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      Modular pathway engineering for the microbial production of branched-chain fatty alcohols

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          Abstract

          The intrinsic structural properties of branched long-chain fatty alcohols (BLFLs) in the range of C12 to C18 make them more suitable as diesel fuel replacements and for other industrial applications than their straight-chain counterparts. While microbial production of straight long-chain fatty alcohols has been achieved, biosynthesis of BLFLs has never been reported. In this work, we engineered four different biosynthetic pathways in Escherichia coli to produce BLFLs. We then employed a modular engineering approach to optimize the supply of α-keto acid precursors and produced either odd-chain or even-chain BLFLs with high selectivity, reaching 70 and 75% of total fatty alcohols, respectively. The acyl-ACP and alcohol-producing modules were also extensively optimized to balance enzyme expression level and ratio, resulting in a 6.5-fold improvement in BLFL titers. The best performing strain overexpressed 14 genes from 6 engineered operons and produced 350 mg/L of BLFLs in fed-batch fermenter. The modular engineering strategy successfully facilitated microbial production of BLFLs and allowed us to quickly optimize new BLFL pathway with high titers and product specificity. More generally, this work provides pathways and knowledge for the production of BLFLs and BLFL-related, industry-relevant chemicals in high titers and yields.

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          The online version of this article (doi:10.1186/s13068-017-0936-4) contains supplementary material, which is available to authorized users.

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          Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels.

          Global energy and environmental problems have stimulated increased efforts towards synthesizing biofuels from renewable resources. Compared to the traditional biofuel, ethanol, higher alcohols offer advantages as gasoline substitutes because of their higher energy density and lower hygroscopicity. In addition, branched-chain alcohols have higher octane numbers compared with their straight-chain counterparts. However, these alcohols cannot be synthesized economically using native organisms. Here we present a metabolic engineering approach using Escherichia coli to produce higher alcohols including isobutanol, 1-butanol, 2-methyl-1-butanol, 3-methyl-1-butanol and 2-phenylethanol from glucose, a renewable carbon source. This strategy uses the host's highly active amino acid biosynthetic pathway and diverts its 2-keto acid intermediates for alcohol synthesis. In particular, we have achieved high-yield, high-specificity production of isobutanol from glucose. The strategy enables the exploration of biofuels beyond those naturally accumulated to high quantities in microbial fermentation.
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            Microbial production of fatty-acid-derived fuels and chemicals from plant biomass.

            Increasing energy costs and environmental concerns have emphasized the need to produce sustainable renewable fuels and chemicals. Major efforts to this end are focused on the microbial production of high-energy fuels by cost-effective 'consolidated bioprocesses'. Fatty acids are composed of long alkyl chains and represent nature's 'petroleum', being a primary metabolite used by cells for both chemical and energy storage functions. These energy-rich molecules are today isolated from plant and animal oils for a diverse set of products ranging from fuels to oleochemicals. A more scalable, controllable and economic route to this important class of chemicals would be through the microbial conversion of renewable feedstocks, such as biomass-derived carbohydrates. Here we demonstrate the engineering of Escherichia coli to produce structurally tailored fatty esters (biodiesel), fatty alcohols, and waxes directly from simple sugars. Furthermore, we show engineering of the biodiesel-producing cells to express hemicellulases, a step towards producing these compounds directly from hemicellulose, a major component of plant-derived biomass.
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              Microbial biosynthesis of alkanes.

              Alkanes, the major constituents of gasoline, diesel, and jet fuel, are naturally produced by diverse species; however, the genetics and biochemistry behind this biology have remained elusive. Here we describe the discovery of an alkane biosynthesis pathway from cyanobacteria. The pathway consists of an acyl-acyl carrier protein reductase and an aldehyde decarbonylase, which together convert intermediates of fatty acid metabolism to alkanes and alkenes. The aldehyde decarbonylase is related to the broadly functional nonheme diiron enzymes. Heterologous expression of the alkane operon in Escherichia coli leads to the production and secretion of C13 to C17 mixtures of alkanes and alkenes. These genes and enzymes can now be leveraged for the simple and direct conversion of renewable raw materials to fungible hydrocarbon fuels.
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                Author and article information

                Contributors
                w.jiang@wustl.edu
                jbqiao@wustl.edu
                Gayle.Bentley@nrel.gov
                liudi@wustl.edu
                314-935-7671 , fzhang@seas.wustl.edu
                Journal
                Biotechnol Biofuels
                Biotechnol Biofuels
                Biotechnology for Biofuels
                BioMed Central (London )
                1754-6834
                27 October 2017
                27 October 2017
                2017
                : 10
                : 244
                Affiliations
                [1 ]ISNI 0000 0001 2355 7002, GRID grid.4367.6, Department of Energy, Environmental and Chemical Engineering, , Washington University in St. Louis, ; 1 Brookings Drive, Campus Box 1180, Saint Louis, MO 63130 USA
                [2 ]ISNI 0000 0001 2355 7002, GRID grid.4367.6, Division of Biological & Biomedical Sciences, , Washington University in St. Louis, ; Saint Louis, MO 63130 USA
                [3 ]ISNI 0000 0001 2355 7002, GRID grid.4367.6, Institute of Materials Science & Engineering, , Washington University in St. Louis, ; Saint Louis, MO 63130 USA
                [4 ]ISNI 0000 0001 2199 3636, GRID grid.419357.d, Present Address: National Bioenergy Center, , National Renewable Energy Laboratory, ; Golden, CO 80401 USA
                Author information
                http://orcid.org/0000-0001-6979-7909
                Article
                936
                10.1186/s13068-017-0936-4
                5658922
                29090017
                6e8b395a-98b4-4597-af4f-adf9dcf96a23
                © The Author(s) 2017

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                : 9 July 2017
                : 19 October 2017
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/100000185, Defense Advanced Research Projects Agency;
                Award ID: D13AP00038
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100000152, Division of Molecular and Cellular Biosciences;
                Award ID: MCB1453147
                Award Recipient :
                Categories
                Research
                Custom metadata
                © The Author(s) 2017

                Biotechnology
                branched long-chain fatty alcohols,branched-chain fatty acids,advanced biofuels,modular pathway engineering

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