Molecular evolution of the plant ECERIFERUM1 and ECERIFERUM3 genes involved in aliphatic hydrocarbon production

Highlights

• Chlorophytes have a common homolog of the genes CER1 and CER3.

• Chlorophytes may be one of the earliest plant taxa to contain CER1 and CER3.

• CER1 and CER3 proteins are structurally similar, but CER1 proteins have more conserved histidine-containing motifs.

• For CER1 proteins, there was no significant loss or gain of protein motifs after ancient and recent duplications.

• CER1 proteins are highly conserved throughout evolution with no evidence of positive selection.

Abstract

The Arabidopsis ECERIFERUM1 (CER1) protein is a decarbonylase that converts fatty acid metabolites into alkanes. Alkanes are components of waxes in the plant cuticle, a waterproof barrier serving to protect land plants from both biotic and abiotic stimuli. CER1 enzymes can be used to produce alternative and sustainable hydrocarbons in eukaryotic systems. In this report we identified 193 CER1 and 128 CER3 sequences from 56 land plants respectively. CER1 and CER3 proteins have high amino acid similarity and both are involved in alkane synthesis in Arabidopsis. The common homologues of CER1 and CER3 genes were identified in three species of chlorophytes, which may be one of the earliest plant taxa that possess CER1 and CER3 genes. To facilitate potential applications, the 3-dimensional structure and conserved motifs of CER1 proteins were also characterized. CER1 and CER3 proteins are structurally similar, but CER1 proteins have more conserved histidine-containing motifs common to fatty acid hydroxylases and stearoyl-CoA desaturases. There was no significant loss or gain of protein motifs after ancient and recent duplications, suggesting that varied properties of CER1 proteins may be associated with less-conserved regions. Among 56 land plants, the codon-based assessments of selection modes revealed that neither entire proteins nor individual amino acids of CER1 proteins were significantly subjected to positive selection, indicating that CER1 proteins are highly conserved throughout evolution.

Introduction

Alkanes are acyclic saturated hydrocarbons and have the general chemical formula C_nH_2n+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.