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A strategy for L-isoleucine dioxygenase screening and 4-hydroxyisoleucine production by resting cells.

ABSTRACT: L-Isoleucine dioxygenase (IDO) specifically converts L-isoleucine(L-Ile) to 4-hydroxyisoleucine(4-HIL). To obtain IDO with improved activity, a strategy was developed that is dependent on the restoration of succinate-minus E. coli cell growth by the coupling of L-Ile hydroxylation and the oxidation of ?-ketoglutarate(?-KGA) to succinate. Five mutants were obtained with this strategy, and the characteristics of IDOM3, which exhibited the highest activity, were studied. The catalytic efficiency, thermal stability and catalytic rate of IDOM3 were significantly improved compared with those of wild-type IDO. Moreover, an efficient method for the biotransformation of 4-HIL by resting cells expressing IDOM3 was developed, with which 151.9 mmol of 4-HIL/L (22.4 g/L) was synthesized in 12 h while the substrates seldom exhibited additional consumption.

SUBMITTER: Zhang C

PROVIDER: S-EPMC5972919 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

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A strategy for L-isoleucine dioxygenase screening and 4-hydroxyisoleucine production by resting cells.

Zhang Chenglin C Ma Jie J Li Zhixiang Z Liang Yunlong Y Xu Qingyang Q Xie Xixian X Chen Ning N

Bioengineered 20170428 1

L-Isoleucine dioxygenase (IDO) specifically converts L-isoleucine(L-Ile) to 4-hydroxyisoleucine(4-HIL). To obtain IDO with improved activity, a strategy was developed that is dependent on the restoration of succinate-minus E. coli cell growth by the coupling of L-Ile hydroxylation and the oxidation of α-ketoglutarate(α-KGA) to succinate. Five mutants were obtained with this strategy, and the characteristics of IDO<sup>M3</sup>, which exhibited the highest activity, were studied. The catalytic ef ...[more]

PMID: 28430004

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Project description:BackgroundMembrane proteins play a key role in many fundamental cellular processes such as transport of nutrients, sensing of environmental signals and energy transduction, and account for over 50% of all known drug targets. Despite their importance, structural and functional characterisation of membrane proteins still remains a challenge, partially due to the difficulties in recombinant expression and purification. Therefore the need for development of efficient methods for heterologous production is essential.Methodology/principal findingsFifteen integral membrane transport proteins from Archaea were selected as test targets, chosen to represent two superfamilies widespread in all organisms known as the Major Facilitator Superfamily (MFS) and the 5-Helix Inverted Repeat Transporter superfamily (5HIRT). These proteins typically have eleven to twelve predicted transmembrane helices and are putative transporters for sugar, metabolite, nucleobase, vitamin or neurotransmitter. They include a wide range of examples from the following families: Metabolite-H(+)-symporter; Sugar Porter; Nucleobase-Cation-Symporter-1; Nucleobase-Cation-Symporter-2; and neurotransmitter-sodium-symporter. Overproduction of transporters was evaluated with three vectors (pTTQ18, pET52b, pWarf) and two Escherichia coli strains (BL21 Star and C43 (DE3)). Thirteen transporter genes were successfully expressed; only two did not express in any of the tested vector-strain combinations. Initial trials showed that seven transporters could be purified and six of these yielded quantities of ≥ 0.4 mg per litre suitable for functional and structural studies. Size-exclusion chromatography confirmed that two purified transporters were almost homogeneous while four others were shown to be non-aggregating, indicating that they are ready for up-scale production and crystallisation trials.Conclusions/significanceHere, we describe an efficient strategy for heterologous production of membrane transport proteins in E. coli. Small-volume cultures (10 mL) produced sufficient amount of proteins to assess their purity and aggregation state. The methods described in this work are simple to implement and can be easily applied to many more membrane proteins.

Dataset Information

A strategy for L-isoleucine dioxygenase screening and 4-hydroxyisoleucine production by resting cells.

Publications

A strategy for L-isoleucine dioxygenase screening and 4-hydroxyisoleucine production by resting cells.

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