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Overexpression of the lat gene in Nocardia lactamdurans from strong heterologous promoters results in very high levels of lysine-6-aminotransferase and up to two-fold increase in cephamycin C production. 2000

V K Chary, and J L de la Fuente, and A L Leitão, and P Liras, and J F Martín
Area of Microbiology, Faculty of Biology, University of León, Spain.

The level of lysine-6-aminotransferase (encoded by the lat gene), an enzyme that commits lysine to the cephamycin biosynthesis pathway, is very low in wild type Nocardia lactamdurans. Two lat overexpression systems (pAMEXlat and pSAFlat) were constructed to express the promoterless lat gene of N. lactamdurans from the strong promoters amyP (of the alpha-amylase gene) and safP (of the secretion activating factor gene) of Streptomyces griseus. Both constructions led to very high levels of lysine-6-aminotransferase (between 8- and 15-fold) in the cells. Expression of lat from the amy promoter was optimal in glycerol-containing medium and was negatively regulated by glucose. The high levels of lysine-6-aminotransferase resulted in a 50-200% increase in cephamycin C production in the standard fermentation conditions. Onset of cephamycin C biosynthesis occurred at the same time in control and in lat-overexpressing strains, but the cephamycin production rate was clearly higher in transformants overexpressing the lat gene. Furthermore, HPLC analysis of cephamycin C in the culture broths revealed an early depletion of biosynthetic intermediates and an accumulation of cephamycin C when the lat gene was overexpressed. These results indicate that lysine-6-aminotransferase activity is limiting for cephamycin C biosynthesis under some culture conditions.

UI MeSH Term Description Entries
D009615 Nocardia A genus of gram-positive, aerobic bacteria whose species are widely distributed and are abundant in soil. Some strains are pathogenic opportunists for humans and animals. Micropolyspora
D011401 Promoter Regions, Genetic DNA sequences which are recognized (directly or indirectly) and bound by a DNA-dependent RNA polymerase during the initiation of transcription. Highly conserved sequences within the promoter include the Pribnow box in bacteria and the TATA BOX in eukaryotes. rRNA Promoter,Early Promoters, Genetic,Late Promoters, Genetic,Middle Promoters, Genetic,Promoter Regions,Promoter, Genetic,Promotor Regions,Promotor, Genetic,Pseudopromoter, Genetic,Early Promoter, Genetic,Genetic Late Promoter,Genetic Middle Promoters,Genetic Promoter,Genetic Promoter Region,Genetic Promoter Regions,Genetic Promoters,Genetic Promotor,Genetic Promotors,Genetic Pseudopromoter,Genetic Pseudopromoters,Late Promoter, Genetic,Middle Promoter, Genetic,Promoter Region,Promoter Region, Genetic,Promoter, Genetic Early,Promoter, rRNA,Promoters, Genetic,Promoters, Genetic Middle,Promoters, rRNA,Promotor Region,Promotors, Genetic,Pseudopromoters, Genetic,Region, Genetic Promoter,Region, Promoter,Region, Promotor,Regions, Genetic Promoter,Regions, Promoter,Regions, Promotor,rRNA Promoters
D011994 Recombinant Proteins Proteins prepared by recombinant DNA technology. Biosynthetic Protein,Biosynthetic Proteins,DNA Recombinant Proteins,Recombinant Protein,Proteins, Biosynthetic,Proteins, Recombinant DNA,DNA Proteins, Recombinant,Protein, Biosynthetic,Protein, Recombinant,Proteins, DNA Recombinant,Proteins, Recombinant,Recombinant DNA Proteins,Recombinant Proteins, DNA
D002513 Cephamycins Naturally occurring family of beta-lactam cephalosporin-type antibiotics having a 7-methoxy group and possessing marked resistance to the action of beta-lactamases from gram-positive and gram-negative organisms. Antibiotics, Cephamycin,Cephamycin,Cephamycin Antibiotics
D003470 Culture Media Any liquid or solid preparation made specifically for the growth, storage, or transport of microorganisms or other types of cells. The variety of media that exist allow for the culturing of specific microorganisms and cell types, such as differential media, selective media, test media, and defined media. Solid media consist of liquid media that have been solidified with an agent such as AGAR or GELATIN. Media, Culture
D005822 Genetic Vectors DNA molecules capable of autonomous replication within a host cell and into which other DNA sequences can be inserted and thus amplified. Many are derived from PLASMIDS; BACTERIOPHAGES; or VIRUSES. They are used for transporting foreign genes into recipient cells. Genetic vectors possess a functional replicator site and contain GENETIC MARKERS to facilitate their selective recognition. Cloning Vectors,Shuttle Vectors,Vectors, Genetic,Cloning Vector,Genetic Vector,Shuttle Vector,Vector, Cloning,Vector, Genetic,Vector, Shuttle,Vectors, Cloning,Vectors, Shuttle
D000516 alpha-Amylases Enzymes that catalyze the endohydrolysis of 1,4-alpha-glycosidic linkages in STARCH; GLYCOGEN; and related POLYSACCHARIDES and OLIGOSACCHARIDES containing 3 or more 1,4-alpha-linked D-glucose units. Taka-Amylase A,alpha-Amylase,Alpha-Amylase Bayer,Maxilase,Mégamylase,alpha-1,4-D-Glucanglucanohydrolase,Alpha Amylase Bayer,AlphaAmylase Bayer,Taka Amylase A,TakaAmylase A,alpha 1,4 D Glucanglucanohydrolase,alpha Amylase,alpha Amylases
D000637 Transaminases A subclass of enzymes of the transferase class that catalyze the transfer of an amino group from a donor (generally an amino acid) to an acceptor (generally a 2-keto acid). Most of these enzymes are pyridoxyl phosphate proteins. (Dorland, 28th ed) EC 2.6.1. Aminotransferase,Aminotransferases,Transaminase
D051306 L-Lysine 6-Transaminase A PYRIDOXAL PHOSPHATE containing enzyme that catalyzes the transfer of amino group of L-LYSINE onto 2-oxoglutarate to generate 2-aminoadipate 6-semialdehyde and L-GLUTAMATE. Lysine 2-Oxoglutarate 6-Aminotransferase,Lysine 6-Aminotransferase,Lysine epsilon-Transaminase,Lysine-6-Aminotransferase,Lysine-epsilon Aminotransferase,2-Oxoglutarate 6-Aminotransferase, Lysine,6-Aminotransferase, Lysine,6-Aminotransferase, Lysine 2-Oxoglutarate,6-Transaminase, L-Lysine,Aminotransferase, Lysine-epsilon,L Lysine 6 Transaminase,Lysine 2 Oxoglutarate 6 Aminotransferase,Lysine 6 Aminotransferase,Lysine epsilon Aminotransferase,Lysine epsilon Transaminase,epsilon-Transaminase, Lysine

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