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Abstract

We have designed protein molecules based on an alpha-helical coiled-coil structure. These proteins can be tailored to complement nutritionally unbalanced seed meals. In particular, these proteins may contain up to 43% mol/mol of the essential amino acid lysine. Genes encoding such proteins were constructed using synthetic oligonucleotides and the protein stability was tested for in vivo by expression in an Escherichia coli model system. A protein containing 31% lysine and 20% methionine (CP 3-5) was expressed in transgenic tobacco seeds utilizing the seed specific bean phaseolin and soybean beta-conglycinin promoters. Both promoters provided a level of expression in the mature transgenic tobacco seeds which resulted in a significant increase in the total lysine content of the seeds. Several of these transgenic lines were analyzed for three generations to determine the stability of gene expression. Plants transformed with the soybean beta-conglycinin promoter/CP 3-5 gene consistently expressed the high-lysine phenotype through three generations. However, expression of the high-lysine phenotype in plants transformed with the bean phaseolin/CP 3-5 was variable. This is the first report of a significant increase in seed lysine content due to the seed-specific expression of a de novo protein sequence.

Improving protein content and quality by over-expressing artificially synthetic fusion proteins with high lysine and threonine constituent in rice plants

“…As a result, lysine content in seeds was increased in these transgenic tobacco seeds carrying one of these genes. However, lysine content was increased by less than 20% 26 , which might be due to that these genes encoded high lysine proteins with very small molecular weight (3–7 kDa). In this study, lysine/threonine-rich fragments consist of 73% and 83% of total amino acids with molecular weight at 78.7 kDa and 68.7 kDa in both TKTKK1 and TKTKK2 genes, respectively.…”

“…The application of synthetic biology on improving protein quality has been carried out long time ago. For example, several genes have been designed according to an alpha-helical coiled-coil structure and these genes encoded high lysine proteins 26 . As a result, lysine content in seeds was increased in these transgenic tobacco seeds carrying one of these genes.…”

“…However, even in artificially modified genes, the percentage of lysine or threonine among the total amino acids was still not high due to the instability of the modified proteins. For example, in the synthetic gene CP 3-5 , it encodes only 31% lysine and 20% methionine 26 . In this study, we increased lysine and threonine percentages to 48.1% and 25.0%, respectively, by fusing a TKTKK coding motifs to endogenous rice genes.…”

“…They were used for improving lysine content in rice and transgenic plants showed up to 35% increase in its content 24 . Besides these naturally evolved lysine-rich encoding genes, artificially synthetic or modified genes were also used for lysine, methionine, or cysteine enrichment in potato 25 , tobacco 26 or soybean 27 .…”

Transgenic high-lysine rice – a realistic solution to malnutrition?

“…Expression in soybean and canola seeds led to a notable increase of seed free lysine ( Falco et al , 1995 ). Among these early studies, Keeler et al (1997) also indicated that de novo expression of alpha-helical coiled-coil proteins might also increase the accumulation of lysine in mature tobacco seeds.…”