The Prokaryotic Microalga Limnothrix redekei KNUA012 to Improve Aldehyde Decarbonylase Expression for Use as a Biological Resource

L. redekei KNUA012 was obtained from the KNUA culture collection (Hong et al. 2016). This species was originally isolated in September 2010 from a freshwater bloom sample obtained from Lake Hapcheon (Bongsanmyeon, Hapcheon-gun, Gyeongsangnamdo, South Korea; 35°37′N, 128°02′E) and was named as Korean domestic L. redekei KNUA012 (Hong et al. 2016). This strain was cultured axenically for 20 days on nitrate-containing BG-11 medium (Chang et al. 2013; 2016).

Sequences of the 16S ribosomal RNA gene were compared with those of L. redekei microalgae identified using a BLAST search (NCBI website) for L. redekei KNUA012 (Fig. 1). The primer sets 16S-F (5′-CGGA CGGGT GAGTAACGCGTGA-3′) and 16S-R (5′-GACTACTGGGGTATCTAATCCCATT-3′) were used to amplify the 16s rRNA region. A phylogenetic tree was constructed using Mega 5.2 software (Tamura et al. 2004; 2011) with the maximum composite likelihood model of the neighbor-joining algorithm (Gkelis et al. 2005; Shimura et al. 2015; Tan et al. 2016; Tajima et al. 2018). To optimize the growth conditions, L. redekei KNUA012 cultures were incubated using an Optizen 2120 UV spectrophotometer (Mecasys, South Korea) until their optical densities (ODs) at 750 nm reached 1.2 (OD₇₅₀). This procedure was performed in triplicate. L. redekei KNUA012 cells were cultured over 5–35°C (at intervals of 5°C) to determine the optimal culture temperature.

Fig. 1.

AD (Gen-Bank accession no. KU341741) was amplified from the genomic DNA of Korean domestic L. redekei KNUA012 using 34 cycles of PCR at 94°C for 40 s, 54°C for 40 s, and 72°C for 50 s. The following primers were used: AD-EcoRI–F (5′-GAATTCACCATGCCGCAACTCGAGG-3′) and AD-SalI-R (5′-GTCGACTGGTGGTTAAGCGGCAGCG-3′). The amplicon was ligated into the EcoRI and SalI sites downstream of the T7 promoter in vector pET-28a. We had previously determined the crystal structure of AD in Limnothrix sp. KNUA012 (Park et al. 2016). Based on these results, E. coli BL21 (DE3)-competent cells were transformed with a pET-28a expression vector carrying AD (pET28-AD). To assess the expression of AD, the transformed cells were cultured with vigorous shaking at 20°C in 500 ml of Luria-Bertani broth supplemented with 50 μg/ml kanamycin. The culture was induced with 0.2 mM isopropyl-D-1-thiogalactopyranoside when the OD₆₀₀ reached 0.6. The culture was further incubated for approximately 20 h. The cell pellet was harvested via centrifugation at 4,000 rpm for 20 min and then resuspended in cold lysis buffer (50 mM sodium phosphate, pH 8.0; 300 mM NaCl; and 5 mM imidazole) freshly supplemented with 0.2 mg/ml lysozyme, 0.5 mM phenylmethylsulfonyl fluoride, and EDTA-free Protease Inhibitor Cocktail (Sigma-Aldrich, USA). After incubation for 20 min on ice, the cells were disrupted via ultrasonication (Sonic Disembrator 550; Thermo Fisher Scientific, Inc., USA) with short pulses (10 s) and pauses (10 s) on ice over a 1 h period. Cell debris was removed via centrifugation at 10,000 rpm for 30 min. The clear supernatant was then poured into a gravity-flow column prepacked with Ni-NTA resin (Qiagen, Germany) to isolate the His-tagged AD in the supernatant. Unbound proteins were washed with a 10-column volume of wash buffer (50 mM sodium phosphate, pH 8.0; 300 mM NaCl; and 20 mM imidazole). Subsequently, the protein bound to the Ni-NTA resin was eluted with the wash buffer. To desalt and concentrate the eluted AD protein solution, it was first mixed with the EDTA-free Protease Inhibitor Cocktail and cold 20 mM Tris-HCl (pH 8.0) and then filtered using Amicon^® Ultra Centrifugal Filters (Ultracel-10K, Millipore^®; Merck KGaA, Germany). The resultant protein concentration was spectrophotometrically analyzed using a Protein Dye Reagent (Bio-Rad Laboratories, Inc., USA).

To determine the fatty acid composition, lipids were extracted and analyzed using gas chromatography/mass spectrophotometry (Bligh and Dyer 1959). L. redekei KNUA012 was cultured for 18 days and then harvested via centrifugation at 4,000 rpm for 3 min. Lipid yields with equal biomasses were calculated based on OD, as described by Yeo et al. (2013). The dried sample (100 mg) was mixed with the chloroform: methanol (2 : 1) solution, and the mixture was incubated at 25°C for 16 h. Subsequently, the chloroform-soluble portion was isolated and dried using a rotary evaporator. The crude lipid was treated with methanol and potassium hydroxide to facilitate transesterification. To isolate fatty acid methyl esters (FAMEs), 2 ml of hexane was added to the crude lipid mixture, and the reaction mixture was stirred at 30°C for 10 h (Hong et al. 2012; Hong et al. 2016). Subsequently, the hexane layer was isolated from the reaction mixture, analyzed using GLC-90 (Supelco^®, Merck KGaA, Germany), and used as an external standard to identify FAME contents. The FAME contents in the sample were evaluated using a 6890N gas chromatograph (Agilent Technologies Inc., USA) equipped with a 5973N mass selective detector (Agilent Technologies Inc., USA) and HP-5MS capillary column (30 m × 0.25 mm internal diameter × 0.25 μm film thickness; Agilent Technologies Inc., USA). The temperature of the GC oven was maintained at 120°C for 2 min, increased to 300°C at a rate of 5°C/min, and then maintained at 300°C for 22 min. The injection volume was set at 1 μl, with a split ratio 20 : 1. Helium was used as a carrier gas at a constant flow rate of 1 ml/min. The injector and MS source temperatures were set at 250°C and 230°C, respectively. In the electron impact mode, an acceleration voltage of 70 eV was used for sample ionization, and the scanning range was increased from 50 to 550 m/z. Wiley/NBS libraries were used as the reference databases (McLafferty and Stauffer 1989). The calculated GC/MS values were represented as percentages relative to the values presented in Fig. 4 and S1 (calibration curves). The standard GLC-90 control was used to obtain the GC/MS total ion chromatogram of standard FAMEs (Fig. 4a).

We compared individual data points using Student's t-test, and a p-value of < 0.05 was considered to indicate statistical significance. All experiments were performed at least in triplicate, and the results were expressed as means ± standard deviation. We expressed all biochemical results corresponding to pET28-AD and pET28 empty vector relative to those corresponding to L. redekei KNUA012, which we defined as 100%.

Global warming and petroleum depletion have prompted researchers to search for renewable energy sources. Microalgae autotrophically produce alkanes (C_nH_n) via an AD that can be used directly as a biodiesel (Regalbuto 2009). Commercial alkanes such as pentadecane (C₁₅H₃₂), 8-heptadecane (C₁₇H₃₄), and heptadecane (C₁₇H₃₆) could be directly used as gasoline, diesel and jet fuel (Lin and Lin 2011; Zhang et al. 2013; Hayashi et al. 2015; Kang et al. 2017; Mathur et al. 2022). To produce alkanes in microalgae, two alkane biosynthesis genes are needed to produce AAR and AD (Schirmer et al. 2010). AD catalyzes the decarbonylation of fatty aldehydes, and fatty acid intermediates are produced via the photosynthetic energy of the microalgae (Marsh and Waugh 2013; Vélez et al. 2015; Gao et al. 2020). Alkane-producing genes have not previously been reported in microalgae isolated in Korea but have been observed in microalgae from other countries. We sought to identify alkane-producing genes in Korean domestic L. redekei KNUA012. Based on its 16s rRNA gene sequence data (Fig. 1), L. redekei KNUA012 is closely related to L. redekei (GenBank accession no. GQ848190.1), with 99% identity (Fig. 1 and Table SII), and clustered with Limnothrix sp. cultures isolated from previous studies (Gkelis et al. 2005; Hong et al. 2016). Although the previous study (Hong et al. 2016) showed that KNUA012 belonged to Limnothrix, we reconfirmed the 16s rRNA sequencing and showed (using a phylogenetic tree) that KNUA012 Limnothrix was a cyanobacterium and could be differentiated from Oscillatoria, Geitlerinema, Leptolyngbya, and Phormidium. Additionally, as aldehyde decarbonylase (AD) is the primary focus of our research, its existence in Korean microalgae was not previously reported; thus, KNUA012 genetic identity was ascertained using 16s rRNA instead of AD genetic information. As a result, the culture isolate was identified as L. redekei KNUA012.

Several microalgae produce polysaccharides and small quantities of proteins, lipids, and glycoproteins (Wingender et al. 1999; Khattar et al. 2010; Spain and Funk 2022). Depending on the location, biofuel constituents, and gas vacuoles occur as capsular polysaccharides with the polymer loosely attached to the cell surface (Trabelsi et al. 2009; Khattar et al. 2010). Microbial biofuel constituents and gas vacuoles are produced by prokaryotes (Parikh and Madamwar 2006; Zou et al. 2006; Chi et al. 2007; Duan et al. 2008; Khattar et al. 2010; Cheah and Chan 2022). Microbial polysaccharides play a crucial role in several microbial traits, including pathogenesis, symbiotic ability, biofuel production, and stress resistance (Parikh and Madamwar 2006; Khattar et al. 2010; Spain and Funk 2022). The Korean domestic L. redekei KNUA012 strain exhibited the common features of Limnothrix sp. strains found in Lake Hapcheon, South Korea. Furthermore, it exhibited gas vacuoles as one of the common features (Fig. 2a). In addition, L. redekei KNUA012 shared several traits with other Limnothrix spp. and members of the family Pseudanabaenacea and was morphologically similar to L. redekei, also known as Oscillatoria redekei (Gkelis et al. 2005). In this strain, the cells were straight and not bent or spiraled. The cells possessed filamentous and free-floating characteristics. According to the study by Meffert (1988), this strain had following features: a straight trichome without a sheath, the trichome unattenuated toward the ends; rectangular rather than round cells; cells observed in the cross wall at the ends; and gas vacuoles localized on either side of the cross wall on the inner cell surface. A previous study (Hong et al. 2016) stated that KNUA012 belonged to the Limnothrix group, but no morphological characteristics were referenced. This manuscript included references to the morphological characteristics to show that KNUA012 belonged to the Limnothrix group. As shown in Fig. 2b, the optimal temperature required for L. redekei KNUA012 growth was determined to be 25°C, i.e., the temperature at which the cells attained confluency after 20 days; however, their growth was delayed at 5°C, 10°C, 15°C, 20°C, 30°C, and 35°C (Fig. 2b). The optimal growth temperature allows the rapid growth of cells to increase biomass yield. Microalgal growth is affected by various environmental factors, such as temperature and light intensity, suggesting that the properties of the biomass can be regulated by identifying the optimal culture settings under various environmental conditions (Dalirynezhad et al. 2017; Cheah and Chan 2022). Therefore, microalgae can produce a range of value-added products, including proteins, lipids, carbohydrates, and pigments, and the production of such organic compounds under stressful environmental conditions can be enhanced (Bhalamurugan et al. 2018). In the current study, when L. redekei KNUA012 was cultured for 20 days, the culture at 25°C showed optimal overall growth compared with that at 5°C, 10°C, 15°C, 20°C, 30°C, and 35°C (Fig. 2b). The previous study (Hong et al. 2016) provided information on the optimal growth temperature for KNUA012 at 5°C, 10°C, 15°C, 20°C, and 25°C along with the optical density at the stationary phase. In the present study, we provided optimal growth temperatures at 30°C and 35°C, generating the highest optical density values and presenting growth patterns for 20 days. Additionally, KNUA012 grows easily at 30°C and 35°C, although 25°C is the optimal growth temperature. Based on these results, the optimal growth conditions for L. redekei KNUA012 were set at 25°C for 20 days for subsequent experiments. The optimal growth conditions for maximum lipid yield can be used to achieve the highest biomass yield.

Fig. 2.

Microalgae can produce straight-chain alkanes/alkenes from fatty acyl-ACP via the alkane biosynthesis pathway using the enzymes AAR and AD (Tillman et al. 1999; Hayashi et al. 2015; Kudo et al. 2019; Kittel et al. 2023). AAR reduces fatty acyl-ACP to fatty aldehyde, which is reduced to alkanes/alkenes by AD. In previous studies, the presented alkane operons in E. coli cells led to the production of AAR and AD (Schirmer et al. 2010; Andre et al. 2013; Choi and Lee 2013; Harger et al. 2013; Rahmana et al. 2014; Coursolle et al. 2015; Moulin et al. 2019; Hayashi and Arai 2022). The alkane operons were expressed in E. coli cells, and the production of alkanes was related to the amount of AAR and AD during construction by alkane-producing enzymes for efficient alkane biosynthesis in E. coli cells. Moreover, the production of alkanes has been reported most recently in metabolic reactions for the photoproduction of hydrocarbons by E. coli cells (Schirmer et al. 2010; Andre et al. 2013; Choi and Lee 2013; Harger et al. 2013; Rahmana et al. 2014; Coursolle et al. 2015; Moulin et al. 2019; Gao et al. 2020). Therefore, heterologous and endogenous expression of the alkane operon in E. coli cells leads to the production and secretion of both AAR and AD containing C₁₃–C₁₇ alkanes (Schirmer et al. 2010; Rahmana et al. 2014; Coursolle et al. 2015; Moulin et al. 2019; Gao et al. 2020), indicating the potential of these enzymes for the industrial biosynthesis of hydrocarbons using native microorganisms (Schirmer et al. 2010; Choi and Lee 2013; Harger et al. 2013; Hayashi et al. 2015; Hayashi and Arai 2022). Furthermore, the addition of AD expression to a fatty aldehyde solution is a promising method of facilitating hydrocarbon production by E. coli cells (Schirmer et al. 2010; Choi and Lee 2013; Harger et al. 2013; Rahmana et al. 2014; Coursolle et al. 2015; Moulin et al. 2019; Cheah and Chan 2022). Heterologously expressed AD can produce straight-chain alkanes from fatty acyl-ACP via the alkane biosynthesis pathway, complemented by the AAR of E. coli cells (Fig. 3 and Table I). As shown in Fig. 3, heterologous AD expression by pET28-AD in E. coli was verified through purification and SDS-PAGE of the AD protein, and cells transformed with the empty vector pET28 were used as the background (negative) control (Fig. 3a). The SDS-PAGE results revealed that the size of the purified AD protein corresponded to the size of the L. redekei KNUA012 AD protein (28 kDa; 233-amino-acid long according to GenBank accession no. ANI69989.1) (Fig. 3b). This indicates that heterologously expressed AD could be used to produce alkanes and significant fatty acids for use as biofuel constituents.

Fig. 3.

The alkanes and major fatty acid components of L. redekei KNUA012 are summarized in Supplementary Table SI. The GC peaks are presented in Fig. 4b. L. redekei KNUA012 could synthesize alkanes (C₁₃–C₁₇) and fatty acids (C₈–C₁₈), including pentadecane (C₁₅H₃₂), heptadecane (C₁₇H₃₆), 8-heptadecene (C₁₇H₃₄), tetradecanoic acid methyl ester (C₁₅H₃₀O₂), 9-hexadecenoic acid methyl ester (C₁₇H₃₂O₂), hexadecanoic acid methyl ester (C₁₇H₃₄O₂), 9-octadecenoic acid methyl ester (C₁₉H₃₆O₂), and octadecanoic acid methyl ester (C₁₉H₃₈O₂) (Table SI). Our GC/MS analysis results indicated that compared with empty vector pET28, the heterologous expression of L. redekei KNUA012 AD in E. coli increased the levels of alkanes and major fatty acids (Table I). GC/MS results revealed that compared with the pET28 empty vector, the heterologous expression of AD in the pET28-AD construct increased the production of pentadecane (C₁₅H₃₂), heptadecane (C₁₇H₃₆), and 8-heptadecene (C₁₇H₃₄) (Table I). Thus, compared with L. redekei KNUA012 and pET28 empty vector, the heterologous expression of AD in pET28-AD can lead to the production of alkanes and major fatty acids using fatty acyl-ACP via the alkane biosynthesis pathway by complementing E. coli AAR. These findings demonstrate that the heterologous expression of AD in the pET28-AD construct of E. coli was efficient when E. coli cells transformed with pET28 empty vector were used as the background (negative control) (Fig. 3). Thus, the homologous expression of AD in L. redekei KNUA012 and pET28-AD in E. coli may lead to the production of major fatty acids using alkanes (pentadecane, heptadecane, and 8-heptadecene) as biofuels. These findings demonstrate that L. redekei KNUA012 can produce lipids compatible with biofuel production.

Fig. 4.