Expression of threonine-biosynthetic genes in mammalian cells and transgenic mice

Abstract

Threonine is a nutritionally essential amino acid (EAA) for the growth and development of humans and other nonruminant animals and must be provided in diets to sustain life. The aim of this study was to synthesize threonine in mammalian cells through transgenic techniques. To achieve this goal, we combined the genes involved in bacterial threonine biosynthesis pathways into a single open reading frame separated by self-cleaving peptides (2A) and then linked it into a transposon system (piggyBac). The plasmids pEF1a-IRES-GFP-E2F-his and pEF1a-IRES-GFP-M2F-his expressed Escherichia coli homoserine kinase and threonine synthase efficiently in mouse cells and enabled cells to synthesize threonine from homoserine. This biosynthetic pathway occurred with a low level of efficiency in transgenic mice. Three transgenic mice were identified by Southern blot from 72 newborn mice, raising the possibility that a high level of expression of these genes in mouse embryos might be lethal. The results indicated that it is feasible to synthesize threonine in animal cells using genetic engineering technology. Further work is required to improve the efficiency of this method for introducing genes into mammals. We propose that the transgenic technology provides a promising means to enhance the synthesis of nutritionally EAAs in farm animals and to eliminate or reduce supplementation of these nutrients in diets for livestock, poultry and fish.

Highly efficient transgenic mouse production using piggyBac and its application to rapid phenotyping at the founder generation

“…The previously described methods and ours differ in terms of how mPBase is introduced. In several previous studies, mPBase expression plasmid vector was introduced into the cytoplasm of unfertilized eggs or the pronucleus of fertilized eggs 15,[40][41][42] . Urschits et al showed that mPBase protein expression was at the background level until 6 h after injection of the expression plasmid, and peak expression was observed at about 30 h 40 .…”

Application of new biotechnologies for improvements in swine nutrition and pork production

“…Potentially, pigs that express plant or microbial genes for the synthesis of nutritionally essential amino acids can permit the feeding of low-P and low-protein diets without the need for dietary supplementation with P or crystalline amino acids. One of those amino acids is threonine [57], which is low in plant-source feedstuffs relative to piglet growth [58].…”

Resurrecting essential amino acid biosynthesis in mammalian cells

“…Interestingly, independent evidence for BCAA biosynthesis has also been obtained for sap-feeding whitefly bacteriocytes that host bacterial endosymbionts; metabolite sharing between these cells is predicted to lead to biosynthesis of BCAAs that are limiting in their restricted diet. The malleability of mammalian metabolism to accept heterologous core pathways opens up the possibility of animals with designer metabolisms and enhanced capacities to thrive under environmental stress and nutritional starvation ( Zhang et al, 2014 ). Yet, our failure to functionalize designed methionine, threonine, and isoleucine pathways highlights outstanding challenges and future directions for synthetic metabolism engineering in animal cells and animals.…”