| 其他摘要 | Given the concerns about fossil fuel depletion and climatic change, exploiting renewable and environment-friendly energy has become attractive. Short-chain alkane propane has widespread applications in vehicles, cooking, and ambient heating owning to its excellent features such as higher energy density, cleaner combustion, less greenhouse gas emission, and fine compatibility. Hence, producing renewable propane from microbial platforms attracts increasing concerns from academia and industrial community. Previous propane biosynthetic pathways based on fatty acid biosynthesis (FASII) of E. coli or CoA-dependent butanol pathway from Clostridium sp. were restricted by the low availability of precursor. To address this obstacle, we constructed a new biosynthetic pathway for propane production by assembling the engineered valine pathway of E. coli and cyanobacterial aldehyde-deformylating oxygenase (ADO). The new propane pathway can produce abundant isobutyraldehyde and overcomes the low availability of precursor in propane production. After improving the activity of ADO (Prochlorococcus marinus MIT 9313_PMT1231) via rational design aiming at substrate channel, we increased the propane productivity by three times. We also developed a fluorescent biosensor for rapid and in situ monitoring of alkane synthesis in E. coli and carried out several rounds of directed evolution on ADO (Synechococcus elongates PCC7942_orf1593). The main results and conclusions are summarized as follows:
(1) A two-step markerless recombination method was applied to delete 13 genes of BW25113, namely yqhD, adhE, adhP, eutG, yiaY, yjgB, fucO, yahK, DkgA, frdABCD, ldhA, pflB, and fnr, resulting an aldehyde reductases (ALR)-deprived strain BW25113(DE3) Δ13. The strain produced sufficient isobutyraldehyde precursor (1.1 g/L) and finally achieved de novo synthesis of propane (91 μg/L) by assembling the engineered valine pathway and cyanobacterial ADO.
(2) To overcome the poor activity of ADO on isobutyraldehyde, rational design was applied to engineer the ADO active center to accommodate branched-chain isobutyraldehyde. After extensive screening of ADO mutants via whole-cell assay method, we identified two ADO mutants (I127G, I127G/A48G) which exhibited higher catalytic activity for isobutyraldehyde and improved propane productivity by three times (267 μg/L).
(3) Given the lack of fast and high-throughput intracellular alkane detection methods in biofuels research field, we reassembled the two natural alkane-responsive plugins AlkR-PalkM from Acinetobacter baylyi and AlkS-PalkB from Pseudomonas oleovorans to develop a synthetic chimera alkane response biosensor in E. coli with green fluorescent protein (GFP) as reporter. The alkane biosensor provides a visualized and high-throughput way of monitoring the alkane synthesis in E. coli.
(4) Combining the alkane biosensor, error-prone PCR, and fluorescence activated cell sorting (FACS) together, several rounds of directed evolution were carried out on ADO, resulting in about three-fold increase on fatty alkane productivity. |
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