A two-staged bacterial process coupling methanotrophic and heterotrophic bacteria for 1-alkene production from methane

Methane (CH4) is a sustainable carbon feedstock source for aerobic CH4-oxidizing bacteria (methanotrophs) to produce value-added chemicals. Under O2-limiting conditions, CH4 oxidation results in the production of various short-chain organic acids and platform chemicals. However, these CH4-derived products are still limited to C2-C6 and could be extended by utilizing them as a feedstock for heterotrophic bacteria. A two-stage system for CH4 abatement and 1-alkene production was developed in this study. Gamma- and alphaproteobacterial methanotrophs, i.e. Methylobacter tundripaludum SV96 and Methylocystis rosea SV97, respectively, were investigated in batch tests under different gas supplementation schemes. Under O2 limiting conditions (O2/CH4 molar ratio ~0.3), M. tundripaludum SV96 could potentially produce formate, acetate, succinate, and malate, accounted for ~7.4% of mol-CH4 consumed. For the first time, the organic acids-rich spent media derived from O2 limited-methanotrophic cultivation were successfully used for 1-alkene production using engineered Acinetobacter baylyi ADP1 (′tesA-undA) cells.

Harnessing a Novel P450 Fatty Acid Decarboxylase from Macrococcus caseolyticus for Microbial Biosynthesis of Odd Chain Terminal Alkenes

ABSTRACTAlkenes are industrially important platform chemicals with broad applications. In this study, we report a microbial conversion route for direct biosynthesis of medium and long chain terminal alkenes from fermentable sugars by harnessing a novel P450 fatty acid (FA) decarboxylase from Macrococcus caseolyticus (OleTMC). We first characterized OleTMC and demonstrated its in vitro H2O2-independent activities towards linear and saturated C10:0-C18:0 FAs, with the highest activity for C16:0 and C18:0 FAs. Combining protein homology modeling, in silico residue mutation analysis, and docking simulation with direct experimental evidence, we elucidated the underlying mechanism for governing the observed substrate preference of OleTMC, which depends on the size of FA binding pocket, not the catalytic site. Next, we engineered the terminal alkene biosynthesis pathway, consisting of an engineered E. coli thioesterase (TesA*) and OleTMC, and introduced this pathway into E. coli for direct terminal alkene biosynthesis from glucose. The recombinant strain E. coli EcNN101 produced a total of 17.78 ± 0.63 mg/L odd-chain terminal alkenes, comprising of 0.9% ± 0.5% C11 alkene, 12.7% ± 2.2% C13 alkene, 82.7% ± 1.7% C15 alkene, and 3.7% ± 0.8% C17 alkene, and a yield of 0.87 ± 0.03 (mg/g) on glucose after 48 h in baffled shake flasks. To improve the terminal alkene production, we identified and overcame the electron transfer limitation in OleTMC, by introducing a two-component redox system, consisting of a putidaredoxin reductase CamA and a putidaredoxin CamB from Pseudomonas putida, into EcNN101, and demonstrated the terminal alkene production increased ∼2.8 fold after 48 h. Overall, this study provides a better understanding of the function of P450 FA decarboxylases and helps guide future protein and metabolic engineering for enhanced microbial production of target designer alkenes in a recombinant host.

Combinatorial metabolic engineering of Saccharomyces cerevisiae for terminal alkene production

Biological production of terminal alkenes has garnered a significant interest due to their industrial applications such as lubricants, detergents and fuels. Here, we engineered the yeast Saccharomyces cerevisiae to produce terminal alkenes via a one-step fatty acid decarboxylation pathway and improved the alkene production using combinatorial engineering strategies. In brief, we first characterized eight fatty acid decarboxylases to enable and enhance alkene production. We then increased the production titer 7-fold by improving the availability of the precursor fatty acids. We additionally increased the titer about 5-fold through genetic cofactor engineering and gene expression tuning in rich medium. Lastly, we further improved the titer 1.8-fold to 3.7 mg/L by optimizing the culturing conditions in bioreactors. This study represents the first report of terminal alkene biosynthesis in S. cerevisiae, and the abovementioned combinatorial engineering approaches collectively increased the titer 67.4-fold. We envision that these approaches could provide insights into devising engineering strategies to improve the production of fatty acid-derived biochemicals in S. cerevisiae.

Beyond the use of modifiers in selective alkyne hydrogenation: silver and gold nanocatalysts in flow mode for sustainable alkene production

Supported silver and gold nanoparticles are highly stereo and chemoselective catalysts for the three-phase hydrogenation of alkynes in continuous mode.