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Digitalizing heterologous gene expression in Gram-negative bacteria with a portable ON/OFF module.

ABSTRACT: While prokaryotic promoters controlled by signal-responding regulators typically display a range of input/output ratios when exposed to cognate inducers, virtually no naturally occurring cases are known to have an OFF state of zero transcription-as ideally needed for synthetic circuits. To overcome this problem, we have modelled and implemented a simple digitalizer module that completely suppresses the basal level of otherwise strong promoters in such a way that expression in the absence of induction is entirely impeded. The circuit involves the interplay of a translation-inhibitory sRNA with the translational coupling of the gene of interest to a repressor such as LacI. The digitalizer module was validated with the strong inducible promoters Pm (induced by XylS in the presence of benzoate) and PalkB (induced by AlkS/dicyclopropyl ketone) and shown to perform effectively in both Escherichia coli and the soil bacterium Pseudomonas putida. The distinct expression architecture allowed cloning and conditional expression of, e.g. colicin E3, one molecule of which per cell suffices to kill the host bacterium. Revertants that escaped ColE3 killing were not found in hosts devoid of insertion sequences, suggesting that mobile elements are a major source of circuit inactivation in vivo.

SUBMITTER: Calles B

PROVIDER: S-EPMC6920698 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Digitalizing heterologous gene expression in Gram-negative bacteria with a portable ON/OFF module.

Calles Belén B Goñi-Moreno Ángel Á de Lorenzo Víctor V

Molecular systems biology 20191201 12

While prokaryotic promoters controlled by signal-responding regulators typically display a range of input/output ratios when exposed to cognate inducers, virtually no naturally occurring cases are known to have an OFF state of zero transcription-as ideally needed for synthetic circuits. To overcome this problem, we have modelled and implemented a simple digitalizer module that completely suppresses the basal level of otherwise strong promoters in such a way that expression in the absence of indu ...[more]

PMID: 31885200

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Project description:Alkene monooxygenases (MOs) are soluble di-iron-containing enzymes found in bacteria that grow on alkenes. Here, we report improved heterologous expression systems for the propene MO (PmoABCD) and ethene MO (EtnABCD) from Mycobacterium chubuense strain NBB4. Strong functional expression of PmoABCD and EtnABCD was achieved in Mycobacterium smegmatis mc2155, yielding epoxidation activities (62 and 27 nmol/min/mg protein, respectively) higher than any reported to date for heterologous expression of a di-iron MO system. Both PmoABCD and EtnABCD were specialized for the oxidation of gaseous alkenes (C2 to C4), and their activity was much lower on liquid alkenes (C5 to C8). Despite intensive efforts to express the complete EtnABCD enzyme in Escherichia coli, this was not achieved, although recombinant EtnB and EtnD proteins could be purified individually in soluble form. The biochemical function of EtnD as an oxidoreductase was confirmed (1.36 μmol cytochrome c reduced/min/mg protein). Cloning the EtnABCD gene cluster into Pseudomonas putida KT2440 yielded detectable epoxidation of ethene (0.5 nmol/min/mg protein), and this could be stimulated (up to 1.1 nmol/min/mg protein) by the coexpression of cpn60 chaperonins from either Mycobacterium spp. or E. coli Successful expression of the ethene MO in a Gram-negative host was validated by both whole-cell activity assays and peptide mass spectrometry of induced proteins seen on SDS-PAGE gels.IMPORTANCE Alkene MOs are of interest for their potential roles in industrial biocatalysis, most notably for the stereoselective synthesis of epoxides. Wild-type bacteria that grow on alkenes have high activities for alkene oxidation but are problematic for biocatalysis, since they tend to consume the epoxide products. Using recombinant biocatalysts is the obvious alternative, but a major bottleneck is the low activities of recombinant alkene MOs. Here, we provide new high-activity recombinant biocatalysts for alkene oxidation, and we provide insights into how to further improve these systems.

Dataset Information

Digitalizing heterologous gene expression in Gram-negative bacteria with a portable ON/OFF module.

Publications

Digitalizing heterologous gene expression in Gram-negative bacteria with a portable ON/OFF module.

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