Global climate change linked to the accumulation of greenhouse gases has caused concerns regarding the use of fossil fuels as the major energy source. To mitigate climate change while keeping energy supply sustainable, one solution is to rely on the ability of microorganisms to use renewable resources for biofuel synthesis. In this Review, we discuss how microorganisms can be explored for the production of next-generation biofuels, based on the ability of bacteria and fungi to use lignocellulose; through direct CO2 conversion by microalgae; using lithoautotrophs driven by solar electricity; or through the capacity of microorganisms to use methane generated from landfill. Furthermore, we discuss how to direct these substrates to the biosynthetic pathways of various fuel compounds and how to optimize biofuel production by engineering fuel pathways and central metabolism.

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Fuelling the future: microbial engineering for the production of sustainable biofuels

/pdf/transformation-of-biomass-into-commodity-chemicals-using-2a93itd2a6.pdf

Transformation of biomass into commodity chemicals using enzymes or cells

Systems Metabolic Engineering Strategies: Integrating Systems and Synthetic Biology with Metabolic Engineering

Genetic determinants

https://backend.orbit.dtu.dk/ws/files/189263285/1_s2.0_S1096717619301533_main.pdf

The emergence of adaptive laboratory evolution as an efficient tool for biological discovery and industrial biotechnology.

Carboxylic acids are an attractive biorenewable chemical. However, like many other fermentatively produced compounds, they are inhibitory to the biocatalyst. An understanding of the mechanism of toxicity can aid in mitigating this problem. Here, we show that hexanoic and octanoic acids are completely inhibitory to Escherichia coli MG1655 in minimal medium at a concentration of 40 mM, while decanoic acid was inhibitory at 20 mM. This growth inhibition is pH-dependent and is accompanied by a significant change in the fluorescence polarization (fluidity) and integrity. This inhibition and sensitivity to membrane fluidization, but not to damage of membrane integrity, can be at least partially mitigated during short-term adaptation to octanoic acid. This short-term adaptation was accompanied by a change in membrane lipid composition and a decrease in cell surface hydrophobicity. Specifically, the saturated/unsaturated lipid ratio decreased and the average lipid length increased. A fatty acid-producing strain exhibited an increase in membrane leakage as the product titer increased, but no change in membrane fluidity. These results highlight the importance of the cell membrane as a target for future metabolic engineering efforts for enabling resistance and tolerance of desirable biorenewable compounds, such as carboxylic acids. Knowledge of these effects can help in the engineering of robust biocatalysts for biorenewable chemicals production.

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The damaging effects of short chain fatty acids on Escherichia coli membranes

Carboxylic acids are an attractive biorenewable chemical in terms of their flexibility and usage as precursors for a variety of industrial chemicals. It has been demonstrated that such carboxylic acids can be fermentatively produced using engineered microbes, such as Escherichia coli and Saccharomyces cerevisiae. However, like many other attractive biorenewable fuels and chemicals, carboxylic acids become inhibitory to these microbes at concentrations below the desired yield and titer. In fact, their potency as microbial inhibitors is highlighted by the fact that many of these carboxylic acids are routinely used as food preservatives. This review highlights the current knowledge regarding the impact that saturated, straight-chain carboxylic acids, such as hexanoic, octanoic, decanoic, and lauric acids can have on E. coli and S. cerevisiae, with the goal of identifying metabolic engineering strategies to increase robustness. Key effects of these carboxylic acids include damage to the cell membrane and a decrease of the microbial internal pH. Certain changes in cell membrane properties, such as composition, fluidity, integrity, and hydrophobicity, and intracellular pH are often associated with increased tolerance. The availability of appropriate exporters, such as Pdr12, can also increase tolerance. The effect on metabolic processes, such as maintaining appropriate respiratory function, regulation of Lrp activity and inhibition of production of key metabolites such as methionine, are also considered. Understanding the mechanisms of biocatalyst inhibition by these desirable products can aid in the engineering of robust strains with improved industrial performance.

/pdf/understanding-biocatalyst-inhibition-by-carboxylic-acids-29t9zwm3nz.pdf

Understanding biocatalyst inhibition by carboxylic acids

Evolution for exogenous octanoic acid tolerance improves carboxylic acid production and membrane integrity

Metabolic Engineering has enabled the production of biorenewable fuels and chemicals from biomass using recombinant bacteria. The economic viability of these processes is often limited by inhibition of the biocatalyst by the metabolic product, such as a carboxylic acid or alcohol, or by contaminant compounds in the biomass-derived sugars, such as acetic acid or furans. Historically, selection-based methods have been used to improve biocatalyst tolerance to these inhibitors. But recently, genome-wide analysis has been used to both identify the mechanism of inhibition and reverse engineer inhibitor-tolerant strains, enabling the rational, predictive manipulation of bacteria in order to increase inhibitor tolerance. Here we review recent work in this area, particularly in relation to carboxylic acids, furfural and butanol.

https://lib.dr.iastate.edu/cgi/viewcontent.cgi?article=1175&context=cbe_pubs

Engineering inhibitor tolerance for the production of biorenewable fuels and chemicals

Carboxylic acids are an attractive biorenewable chemical. Enormous progress has been made in engineering microbes for production of these compounds though titers remain lower than desired. Here we used transcriptome analysis of Escherichia coli during exogenous challenge with octanoic acid (C8) at pH 7.0 to probe mechanisms of toxicity. This analysis highlights the intracellular acidification and membrane damage caused by C8 challenge. Network component analysis identified transcription factors with altered activity including GadE, the activator of the glutamate-dependent acid resistance system (AR2) and Lrp, the amino acid biosynthesis regulator. The intracellular acidification was quantified during exogenous challenge, but was not observed in a carboxylic acid producing strain, though this may be due to lower titers than those used in our exogenous challenge studies. We developed a framework for predicting the proton motive force during adaptation to strong inorganic acids and carboxylic acids. This model predicts that inorganic acid challenge is mitigated by cation accumulation, but that carboxylic acid challenge inverts the proton motive force and requires anion accumulation. Utilization of native acid resistance systems was not useful in terms of supporting growth or alleviating intracellular acidification. AR2 was found to be non-functional, possibly due to membrane damage. We proposed that interaction of Lrp and C8 resulted in repression of amino acid biosynthesis. However, this hypothesis was not supported by perturbation of lrp expression or amino acid supplementation. E. coli strains were also engineered for altered cyclopropane fatty acid content in the membrane, which had a dramatic effect on membrane properties, though C8 tolerance was not increased. We conclude that achieving higher production titers requires circumventing the membrane damage. As higher titers are achieved, acidification may become problematic.

/pdf/transcriptomic-analysis-of-carboxylic-acid-challenge-in-15zs1t34t2.pdf

Liam A. Royce

Papers

The damaging effects of short chain fatty acids on Escherichia coli membranes

Understanding biocatalyst inhibition by carboxylic acids

Evolution for exogenous octanoic acid tolerance improves carboxylic acid production and membrane integrity

Engineering inhibitor tolerance for the production of biorenewable fuels and chemicals

Transcriptomic Analysis of Carboxylic Acid Challenge in Escherichia coli: Beyond Membrane Damage