Monoterpenes (C 10 isoprenoids) are the main components of essential oils and are possible precursors for many commodity chemicals and high energy density fuels. Monoterpenes are synthesized from geranyl diphosphate (GPP), which is also the precursor for the biosynthesis of farnesyl diphosphate (FPP). FPP biosynthesis diverts the carbon flux from monoterpene production to C 15 products and quinone biosynthesis. In this study, we tested a chromosomal mutation of E. coli's native FPP synthase (IspA) to improve GPP availability for the production of monoterpenes using a heterologous mevalonate pathway. Monoterpene production at high levels required not only optimization of GPP production but also a basal level of FPP to maintain growth. The optimized strains produced two jet fuel precursor monoterpenoids 1,8-cineole and linalool at the titer of 653 mg/L and 505 mg/L, respectively, in batch cultures with 1% glucose. The engineered strains developed in this work provide useful resources for the production of high-value monoterpenes. This article is protected by copyright. All rights reserved Acc e p ted P r e p r i nt

Metal-reducing microorganisms such as Shewanella oneidensis MR-1 reduce highly soluble species of hexavalent uranyl (U(VI)) to less mobile tetravalent uranium (U(IV)) compounds. The biologically mediated immobilization of U(VI) is being considered for the remediation of U contamination. However, the mechanistic underpinnings of biological U(VI) reduction remain unresolved. It has become clear that a first electron transfer occurs to form pentavalent (U(V)) intermediates, but it has not been definitively established whether a second one-electron transfer can occur or if disproportionation of U(V) is required. Here, we utilize the unusual properties of dpaea 2– ((dpaeaH 2 =bis(pyridyl-6-methyl-2-carboxylate)-ethylamine)), a ligand forming a stable soluble aqueous complex with U(V), and investigate the reduction of U(VI)–dpaea and U(V)–dpaea by S. oneidensis MR-1. We establish U speciation through time by separating U(VI) from U(IV) by ion exchange chromatography and characterize the reaction end-products using U M 4 -edge high resolution X-ray absorption near-edge structure (HR-XANES) spectroscopy. We document the reduction of solid phase U(VI)–dpaea to aqueous U(V)–dpaea but, most importantly, demonstrate that of U(V)–dpaea to U(IV). This work establishes the potential for biological reduction of U(V) bound to a stabilizing ligand. Thus, further work is warranted to investigate the possible persistence of U(V)–organic complexes followed by their bioreduction in environmental systems.

The biological reduction of soluble U(VI) complexes to form immobile U(IV) species has been proposed to remediate contaminated sites. It is well established that multiheme c-type cytochromes (MHCs) are key mediators of electron transfer to aqueous phase U(VI) complexes for bacteria such as Shewanella oneidensis MR-1. Recent studies have confirmed that the reduction proceeds via a first electron transfer forming pentavalent U(V) species that readily disproportionate. However, in the presence of the stabilizing aminocarboxylate ligand, dpaea 2− (dpaeaH 2 �bis(pyridyl-6-methyl-2-carboxylate)-ethylamine), biologically produced U(V) persisted in aqueous solution at pH 7. We aim to pinpoint the role of MHC in the reduction of U(V)-dpaea and to establish the mechanism of solidphase U(VI)-dpaea reduction. To that end, we investigated U-dpaea reduction by two deletion mutants of S. oneidensis MR-1−one lacking outer membrane MHCs and the other lacking all outer membrane MHCs and a transmembrane MHC−and by the purified outer membrane MHC, MtrC. Our results suggest that solid-phase U(VI)-dpaea is reduced primarily by outer membrane MHCs. Additionally, MtrC can directly transfer electrons to U(V)-dpaea to form U(IV) species but is not strictly necessary, underscoring the primary involvement of outer membrane MHCs in the reduction of this pentavalent U species but not excluding that of periplasmic MHCs.

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