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Overexpression and functional characterization of an Aspergillus niger phytase in the fat body of transgenic silkworm, Bombyx mori

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Abstract

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.

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Acknowledgments

We thank Dr. Marcé D. Lorenzen of North Carolina State University, USA, for improving this manuscript. This work was supported by the Grant (No. 2012CB114600) from the National Basic Research Program of China, Grant (No. 31000981) from National Natural Science Foundation of China, and Grant (No. CSTC2010BB1144) from the Natural Science Foundation of Chongqing.

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Authors and Affiliations

  1. State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing, 400715, China

    Hanfu Xu, Yaowen Liu, Feng Wang, Lin Yuan, Yuancheng Wang, Sanyuan Ma & QingYou Xia

  2. State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, 650223, China

    Yaowen Liu

  3. Department of Neurobiology and Developmental Sciences, College of Medicine, University of Arkansas for Medical Sciences, 4301 West Markham St., #510, Little Rock, AR, 72205, USA

    Helen Beneš

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  1. Hanfu Xu
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  2. Yaowen Liu
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  3. Feng Wang
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  4. Lin Yuan
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Corresponding authors

Correspondence to Helen Beneš or QingYou Xia.

Additional information

Hanfu Xu and Yaowen Liu have contributed equally to this work.

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11248_2014_9797_MOESM1_ESM.tif

Fig. S1 Sequence of the codon-optimized A. niger phytase gene. (A) Synthesized nucleotide sequence of the phyA gene. Lower-case letters indicate BamH I and Not I restriction enzyme sites. Underlined sequences indicate the six His-tags. (B) Deduced amino acid sequence of phyA. Putative N-glycosylation sites, active sites of phyA and six His-tags are shown in green, red and yellow color, respectively. (TIFF 3994 kb)

11248_2014_9797_MOESM2_ESM.tif

Fig. S2 Southern blot and insertion analysis of transgenic silkworms. (A) Samples of genomic DNA extracted from G1 transgenic and wild-type silkworms were fully digested with Bgl II (lane 1, control), Bgl II (lane 2) and BamH I (lane 3), and subjected to Southern blot analysis with a DsRed probe. (B) The inverse PCR products (lane 1) was separated on a 1 % agarose gel and visualized by staining with ethidium bromide. (C) The flanking genomic sequences obtained with insertion site TTAA on the piggyBac left arm and piggyBac right arm. The insertion site was located in the Scaffold nscaf416 of Chromosome 14 of a transgenic silkworm. (TIFF 335 kb)

11248_2014_9797_MOESM3_ESM.tif

Fig. S3 Western blot analysis of recombinant phyA in transgenic silkworms. Protein samples were extracted from the fat body of G2 transgenic and wild-type day-2 pupae. The filter was probed using an anti-phyA antibody, then stripped and re-probed using an anti α-tubulin antibody (α-tub). M, protein molecular weight marker; Lane 1, wild-type pupae; Lane 2, transgenic pupae. (TIFF 767 kb)

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Xu, H., Liu, Y., Wang, F. et al. Overexpression and functional characterization of an Aspergillus niger phytase in the fat body of transgenic silkworm, Bombyx mori . Transgenic Res 23, 669–677 (2014). https://doi.org/10.1007/s11248-014-9797-9

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