Transcriptional Tuning of Mevalonate Pathway Enzymes to Identify the Impact on Limonene Production in Escherichia coli

Shin, Jonghyeon; South, Eric J.; Dunlop, Mary J.

doi:10.1021/acsomega.2c00483

Title: Transcriptional Tuning of Mevalonate Pathway Enzymes to Identify the Impact on Limonene Production in Escherichia coli

Journal Article · Thu May 26 00:00:00 EDT 2022 · ACS Omega

DOI:https://doi.org/10.1021/acsomega.2c00483· OSTI ID:1870027

Shin, Jonghyeon ^[1];

^[2];

^[3]

Biomedical Engineering Department, Boston University, Boston, Massachusetts 02215, United States
Molecular Biology, Cell Biology & Biochemistry Program, Boston University, Boston, Massachusetts 02215, United States
Biomedical Engineering Department, Boston University, Boston, Massachusetts 02215, United States, Molecular Biology, Cell Biology & Biochemistry Program, Boston University, Boston, Massachusetts 02215, United States

Heterologous production of limonene in microorganisms through the mevalonate (MVA) pathway has traditionally imposed metabolic burden and reduced cell fitness, where imbalanced stoichiometries among sequential enzymes result in the accumulation of toxic intermediates. Although prior studies have shown that changes to mRNA stability, RBS strength, and protein homology can be effective strategies for balancing enzyme levels in the MVA pathway, testing different variations of these parameters often requires distinct genetic constructs, which can exponentially increase assembly costs as pathways increase in size. Here, we developed a multi-input transcriptional circuit to regulate the MVA pathway, where four chemical inducers, L-arabinose (Ara), choline chloride (Cho), cuminic acid (Cuma), and isopropyl β-D-1- thiogalactopyranoside (IPTG), each regulate one of four orthogonal promoters. We tested modular transcriptional regulation of the MVA pathway by placing this circuit in an engineered Escherichia coli “marionette” strain, which enabled systematic and independent tuning of the first three enzymes (AtoB, HMGS, and HMGR) in the MVA pathway. By systematically testing combinations of chemical inducers as inputs, we investigated relationships between the expressions of different MVA pathway submodules, finding that limonene yields are sensitive to the coordinated transcriptional regulation of HMGS and HMGR.

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Research Organization:: Boston Univ., MA (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER); National Science Foundation (NSF); National Institutes of Health (NIH)

Grant/Contract Number:: SC0019387; CBET-1804096; T32GM130546

OSTI ID:: 1870027

Alternate ID(s):: OSTI ID: 1871502

Journal Information:: ACS Omega, Journal Name: ACS Omega Vol. 7 Journal Issue: 22; ISSN 2470-1343

Publisher:: American Chemical SocietyCopyright Statement

Country of Publication:: United States

Language:: English

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