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Analysis of polyhydroxyalkanoate (PHA) synthase gene in activated sludge that produces PHA containing 3‐hydroxy‐2‐methylvalerate
Author(s) -
Michinaka Atsuko,
Arou Jun,
Onuki Motoharu,
Satoh Hiroyasu,
Mino Takashi
Publication year - 2006
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.21085
Subject(s) - activated sludge , polyhydroxyalkanoates , restriction fragment length polymorphism , biology , terminal restriction fragment length polymorphism , cloning (programming) , gene , molecular cloning , biochemistry , microbiology and biotechnology , polymerase chain reaction , genetics , bacteria , peptide sequence , sewage treatment , computer science , waste management , engineering , programming language
The identification of phaC which encodes PHA synthase, that is involved in the formation of polyhydroxyalkanoate (PHA) containing 3‐hydroxy‐2‐methylvalerate (3H2MV), was attempted. As of now, PHA containing 3H2MV has been reported to be produced only by mixed microbial cultures in activated sludge, but no pure bacterial cultures. A laboratory‐scale activated sludge process was operated for 67 days. During the operation of the activated sludge process, its capacity to produce PHA containing 3H2MV, and the diversity of the partial phaC genes in the activated sludge microorganisms were monitored periodically. Analysis of the partial phaC genes was conducted by PCR followed by cloning and DNA sequencing, or by PCR followed by terminal‐restriction fragment length polymorphism (T‐RFLP). The cloning‐sequencing of the 263 clones gave 11 distinct genetic groups (GGs). All of the genetic groups had similarities to known phaC higher than 48%, and one of them had similarity as high as 96% to that of Alcaligenes sp. The behavior of each of the genetic groups during the operation of the activated sludge process was monitored by the T‐RFLP method. The restriction enzyme Acc II, with the help of Mbo I, enabled the monitoring of each of the genetic groups. One of the genetic groups was found to have a strong correlation with the capability of the activated sludge to produce PHA containing 3H2MV, and its DNA sequence together with its amino acid sequence are reported. Biotechnol. Bioeng. 2007;96:871–880. © 2006 Wiley Periodicals, Inc.