Engineering microbes to produce biofuels

Wackett, Lawrence P.

doi:10.1016/j.copbio.2010.10.010

Cited by 42 publications

(23 citation statements)

References 43 publications

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“…These advances, in combination with the development of reliable genetic transformation protocols for photosynthetic organisms with high innate biomass accumulation rates will enable the engineering of improved strains that not only have high production rates but also produce tailored precursors, or even finished products of biotechnological interest2567.…”

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confidence: 99%

Draft genome sequence and genetic transformation of the oleaginous alga Nannochloropsis gaditana

et al. 2012

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The potential use of algae in biofuels applications is receiving significant attention. However, none of the current algal model species are competitive production strains. Here we present a draft genome sequence and a genetic transformation method for the marine microalga Nannochloropsis gaditana CCMP526. We show that N. gaditana has highly favourable lipid yields, and is a promising production organism. The genome assembly includes nuclear (~29 Mb) and organellar genomes, and contains 9,052 gene models. We define the genes required for glycerolipid biogenesis and detail the differential regulation of genes during nitrogen-limited lipid biosynthesis. Phylogenomic analysis identifies genetic attributes of this organism, including unique stramenopile photosynthesis genes and gene expansions that may explain the distinguishing photoautotrophic phenotypes observed. The availability of a genome sequence and transformation methods will facilitate investigations into N. gaditana lipid biosynthesis and permit genetic engineering strategies to further improve this naturally productive alga.

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confidence: 99%

Draft genome sequence and genetic transformation of the oleaginous alga Nannochloropsis gaditana

et al. 2012

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“…The transition from reliance on fossil fuels to developing renewable hydrocarbons for biofuels and commodity chemicals has been a major focus in recent biotechnology research [1-3]. Microbial production of specialty hydrocarbons represents a viable option for this transition [1].…”

Section: Introductionmentioning

confidence: 99%

“…Microbial production of specialty hydrocarbons represents a viable option for this transition [1]. Much research has been conducted to identify candidate organisms for heterologous expression and synthesis of these compounds; however, a great deal is yet unknown about how and why the native organisms produce these hydrocarbons.…”

Section: Introductionmentioning

confidence: 99%

OleA Glu117 is key to condensation of two fatty-acyl coenzyme A substrates in long-chain olefin biosynthesis

Jensen

Goblirsch

Christenson

et al. 2017

Biochemical Journal

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In the interest of decreasing dependence on fossil fuels, microbial hydrocarbon biosynthesis pathways are being studied for renewable, tailored production of specialty chemicals and biofuels. One candidate is long-chain olefin biosynthesis, a widespread bacterial pathway that produces waxy hydrocarbons. Found in three- and four-gene clusters, oleABCD encode the enzymes necessary to produce cis-olefins that differ by alkyl chain length, degree of unsaturation, and alkyl chain branching. The first enzyme in the pathway, OleA, catalyzes the Claisen condensation of two fatty acyl-coenzyme A molecules to form a β-keto acid. In this report, the mechanistic role of Xanthomonas campestris OleA Glu117 is investigated through mutant enzymes. Crystal structures were determined for each mutant as well as their complex with the inhibitor cerulenin. Complemented by substrate modelling, these structures suggest that Glu117 aids in substrate positioning for productive carbon-carbon bond formation. Analysis of acyl-coenzyme A substrate hydrolysis shows diminished activity in all mutants. When the active site lacks an acidic residue in the 117 position, OleA cannot form condensed product, demonstrating Glu117 has a critical role upstream of the essential condensation reaction. Profiling of pH dependence shows that the apparent pKa for Glu117 is impacted by mutagenesis. Taken together, we propose that Glu117 is the general base needed to prime condensation via deprotonation of the second, non-covalently bound substrate during turnover. This is the first example of a member of the thiolase superfamily of condensing enzymes to contain an active site base originating from the second monomer of the dimer. Summary Statement OleA is the first reported thiolase enzyme whose active site general base, a glutamic acid (Glu117), is derived from a homodimeric interface. Structural and functional characterization of glutamatic acid mutants establishes its mechanistic role in initiating olefin biosynthesis.

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“…An alternative method for the microbial production of both fuels and chemicals is to use low potential reducing power from sources such as hydrogen gas, reduced metals, or electricity to reduce carbon dioxide directly to useful products. This circumvents the overall low efficiency experienced in generating both plant and algal photosynthetic products (5). Moreover, such electron sources can potentially be used to reduce carbon dioxide directly to produce liquid fuels or "electrofuels" (6) or to produce industrial chemicals without a sugar intermediate.…”

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confidence: 99%

Exploiting microbial hyperthermophilicity to produce an industrial chemical, using hydrogen and carbon dioxide

Keller

Schut

Lipscomb

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

120

103

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Microorganisms can be engineered to produce useful products, including chemicals and fuels from sugars derived from renewable feedstocks, such as plant biomass. An alternative method is to use low potential reducing power from nonbiomass sources, such as hydrogen gas or electricity, to reduce carbon dioxide directly into products. This approach circumvents the overall low efficiency of photosynthesis and the production of sugar intermediates. Although significant advances have been made in manipulating microorganisms to produce useful products from organic substrates, engineering them to use carbon dioxide and hydrogen gas has not been reported. Herein, we describe a unique temperature-dependent approach that confers on a microorganism (the archaeon Pyrococcus furiosus, which grows optimally on carbohydrates at 100°C) the capacity to use carbon dioxide, a reaction that it does not accomplish naturally. This was achieved by the heterologous expression of five genes of the carbon fixation cycle of the archaeon Metallosphaera sedula, which grows autotrophically at 73°C. The engineered P. furiosus strain is able to use hydrogen gas and incorporate carbon dioxide into 3-hydroxypropionic acid, one of the top 12 industrial chemical building blocks. The reaction can be accomplished by cell-free extracts and by whole cells of the recombinant P. furiosus strain. Moreover, it is carried out some 30°C below the optimal growth temperature of the organism in conditions that support only minimal growth but maintain sufficient metabolic activity to sustain the production of 3-hydroxypropionate. The approach described here can be expanded to produce important organic chemicals, all through biological activation of carbon dioxide.

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Engineering microbes to produce biofuels

Cited by 42 publications

References 43 publications

Draft genome sequence and genetic transformation of the oleaginous alga Nannochloropsis gaditana

Draft genome sequence and genetic transformation of the oleaginous alga Nannochloropsis gaditana

OleA Glu117 is key to condensation of two fatty-acyl coenzyme A substrates in long-chain olefin biosynthesis

Exploiting microbial hyperthermophilicity to produce an industrial chemical, using hydrogen and carbon dioxide

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