Research articleMolecular evolution of the plant ECERIFERUM1 and ECERIFERUM3 genes involved in aliphatic hydrocarbon production
Graphical abstract
Introduction
Alkanes are acyclic saturated hydrocarbons and have the general chemical formula CnH2n+2 (Gutman, 2008). Alkanes are the major constituents of fossil fuels including petroleum and natural gas and exist in the form of a gas, liquid or wax, depending on the length and structure of the carbon backbone (Arora, 2006). Alkanes can also be naturally produced by living organisms, including bacteria, animals, fungi, and plants (Marsh and Waugh, 2013). In general, biological alkanes have an odd number of carbon atoms, indicating that they are derived by the loss of one carboxyl carbon from fatty acids with even numbers of carbons. The enzymatic decarbonylation processes in various living organisms convert fatty acid metabolites into alkanes (Marsh and Waugh, 2013).
The 2000s energy crisis triggered extensive research efforts in identifying genes that encode enzymes involved in enzymatic decarbonylation of fatty aldehydes, a promising biocatalytic conversion for the production of alternative and sustainable hydrocarbon fuels from renewable sources (Bhujade et al., 2017; Schirmer et al., 2010). As a result, an alkane biosynthesis pathway consisting of an acyl-acyl carrier protein reductase and an aldehyde decarbonylase in cyanobacterium Synechococcus elongatus PCC7942 was first identified and characterized (Schirmer et al., 2010). This microbial operon has been utilized to modify an Escherichia coli strain to produce C13 to C17 alkanes from renewable sources in a single fermentation step (d’Espaux et al., 2015; Schirmer et al., 2010). Although the first decarbonylase was discovered in bacteria, alkane biosynthesis in plants was investigated over three decades ago (Cheesbrough and Kolattukudy, 1984). The aerial epidermis of terrestrial plants produces and secretes alkane waxes, which are very-long-chain molecules (between 20–36 carbons) derived from very-long-chain fatty acids (Fu et al., 2015). The alkane waxes are deposited in the plant cuticle, a waterproof barrier that plays a preventive role against both biotic and abiotic stimuli in terrestrial plants.
The Arabidopsis thaliana ECERIFERUM1 (CER1) gene was proposed, over two decades ago, to encode a plant alkane-producing decarbonylase (Aarts et al., 1995). However, how the CER1 protein works was not well understood until recently. A successful reconstitution of alkane biosynthesis in yeast found that CER1 and A. thaliana CER3 proteins interact to produce alkanes together (Bernard et al., 2012). The CER3 protein serves as a fatty acyl reductase to produce metabolic precursors such as fatty aldehydes or other intermediates that are currently unknown. CER1, which serves as a decarbonylase, converts the precursors into alkanes. Interestingly, CER1 and CER3 proteins share 35% amino acid identity, indicating that they likely originated from a common ancestor gene (Bernard et al., 2012).
Research has attempted to improve the catalytic efficiency of these enigmatic alkane-producing enzymes based on the understandings of their catalytic mechanisms (Hayashi et al., 2015; Wang et al., 2014). Protein structure information would facilitate the efforts. Decarbonylases in cyanobacteria are soluble proteins and crystal structures of several cyanbacterial decarbonylases are now available (Buer et al., 2014; Jia et al., 2015; Park et al., 2016). In contrast, Arabidopsis CER1 and fruit fly decarbonylase CYP4G1 are membrane proteins, of which overexpression and purification are a considerable challenge (Marsh and Waugh, 2013). In addition, the decarbonylases from different organisms or species exhibit differential preferences of substrates in alkane production (Qiu et al., 2012; Schirmer et al., 2010). Various decarbonylases are required to produce gasoline (C4-C9), jet fuel (C8-C16), diesel (C10-C18), and lubricants (C16-C30). It would therefore be necessary to carry out further screening for the identification of efficient decarbonylases that produce alkanes in different chain lengths for various applications.
To our knowledge, the alkane-producing enzymes in plants have not been comprehensively investigated. In this report, we examined the phylogeny and evolution of plant CER1 and CER3 genes. To facilitate the potential applications, the protein structures and motifs of CER1 proteins were investigated as well. This in silico analysis would lay a foundation for further experimental study.
Section snippets
Acquisition of CER1 and CER3 sequences
The identifications of CER1 and CER3 sequences were conducted in Phytozome v12.1.5 (https://phytozome.jgi.doe.gov/), using the Arabidopsis CER1 (Locus AT1G02205) and CER3 (Locus AT5G57800) protein sequences in The Arabidopsis Information Resource (TAIR) (https://www.arabidopsis.org/) as the Basic Local Alignment Search Tool-protein (BLASTp) queries, respectively.
We identified and retrieved CER1 and CER3 sequences from 56 out of 57 land plant species, including Amaranthus hypochondriacus (Ahy),
CER1 and CER3 genes were obtained from 56 land plants and 3 chlorophyte species
193 CER1 proteins and their corresponding coding sequences were identified and downloaded for 56 land plant species (Additional file 1). Like the Arabidopsis CER1 gene, the identified CER1 sequences encode histidine-containing motifs and the putative WAX2 C-terminal domain. Histidine clusters are essential for the catalytic activity of hydroxylases and desaturases (Shanklin and Cahoon, 1998). We failed to identify a CER1 homologue in duckweed Spirodela polyrhiza. Since the aquatic species has
Conclusion
We identified 193 CER1 and 128 CER3 gene sequences from 56 land plant respectively, and three common homologues (i.e. CER1/3) of CER1 and CER3 from three chlorophytes. This is the first survey of CER1 and CER3 genes covering a wide range of plant taxa. The presence of common homologue of CER1 and CER3 in chlorophytes and the structural similarity of their encoding proteins shown in this study support the common origin of CER1 and CER3 genes. Although alkane synthesis already occurs in
Competing interest
We have no competing interests.
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Origin and diversification of ECERIFERUM1 (CER1) and ECERIFERUM3 (CER3) genes in land plants and phylogenetic evidence that the ancestral CER1/3 gene resulted from the fusion of pre-existing domains
2021, Molecular Phylogenetics and EvolutionCitation Excerpt :CER1 and CER3 proteins have a characteristic bi-domain structure (ERG3/FAH and WAX2) that may show differential divergence (Chen et al., 2003; Bernard et al., 2012). Our phylogenetic analyses of these proteins from green plants provide insights into the origin, diversification and variation in copy number of the genes across land plants that are consistent with results of a recent study (Wang et al., 2019). Shedding new light on the question, we present phylogenetic evidence that: (a) a single ancestral gene, CER1/3, duplicated in the ancestral land plants to generate the paralogs; CER1 and CER3, supporting a critical role for cuticular waxes in early colonization of land (Niklas et al., 2017), (b) WAX2-WxAR sequences are limited to relatively few lineages across the tree of life, and the CER1/3 protein may have been assembled from the WAX2-WxAR and ERG3/FAH domains in green plants and two other lineages.
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Present address at: U.S. Department of Agriculture, Agricultural Research Service, Hard Winter Wheat Genetics Research, Throckmorton Hall, Manhattan, KS 66506.