Harnessing a Novel P450 Fatty Acid Decarboxylase fromMacrococcus caseolyticusfor Microbial Biosynthesis of Odd Chain Terminal Alkenes

Abstract

23Alkenes are industrially important platform chemicals with broad applications. In this 24 study, we report a microbial conversion route for direct biosynthesis of medium and long chain 25 terminal alkenes from fermentable sugars by harnessing a novel P450 fatty acid (FA) 26 decarboxylase from Macrococcus caseolyticus (OleTMC). We first characterized OleTMC and 27 demonstrated its in vitro H2O2-independent activities towards linear and saturated C10:0-C18:0 28 FAs, with the highest activity for C16:0 and C18:0 FAs. Combining protein homology 29 modeling, in silico residue mutation analysis, and docking simulation with direct experimental 30 evidence, we elucidated the underlying mechanism for governing the observed substrate 31 preference of OleTMC, which depends on the size of FA binding pocket, not the catalytic site. 32Next, we engineered the terminal alkene biosynthesis pathway, consisting of an engineered E. 33 coli thioesterase (TesA*) and OleTMC, and introduced this pathway into E. coli for direct 34 terminal alkene biosynthesis from glucose. The recombinant strain E. coli EcNN101 produced 35 a total of 17.78 ± 0.63 mg/L odd-chain terminal alkenes, comprising of 0.9% ± 0.5% C11 36 alkene, 12.7% ± 2.2% C13 alkene, 82.7% ± 1.7% C15 alkene, and 3.7% ± 0.8% C17 alkene, 37 and a yield of 0.87 ± 0.03 (mg/g) on glucose after 48 h in baffled shake flasks. To improve the 38 terminal alkene production, we identified and overcame the electron transfer limitation in 39OleTMC, by introducing a two-component redox system, consisting of a putidaredoxin 40 reductase CamA and a putidaredoxin CamB from Pseudomonas putida, into EcNN101, and 41 demonstrated the terminal alkene production increased ~2.8 fold after 48 h. Overall, this study 42 provides a better understanding of the function of P450 FA decarboxylases and helps guide 43 future protein and metabolic engineering for enhanced microbial production of target designer 44 alkenes in a recombinant host.