초록
<P>Metabolic engineering of <I>Escherichia coli</I> was performed to construct a 100% rationally engineered strain capable of overproducing <SMALL>l</SMALL>-isoleucine, an important branched-chain amino acid. The <I>thrABC</I> (encoding <SMALL>l</SMALL>-threonine biosynthetic enzymes), <I>ilvA</I> (encoding feedback-resistant threonine dehydratase), <I>ilvIH</I> (encoding feedback-resistant acetohydroxy acid synthase III), and <I>ygaZH</I> (encoding branched-chain amino acid exporter) genes were amplified by plasmid-based overexpression. The <I>ilvCED</I> (encoding <SMALL>l</SMALL>-isoleucine biosynthetic enzymes) and <I>lrp</I> (encoding global regulator Lrp) genes were also amplified by chromosomal promoter replacement in order to further increase the flux toward <SMALL>l</SMALL>-isoleucine. The final engineered <I>E. coli</I> strain was able to produce 9.46 g/L of <SMALL>l</SMALL>-isoleucine with a yield of 0.14 g/g of glucose by fed-batch culture. The overall design principles described here for the production of highly regulated product should be useful in designing strains for the production of other similar bioproducts.</P><P><B>Graphic Abstract</B><BR><IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/asbcd6/2012/asbcd6.2012.1.issue-11/sb300071a/production/images/medium/sb-2012-00071a_0005.gif'></P>