Cloning and characterization of the mutated threonine operon (thrA(1)5A(2)5BC) of Serratia marcescens

The entire threonine operon (thrA(1)5A(2)5BC) of Serratia marcescens TLr156, which lacks threonine-mediated feedback inhibition of both aspartokinase I (AK I) and homoserine dehydrogenase I (HD I), was cloned on a multicopy plasmid pLG339. Hybrid plasmid pSK301 carried a 6.5-kb chromosomal DNA. Several derivatives of pSK301 with Tn1000 insertions were obtained. By examining the phenotypes and the physical maps of these plasmids, we could define the loci of the thrA(1)5A(2)5, thrB, and thrC genes. The thrA(1)5A(2)5 and thrC gene products were identified by the maxicell method as proteins with Mrs of 85,000 and 43,000, respectively. The thrA(1)5A(2)5 genes encode a single polypeptide similar to the thrA1A2 genes of Escherichia coli. Plasmid pSK301 was introduced into S. marcescens T-1112, in which both AK I and HD I are produced constitutively. The resulting transformant carried five to six copies of pSK301 per chromosome and produced the AK I and HD I enzymes at three to four times higher level than control strain T-1112pLG339]. Strain T-1112pSK301] produced four times higher levels of threonine than strain T-1112pLG339], yielding about 35 mg of threonine per ml of a medium containing sucrose and urea.