An Artificial Tca Cycle Selects For Efficient -Ketoglutarate Dependent Hydroxylase Catalysis In Engineered Escherichia Coli

Amino acid hydroxylases depend directly on the cellular TCA cycle via their cosubstrate -ketoglutarate (-KG) and are highly useful for the selective biocatalytic oxyfunctionalization of amino acids. This study evaluates TCA cycle engineering strategies to force and increase -KG flux through proline-4-hydroxylase (P4H). The genes sucA (-KG dehydrogenase E1 subunit) and sucC (succinyl-CoA synthetase subunit) were alternately deleted together with aceA (isocitrate lyase) in proline degradation-deficient Escherichia coli strains (putA) expressing the p4h gene. Whereas, the sucCaceAputA strain grew in minimal medium in the absence of P4H, relying on the activity of fumarate reductase, growth of the sucAaceAputA 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 putP, encoding the Na+/l-proline transporter, was overexpressed in the sucAaceAputA strain. This is in contrast to wildtype and putA single-knock out strains, in which -KG availability obviously limited proline hydroxylation. Such -KG limitation was relieved in the sucAaceAputA strain. Furthermore, the sucAaceAputA strain was used to demonstrate an agar plate-based method for the identification and selection of active -KG dependent hydroxylases. This together with the possibility to waive selection pressure and overcome -KG limitation in respective hydroxylation processes based on living cells emphasizes the potential of TCA cycle engineering for the productive application of -KG dependent hydroxylases. Biotechnol. Bioeng. 2017;114: 1511-1520. (c) 2017 Wiley Periodicals, Inc.