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

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.