Overexpression and functional characterization of an Aspergillus niger phytase in the fat body of transgenic silkworm, Bombyx mori.
In a previous study, we isolated 1,119 bp of upstream promoter sequence from Bmlp3, a gene encoding a member of the silkworm 30 K storage protein family, and demonstrated that it was sufficient to direct fat body-specific expression of a reporter gene in a transgenic silkworm, thus highlighting the potential use of this promoter for both functional genomics research and biotechnology applications. To test whether the Bmlp3 promoter can be used to produce recombinant proteins in the fat body of silkworm pupae, we generated a transgenic line of Bombyx mori which harbors a codon-optimized Aspergillus niger phytase gene (phyA) under the control of the Bmlp3 promoter. Here we show that the Bmlp3 promoter drives high levels of phyA expression in the fat body, and that the recombinant phyA protein is highly active (99.05 and 54.80 U/g in fat body extracts and fresh pupa, respectively). We also show that the recombinant phyA has two optimum pH ranges (1.5-2.0 and 5.5-6.0), and two optimum temperatures (55 and 37 °C). The activity of recombinant phyA was lost after high-temperature drying, but treating with boiling water was less harmful, its residual activity was approximately 84% of the level observed in untreated samples. These results offer an opportunity not only for better utilization of large amounts of silkworm pupae generated during silk production, but also provide a novel method for mass production of low-cost recombinant phytase using transgenic silkworms.
State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing, 400715, China.
Hanfu
H
Xu
Yaowen
Y
Liu
Feng
F
Wang
Lin
L
Yuan
Yuancheng
Y
Wang
Sanyuan
S
Ma
Helen
H
Beneš
QingYou
Q
Xia
D010832:6-Phytase / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D001234:Aspergillus niger / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D015139:Blotting, Southern; D015153:Blotting, Western; D012831:Bombyx / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D005216:Fat Body / Q000378:metabolism*; D005822:Genetic Vectors; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D012333:RNA, Messenger; D011994:Recombinant Proteins; D010832:6-Phytase