Abstract
A novel technically compliant expression system was developed for
heterologous protein production in Bacillus subtilis with the aim of
increasing product yields at the same time as decreasing production
costs. Standard systems involve the positively regulated manP promoter
of the mannose operon, which led to relatively high product yields of
5.3\% (5.3 g enhanced green fluorescent protein eGFP per 100 g cell
dry weight CDW) but required large quantities of mannose to induce
the reactions, thus rendering the system's technical application rather
expensive. To improve this situation, mutant B. subtilis strains were
used: the Delta manA (mannose metabolism) strain TQ281 and the Delta
manP (mannose uptake) strain TQ356. The total amount of inducer could be
reduced with TQ281, which, however, displayed sensitivity to mannose. An
inducer-independent self-induction system was developed with TQ356 to
further improve the cost efficiency and product yield of the system, in
which glucose prevents induction by carbon catabolite repression. To
create optimal self-induction conditions, a glucose-limited process
strategy, namely, a fed-batch process, was utilized as follows. The
initiation of self-induction at the beginning of the glucose-restricted
transition phase between the batch and fed-batch phase of fermentation
and its maintenance throughout the glucose-limiting fed-batch phase led
to a nearly 3-fold increase of product yield, to 14.6\%. The novel B.
subtilis self-induction system thus makes a considerable contribution to
improving product yield and reducing the costs associated with its
technical application.
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