Isobutene formation from 3-hydroxy-3-methylbutyrate (3-HMB) by the Saccharomyces cerevisiae diphosphomevalonate decarboxylase (ScMDD) and directed enzyme evolution to improve enzyme function

Gogerty, David S.

doi:10.31274/etd-180810-1943

Cited by 1 publication

(1 citation statement)

References 115 publications

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“…In this work, TbuT served as the transcriptional regulator to detect the presence of an isoprene and to induce the expression of a fluorescent reporter protein. Gogerty et al previously constructed a TbuT-based isobutene biosensor in E. coli , in which the LacZ (β-galactosidase)-encoding gene was under the transcriptional control of P tbuA1 promoter region in a medium-copy number plasmid . The TbuT-based isobutene biosensor was also induced by isoprene (1.3-fold induction), which is relatively smaller than those by isobutene (1.64-fold) and toluene (3.58-fold).…”

Section: Resultsmentioning

confidence: 99%

A Genetically Encoded Biosensor for Monitoring Isoprene Production in Engineered Escherichia coli

Kim

Subhadra

et al. 2018

ACS Synth. Biol.

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Isoprene is a valuable precursor for synthetic rubber and a signature product of terpenoid pathways. Here, we developed an isoprene biosensor by employing a TbuT transcriptional regulator of Ralstonia pickettii to express a fluorescent reporter gene in response to intracellular isoprene in engineered Escherichia coli. The TbuT regulator recognizes isoprene as its less-preferred effector molecule; thus, we amplified the reporter gene expression using a T7 RNA polymerase-mediated transcriptional cascade and iteratively tuned the promoter transcribing tbuT to improve the sensitivity for detecting isoprene. When the engineered E. coli cells expressed heterologous genes for isoprene biosynthesis, the intracellular isoprene was expelled and the tbuT transcription factor was subsequently activated, leading to gfp expression. The chromosomal isoprene biosensor showed a linear correlation between GFP fluorescence and intracellular isoprene concentration. Using this chromosomal isoprene biosensor, we successfully identified the highest isoprene producer among four different E. coli strains producing different amounts of isoprene. The isoprene biosensor presented here can enable high-throughput screening of isoprene synthases and metabolic pathways for efficient and sustainable production of bioisoprene in engineered microbes.

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Section: Resultsmentioning

confidence: 99%