinds of membrane transporters in Ca2+ group,which was good for malic acid and PMLA intracellular transport and exocytosis.In this thesis,a high-throughput method was established to screen PQQ producing stain,and the followed medium and fermentation optimization,as well as the mutation breeding were successfully carried out to improve the PQQ yield.On the other hand,the cell recycle fermentation was developed to produce PMLA.Besides,the biosynthetic mechanism of PMLA was further investigated by proteomic analyses.Our work has provided a useful guiding for efficient biosynthesis of these two products.