Current energy sources are facing increasing challenges related to long-term sustainability because of climate changes and the inevitable depletion of fossil resources; this situation has prompted the development of alternative energy sources such as biofuels from renewable biomass. Bio-alka(e)nes are potentially high-value chemicals with several industrial applications such as drop-in biofuels [1]. They chemically and structurally resemble petroleum hydrocarbons. Moreover, they show superior energy density and hydrophobicity than other biofuels.

Alka(e)ne synthesis by engineered Escherichia coli occurs through a heterologous pathway based on two sequential enzymatic mechanisms. First, fatty acyl-ACP (acyl carrier protein) is reduced by ACP reductase (AAR) to fatty aldehydes. Second, fatty aldehydes are converted to alka(e)nes by aldehyde deformylating oxygenase (ADO) [2] (Fig. 1). The synthesis of alka(e)nes in cyanobacteria is catalyzed by AAR and ADO. The yield of alka(e)nes depends on the activity and concentration of soluble AAR and ADO. Various cyanobacterial strains are capable of producing alka(e)nes. SeAAR obtained from Synechococcus elongatus PCC 7942 demonstrates the highest yield; however, even the most potent SeAAR exhibits reduced activity due to enzymatic aggregation, resulting in poor solubility of proteins expressed in E. coli [3,4,5,6,7]. Previous studies compared the solubility and activity of AARs and ADOs from multiple representative cyanobacteria, it was observed that AAR has markedly lower solubility than ADO [8,9]. This led to the limitation of the AAR enzymatic reaction as the rate-limiting step in alkane synthesis during the co-expression of the AAR and ADO genes in E. coli [10]. Hence, it is essential to increase the amount as well as activity of soluble AAR for improving bioalka(e)ne yield [3].

Fusion tags increase the solubility as well as stability of target proteins and peptides synthesized in E. coli [11]. Therefore, in this study, fusion tags were used to further enhance the activity or solubility of AAR and thus increase the production of alka(e)nes. Polyhistidine, also known as His6 tag, is an affinity tag comprising six histidine residues. Solubility-enhancing tags also include the well-known thioredoxin (Trx), maltose-binding protein (MBP), and N-utilization substance protein A (NusA) [12].

His tag comprises at least six consecutive histidine residues located at the C- or N-terminal or in the fusion protein’s center. The 0.8-kDa His6 tag reduces the interference with the folding and structure of the target protein. Previous studies have combined His tag with His tag-specific antibodies to detect proteins [13,14,15,16]. His tag-carrying imidazole rings interact with divalent transition metal ions such as Co2+, Ni2+, Zn2+, Ca2+, and Cu2+ by forming reversible coordinate bonds [17].

Trx is a 12-kDa cytoplasmic and intracellular protein found in E. coli and is thermostable and highly soluble [12]. It can be synthesized along with a target protein, resulting in improved protein solubility [18]. Trx exhibits inherent redox activity, as it reduces disulfide bonds by exchanging thiol and disulfide groups. It is generally employed as a fusion tag to prevent inclusion body formation during the synthesis of recombinant proteins [19,20].

MBP is a 43-kD a highly soluble periplasmic protein that is highly expressed in E. coli; it is used as a solubility enhancer tag [21]. As a fusion tag, MBP enhances target protein solubility by exhibiting the intrinsic activity of chaperones [22]. This tag facilitates target protein folding by preventing its self-association. The large hydrophobic surface area of MBP enables binding to other maltose transport proteins during protein folding [21,23].

NusA is a large (55 kDa) protein involved in transcription. NusA promotes the termination of protein synthesis by pausing the activity of RNA polymerase when acting alone. It also acts as an anti-termination protein and is thus employed as a fusion tag to ensure stability as well as to increase target protein solubility [24,25,26]. NusA reduces translation during the transcriptional pauses, thereby enhancing the duration of the protein folding mechanism [27]. However, the large size of MBP and NusA affects the fusion process [17].

The location of fusion tags at the C- or N-terminal of the passenger protein affects the outcome. Generally, N-terminal tags are preferred because of their reliability and efficiency in translation initiation. The efficient translation initiation sites present on the tag assist in the synthesis of fusion proteins. These sites can be deleted with or without extra residues at the target protein’s native N-terminal sequence, as most endoproteases cleave near or at the C-terminal of their recognition sites [28].

The biosynthetic pathway for producing alkanes can be introduced into non-native hosts, such as yeast and E. coli, to increase alkane production. In a previous study, cyanobacterial alkane biosynthetic genes were expressed in E. coli, and a mixture of alka(e)nes was produced by directly using free fatty acids as the substrate; the yield was approximately 5.8 mg/L [29]. In another study, alkane production was increased by enhancing AAR and ADO enzymes’ catalytic activity using the protein engineering approach. They constructed a chimeric ADO-AAR fusion protein assembly on a DNA scaffold to regulate their spatial arrangement and stoichiometric ratios when expressed in E. coli; the yield achieved was 44 mg/L alkane [30]. In the third approach, alkane production was increased by blocking the competing alcohol production pathway through the deletion of the yqhD gene encoding aldehyde reductase and through the overexpression of the transcription factor FadR-encoding gene, which participates in fatty acid synthesis. This approach increased the yield to approximately 250 mg/L alkane [31]. Among yeast species, Saccharomyces cerevisiae was used for alkane production; a comparison of various aldehyde decarbonylases showed that cyanobacterial ADO was the most optimal enzyme to produce alkanes, resulting in 0.11 mg/L of alkane yield [32]. Researchers engineered yeast strains for increasing free fatty acid accumulation and for expressing the alkane biosynthetic gene CAR obtained from Mycobacterium marinum and the gene ADO obtained from Nostoc punctiforme; this approach significantly increased alkane production to 0.8 mg/L [33].

In the present study, Synechococcus elongatus PCC 7942 AAR (SeAAR) and N. punctiforme PCC 73102 ADO (NpADO) were used for alka(e)ne biosynthesis. Enzyme expression was induced in the E. coli BL21 (DE3) strain, which was used as a host for alka(e)ne production. We compared the fusion tags His6, 2 ×His6, His12, Trx, MBP, and NusA at the N-terminal of SeAAR to demonstrate their utility in fusion protein expression and solubility and in bio-alka(e)ne synthesis.