초록
<P><B>ABSTRACT</B></P><P>Amino acid hydroxylases depend directly on the cellular TCA cycle via their cosubstrate α‐ketoglutarate (α‐KG) and are highly useful for the selective biocatalytic oxyfunctionalization of amino acids. This study evaluates TCA cycle engineering strategies to force and increase α‐KG flux through proline‐4‐hydroxylase (P4H). The genes <I>sucA</I> (α‐KG dehydrogenase E1 subunit) and <I>sucC</I> (succinyl‐CoA synthetase β subunit) were alternately deleted together with <I>aceA</I> (isocitrate lyase) in proline degradation‐deficient <I>Escherichia coli</I> strains (Δ<I>putA</I>) expressing the <I>p4h</I> gene. Whereas, the Δ<I>sucC</I>Δ<I>aceA</I>Δ<I>putA</I> strain grew in minimal medium in the absence of P4H, relying on the activity of fumarate reductase, growth of the Δ<I>sucA</I>Δ<I>aceA</I>Δ<I>putA</I> strictly depended on P4H activity, thus coupling growth to proline hydroxylation. P4H restored growth, even when proline was not externally added. However, the reduced succinyl‐CoA pool caused a 27% decrease of the average cell size compared to the wildtype strain. Medium supplementation partially restored the morphology and, in some cases, enhanced proline hydroxylation activity. The specific proline hydroxylation rate doubled when <I>putP</I>, encoding the Na<SUP>+</SUP>/<SMALL>l</SMALL>‐proline transporter, was overexpressed in the Δ<I>sucA</I>Δ<I>aceA</I>Δ<I>putA</I> strain. This is in contrast to wildtype and Δ<I>putA</I> single‐knock out strains, in which α‐KG availability obviously limited proline hydroxylation. Such α‐KG limitation was relieved in the Δ<I>sucA</I>Δ<I>aceA</I>Δ<I>putA</I> strain. Furthermore, the Δ<I>sucA</I>Δ<I>aceA</I>Δ<I>putA</I> strain was used to demonstrate an agar plate‐based method for the identification and selection of active α‐KG dependent hydroxylases. This together with the possibility to waive selection pressure and overcome α‐KG limitation in respective hydroxylation processes based on living cells emphasizes the potential of TCA cycle engineering for the productive application of α‐KG dependent hydroxylases. Biotechnol. Bioeng. 2017;114: 1511–1520. © 2017 Wiley Periodicals, Inc.</P>