Expression And Function Of Threonine-biosynthetic Genes In C2C12,NIH-3T3Cells And Transgenic Mice

Metabolic pathways for the synthesis of threonine in animal cells are lost during the evolutionary process, it must be present in diets to support the growth, development and survival of animals. Regarding European diets, threonine is the2nd limiting amino acid and the3rd in broilers. Except for its utilization for supporting normal growth, threonine also improves the immunity function of the animals by promoting the synthesis of intestinal mucin and plasma globulin.In bacteria, five enzymes:aspartate kinase, aspartylsemiaidehyde dehydrogenase, homoserine dehydrogenase, homoserine kinase and threonine synthase are involved in the biosynthesis pathway of L-threonine from L-aspartate. In E. coli, there are three aspartate kinase isoenzymes, and aspartate kinase I is the most abundant which is encoded by thrA. Aspartate kinases Ⅰ and Ⅱ are bifunctional enzymes, with both aspartate kinase activity and homoserine dehydrogenase activity. In C. glutamicum, aspartate kinase encoded by lysC and homoserine dehydrogenase encoded by hom are monofunctional enzymes. In both E. coli and C. glutamicum, aspartylsemiaidehyde dehydrogenase is encoded by asd, homoserine kinase is encoded by thrB and threonine synthase is encoded by thrC.The development of transgenic technology provides a method to repair the nonfunctional biochemical pathways in animals that still exist in bacteria and plants. Here, we tried to introduce the genes involved in the threonine-biosynthetic pathway into mammalian cell lines and mice. We constructed two plasmid systems (pEF1α-IRES-GFP-E4F-his and PEF1α-IRES-GFP-M4F-his), but the genes of E. coli could not express in C2C12or NIH-3T3cells. The sequences optimized for mammals could express in C2C12and NIH-3T3cells, but could not synthesize threonine in mouse cells. We divided the whole pathway into two parts, and added homoserine in the systems. The results showed that threonine could be synthesized from homoserine in NIH-3T3and C2C12cells efficiently. To compare the efficiency of E. coli genes and synthetic genes optimized for mammals, we linked each source of genes encoding homoserine kinase and threonine synthase with the2A peptide to construct pEF1α-IRES-GFP-E2F-his and pEF1α-IRES-GFP-M2F-his plasmids. We electroporated these vectors into C2C12and NIH-3T3cells, and the results showed that there was no difference between these plasmids on the expression. Threonine was formed readily from homoserine using E. coli genes and synthetic genes optimized for mammals. In the C2C12group, the rate of threonine synthesis was much greater in response to transfection with pEF1α-IRES-GFP-E2F-his compared to pEFla-IRES-GFP-M2F-his, but the results were diametrically opposed in the NIH-3T3group. To verify the possibility of synthesis of homoserine from aspartate, we tested the effect of NADPH, cleavage efficiency of2A peptide and monofunctional enzymes. The results showed that the two polypeptides were expressed and cleaved with a high degree of efficiency. When added NADPH, there was no significant increase in threonine production, but peaks representing homoserine appeared in the HPLC trace of transgenic cells provided with NADPH. This suggested that homoserine could be synthesized from aspartate in an NADPH-dependent manner. When introducing the genes involved in pathway of asparate to homoserine in C. glutamicum into mouse cells, no homoserine detected. We generated transgenic mice carrying synthetic genes that optimized thrB and thrC genes for mammals but only one male (No.3) and two female (No.16and No.26) transgenic founders were identified by Southern blot from72new born mice. The efficiency of transgene (4.2%) was very low compared to other genes in our laboratory even with the same plasmid backbone. Analysis of RT-PCR, western blot and feeding experiments showed that foreign genes were not expressed in transgenic mice. This result could be explained if high levels of expression of the threonine-biosynthetic genes in mouse embryos were lethal. The only transgenic animals obtained were those in which the genes had been inserted into the region that prevented their expression. We determined the insertion site of foreign genes by inverse PCR, the results showed that genes inserted in lacZ-tagged of chromosome14and RP23-451D4of chromosome4.We propose that the transgenic technology provides a promising means to enhance the synthesis of nutritionally essential amino acids in farm animals.