Targeted proteomics is a convenient method determining enzyme expression levels, but a quantitative analysis of these proteomic data has not been fully explored yet. Here, we present and demonstrate a computational tool (principal component analysis of proteomics, PCAP) that uses quantitative targeted proteomics data to guide metabolic engineering and achieve higher production of target molecules from heterologous pathways. The method is based on the application of principal component analysis to a collection of proteomics and target molecule production data to pinpoint specific enzymes that need to have their expression level adjusted to maximize production. We illustrated the method on the heterologous mevalonate pathway in Escherichia coli that produces a wide range of isoprenoids and requires balanced pathway gene expression for high yields and titers. PCAP-guided engineering resulted in over a 40% improvement in the production of two valuable terpenes. PCAP could potentially be productively applied to other heterologous pathways as well.

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

Lipid A on the Gram-negative outer membrane (OM) is synthesized in the cytoplasm by the Lpx pathway and translocated to the OM by the Lpt pathway. Some Acinetobacter baumannii strains can tolerate the complete loss of lipopolysaccharide (LPS) resulting from the inactivation of early LPS pathway genes such as lpxC. Here, we characterized a mutant deleted for lptD, which encodes an OM protein that mediates the final translocation of fully synthesized LPS to the OM. Cells lacking lptD had a growth defect comparable to that of an lpxC deletion mutant under the growth conditions tested but were more sensitive to hydrophobic antibiotics, revealing a more significant impact on cell permeability from impaired LPS translocation than from the loss of LPS synthesis. Consistent with this, ATP leakage and N-phenyl-1-naphthylamine (NPN) fluorescence assays indicated a more severe impact of lptD deletion than of lpxC deletion on inner and outer membrane permeability, respectively. Targeted In most Gram-negative bacteria, many of the proteins responsible for lipopolysaccharide (LPS) synthesis and transport are essential, making them potential targets for antibacterial drug development. In particular, the nine conserved Lpx enzymes are considered promising for the development of novel antibiotics, since (3-deoxy-D-manno-oct-2-ulosonic acid) 2 -lipid A (Kdo 2 -lipid A) is the minimal LPS structure that supports a functional outer membrane (OM) and cell viability for most Gram-negative bacteria (1, 2). Indeed, the optimization of LpxC inhibitors has been an ongoing effort in antimicrobial research for over 2 decades, which has yielded compounds with impressive antibacterial activity against Gram-negative organisms such as Pseudomonas aeruginosa (1,(3)(4)(5)(6)(7)(8)(9). The identification of RJPXD33, an antimicrobial peptide that inhibits both Escherichia coli LpxA and LpxD, and a recently identified LpxH inhibitor suggests that other LPS biosynthetic steps could also be successfully targeted (10-12). POL7001, a peptidomimetic antibiotic that inhibits LptD, the final essential step of the LPS transport (Lpt) system, has potent and specific antibacterial activity against P. aeruginosa, which, importantly, indicates that the LPS transport and OM assembly machinery may be attractive targets for antibacterial discovery (13)(14)(15).Acinetobacter baumannii is an emerging opportunistic bacterial pathogen of increasing concern due to multidrug resistance (16). A. baumannii is noted for its ability to develop resistance against most conventional antibiotics through mechanisms such as the upregulation of efflux pumps and horizontal transfer of resistance genes (17)(18)(19). Because of this, clinical resistance is becoming a serious issue for this organism (as well as for other Gram-negative pathogens), often necessitating the use of antibiotics of last resort, such as polymyxins (19,20). Understanding resistance to polymyxins, which are cationic and utilize LPS to gain access to cells, has therefore become a focus of renewed in-