A novel mammalian expression system derived from components coordinating nicotine degradation in arthrobacter nicotinovorans pAO1

Malphettes, Laetitia

doi:10.1093/nar/gni107

Cited by 34 publications

(13 citation statements)

References 70 publications

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“…Knowledge of the enzymes involved in nicotine catabolism will have applications not only in the bioremediation of nicotine waste, but also in the supply of nicotine derivatives as starting materials for the synthesis of new products of industrial and pharmaceutical importance [3,4]. Construction of inducible mammalian systems responsive to nicotine and nicotine metabolites are feasible [5]. To achieve such goals, an in‐depth understanding of the enzymology of nicotine catabolism is required.…”

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confidence: 99%

Final steps in the catabolism of nicotine

et al. 2006

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New enzymes of nicotine catabolism instrumental in the detoxification of the tobacco alkaloid by Arthrobacter nicotinovorans pAO1 have been identified and characterized. Nicotine breakdown leads to the formation of nicotine blue from the hydroxylated pyridine ring and of γ‐N‐methylaminobutyrate (CH3‐4‐aminobutyrate) from the pyrrolidine ring of the molecule. Surprisingly, two alternative pathways for the final steps in the catabolism of CH3‐4‐aminobutyrate could be identified. CH3‐4‐aminobutyrate may be demethylated to γ‐N‐aminobutyrate by the recently identified γ‐N‐methylaminobutyrate oxidase [Chiribau et al. (2004) Eur J Biochem271, 4677–4684]. In an alternative pathway, an amine oxidase with noncovalently bound FAD and of novel substrate specificity removed methylamine from CH3‐4‐aminobutyrate with the formation of succinic semialdehyde. Succinic semialdehyde was converted to succinate by a NADP+‐dependent succinic semialdehyde dehydrogenase. Succinate may enter the citric acid cycle completing the catabolism of the pyrrolidine moiety of nicotine. Expression of the genes of these enzymes was dependent on the presence of nicotine in the growth medium. Thus, two enzymes of the nicotine regulon, γ‐N‐methylaminobutyrate oxidase and amine oxidase share the same substrate. The Km of 2.5 mm and kcat of 1230 s−1 for amine oxidase vs. Km of 140 µm and kcat of 800 s−1 for γ‐N‐methylaminobutyrate oxidase, determined in vitro with the purified recombinant enzymes, may suggest that demethylation predominates over deamination of CH3‐4‐aminobutyrate. However, bacteria grown on [14C]nicotine secreted [14C]methylamine into the medium, indicating that the pathway to succinate is active in vivo.

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confidence: 99%

Final steps in the catabolism of nicotine

et al. 2006

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“…Increasing the number of tandem operator modules within a trigger-inducible promoter may increase maximum expression levels as more transactivators can be recruited to the promoter ( 57 ). The transcriptional activity of the promoters harboring the operator in a monomeric (pSH117, P ARTm1 -SEAP-pA; P ARTm1 , Asc I-O ARG - Mlu I-0bp-P hCMVmin ), dimeric (pSH119, P ARTm2 -SEAP-pA; P ARTm2 , Asc I-O ARG -7bp-O ARG – Mlu I-0bp-P hCMVmin ), trimeric (pSH126, P ARTm3 -SEAP-pA; P ARTm3 , Asc I-O ARG -7bp-O ARG -7bp-O ARG – Mlu I-0bp-P hCMVmin ) or tetrameric (pSH127, P ARTm4 -SEAP-pA; P ARTm4 , Asc I-O ARG -7bp-O ARG -7bp-O ARG –7bp-O ARG - Mlu I-0bp-P hCMVmin ) configuration were assessed in CHO-K1 cells co-transfected with pSH91 (pSH91, P SV40 -ARG2-pA).…”

Section: Resultsmentioning

confidence: 99%

An engineered L-arginine sensor of Chlamydia pneumoniae enables arginine-adjustable transcription control in mammalian cells and mice

Hartenbach

Baba

Weber

et al. 2007

Nucleic Acids Research

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For optimal compatibility with biopharmaceutical manufacturing and gene therapy, heterologous transgene control systems must be responsive to side-effect-free physiologic inducer molecules. The arginine-inducible interaction of the ArgR repressor and the ArgR-specific ARG box, which synchronize arginine import and synthesis in the intracellular human pathogen Chlamydia pneumoniae, was engineered for arginine-regulated transgene (ART) expression in mammalian cells. A synthetic arginine-responsive transactivator (ARG), consisting of ArgR fused to the Herpes simplex VP16 transactivation domain, reversibly adjusted transgene transcription of chimeric ARG box-containing mammalian minimal promoters (PART) in an arginine-inducible manner. Arginine-controlled transgene expression showed rapid induction kinetics in a variety of mammalian cell lines and was adjustable and reversible at concentrations which were compatible with host cell physiology. ART variants containing different transactivation domains, variable spacing between ARG box and minimal promoter and several tandem ARG boxes showed modified regulation performance tailored for specific expression scenarios and cell types. Mice implanted with microencapsulated cells engineered for ART-inducible expression of the human placental secreted alkaline phosphatase (SEAP) exhibited adjustable serum phosphatase levels after treatment with different arginine doses. Using a physiologic inducer, such as the amino acid l-arginine, to control heterologous transgenes in a seamless manner which is devoid of noticeable metabolic interference will foster novel opportunities for precise expression dosing in future gene therapy scenarios as well as the manufacturing of difficult-to-produce protein pharmaceuticals.

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“…In parallel, orthogonal promoters that can respond to other signal molecules, such as cumate [ 38 ] or 6-hydroxy-nicotine [ 39 ], have been developed. Likewise, two novel antibiotic-regulatable systems based on the Tet-system principle have focused much attention.…”

Section: Induction Of Transgene Expression By External Stimulimentioning

confidence: 99%

Toward Tightly Tuned Gene Expression Following Lentiviral Vector Transduction

Page

Fusil

Cosset

2020

Viruses

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Lentiviral vectors are versatile tools for gene delivery purposes. While in the earlier versions of retroviral vectors, transgene expression was controlled by the long terminal repeats (LTRs), the latter generations of vectors, including those derived from lentiviruses, incorporate internal constitutive or regulated promoters in order to regulate transgene expression. This allows to temporally and/or quantitatively control transgene expression, which is required for many applications such as for clinical applications, when transgene expression is required in specific tissues and at a specific timing. Here we review the main systems that have been developed for transgene regulated expression following lentiviral gene transfer. First, the induction of gene expression can be triggered either by external or by internal cues. Indeed, these regulated vector systems may harbor promoters inducible by exogenous stimuli, such as small molecules (e.g., antibiotics) or temperature variations, offering the possibility to tune rapidly transgene expression in case of adverse events. Second, expression can be indirectly adjusted by playing on inserted sequence copies, for instance by gene excision. Finally, synthetic networks can be developed to sense specific endogenous signals and trigger defined responses after information processing. Regulatable lentiviral vectors (LV)-mediated transgene expression systems have been widely used in basic research to uncover gene functions or to temporally reprogram cells. Clinical applications are also under development to induce therapeutic molecule secretion or to implement safety switches. Such regulatable approaches are currently focusing much attention and will benefit from the development of other technologies in order to launch autonomously controlled systems.

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A novel mammalian expression system derived from components coordinating nicotine degradation in arthrobacter nicotinovorans pAO1

Cited by 34 publications

References 70 publications

Final steps in the catabolism of nicotine

Final steps in the catabolism of nicotine

An engineered L-arginine sensor of Chlamydia pneumoniae enables arginine-adjustable transcription control in mammalian cells and mice

Toward Tightly Tuned Gene Expression Following Lentiviral Vector Transduction

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