Combined Drug Resistance Mutations Substantially Enhance Enzyme Production in Paenibacillus agaridevorans

Abstract

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.

The Role ofPseudomonas aeruginosaRNA Methyltransferases in Antibiotic Resistance

“…RsmG well known as 16S rRNA (guanine 527 -N 7 )-methyltransferase methylates guanine 527 at N 7 in 16S rRNA [36,52] (Table 1) and catalyzes S-adenosyl-L-methionine + guanine 527 in 16S rRNA → S-adenosyl-L-homocysteine + N 7 -methylguanine527 in 16S rRNA (see reaction in UniProt, KEGG, or MetaCyc). Researches in M. tuberculosis reveal that rsmG mutations confer low-level streptomycin resistance; moreover, it has been reported that combining drug resistance mutations of rsmG gene remarkably enhances enzyme production in Paenibacillus agaridevorans [53]. Likewise, for P. aeruginosa, rsmG is conserved in both aminoglycoside-resistant and aminoglycoside-susceptible strains.…”

Substantial improvement of tetraene macrolide production in Streptomyces diastatochromogenes by cumulative drug resistance mutations

“…Nevertheless, quintuple mutant G5-59 generated in the present work grew very well and the mycelia dry weight was even greater than that of wild-type 1628 as examined with 5-L fermentor. The P. agaridevorans triple mutant strain YT 478 also grew as well as the wildtype strain and produced CITase even after entering into stationary-growth phase [32]. These findings suggest that growth characteristic is dependent of strain, although mutant strains were introduced with different drug-resistance.…”

“…Tanaka et al constructed rpsL rsmG double mutants by generating high-level streptomycin-resistant mutants from rsmG mutants [31]. Notably, production of the enzyme cycloisomaltooligosaccharide glucanotransferase (CITase) by Paenibacillus agaridevorans was enhanced dramatically by introducing rsmG, rpsL, and rpoB mutations successively [32]. In S. diastatochromogenes, rsmG mutation was successfully introduced into G3-13 mutant, which has a mutation in rpsL gene.…”

Efficiently activated ε‐poly‐L‐lysine production by multiple antibiotic‐resistance mutations and acidic pH shock optimization in Streptomyces albulus

“…Ribosome engineering is a novel and remarkably efficient tool for screening high‐producing strains (Funane et al., ; Hu & Ochi, ). It can modulate the ribosome by simply inducing mutations that confer resistance to antibiotics (streptomycin, paromomycin, etc.)…”