Continuous photoproduction of hydrocarbon drop-in fuel by microbial cell factories

Abstract

Use of microbes to produce liquid transportation fuels is not yet economically viable. A key point to reduce production costs is the design a cell factory that combines the continuous production of drop-in fuel molecules with the ability to recover products from the cell culture at low cost. Medium-chain hydrocarbons seem ideal targets because they can be produced from abundant fatty acids and, due to their volatility, can be easily collected in gas phase. However, pathways used to produce hydrocarbons from fatty acids require two steps, low efficient enzymes and/or complex electron donors. Recently, a new hydrocarbon-forming route involving a single enzyme called fatty acid photodecarboxylase (FAP) was discovered in microalgae. Here, we show that in illuminated E. coli cultures coexpression of FAP and a medium-chain fatty acid thioesterase results in continuous release of volatile hydrocarbons. Maximum hydrocarbon productivity was reached under low/medium light while higher irradiance resulted in decreased amounts of FAP. It was also found that the production rate of hydrocarbons was constant for at least 5 days and that 30% of total hydrocarbons could be collected in the gas phase of the culture. This work thus demonstrates that the photochemistry of the FAP can be harnessed to design a simple cell factory that continuously produces hydrocarbons easy to recover and in pure form.

Optimization and Engineering of Fatty Acid Photodecarboxylase for Substrate Specificity

“…Our results show that there is no detectable difference on the total protein expression levels (Figure S2). However, the activity of the whole cell catalyst protected from light during expression was higher than that exposed to ambient light for C16 : 0 and C18 : 1 substrates (Figure 1), indicating deactivation (due to photo‐damage) of the protein to some extent, consistent with the earlier study [6c] . Substrate addition to protein expression medium, to avoid deactivation, was analyzed as well.…”

“…According to a previous study, [6c] the expression of soluble Cv FAP is negatively affected by high intensity light exposure. This has been attributed to photo‐damage caused by FAD‐mediated free radical generation in the absence of substrate, as confirmed in a more recent study by EPR spectroscopy [9] .…”

A Reconstructed Common Ancestor of the Fatty Acid Photo‐decarboxylase Clade Shows Photo‐decarboxylation Activity and Increased Thermostability

“…[14,15] Unfortunately, synthetic applications of CvFAP are limited by its relatively poor production in Escherichia coli, its light stability and its sensitivity to solvents. [11,16,17] All this results in a limited product formation, which cannot pay off the effort for the production of the enzyme. On top of the low stability, production yields remain low, which is an obstacle for both the enzyme's biochemical characterization and industrial application -even though the purification under red light was beneficial.…”

“…Unfortunately, synthetic applications of CvFAP are limited by its relatively poor production in Escherichia coli , its light stability and its sensitivity to solvents [11,16,17] . All this results in a limited product formation, which cannot pay off the effort for the production of the enzyme.…”

CYP505E3: A Novel Self‐Sufficient ω‐7 In‐Chain Hydroxylase

“…In summary, we have reported the first catalyst for the regioselective in‐chain hydroxylation of n ‐alkanes. Given the high TTNs obtained and already high regioselectivity, CYP505E3 is a promising biocatalyst for the further development of in‐chain hydroxylases with improved or adjusted regioselectivity and substrate scope to enable the synthesis of unique “natural” alcohols from “natural” biosynthesized n ‐alkanes . This could, for instance, be done by directed evolution through iterative saturation mutagenesis of selected residues as reported for P450pyr (CYP153A7) .…”