[Breeding of Hyphomicrobium denitrificans for high production of pyrroloquinoline quinone by adaptive directed domestication]

Abstract

Pyrroloquinoline quinone (PQQ) is widely distributed in organisms and has physiological functions such as boosting body growth, maintaining mitochondrial function, promoting synthesis of nerve growth factor and regulating free radical levels in the body. It has broad application prospects in the fields of medicine, food and cosmetics. In order to improve the PQQ production of Hyphamicrobium denitrificans FJNU-6, the high-concentration methanol was used as the antagonistic factor for laboratory adaptive domestication. The PQQ positive mutants were selected using rapid screening system by spectroscopy. After 6 rounds of adaptive domestication, about 10% mutants were acquired with a doubled yield, and over 90% positive mutation rate of each round of domestication was reached. Subsequently, the mutant strain FJNU-R8 was fermented by 5 L fermenter. Compared with the original strain, the expression of pqq and moxF gene clusters were higher at different methanol concentrations and similar to each other. Meanwhile, the methanol consumption rate and growth rate were slower than the original strain. Finally, the PQQ yield was increased by 1.42 times to 1 087.81 mg/L (143 h), indicating good industrial application potential. The adaptive domestication combined with rapid screening system described in this study can easily and rapidly obtain mutants with high yield of PQQ, which can be used as reference for high-throughput screening of other high-yield PQQ mutants of methylotrophic bacteria.

Keywords: Hyphomicrobium denitrificans; adaptive domestication; high-throughput screening; pyrroloquinoline quinone.