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.
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.
Chary
V K
VK
Area of Microbiology, Faculty of Biology, University of León, Spain.
de la Fuente
J L
JL
Leitão
A L
AL
Liras
P
P
Martín
J F
JF
D002513:Cephamycins / Q000096:biosynthesis*; D003470:Culture Media; D005822:Genetic Vectors; D051306:L-Lysine 6-Transaminase; D009615:Nocardia / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000378:metabolism; D000637:Transaminases / Q000235:genetics / Q000378:metabolism*; D000516:alpha-Amylases / Q000235:genetics
D002513:Cephamycins; D003470:Culture Media; D011994:Recombinant Proteins; D000637:Transaminases; D051306:L-Lysine 6-Transaminase; D000516:alpha-Amylases; C024925:cephamycin C