Directed molecular evolution of nitrite oxido-reductase by DNA-shuffling.
OBJECTIVE
To develop directly molecular evolution of nitrite oxido-reductase using DNA-shuffling technique because nitrobacteria grow extremely slow and are unable to nitrify effectively inorganic nitrogen in wastewater treatment.


METHODS
The norB gene coding the ndtrite oxido-reductase in nitrobacteria was cloned and sequenced. Then, directed molecular evolution of nitrite oxido-reductase was developed by DNA-shuffling of 15 norB genes from different nitrobacteria.


RESULTS
After DNA-shuffling with sexual PCR and staggered extension process PCR, the sequence was different from its parental DNA fragments and the homology ranged from 98% to 99%. The maximum nitrification rate of the modified bacterium of X16 by DNA-shuffling was up to 42.9 mg/L x d, which was almost 10 times higher than that of its parental bacteria. Furthermore, the modified bacterium had the same characteristics of its parental bacteria of E. coli and could grow rapidly in normal cultures.


CONCLUSIONS
DNA-shuffling was successfully used to engineer E. coli, which had norB gene and could degrade inorganic nitrogen effectively.
Li
Jun-Wen
JW
Institute of Environment and Health, 1 Dali Road, Tianjin 300050, China. junwen9999@hotmail.com
Zheng
Jin-Lai
JL
Wang
Xin-Wei
XW
Jin
Min
M
Chao
Fu-Huan
FH
D003001:Cloning, Molecular; D032621:DNA Shuffling; D020564:Deltaproteobacteria / Q000201:enzymology* / Q000235:genetics; D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000235:genetics*; D020563:Gammaproteobacteria / Q000201:enzymology* / Q000235:genetics; D009572:Nitrite Reductases / Q000737:chemistry / Q000235:genetics*; D009584:Nitrogen / Q000378:metabolism; D010802:Phylogeny; D017422:Sequence Analysis, DNA
D009572:Nitrite Reductases; D009584:Nitrogen