Production of jet fuel precursor monoterpenoids from engineered Escherichia coli

Abstract

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

Techno-economic analysis and life-cycle greenhouse gas mitigation cost of five routes to bio-jet fuel blendstocks

“…Monoterpenes (C10 isoprenoids), such as limonene, 1,8cineole, and linalool are promising potential precursors for jet fuel [8][9][10] due to their low freezing point and high energy density. A recent study 9 investigated properties of jet fuel and several hydrocarbons such as n-butanol, n-hexanol, butyl levulinate, butyl butyrate, ethyl octanoate, methyl linolenate, farnesene, ethyl cyclohexane, and limonene.…”

“…There are two major natural biosynthetic pathways to produce isoprenoids: (i) mevalonate-dependent isoprenoid pathway (MVA pathway) for eukaryotes (except some plants and some bacteria) and the mevalonate-independent 1-deoxy-D-xylulose-5-phosphate pathway (DXP pathway) for most prokaryotes. 10,15 These pathways have been engineered in two host microorganisms, Saccharomyces cerevisiae and Escherichia coli, to increase biofuel yield and titer, increase sugar loading levels, and reduce the cytotoxicity of the targeted biofuel. 10,15 For instance, tolerance mechanisms , such as efflux pumps that export toxins from the cell using the proton motive force, reduce the toxicity from limonene accumulation and improves yield.…”

“…10,15 These pathways have been engineered in two host microorganisms, Saccharomyces cerevisiae and Escherichia coli, to increase biofuel yield and titer, increase sugar loading levels, and reduce the cytotoxicity of the targeted biofuel. 10,15 For instance, tolerance mechanisms , such as efflux pumps that export toxins from the cell using the proton motive force, reduce the toxicity from limonene accumulation and improves yield. 8 Additionally, E. coli is a promising host due to its potential to utilize a wide range of sugars (both glucose and xylose) under aerobic (aeration rate of 1-2 volume of air per volume of liquid solution per minute, referred to as vvm) 10,16,17 and microaerobic conditions (aeration rate of 0.2-0.6 vvm).…”

Metabolic Engineering for Advanced Biofuels Production and Recent Advances Toward Commercialization

“…Optimization of ribosome binding site for NudB [38] E. coli Geraniol 2.0 g L À1 Fed-batch, 68 h Two-phase fermentation platform [43] S. cerevisiae Geraniol 293 mg L À1 Fed-batch, 48 h Overexpression of idi1 and tHMG1 [44] E. coli Limonene 650 mg L À1 Batch, 72 h Principal Component Analysis of proteomics data to optimize MVA pathway protein expression levels [46] E. coli Myrcene 58 mg L À1 Batch, 72 h Heterologous expression of myrcene synthase from Quecrus ilex [96] E. coli Cineol 653 mg L À1 Batch, 48 h Chromosomal mutation of ispA; heterologous expression of cineol synthase from Streptomyces clavuligerus [49] E. coli Linalool 505 mg L À1 Batch, 48 h Chromosomal mutation of ispA; heterologous expression of cineol synthase from Streptomyces clavuligerus [49] E. coli Pinene 140 mg L À1 Batch, 24 h Evolved pinene synthase from Pinus taeda to decrease substrate inhibition [48] E. coli Sabinene 2.7 g L À1 Fed-batch, 24 h Heterologous expression of gpps2 from Abies grandis and sabinene synthase from Salvia pomifera [52] S. cerevisiae Sabinene 18 mg L À1 Batch a) Altering a squalene synthase (erg20p) for GPP specificity [53] E. coli Farnesene 1.1 g L À1 Batch, 96 h In vitro measurement of MVA enzyme activity; balanced expression based on in vitro activity of heterologous pathway [55] S. cerevisiae Farnesene 130 g L À1 Fed-batch, 5-6 d a) Rewiring central carbon metabolism to enhance cytosolic CoA availability [56] E. coli Bisabolene 1.2 g L À1 Batch, 72 h Principal Component Analysis of proteomics data to optimize MVA pathway protein expression levels [46] E. coli b-Caryophyllene 1.5 g L À1 Fed-batch, 72 h Balanced overexpression of MVA and DXP pathway enzymes [58] Fatty acids E. coli Fatty acids 5.2 g L À1 Batch, 72 h Dynamic regulation and control; tuning expression of FadR [62,66] E. coli Fatty acids 8.6 g L À1 Fed-batch, 70 h Optimization of transcription levels in three arbitrary modules within fatty-acid biosynthesis [67] E. coli Fatty acids 3.9 g L À1 Fed-batch, 44 h Dynamic control using transcriptional regulator FapR [61] E. coli Fatty acids 7 g L À1 Batch, 24 h Reversed b-oxidation cycle; overexpression of FadBA and select thioesterases in strain RB03 (RB02 DyqhD DfucO DfadD) [64] E. coli Branched fatty acids 276 mg L À1 Batch, 48 h Incomplete lipoylation of 2-oxoacid dehydrogenases [76] E. coli Fatty acids 694 mg L À1 Batch, 48 h Heterologous expression of Val, Leu, Ile biosynthetic pathways; overexpression of bFabH2 and 'TesA [74] E. coli Fatty acids 21.5 g L À1 Fed-batch, 43 h Ensemble-based selection of bacterial strains u...…”

“…[46] Recently, linalool and cineol were produced in E. coli at relatively high titers (505 and 653 mg L À1 , respectively) by mutating E. coli's genomic copy of FPP synthase (ispA) and increasing the availability of GPP for monoterpene synthases. [49] Efforts to produce linalool in S. cerevisiae were initiated by incorporating a linalool synthase (lis) from Clarkia breweri, but the strain produced linalool at titers of less than 1 mg L À1 due to issues with hmgr overexpression upstream within the MVA pathway. [50] Further attempts to improve S. cerevisiae production by down regulating squalene synthase (erg9) also resulted in similarly low titers of linalool, which was ultimately attributed to its high toxicity in yeast.…”