Gene manipulation in the Mucorales fungus Rhizopus oryzae using TALENs with exonuclease overexpression.

The Mucorales fungal genus Rhizopus is used for the industrial production of organic acids, enzymes and fermented foods. The metabolic engineering efficiency of Rhizopus could be improved using gene manipulation; however, exogenous DNA rarely integrates into the host genome. Consequently, a genetic tool for Mucorales fungi needs to be developed. Recently, programmable nucleases that generate DNA double-strand breaks (DSBs) at specific genomic loci have been used for genome editing in various organisms. In this study, we examined gene disruption in Rhizopus oryzae using transcription activator-like effector nucleases (TALENs), with and without exonuclease overexpression. TALENs with an overexpressing exonuclease induced DSBs, followed by target site deletions. Although DSBs are repaired mainly by nonhomologous end joining in most organisms, our results suggested that in R. oryzae microhomology-mediated end joining was the major DSB repair system. Our gene manipulation method using TALENs coupled with exonuclease overexpression contributes to basic scientific knowledge and the metabolic engineering of Rhizopus.

Dependence of swarming in Escherichia coli K-12 on spermidine and the spermidine importer.

In a previous work, it was observed that the swarming of polyamine-deficient Proteus mirabilis (speB::sm) was severely inhibited on Luria-Bertani (LB) swarming plates (LBSw) (LB, 0.5% glucose, 0.5% agar), and it was clarified that extracellular putrescine was important as a signaling molecule for the induction of swarming in P. mirabilis. However, a polyamine-deficient strain (delta-speAB delta-speC) of Escherichia coli swarmed as well as the parental strain on LBSw plates. We report that the swarming phenotype of a polyamine-deficient E. coli strain is dependent on spermidine and PotABCD, a spermidine importer.

The putrescine Importer PuuP of Escherichia coli K-12.

The Puu pathway is a putrescine utilization pathway involving gamma-glutamyl intermediates. The genes encoding the enzymes of the Puu pathway form a gene cluster, the puu gene cluster, and puuP is one of the genes in this cluster. In Escherichia coli, three putrescine importers, PotFGHI, PotABCD, and PotE, were discovered in the 1990s and have been studied; however, PuuP had not been discovered previously. This paper shows that PuuP is a novel putrescine importer whose kinetic parameters are equivalent to those of the polyamine importers discovered previously. A puuP(+) strain absorbed up to 5 mM putrescine from the medium, but a DeltapuuP strain did not. E. coli strain MA261 has been used in previous studies of polyamine transporters, but PuuP had not been identified previously. It was revealed that the puuP gene of MA261 was inactivated by a point mutation. When E. coli was grown on minimal medium supplemented with putrescine as the sole carbon or nitrogen source, only PuuP among the polyamine importers was required. puuP was expressed strongly when putrescine was added to the medium or when the puuR gene, which encodes a putative repressor, was deleted. When E. coli was grown in M9-tryptone medium, PuuP was expressed mainly in the exponential growth phase, and PotFGHI was expressed independently of the growth phase.

gamma-Glutamylputrescine synthetase in the putrescine utilization pathway of Escherichia coli K-12.

Glutamate-putrescine ligase (gamma-glutamylputrescine synthetase, PuuA, EC 6.3.1.11) catalyzes the gamma-glutamylation of putrescine, the first step in a novel putrescine utilization pathway involving gamma-glutamylated intermediates, the Puu pathway, in Escherichia coli. In this report, the character and physiological importance of PuuA are described. Purified non-tagged PuuA catalyzed the ATP-dependent gamma-glutamylation of putrescine. The K(m) values for glutamate, ATP, and putrescine are 2.07, 2.35, and 44.6 mm, respectively. There are two putrescine utilization pathways in E. coli: the Puu pathway and the pathway without gamma-glutamylation. Gene deletion experiments of puuA, however, indicated that the Puu pathway was more critical in utilizing putrescine as a sole carbon or nitrogen source. The transcription of puuA was induced by putrescine and in a puuR-deleted strain. The amino acid sequences of PuuA and glutamine synthetase (GS) show high similarity. The molecular weights of the monomers of the two enzymes are quite similar, and PuuA exists as a dodecamer as does GS. Moreover the two amino acid residues of E. coli GS that are important for the metal-catalyzed oxidation of the enzyme molecule involved in protein turnover are conserved in PuuA, and it was experimentally shown that the corresponding amino acid residues in PuuA were involved in the metal-catalyzed oxidation similarly to GS. It is suggested that the intracellular concentration of putrescine is optimized by PuuA transcriptionally and posttranslationally and that excess putrescine is converted to a nutrient source by the Puu pathway.