CRISPR-Cas9 mediated construction of a food-grade pyrroloquinoline quinone high-yielding Acetobacter pasteurianus Ab3 strain

Abstract

Pyrroloquinoline quinone (PQQ) is believed to be a new B vitamin compound, and dietary PQQ has received more attention as a potential treatment for diseases including inflammatory injury and obesity. It widely exists in foods related to acetic acid bacteria fermentation like fermented vinegars. To obtain a food-grade PQQ high-yielding strain, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (CRISPR/Cas)-based genome editing system was applied to genetically manipulate Acetobacter pasteurianus Ab3 for utilizing in industrial vinegar fermentation. Its PQQ biosynthetic genes were first characterized with conserved pqqA gene and pqqBCDE operon, and tldD gene instead of pqqF/G. Besides, our results revealed that pqqBCDE but not pqqA was the major limiting factor of improving PQQ production. Furthermore, endogenous promoters were screened from transcriptome data and evaluated their strength of improving PQQ production. The promoter P2690 showed the best efficiency and was selected to overexpress pqqBCDE operon. The engineered strain A. pasteurianus Ab3:P2690 produced 12.74 μg/mL of PQQ, which was 9.1-fold higher than that of WT strain. The potential of PQQ production of the engineering strain A. pasteurianus Ab3:P2690 was further investigated by the semi-continuous fermentation in a 3 L fermentation tank. After 240 h of fermentation, the concentration of PQQ reached 41.96 μg/mL. To our knowledge, this is the greatest PQQ yield reported so far using A. pasteurianus as a host strain. This study provides a practical approach to construct PQQ high-yielding A. pasteurianus strain that can be used in food industry and enhanced the health-promoting properties of vinegars.