Use of lac Fusion to Approach the High TyrB Production with a Runaway-Replication Vector

Abstract

An easy and efficient method was proposed to achieve high production of the tyrB gene product on a runaway-replication vector. The DNA containing the tyrB promoter fused transcriptionally with the lacZ gene was cloned into a runaway-replication plasmid, pRA90. With the aid of this gene fusion, the expression level of tyrB in response to temperature alter-ations, the induction cell density, the temperature upshift rate, and various culture medium was investigated by measuring LacZ activities. The optimal condition thus set up for LacZ production was adopted to yield TyrB by a similar clone harboring an intact tyrE gene. As a result, TyrB was over-amplified 135-fold in tenns of specific activity in comparison with the wild-type level. This work demonstrates the potential use of lacZ fusion to probe the sound condition for high TryB production with uncontrolled-replication plasmids.