Combined Drug Resistance Mutations Substantially Enhance Enzyme Production in Paenibacillus agaridevorans

• Published in: Journal of bacteriology Vol. 200; no. 17
• Main Authors: Funane, Kazumi, Tanaka, Yukinori, Hosaka, Takeshi, Murakami, Kiriko, Miyazaki, Takatsugu, Shiwa, Yuh, Gibu, Shigehachi, Inaoka, Takashi, Kasahara, Ken, Fujita, Nobuyuki, Yoshikawa, Hirofumi, Hiraga, Yoshikazu, Ochi, Kozo
• Format: Journal Article
• Language: English
• Published: United States American Society for Microbiology 01-09-2018
• Subjects: Adenosylmethionine; Bacteriology; Drug resistance; Enzymes; Extracellular matrix; Gene expression; Glycolysis; Metabolites; Mutants; Mutation; Nucleic acids; Paenibacillus; Protein biosynthesis; Protein synthesis; Proteins; Rare earth elements; Ribonucleic acid; Rifampin; RNA; RpoB protein; S-Adenosylmethionine; Scandium; Stationary phase; Stop codon; Streptomycin; ribosome engineering; metabolome analysis; enzyme production; silent genes; drug resistance
• ISSN: 0021-9193; 1098-5530
• DOI: 10.1128/jb.00188-18

This study shows that sequential introduction of drug resistance mutations substantially increased enzyme production in
The triple mutant YT478 (
Gln225→stop codon,
K56R, and
R485H), generated by screening for resistance to streptomycin and rifampin, expressed a 1,100-fold-larger amount of the extracellular enzyme cycloisomaltooligosaccharide glucanotransferase (CITase) than the wild-type strain. These mutants were characterized by higher intracellular
-adenosylmethionine concentrations during exponential phase and enhanced protein synthesis activity during stationary phase. Surprisingly, the maximal expression of CITase mRNA was similar in the wild-type and triple mutant strains, but the mutant showed greater CITase mRNA expression throughout the growth curve, resulting in enzyme overproduction. A metabolome analysis showed that the triple mutant YT478 had higher levels of nucleic acids and glycolysis metabolites than the wild type, indicating that YT478 mutant cells were activated. The production of CITase by the triple mutant was further enhanced by introducing a mutation conferring resistance to the rare earth element, scandium. This combined drug resistance mutation method also effectively enhanced the production of amylases, proteases, and agarases by
and
This method also activated the silent or weak expression of the
CITase gene, as shown by comparisons of the CITase gene loci of
T-3040 and another cycloisomaltooligosaccharide-producing bacterium,
sp. strain 598K. The simplicity and wide applicability of this method should facilitate not only industrial enzyme production but also the identification of dormant enzymes by activating the expression of silent or weakly expressed genes.
Enzyme use has become more widespread in industry. This study evaluated the molecular basis and effectiveness of ribosome engineering in markedly enhancing enzyme production (>1,000-fold). This method, due to its simplicity, wide applicability, and scalability for large-scale production, should facilitate not only industrial enzyme production but also the identification of novel enzymes, because microorganisms contain many silent or weakly expressed genes which encode novel antibiotics or enzymes. Furthermore, this study provides a new mechanism for strain improvement, with a consistent rather than transient high expression of the key gene(s) involved in enzyme production.