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原文The overexpression of folA caused a two- fold decrease in folate production compared to that in a con- trol strain or a noninduced strain (Fig. 3). The intracellular folate distribution and the relative amount of polyglutamyl folates remained unchanged. In a similar experiment we stud- ied the effect of the production of antisense RNA encoding dihydrofolate reductase. The complementary sequence of the coding strand of folA was cloned under the control of the nisin promoter nisA in pNZ8048, starting at the 5 end with the complementary sequence of the stop codon of folA and finish- ing at the 3 end with the complementary start codon of the gene. The plasmid generated, pNZ7014, was transformed into L. lactis NZ9000. Under inducing conditions, a small but re- producible increase of approximately 20% in the total folate production was observed compared to that in a control strain (results not shown). To confirm the effect of the transcription of folA antisense RNA, the enzymatic activity of dihydrofolate reductase was determined. Cell extracts of strain NZ9000 har- boring pNZ7014, transcribing antisense RNA of folA, showed a twofold decrease in dihydrofolate reductase activity (Fig. 4). In contrast, cell extracts of L. lactis strains overexpressing folA showed a more-than-1,000-fold increase in dihydrofolate re- ductase activity compared to a control strain |
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