Transgenic barley expressing a protein-engineered, thermostable (1,3-1,4)-beta-glucanase during germination.

Abstract

The codon usage of a hybrid bacterial gene encoding a thermostable (1,3-1,4)-13-glucanase was modified to match that of the barley (1,3-1,4)-p-glucanase isoenzyme (4) and their degradation is a prerequisite for the enzymatic mobilization of endosperm storage components, which serve as nutrients for the growing embryo. Efficient degradation of endosperm cell walls is also important for utilization of barley as a monogastric animal feed (5, 6) and in industrial processes such as malting and brewing (7). Furthermore, extraction of non-food products deposited in the endosperm of transgenic barley would be facilitated by the action of highly efficient, heat stable cell wall-degrading enzymes. The (1,3-1,4)-3-glucanases (EC 3.2

The production of recombinant proteins in transgenic barley grains

“…Two of the three Nglycosylation sequons of the heat-stable hybrid (1, 3-1, 4)-␤-glucanases H(A16-M) and H(A12-M)⌬Y13 are glycosylated to different degrees when the protein is expressed in yeast (28,29) or in barley aleurone protoplasts (9). It was therefore of interest to see whether the H(A12-M) ⌬Y13 protein is glycosylated when targeted via the ER and Golgi apparatus into the protein bodies of the endosperm.…”

“…The heatstable ␤-glucanase gene was PCR amplified from plasmid pEII-␣H(A12-M)⌬Y13-GC-NOS (10) by using primer hor͞glu (5Ј-CGAGATGCAGACCGGCGGCAGCTTC-3Ј) and the M13 reverse primer (5Ј-GGTTTTCCCAGTCACGAC-3Ј). These two PCR fragments were spliced together by overlap extension (SOE) (9). The resultant fragment was digested with BamHI and EcoRI and cloned into pUC18 digested with the same two enzymes.…”

“…USA, 10.1073͞pnas.030527497. Article and publication date are at www.pnas.org͞cgi͞doi͞10.1073͞pnas.030527497 codon-optimized (1, 3-1, 4)-␤-glucanase gene (9). The D hordein gene promoter fragment was PCR amplified from a Hor3-1 genomic clone (19) by using the universal forward primer (5Ј-GTAAAACGACGGCCAGTG-3Ј) and primer dhor3 (5ЈCGGTCTGCATCTCGGTGGACTGTC-3Ј).…”

“…The stored carbohydrates, oils, and proteins in the kernels are exploited for feed, food, and beverages, both in the form of mature grain and as malt produced in large scale by steeping, germination, and kiln drying (1,2). Advances in the molecular genetics of barley storage proteins (3,4), elucidation of mechanisms by which storage proteins are synthesized, targeted, and sequestered for long-term storage in the endosperm cells (5,6), and establishment of genetic transformation procedures (7)(8)(9)(10) provide the basis for studying the production of recombinant proteins in the developing barley grain. The unique capacity of the scutellum and aleurone cells to synthesize hydrolytic enzymes during germination͞malting and secrete them into the starchy endosperm (11,12) provides an additional avenue for the production of recombinant proteins in this cereal (9,10).…”

Synthetic gene design—The rationale for codon optimization and implications for molecular pharming in plants

“…However, altering the gene for preferred (i.e. more frequently used) codons of barley (141 of 215 codons changed) led to successful expression of the enzyme during germination (Jensen et al 1996).…”