Directive Breeding And Fermentation Optimization Of Methylobacterium Extorquens For High Pyrroloquinoline Quinone Production

Pyrroloquinoline quinone?PQQ?is a prosthetic group of quinoprotein dehydrogenase and exhibits different bioactivities in microbial,plants and mammals.It could be applied in health care products,cosmetics,medicine,agriculture,environment and other fields.Due to the high cost of chemical synthesis route,the microbial fermentation for PQQ production attracted more and more attention.In this study,Methylobacterium extorquens I-F2,a PQQ-producing bacterium,was employed as an original strain.A high-titer PQQ-producing mutant was obtained by applying atmospheric and room temperature plasma?ARTP?mutagenesis,flow cytometry sorting and high-throughput screening strategy.Moreover,the titer of PQQ accumulated by M.extorquens was further enhanced by optimizing medium components and fermentation conditions.The main results are listed as following:?1?High-efficiency screening of PQQ-producing strain was established based on ARTP mutagenesis,flow cytometry sorting and high-throughput screening system.The accuracy of sorting was improved by optimizing sample preparation and flow sorting process.The staining conditions of the samples were determined as follows:propidiumiodide(10?g·m L^-1)and samples were mixed with 1:1?v/v?for 30 min under room temperature in the dark.After four rounds of ARTP mutagenesis,a high PQQ-producing mutant M.extorquens 1-C6 with high genetic stability was obtained.The titer of PQQ in shaking flask was 16.0 mg·L^-1,which was increased by 1.5-fold compared with that of the M.extorqunens I-F2.?2?Optimizations of carbon source,nitrogen source,metal ions and growth factors for PQQ production of M.extorquens 1-C6 were conducted in shaking flask.The optimal concentrations in seed culture were as follows:CoCl₂·6H₂O 20 mg·L^-1,FeC₆H₅O₇ 8mg·L^-1,Zn SO₄·7H₂O 4 mg·L^-1,Cu SO₄·5H₂O 0.5 mg·L^-1,riboflavin 4?g·L^-1,pyridoxine 4?g·L^-1,cobalamin 2?g·L^-1,4-aminobenzoic acid 15?g·L^-1,calcium pantothenate 30?g·L^-1,and lipoic acid 10?g·L^-1.The optimal concentration of the key factors in fermentation culture was as follows:methanol 30 m L·L^-1,ammonium sulfate 1.75 g·L^-1,lipoic acid 8?g·L^-1 and riboflavin 2.5?g·L^-1.Finally,the PQQ production of M.extorquens 1-C6 in shaking flask was 29.1 mg·L^-1 by optimizing medium components,which was 81.9%higher than M.extorqunens I-F2.?3?Fermentation controlling for PQQ production of M.extorquens 1-C6 was studied in 3L fermentor.The optimum initial concentration of methanol was determined as 30 m L·L^-11 for batch fermentation.A two-staged pH control strategy was established.The pH was maintained at 7.0 at the beginning of fermentation,then droped spontaneously to 6.0,finally was maintained at 6.0 until the end of fermentation.Furthermore,the feeding fermentation strategy was studied and established a feeding method,that was when the methanol concentration reduced to 20 m L·L^-1 and then maintained in this concentration.Through the pH controlling and feeding strategy,the PQQ production of the M.extorquens 1-C6 in the 3 L fermentor achieved 208.7 mg·L^-1,which was 5.9-fold compared with the batch fermentation.