Characterization and engineering of a dual-function diacylglycerol acyltransferase in the oleaginous marine diatom Phaeodactylum tricornutum

Background Photosynthetic oleaginous microalgae are promising feedstocks for biofuels. Acyl-CoA:diacylglycerol acyltransferases (DGATs) represent rich sources for engineering microalgal lipid production. The principal activity of DGATs has been defined as a single-function enzyme catalyzing the esterification of diacylglycerol with acyl-CoA. Results A dual-function PtWS/DGAT associated with diatom Phaeodactylum tricornutum is discovered in the current study. Distinctive to documented microalgal DGAT types, PtWS/DGAT exhibits activities of both a wax ester synthase (WS) and a DGAT. WS/DGATs are broadly distributed in microalgae, with different topology and phylogeny from those of DGAT1s, DGAT2s, and DGAT3s. In vitro and in vivo assays revealed that PtWS/DGAT, functioning as either a WS or a DGAT, exhibited a preference on saturated FA substrate. Endogenous overexpression of PtWS/DGAT demonstrated that the DGAT activity was dominant, whereas the WS activity was condition dependent and relatively minor. Compared with the wild type (WT), overexpression of PtWS/DGAT in the diatom resulted in increased levels of total lipids (TL) and triacylglycerol (TAG) regardless of nitrogen availability. The stability and scalability of the introduced traits were further investigated at a 10-L photobioreactor, where the mutant growth resembled WT, with moderately increased productivity of TL and TAG. Furthermore, the production of wax esters increased considerably (from undetectable levels to 2.83%) under nitrogen-deplete conditions. Conclusions PtWS/DGAT is a bifunctional enzyme and may serve as a promising target for the engineering of microalga-based oils and waxes for future industrial use. Electronic supplementary material The online version of this article (10.1186/s13068-018-1029-8) contains supplementary material, which is available to authorized users.


Background
Microalgae sequester the carbon dioxide in the atmosphere, converting this gas into green chemicals for scalable production of valuable molecules ranging from therapeutic proteins to biofuels [1]. Triacylglycerol (TAG) is an energy-dense lipid and a promising source for biofuel production from microalgae [2]. However, few natural strains exhibit the demanding traits associated with feedstock for TAG accumulation and, hence, genomic and biological technologies with increased specificity for microalga-based biofuel studies have been sought [3]. Identification of the pathways and crucial enzymes that underlie the oleaginous phenotype should guide the rational genetic engineering of microalgae for the overproduction of TAG [4,5].
Similar to TAG, in a number of plants and microorganisms, wax esters serve as energy reservoir which has been regarded as potential alternative energy sources for biofuel production. A dual-function enzyme wax ester synthase/acyl coenzyme A (acyl-CoA):diacylglycerol acyltransferase (WS/DGAT) has been discovered in some plants and bacteria [31][32][33], catalyzing the final step in TAG and wax esters biosynthesis. However, WS/ DGAT enzyme has been very rarely reported so far in eukaryotic microorganisms [33] and has not been discovered in microalgae. The oleaginous microalga P. tricornutum harbors a single DGAT1 and four putative DGAT2s, of which DGAT1 [8] and DGAT2B [9] have been functionally characterized in vitro. This characterization revealed a distinct substrate preference with PtDGAT1 exhibiting a preference toward saturated C 16 and C 18 fatty acids (FAs) [8] and PtDGAT2B exhibiting specificity on unsaturated C 16 and C 18 FAs [9,10]. In P. tricornutum, we discovered a group of acyl-CoA:diacylglycerol acyltransferases (designated as PtDGATX) with an identity high to WS/DGAT and low to DGAT1s, DGAT2s, and DGAT3s, in amino acid sequence. This suggests that the function of DGATXs differs from those of the remaining types of DGATs. However, identification of the molecular and cellular mechanisms of PtDGATX underlying TAG or wax ester metabolism remains challenging. Therefore, the aim of this study is to investigate PtDGATX function, substrate preference, and regulatory mechanisms in lipid biosynthesis. Distinctive substrate preferences and inconcert choreography in TAG or wax ester biosynthesis of individual DGATs are revealed. Furthermore, a genetically modified diatom with elevated lipid, wax ester, and TAG productivity is created. In particular, the engineered strains yield (although low) wax esters, a class of oleochemicals that can potentially be used for many applications in various industries [34,35].

DGATX distribution in microalgae
The PtDGATX was characterized by first validating its gene model through a polymerase chain reaction (PCR) from cDNA and genomic DNA (Additional file 1: Dataset S1). The lack of intron suggested that the gene structure of PtDGATX was compact. The PtDGATX was distributed separately from (Fig. 1a) and was modestly similar to characterized microalgal DGAT1s, DGAT2s, and DGAT3s (Fig. 1a). This suggested that the origin and function of PtDGATX were distinct from those of DGAT1s, DGAT2s, and DGAT3s. The phylogeny of DGATXs revealed a broad presence and a high level of sequence conservation ranging from bacteria, microalgae to flowering plants (Additional file 2: Figure S1). A relatively high identity in amino acid sequence was revealed between DGATX and WS/DGAT (20%; bacterium Acinetobacter, AF529086; Additional file 2: Figure S2), which has been characterized in bacteria as a bifunctional enzyme WS/DGAT [32]. Moreover, the TIGR02946 domain of WS/DGATs [31] was present in PtDGATX. DGAT1s have at least six transmembrane domains [8] and are larger than DGAT2s, which have (in general) at least two putative transmembrane domains [36], while DGAT3s are cytosolic diacylglycerol acyltransferase in the absence of transmembrane domains [37]. PtDGATX resembled AcWS/DGAT, and was predicated to harbor a single hydrophobic transmembrane region in the N terminal (Val3-Met35) ( Fig. 1b and Additional file 2: Figure  S3a). This was validated by fusing the coding sequence of PtDGATX with a His tag. The fusion protein was expressed endogenously in P. tricornutum and the Histag antibody was used to determine the localization of the PtDGATX protein via western blotting. Soluble and membrane proteins were separated and used for this blotting. Bands were absent from the soluble proteins, but a band occurred in the membrane proteins (Figs. 1c and Additional file 2: Figure S3b), suggesting that PtD-GATX was a transmembrane enzyme. Altogether, in terms of topology and phylogeny, PtDGATX is more similar to WS/DGATs than to DGAT1s, DGAT2s, and DGA T3s.

PtDGATX, DGAT1, and DGAT2B: Similarities and differences
Two DGAT isoforms, i.e., PtDGAT1 [8] and PtDGAT2B [9], which are essential for TAG formation, have been functionally verified in P. tricornutum. The similarities and differences between PtDGATX and other DGAT types were determined via absolute quantitative PCR (qPCR) investigation of the transcriptional dynamics associated with DGAT isoforms in P. tricornutum. Among these three DGAT genes, DGAT1 and DGATX exhibited the highest absolute transcript level and the lowest absolute transcript level, respectively, in P. tricornutum at late logarithmic phase (Fig. 2a). The three PtDGAT genes were all upregulated under nitrogen (N)depleted conditions, although the expression patterns of the individual DGATs were markedly different. PtDGAT1 showed transient upregulation, with its transcript levels peaking at 1 day following the onset of N depletion and declining slightly thereafter (Fig. 2b). PtDGAT2B exhibited the greatest fold change (20.3-fold). Upregulation of PtDGAT2B occurred rapidly within the first day upon N− and remained at a relatively constant level thereafter (Fig. 2b). Compared with those of PtDGAT1 and PtDGAT2B (being transcriptionally unregulated rapidly within the first day), the transcript levels of PtDGATX increased more slowly and continuously, peaking at 4 days (Fig. 2b). This suggested that, upon N−, relative to PtDGAT1 and DGAT2B, DGATX gene may contribute more to TAG synthesis in the latter stage than during the earlier stages.
The correlation between lipid accumulation and gene regulation (Fig. 2c) was determined via lipidomic analysis. For direct comparison of the results, the same independent batches of cells were used for the transcription and lipidomic analyses. N− conditions yielded the following occurrences, namely the (i) total lipid (TL) content increased by approximately twofold (from 11 to 34%) within 14 days (Fig. 2c), (ii) TAG content increased significantly (from 3.85 to 20.87% mg DW −1 , see Fig. 2c), (iii) FA profiles changed significantly, (iv) content of monounsaturated FAs (i.e., C16:1 n = 1 and C18:1 n = 1) increased markedly, whereas the content of saturated FAs (i.e., C14:0 and C18:0) and polyunsaturated (i.e., C20:5) FAs decreased (Fig. 2d), and (v) many TAG molecular species increased considerably with the transcript increase of the three DGATs. The lipid composition changed substantially with an increased percentage of TAG in TLs (from 34.22 to 61.33%; Fig. 2c). Regardless of N availability, C16:0 and C16:1 in P. tricornutum (Fig. 2d) constituted the main FAs of TAG. The transient or progressive upregulation of the DGATs may have resulted in most of the TAG formed during N depletion. Therefore, we assumed that the upregulation of these DGAT genes occurs concomitantly with the increase in TAG under N− conditions. PtDGAT1 and PtDGAT2B play (in general) a major role in TAG synthesis, whereas PtD-GATX may play only a minor role. However, PtDGATX is actively involved in the response to N− conditions and appears crucial in the late stage under N− periods, whereas the role of PtDGATX remains unclear.

Functional rescue of TAG-deficient yeast by PtWS/DGAT
To verify the role of PtDGATX, the gene was expressed in the yeast Saccharomyces cerevisiae H1246 that is deficient in TAG biosynthesis. TLC revealed that TAG was absent from the H-pYES strains (H1246 cells harboring an empty pYES2 vector) and a prominent spot corresponding to TAG in the transformants harboring the pYES2-PtDGATX vector (H1246-PtDGATX) was observed (Table 1 and Fig. 3 left inset). Meanwhile,  (186846) was used as the root at the outer group. Sequence alignment and phylogenetic analysis were performed with BLAST and CLUSTALW, respectively. Bootstrap support values were estimated using 100 pseudo-replicates. b The putative transmembrane construct of PtDGATX. The transmembrane region was analyzed by the online software TMHMM, DAS, and TMpred. c Topology analysis of PtDGATX via western blotting. Soluble and membrane proteins were separated and used for blotting. Actin and DGAT1 which were known soluble protein and membrane protein, respectively, were used as controls. N the N terminal, C the C terminal, Membrane transmembrane proteins, Soluble soluble proteins, PtWDoe the PtDGATX overexpression line of P. tricornutum, WT the wild-type P. tricornutum a substantial amount of wax ester was detected in H1246-PtDGATX (Table 1 and Fig. 3 right inset), indicating the dual function of PtDGATX (hereafter PtWS/ DGAT) with activities of both WS and DGAT. In H1246-PtWS/DGAT, TAG and wax ester accounted for ~ 10.51 and 5.17%, respectively, of the TLs. C16:1 and C18:1 were the dominant FA species in TLs. However, C16:0 and C18:0 FAs were the principle components of TAG and C14:0, C16:0, and C18:0 FAs were the main FAs in the wax ester (Table 1 and Fig. 3). This indicated that PtWS/DGAT, functioning as either a WS or a DGAT, exhibits a preference on saturated FA substrates in yeast. Moreover, unsaturated FAs (i.e., C16:1 and C18:1 FAs) were detected in TAG, but were absent from the ester, suggesting a difference in substrate preference of PtWS/ DGAT functioning as a WS or a DGAT. In addition, the number of TLs accumulated by H1246-PtWS/DGAT was almost triple the number accumulated by the controls (H-pYES strains). This suggested that in vivo manipulating PtWS/DGAT would yield increased lipid production in the oleaginous P. tricornutum.

In vivo TAG-synthetic activities of PtWS/DGAT revealed via endogenous overexpression
To probe the endogenous function, PtWS/DGAT was overexpressed in P. tricornutum (PtWS/DGAToe). The PtWS/DGAT overexpression vector harbored a fulllength PtWS/DGAT gene and a basta resistance (bar) Fig. 2 Dynamics of DGATs and lipidomics of P. tricornutum following nitrogen depletion. a Absolute abundance of DGATs in wild-type P. tricornutum at late logarithmic phase. Y axis means the transcript number. The concentrations of pMD19T-PtDGAT1, pMD19T-PtDGAT2A, and pMD19T-PtWS/ DGAT were determined and diluted to 10 −1 , 10 −2 , 10 −3 , 10 −4 , and 10 −5 as templates in real-time PCR for standard curve. R 2 with values above 0.99 and amplification efficiencies with values above 90% were obtained for PtDGAT1, DGAT2A, and WS/DGAT genes (amplification curves and melting curves are shown in Additional file 2: Figure S4). b Transcriptional dynamics of DGATs upon nitrogen depletion. To impose N deprivation, P. tricornutum cells in the stationary phase were collected, washed, and re-suspended with N-free f/2 medium at room temperature. Samples were collected at indicated periods for transcript analysis. c Dynamics of total lipids and TAG in P. tricornutum after nitrogen depletion. d Profiles of fatty acids in TAG in P. tricornutum after nitrogen depletion gene. The PtWS/DGAT gene fused with a His tag was driven by the fcpA promoter (PfcpA) and terminated by the fcpA terminator (TfcpA) region. Moreover, the bar gene expression was driven by the fcpB promoter (PfcpB) and terminated with the fcpA2 terminator (TfcpA2) (Fig. 4a). Transformants were selected on f/2 plates supplemented with basta. The desired in vivo function of each of these promoters and terminators in P. tricornutum was individually validated. Specifically, the transcriptional level of the PtWS/DGAT gene was determined by the absolute quantitative PCR. Under N-replete (N+) conditions, a significant increase (4.5-fold) of PtWS/DGAT transcription occurred in the PtWS/DGA-Toe cells relative to that occurring in the wild-type cells  (WT) (Fig. 4b). The integration of the PtWS/DGAT vector was validated via Southern blotting, as indicated by a clear band using bar gene as a probe (Fig. 4b inset). Using the PtWS/DGAT gene as a probe during blotting revealed two bands in PtWS/DGAToe and a single band in WT (Fig. 4b inset). This was consistent with a single integration of the exogenous PtWS/DGAT gene into the alga chromosome. Furthermore, in vivo expression of the PtWS/DGAT protein was validated by using Histag antibodies to detect the PtWS/DGAT-6 × His fusion protein via immunoblotting. A band was detected in PtWS/DGAToe, whereas no signal was detected in WT (Fig. 4c). Compared with those of WT, the respective TL content and TAG content of the PtWS/DGAToe increased by (i) 21 and 40% under N+ conditions and (ii) 10 and 25% following N− (see Table 2). The levels of unsaturated FAs (e.g., C16:1 n − 1, C16:1 n − 2, and C18:1 n − 1 FAs) decreased and the levels of saturated FAs (especially the C14:0, C16:0, and C18:0 FAs) increased slightly under both N+ and N− conditions (Table 2). This may have resulted from the substrate preference of PtWS/DGAT on saturated FAs. Therefore, regardless of N availability, PtWS/DGAT exhibited a DGAT activity with a preference on saturated FAs. In contrast, under N−, the WS activity of PtWS/DGAT seemed to be higher in the late stage (than in the early stages). Wax ester was absent from either WT or the PtWS/DGAToe cells under N+ conditions. In contrast, following prolonged N−, a small amount (0.53 ± 0.05% of TLs) of wax ester occurred in the PtWS/DGAToe, but was absent from WT, although a large amount of lipids was loaded for TLC analysis (Fig. 4d). Hence, although the activity as WS is condition dependent and relatively minor, PtWS/DGAT exhibits activities in both wax ester synthesis and TAG synthesis in P. tricornutum.

Engineering PtWS/DGAT for increased lipid productivity
The in vitro and in vivo evidence indicated that perturbation on PtWS/DGAT resulted in increased production of TLs and TAG. However, increased cellular lipid biosynthesis occasionally leads to decreased growth, thereby neutralizing the overall lipid productivity [2]. Biomass accumulation (i.e., growth rate of microalgal strains) is thus crucial for determining the lipid productivity. Therefore, to determine whether the genetically modified (GM) P. tricornutum cells can be exploited for enhanced lipid production, the photosynthetic performance of both WT and PtWS/DGAToe was tracked under either N+ or 14-day N− conditions. Under N+ conditions, both lines demonstrated a high Fv/Fm value (the maximum quantum yield of photosystem II, approximately 0.62, which implies a fit growth condition; a typical result is shown in Fig. 5a). Upon nitrogen depletion, although Fv/Fm values of both lines were decreased, barely any difference was observed between WT and the GM strains ( Fig. 5b and Additional file 3 Table S2). Consistently, the growth of the two lines was very similar under the explored conditions (monitored by detecting cell numbers daily; see Fig. 5c for growth under N+ conditions and see Additional file 2: Figure S5 for that under both N+ and N− conditions). Moreover, the dry weight (DW) of PtWS/ DGAToe strains (397.50 ± 16.25 mg L −1 ) was determined after fourteen-day nitrogen starvation which was similar to that of WT (398.75 ± 26.25 mg L −1 ) and the DW of both was comparable to the levels of documented P. tricornutum strains (Table 3) [38].
Combined with the elevated lipid and TAG content, this indicated that GM P. tricornutum cells were quite capable of photoautotrophic growth, absorbing CO 2 and producing (especially) biomass, lipids, and TAG. The stability and scalability of the GM P. tricornutum were further determined by evaluating the life cycle performance in a 10-L photobioreactor (PBR) (Fig. 5d). The growth rate of the mutant under nitrogen repletion conditions was identical to that of WT, reaching a biomass productivity of 28.4 mg L −1 day −1 (See "Methods" for details). The biomass and lipid content were determined for both WT and GM P. tricornutum cultivated in nitrogen-free f/2 medium in PBR for 14 days. Compared with those of WT, the respective TL content and TAG content of the PtWS/DGAToe increased by 13.8 and 18.4% (371.1 mg g −1 DW TL and 226.4 mg g −1 DW TAG in PtWS/DGAToe) (Table 3), corresponded to a TAG productivity of 6.4 mg L −1 day −1 (an 18.4% increase relative to that of 5.4 mg L −1 day −1 of WT). Moreover, following prolonged N− conditions, a considerable amount of wax ester (from undetectable levels to 28.3 mg g −1 DW) was also generated. Although the productivity remains to be improved, our results provide insight into the customized production of different high-value chemicals via engineering of a single gene [39].

Strains and growth conditions
The P. tricornutum strains were kindly donated by Prof. Mingyan Yin of the Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences. P. tricornutum was grown as previously described [3], i.e., at 23 °C under a 40-50 μmol photons m −2 s −1 light with a 12/12 h light:dark photoperiod. To impose N deprivation, P. tricornutum cells in the stationary phase (8.5 × 10 6 cell mL −1 ) were collected, washed, and re-suspended in nitrogen-free f/2 medium. Samples were collected at indicated periods for transcript and lipid analysis [40]. For the scaled up cultivation of the diatom, 10-L cylinder glass bottles (50 cm diameter and 70 cm height) were used. Cells with an initial concentration of 5 × 10 6 cell mL −1 were cultured using a continuous filtered gas at 1.3 m 3 min −1 and 40-50 μmol photons m −2 s −1 illumination with a 12/12 h light:dark photoperiod. The biomass productivity was calculated 7 days later using the following formula: where DWs is the DW of cells at sampled time point, DWi is the DW of initial culture, T is the duration of culture period, and V is the volume of algal culture. Afterward, cells were collected, washed, and re-suspended in nitrogen-free f/2 medium. Lipid measurements were conducted after 14 days.

Phenotyping and Nile red staining
The growth of microalgae was monitored by measuring the turbidity (OD 750 ) or cell number at indicated intervals. The chlorophyll fluorescence parameter was measured with Diving PAM (WALZ, Germany). The lipid accumulation was monitored by Nile red staining. P. tricornutum cultures (approximately 4 × 10 6 cells) at indicated time points were harvested, stained with 1% (v/v) Nile Red (0.5 mg mL −1 in dimethylsulfoxide), incubated at room temperature for 5 min, and immediately observed by Laser scanning confocal microscopy with an excitation filter at 480 nm (FluoViewFV1000, Olympus, Japan).

Sequence analysis of PtWS/DGAT
Sequence analysis and comparison were undertaken in accordance with previous reports [41]. In brief, sequence alignment and phylogenetic analysis were performed with BLAST (http://www.ncbi.nlm.nih.gov/BLAST ) and CLUSTALW, respectively [42]. Bootstrap support values were estimated using 100 pseudo-replicates.

Gene expression analysis
Genomic DNA, total RNA extraction, and reverse transcription were performed as previously described [3,40,41]. Total RNA was extracted and purified with the Eastep Super Total RNA Extraction Kit (Promega) and then subjected to genomic DNA digestion and reverse transcription using a PrimeScript RT reagent Kit with gDNA Eraser (Takara) [40,41]. Relative qPCR was performed in two steps as given in our earlier description [40]. First, 0.5 μg RNA was mixed with 2 μL of 4×g DNA wiper Mix and Nuclease-free H 2 O was added to a final volume of 8 μL. The mixture was kept at 42 °C for 2 min. Second, 2 μL of 5 × HiScript II Q RT SuperMix IIa was added to the above mix. Reverse transcription (RT) reactions were performed in a GeneAmp ® PCR System 9700 (Applied Biosystems, USA) with a program of 25 °C for 10 min, 50 °C for 30 min, and 85 °C for 5 min. The 10 μL RT reaction mix was then diluted with 10 μL nucleasefree water and held at − 20 °C. Real-time PCR was performed using LightCycler ® 480 II Real-time PCR Instrument (Roche, Swiss) with 10 μL PCR reaction mixtures including 1 μL of cDNA, 5 μL of 2 × Quan-tiFast ® SYBR ® Green PCR Master Mix (Qiagen, Germany), 0.2 μL of forward primer, 0.2 μL of reverse primer, and 3.6 μL of nuclease-free water. Reactions were incubated at 95 °C for 5 min, followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s. At the end of the PCR cycles, melting curve analysis was performed. Each sample was run in triplicate. The expression levels of mRNAs were calculated using the 2 −ΔΔCt method. The mRNA expression level was normalized using the 18S gene as the internal control [43].
For absolute qPCR, RT reaction was conducted following the instruction of Fast Quant RT Kit KR106 (with gDNase; Tiangen, China). The total RNA (500-1000 ng) in 8 μL RNAase-free H 2 O was preheated in 65 °C for 5 min and transferred to ice immediately, mixed with 2 μL 5×g-DNA buffer (containing g-DNase) in 42 °C for 3 min, and transferred to ice immediately. The solution was then mixed with 10 μL premixed buffer containing primers and FastQuant RT Enzyme. RT reaction was performed in a GeneAmp ® PCR System 9700 (Applied Biosystems, USA) at 42 °C for 15 min, 95 °C for 3 min, and 4 °C for 10 min. PtDGAT1, PtDGAT2A, and PtWS/DGAT genes were amplified and cloned into pMD19T vector (TAKARA, China). The concentrations of vectors pMD19T-PtDGAT1, pMD19T-PtDGAT2A, and pMD19T-PtWS/DGAT were measured with Nanodrop ND2000 (Thermo, USA), and diluted to 10 −1 , 10 −2 , 10 −3 , 10 −4 , and 10 −5 as templates for standard curve. The qPCR was performed using ABI7500 (ABI, USA) with a 25 μL PCR reaction mixture (included 1 μL of cDNA, 12.5 μL Bestar Sybr-Green Qpcrmaster mix (DBI Bioscience, Germany), 0.25 μL of forward primer, 0.25 μL of reverse primer, and 11 μL of nuclease-free water). Reactions were incubated at 95 °C for 5 min, followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s. All primers were validated by PCR, sequencing, and gel electrophoresis where a single and clear band was observed for each pair of primers (Additional file 2: Figure S4a). Amplification curves and melting curves are shown in Additional file 2: Figure S4b. As for absolute qPCR, R 2 with values above 0.99 and amplification efficiencies with values above 90% were obtained for PtDGAT1, DGAT2A, and WS/DGAT genes (Additional file 2: Figure S4c).

Expression vector design and P. tricornutum transformation
The PtWS/DGAT overexpression vector harbored a full-length PtWS/DGAT gene and a basta resistance (Bar) gene, each of which was driven by an endogenous promoter and terminated by an endogenous terminator (Fig. 4a). The PtWS/DGAT gene was driven by the Fucoxanthin-chlorophyll protein A promoter (PfcpA) and terminated by the Fucoxanthin-chlorophyll protein A1 termination (TfcpA) region. Expression of the Bar gene was driven by the Fucoxanthin-chlorophyll protein B promoter (PfcpB) and terminated with TfcpA terminator. The fragments were cloned with primers harboring indicated restriction enzyme cutting sites (Additional file 3: Table S1), while the commercial vector pBluescript