Biofuels Production projects
Microbial Production of 1-Undecene and Related Fuel-like Molecules
Bacterial volatiles represent a source for new biofuel compounds in addition to the traditional bioethanol and plant oil-derived biodiesel. The identified relevant volatile compounds include various short- to mediun-chain alkanes, alkenes, alcohols and isoprenoids, which have great potential to replace or supplement petroleum-derived fuels. For example, butanol, isopentanol, undecene and undecane are routinely observed in Pseudomonas cultures. l-Undecene is particularly intriguing attributed to its superior physical properties, which may have direct applications as both a fuel and an industrial chemical. However, little is known about the enzymatic logic for biosynthesis of many volatile organic compounds (VOCs) produced by various bacterial cultures. This project seeks to understand the biosynthetic machinery and to engineer the biosynthesis of l-undecene and related fuel-like molecules. The results will expand our molecular toolbox for hydrocarbon biosynthesis and facilitate the production of new microbial fuels.
We previously discovered the first microbial aliphatic medium-chain 1-alkene (MCAE) biosynthetic enzyme, UndA, which was able to convert medium-chain fatty acids (C10-C14) into their corresponding terminal olefins using an oxygen-activating, non-heme iron dependent mechanism. Further extensive biochemical and X-ray crystal structural analyses suggest a highly unusual mechanism of beta-hydrogen abstraction by a “less reactive” iron center during fatty acid activation. These results together with our preliminary data on the engineered biosynthesis of 1-undecene in microbes pave the way for tailored bioconversion of renewable raw materials to MCAE-based biofuels and chemical commodities.
1-Undecene is naturally produced by Pseudomonads as one of the signature volatile organic compounds and holds interest to researchers as a potential biofuel and “green” feedstock for chemical synthesis. We identified a single gene from P. fluorescens Pf-5 which enabled the 1-undecene production in E. coli. The activity of the encoding enzyme was further reconstituted in vitro: lauric acid was decarboxylated and oxidized to form 1-undecene through an O2-activating, non-heme iron dependent mechanism. This enzyme recognized fatty acid substrates with a medium chain length (C10-C14), and demonstrated promiscuity toward the substitutions on the α-carbon, ω-carbon, and chain branching, showing the potential as a useful biocatalyst. It is well conserved in Pseudomonads and several other closely related species, representing a big family of terminal olefin-producing enzymes with a novel catalytic mechanism. We further solved the crystal structure of holoenzyme which provided insight into its catalytic mechanism and substrate specificity.
1-Undecene is an advanced biodiesel molecule and “green” synthetic precursor for many commercial chemicals. Undecene is naturally produced by several microbes, but the productivity is inadequate for industrial production and applications. Successful improvement of undecene productivity will be enabled by the in-depth understanding of the biosynthetic pathway. Toward this goal, we first developed a sensitive detection method by trapping undecene from the headspace of the bacterial culture onto a specific fiber. Then we attempted to access the biosynthetic pathway through three approaches: 1) We performed the comparative genomic analysis by subtracting the genomes of non-producers from the ones of producers, which generated the candidate genes to guide the screening of a mutant library of a producer, Pseudomonas aeruginosa. The undecene null-producers will reveal the mutated gene essential for the undecene biosynthesis. 2) We randomly sheared the genomic DNA of a producer and introduced the resulting fragments into the non-producer E. coli. An E. coli recipient clone that produces undecene will be identified to reveal the introduced undecene biosynthetic genes. 3) The crude extract of a producer will be fractionized to narrow down the candidate proteins for undecene production.
Published in 2014
Microbial Biosynthesis of Medium-Chain 1-Alkenes by a Non-Heme Iron Oxidase, Z. Rui, Li Xin, X. Zhu, J. Liu, B. Domigan, I. Barr, J. Cate, W. Zhang, Proceedings of the National Academy of Sciences, doi: 10.1073/pnas.1419701112, December 2014.