| Pyrroloquinoline quinone(PQQ)is the third REDOX enzyme coenzyme,widely present in living organisms.The human clinical research have confirmed that PQQ has strong antioxidant capacity,which can help improve mitochondrial performance,reduce chronic inflammation,protect the brain,improve memory and attention,improve sleep,reduce cholesterol and other special physiological activities.It has a huge application prospect in food,medicine,cosmetics and other fields.At present,the synthesis capacity of PQQ producing bacteria is limited,and the cost of separation and purification is high.Therefore,it is of great significance to breed high-yield PQQ strains and develop corresponding fermentation culture strategies as well as simple and low-cost separation and purification process.First,the high-yield strains were screened by mutagenesis,the optimal medium was selected by response surface method,and the two-stage oxygen control strategy(MO)was used in 5L fermenter for fermentation.Starting strain Hyphomicrobium denitrificans FJNU-6 through ARTP mutagenesis and 14/7/4 rounds adaptive evolution laboratory(ALE),PQQ yield up to 48.21 mg/L,and production efficiency increased by 119.4%;The results showed that the optimal mutant FJNU-A26 had the highest content of PQQ fermentation broth,about 68.98 mg/L,and the cell dry weight was 1.458 g/L,which showed good heritability.Response surface method was used to confirm the optimal medium.Shaking flask fermentation showed that the PQQ yield of FJNU-A26 strain was increased to 89.14±2.17 mg/L in optimized medium.FJNU-A26 strain was fermented in5 L fermenting tank for 168 h with two-stage oxygen control strategy.The yield of PQQ was increased to 1372.82 mg/L,the average yield of PQQ was 8.17 mg/(L×h),and the yield per unit cell was 39.52 mg/g DCW,which was increased by 40%.The results of 5 L fermentation showed that the yield of PQQ fermentation using two-stage oxygen control strategy(MO)was 1.69 times and 1.51 times of that using constant low oxygen strategy(LO)and high oxygen strategy(HO),respectively,indicating that oxygen regulation is very important for the synthesis of PQQ.Therefore, in order to study the dynamic regulation mechanism of three oxygen solution control strategies on bacterial growth and product synthesis,transcriptomic and metabolomic analysis of bacteria at growth stage,transition stage and product synthesis stage were conducted.A total of 3476 genes were detected in transcriptome study.During the growth stage,the genes were mainly classified in metabolic process and cellular process.With the increase of dissolved oxygen level,carbon metabolism,nitrogen metabolism,co-factor and amino acid synthesis metabolism,as well as membrane related genes and other pathway genes were up-regulated,which could ensure the rapid growth of bacteria in early stage through sufficient dissolved oxygen level.During the transition period,the main enrichment processes are phosphate assimilation,nitrogen metabolism,sulfur metabolism and energy metabolism.During the product synthesis phase,up-regulated energy metabolism and amino acid pathway are conducive to PQQ synthesis.According to WGCNA analysis,five modules were screened out,which were related to cell growth and PQQ yield.Blue module was positively correlated with cell growth and PQQ synthesis.Purple module is positively correlated with PQQ synthesis;Cyan module and Turquoise module have negative correlation with PQQ output.The metabolomics study screened 399 metabolites,and found that high oxygen condition is conducive to the oxidation of methanol,and can promote the growth of bacteria by enhancing folic acid and nicotinic acid.And form polysaccharides to store energy for later product synthesis;In the product synthesis stage,the level of cell membrane-related metabolites is reduced,the serine cycle and TCA cycle are enhanced,lipids are degraded,and more energy and amino acid precursors are provided for the synthesis of PQQ.Transcription analysis of key genes of methanol oxidation and PQQ synthesis during fermentation showed that mox Fα2 gene was highly expressed and played an important role in the methanol oxidation process.The high expression of pqq A and pqq E is conducive to the accumulation of precursors to promote the synthesis of PQQ,while the low oxygen condition is conducive to the expression of pqq D and pqq E.In order to solve the problem of inconsistent factors affecting cell growth and product synthesis in PQQ semi-even combination,a multi-stage fermentation culture regulation strategy was developed based on fermentation data and transcriptomic and metabolomic analysis results.The results showed that 50% salt concentration and methanol-glycerol compound carbon source as the initial medium could promote the growth of bacteria in the growth stage,and promote the synthesis of PQQ by adding amino acid exogenously.With a two-stage pH control strategy,the fermentation time was shortened from 168 h to 144 h,the yield of PQQ was increased to 1515.35 mg/L and the average yield was 10.52±0.24 mg/(L×h),which was increased by 24.5%.At the same time,it was found that the two-stage strategy was more likely to form the accumulation of formaldehyde and cause cytotoxicity,which changed the balance between growth and production,but was beneficial to the synthesis of PQQ at the later stage of fermentation.Finally,in the decline period,the bacteria died,PQQ was chelated,and the fermentation liquid turned green.With the ORP control and adopting the three-stage DO-ORP regulation strategy,the effective fermentation time was extended to 192 h,the PQQ yield was 2.28 g/L,and the average yield was 11.86 mg/(L×h)through the 5L tank batch experiment.In order to low cost and high purity PQQ products,the separation and purification process of PQQ by membrane system with macroporous resin chromatography was established.By membrane system treatment,98.4% protein can be removed from the fermentation liquid,and the supernatant can be directly adsorbed by resin chromatography.In the chromatographic separation stage,the resin XAD1600 with the largest adsorption capacity of PQQ was first screened.In the process of PQQ separation and purification,the difficult by-product was identified as IPQ,and other derivatives were IPQ/R spontaneously generated by PQQ and different amino acids.In order to remove the by-product IPQ and IPQ/R,PQQ was separated and purified by two column process.Firstly,the precolumn was used to separate PQQ and IPQ at pH3.5,4 BV/h,which solved the problem of difficult separation of impurities such as PQQ derivatives.After amplification verification,about 28 L eluent with a purity of 96.23% and a recovery of 82.57% was obtained.The purity of 99.63% and the recovery of 93.32% were obtained by rapid crystallization at low temperature.In conclusion,in this paper,the PQQ high-yielding strain FJNU-A26 mutant was obtained through ARTP-ALE combined mutagenization and domestication,and the culture system was optimized.According to the difference analysis of transcriptome and metabolome under different oxygen control strategies,the key pathways and genes of oxygen regulation in PQQ synthesis were analyzed.On this basis,the differential regulation was optimized for different stages,and the two-stage pH and three-stage DO-OPR fermentation regulation strategies were established.The crystal samples with purity greater than 99% were obtained by the method of series column and the membrane system.The strain and related technology have indu strialization potential,The physicochemical and microbiological indexes are up to standard the general requirements of the current market for PQQ products.The whole sets of mature and stable process can obtain qualified PQQ products with high efficiency and low cost,with industrialization potential. |
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