[P004] Poster

1:00 PM - 1:50 PM JST(4:00 AM - 4:50 AM UTC)

[P02-908] Molecular breeding of Citrobacter freundii IFO13545 for the improvement of the bioflocculant production using transposon vector pKTLOF

○Masahiro Takeo¹, Yuriko Matsuo¹, Mana Morishige¹, Koki Miyamoto¹, Hidehiro Ishizawa¹, Seiji Negoro¹ (1. Graduate School of Engineering, University of Hyogo (Japan))

Keywords:

bioflocculant,Citrobacter,Gene integration,polysaccharide,production

[Purpose]
Citrobacter freundii IFO13545 can produce a chitosan-like bioflocculant (BF) from acetate, and we are trying the practical production. To improve the BF production, molecular breeding of the BF-producing strain has been conducted using a transposon vector pKTLOF, which was developed for the integration of useful genes into its chromosome. In this study, we introduce the results of the molecular breeding experiments.
[Methods]
C. freundii IFOΔtri, which is a gene-deleted derivative of IFO13545 (ΔpfkA, ΔnagB, and Δzwf), was used in this study. It showed 3.4-fold higher flocculation activity than the original strain. IFOΔtri was routinely cultivated in a mineral salts medium including sodium acetate (10 g/L) and yeast extract (0.1 g/L) at 30 ℃ or 37 ℃ and at 150 rpm on a rotary shaker. Flocculation activity of the culture supernatant was measured using kaolin suspension and expressed as flocculation titer as described previously (Appl. Microbiol. Biotechnol., 97, 9569, 2013). Gene integration into the host chromosome was conducted using pKTLOF (10.2kb) consisting of the temp.-sensitive replicon of pKD46 (PNAS, 97, 6640, 2000), the transposon system of pLOFKm (J. Bacteriol., 172, 6557, 1990), and the arabinose-inducible gene expression system (P_BAD and araC) of pTJ1 (Appl. Environ. Microbiol., 79, 718, 2013). Plasmids were introduced into IFOΔtri by electroporation.
[Results]
The glmUSM (encoding enzymes for the hexosamine synthetic pathway) and bfpABCD (encoding proteins for the polymerization and secretion of the BF) genes of IFO13545 were independently cloned into pKTLOF by PCR and in-fusion cloning techniques. Then, the recombinant plasmids were introduced into IFOΔtri. Through the chromosome-integration and plasmid removal processes, the chromosome-integrated strains, IFOΔtri (glmUSM), IFOΔtri (bfpABCD), and IFOΔtri (glmUSM/bfpABCD) were obtained. They showed 2.3-fold, 1.9-fold, and 4.2-fold higher flocculation activities than that of IFOΔtri under arabinose-induced conditions, respectively. Furthermore, the wspR gene of Pseudomonas aeruginosa (PLOS Biology, 6, e67, 2008), whose gene product produces cyclic-di-GMP stimulating the polymerization and secretion of the BF, was also inserted into the chromosome of IFOΔtri (glmUSM/bfpABCD) in the same manner. The resulting triple-integrated strain, IFOΔtri (glmUSM/bfpABCD/wspR) showed 5.9-fold higher activity than that of IFOΔtri, which corresponds to 20-fold higher activity of the original strain IFO13545.
[Consideration]
We successfully constructed recombinant Citrobacter strains with higher flocculation activity using this chromosome-integration system, which can be repeatedly used in one strain. As the amount of the BF of the original strain IFO13545 was previously evaluated by ELISA using a standard chitosan solution and determined to be approx. 0.46 g-dry weight-chitosan/L (IJBMBS, 4, 76, 2017). Based on this amount, the amount of the triple-integrated strain can be estimated as 9.2 g-dry weight-chitosan/L.
[Conclusion]
In this study, to improve the BF production of C. freundii IFO13545, several useful genes were introduced into the chromosome of its derivative strain IFOΔtri using a transposon vector pKTLOF, and finally a recombinant strain with 20-fold higher flocculation activity than that of IFO13545 was obtained.