Sustained and constitutive high levels of protein production in continuous cultures of bacillus subtilis.

Abstract:

:The feasibility of continuous production of proteins in chemostat cultures of Bacillus subtilis was investigated. An expression system consisting of the bacterium B. subtilis BR151 carrying plasmid p602/19 was used. The plasmid contains the cat (chioramphenicol acetyltrans-ferase) 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 levels of CAT protein (about 4% ofcellular protein) can be sustained. But, experiments at a higher dilution rate of 0.4 h(-1) were unproductive because of high acidformation and washout. Combination of low cell yield, which results from excessive acid formation, and low dilution rate led to a low volumetric CAT productivity. Our recent work with the nonrecombinant cells, has demonstrated that uptake of small amounts of citrate significantly reduces or entirelyeliminates the acid formation. This superior performance in the presence ofcitrate was hypothesized, based on strong experimental evidence, to be the result of a reduction in glycolysis flux through a sequence of events leading to a reduction in pyruvate kinase and phosphof- ructokinase activities, the regulatory enzymes of glycol-ysis. In this study, it is demonstrated that cofeeding of glucose and citrate substantially reduces theorganic acid formation and significantly increases the recombinant culture productivity. The combination of high specific CAT activity and cell density resulted in a total of six- to tenfold higher culture productivitywhen citrate and glucose were cometabolized than when glucose was the only carbon source. (c) 1995 John Wiley & Sons Inc.

journal_name

Biotechnol Bioeng

authors

Vierheller C,Goel A,Peterson M,Domach MM,Ataai MM

doi

10.1002/bit.260470503

subject

Has Abstract

pub_date

1995-09-05 00:00:00

pages

520-4

issue

5

eissn

0006-3592

issn

1097-0290

journal_volume

47

pub_type

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