SUSTAINED AND CONSTITUTIVE HIGH-LEVELS OF PROTEIN-PRODUCTION IN CONTINUOUS CULTURES OF BACILLUS-SUBTILIS

The feasibility of continuous production of proteins in chemostat cult ures of Bacillus subtilis was investigated. An expression system consi sting of the bacterium B. subtilis BR151 carrying plasmid p602/19 was used. The plasmid contains the cat (chloramphenicol acetyltransferase) gene downstream of a strong vegetative T5 promoter. It was found that , at a dilution rate of 0.2 h(-1), production of relatively high level s of CAT protein (about 4% of cellular protein) can be sustained. But, experiments at a higher dilution rate of 0.4 h(-1) were unproductive because of high acid formation and washout. Combination of low cell yi eld, which results from excessive acid formation, and low dilution rat e led to a low volumetric CAT productivity. Our recent work with the n onrecombinant cells, has demonstrated that uptake of small amounts of citrate significantly reduces or entirely eliminates the acid formatio n. This superior performance in the presence of citrate was hypothesiz ed, based on strong experimental evidence, to be the result of a reduc tion in glycolysis flux through a sequence of events leading to a redu ction in pyruvate kinase and phosphofructokinase activities, the regul atory enzymes of glycolysis. In this study, it is demonstrated that co feeding of glucose and citrate substantially reduces the organic acid formation and significantly increases the recombinant culture producti vity. The combination of high specific CAT activity and cell density r esulted in a total of six- to tenfold higher culture productivity when citrate and glucose were cometabolized than when glucose was the only carbon source. (C) 1995 John Wiley and Sons, Inc.