Two possible candidate enzymes from Ulva lactuca-associated epiphytic bacteria obtained through PCR and functional evaluation

Epiphytic bacteria from marine macroalgae synthesize enzymes of industrial and biotechnological interest. In this study, we obtained two DNA candidate fragments for lipid-degrading enzymes from the total DNA of Ulva lactuca-associated epiphytic bacteria. First, we evaluated a method for total bacterial DNA isolation from the surface of U. lactuca thalli. Then, we designed sets of primers and used them directly for PCR amplification. The resulting PCR products were sequence-analyzed and used for expression and functional evaluation with the Escherichia coli pBAD-TOPO system. We obtained high molecular weight and good quality total bacterial DNA that served as a template to identify a fragment corresponding to an Acetyl-CoA C-Acetyltransferase (or Thiolase), and a candidate fragment for a versatile “true” lipase. We expressed the possible “true” lipase gene fragment heterologously in Escherichia coli and obtained proof of hydrolytic activity on Tributyrin, Tween-20, and Olive-oil media. This study resulted in new knowledge on U. lactuca-associated epiphytic bacteria as possible brand-new sources of enzymes such as thiolases and “true” lipases. However, future studies are required to describe the characteristics and important applications of these candidate enzymes.


Introduction
The surfaces of marine macroalgae represent promising candidate sources of novel biocatalysts [1]. These surfaces are exposed to different conditions of temperature and salinity and are also important nutrient-rich environments Lipases are enzymes that act on triglycerides ester bonds to liberate fatty acids and glycerol, thus participating in lipid degradation and biosynthesis pathways [12]. Likewise, lipases have an enormous catalytic versatility including lipid hydrolysis, trans-and inter-esterification, fat and oil acidolysis, aminolysis, and alcoholysis [13,14]. Due to these features, lipases have a variety of applications in the food, pharmaceutical, and cosmetic industries, as well as in the production of agrochemicals, biofuels, and detergents, among others [15,16]. Lipases are divided into two main groups: esterases (EC.3.1.1.1) that prefer water-soluble short-chain fatty acids, and "true" lipases or triacylglycerol hydrolases (EC.3.1.1.3) that prefer low water-soluble long-chain fatty acids [17,18]. All lipases share a high conserved active site (Ser-Asp-His) and a consensus region motif (Gly-Xaa-Ser-Xaa-Gly) [19].
Thiolases (EC.2.3.1. 16), also called Acetyl-CoA C-Acetyltransferases, are transferases that catalyze the reversible cleavage of fatty acids into acyl-CoA and acetyl-CoA throughout the transference and condensation of acyl groups [20,21]. Further, thiolases are involved in lipid transport and assimilation, β-oxidation, as well as in fatty acid, steroid, and polyketide biosynthesis [22]. These enzymes also have several applications in the production of organic solvents and biofuels, synthesis and degradation of antibiotics, and bioremediation processes [23][24][25].
Metagenomics studies have revealed the genetic, metabolic, and functional potential of non-cultivable microorganisms [26,27]. Besides, access to the total DNA of an environmental sample, PCR-based analysis, and functional evaluation have allowed the discovery of lipolytic genes and enzymes from marine environments [28][29][30]. For instance, LipG, EstA, EML1, and EstHE1 lipases, displaying high salt tolerance, thermostability, activity within a broad pH range, and stability in a high concentration of divalent ions and organic solvents, have been recovered from the bacterial metagenomes of intertidal flats, coastal environments, deep-sea sediments, and organism-associated bacteria [31][32][33][34]. LipA, an alkaline "true" lipase, was described from the metagenome of a marine sponge [35]. Likewise, GmEst_7 and Lip5 quorum sensing lipases were discovered in brown algae epiphytic bacteria [36,37]. Further, other studies have reported lipases from the metagenome of the green macroalgae Ulva australis, such as the abg3 gene that encodes a β-lactamase-like lipase that displays lipolytic activity and confers antibacterial properties, as shown in a heterologous expression study [38].
Nonetheless, despite "true" lipases and thiolases being important enzymes in several biotechnology and industrial applications, there is limited information about these enzymes in green macroalgae-associated bacteria; therefore, the question arises whether epiphytic bacteria from U. lactuca produce "true" lipases and thiolases. In consequence, here we present a suitable method for total bacterial DNA isolation from U. lactuca surfaces and a PCR-based identification of these enzymes and closely related enzymes. We found a candidate DNA sequence for a thiolase, and a DNA fragment that displayed "true" lipase activity when was functionally expressed. Our results showed that U. lactuca-associated epiphytic bacteria are potential sources of biocatalysts of marine origin.

Strains, plasmids and media
Lipase-producing strains of Burkholderia cepacia and Pseudomonas aeruginosa were used as positive controls, and Escherichia coli DH5α was used as the negative control. These strains are part of the "Banco de Cepas y Genes del Instituto de Biotecnología de la Universidad Nacional de Colombia". E. coli TOP10 and plasmid pBAD-TOPO (Invitrogen) were used for cloning/expression assays. Luria Bertani agar (10 g/L tryptone, 10 g/L NaCl, 5 g/L yeast extract, 15 g/L bacteriological agar) supplemented with tributyrin 1 % v/v and 0.2 ml/L triton X-100 (emulsifier) was used for lipase screening. To confirm lipolytic activity, Tween 20 (10 g/L peptone, 5 g/L NaCl, 15 g/L bacteriological agar, 1.1 g/L CaCl2 and 10 ml/L Tween 20) and Rhodamine-Olive Oil-Agar (8 g/L nutrient broth, 4 g/L NaCl, 15 g/L bacteriological agar, 10 ml/L Olive Oil and 10 ml/L Rhodamine B) were also used. Some culture media contained 0.1 mg/L ampicillin antibiotic.

Sample collection and total DNA isolation
Thalli of green macroalgae U. lactuca were collected at 'La Punta de la Loma' rocky coast in Santa Marta-Colombia (11 • 07'00.9" N, 74 • 14'01.3" W). The samples were washed several times with sterile seawater and then transferred into sterile refrigerated plastic bags and stored at -80 • C until processing. Afterward, total bacterial DNA from the macroalgal surface was isolated using the ZR Soil Microbe DNA Kit (Zymo Research). Between 0.6 g and 1.0 g of the thallus base section was placed into tubes containing silica-beads and lysis buffer [39]. On a Bead-Beater (Disruptor Genie™, United States) tube holder, physical disruption of bacterial cells was performed at maximum speed for 1.5-min pulses, twice, with a 5-min rest on ice between pulses [40,41]. Then, tubes were centrifuged at maximum speed and the clear supernatant was transferred and further processed according to the manufacturer's protocol.
In the end, total DNA was eluted in 20 µl buffer and then stored at -20 • C. In addition, total DNA was analyzed by electrophoresis on agarose gel, and DNA yield (ng/µl) and quality (Abs at 260/280 nm) were quantified with a Nanodrop 2000C.

Primer design
The Abg3 gene (from U. australis) was used to design primers for homologous sequences. The region between 21704 -22681 bp of the UaAb1 clone fosmid (HQ162719) corresponding to the open reading frame (ORF) [42], was targeted for primer design through the Primer-BLAST (NCBI) tool. In order to include the whole sequence, we picked the most suitable set of primers (labeled UaLip) to be synthesized. In parallel, a set of degenerate primers was designed using a consensus region of nine representative and highly related "true" lipase sequences from different bacteria [43]. First, the alignments were carried using CLUSTAL OMEGA and the consensus region was determined through the Block-Maker tool. Then, the conserved blocks were directly fed to the CODEHOP (Consensus-Degenerate Hybrid Oligonucleotide Primers) program [44,45]. Default values were used except for degeneracy (256), degeneracy strictness (2.5), and codon usage (bacteria and plastids -gbbct-). The primers (labeled LipFam1) were picked in order to include the whole consensus sequence, and were tested for specificity and universality using BLASTx. Lastly, these sets of primers were characterized via Multiple Primer Analyzer (Thermo Fisher Scientific) and Oligo-analyzer (Integrated DNA Technologies) tools and then contrasted with reported optimal values for PCR [46][47][48]. Oligo sequences are listed in Table 1.

Amplification of the target genes by PCR
Hot-Start PCR mixes contained: 1 X Taq Buffer, 0.5 mM dNTPs, 0.5 µM of each primer, 2.0 mM MgCl2, 2 % DMSO (Dimethyl-Sulfoxide), 0.5 mg/µl BSA (Bovine Serum Albumin) and 1 U/µl Taq Polymerase. Total bacterial DNA from U. lactuca surface was used as template (50 -100 ng) for UaLip and LipFam1 primers testing. Amplification using UaLip primers was performed following these conditions: 95 • C for 5 min, 30 cycles of 94 • C for 30 s, 61 • C (optimal) for 30 s and 72 • C for 1 min, plus a final extension step at 72 • C for 10 min. Amplification using LipFam1 primers was carried under the same conditions with a slight modification on annealing temperature: 53 • C (optimal) for 1 min. Then, PCR products were visualized on a 2 % agarose gel and purified using gel extraction with Gene JET Gel Extraction Kit (Thermo Fisher Scientific). Additionally, genomic DNA from "true" lipase producing strains was used as positive control to test LipFam1 primers.
Expression of a possible "true" lipase gene fragment Some purified DNA fragments obtained from total DNA of U. lactuca surface using LipFam1 primers, were cloned and expressed through pBAD TOPO system, following the manufacturer's instructions. The cloning reaction was performed at a vector: insert molar ratio of 3:1 and then, chemically competent cells were transformed by heat-shock and spread on LB agar plates containing tributyrin 1 % v/v and ampicillin. Cells transformed with empty vectors were used as the negative control. Positive clones were randomly selected and confirmed on Tween 20 and Olive-oil agar plates. Activity on tributyrin-LB agar was observed by the formation of a clear zone around the colony [49][50][51]. Enzymatic activity on Tween 20 was identified through the formation of a white precipitate below the colonies [52][53][54]; and on Olive-oil agar by the irradiation of fluorescence under UV light [55].

Sequence Analysis
All the cleaned and purified PCR products were SANGER sequenced at the 'Instituto de Genética de la Universidad Nacional de Colombia'. The sequences obtained were analyzed and edited through the BioEdit program, and the resulting high-quality FASTA files were contrasted against RefSeq Nucleotide database using BLASTn. Functional protein domains were searched using BLASTx against non-redundant UniProtKB/Swiss-Prot Protein database.

Identification of a Thiolase DNA fragment
After PCR reactions with epiphytic bacterial total DNA, a ∼ 1.0 kb fragment (8 -12 ng/µl) ( Fig. 2A) was effectively obtained using the UaLip primers. After sequencing and editing, our BLASTn search revealed that a 519 pb high quality sequence had the most similarity to an Acetyl-CoA C-Acetyltransferase (or Thiolase) from Erythrobacter litoralis (  was detected and corresponded to a 118 aa product. Thiolase (cd00751), Acetyl-CoA acetyltransferase (PRK06025) and PaaJ (COG0183) domains were identified. Additionally, the obtained nucleotide sequence was uploaded to the Genbank with accession number MK418067.

Identification of a "True" lipase DNA fragment
On the other hand, PCR amplification with the degenerate primers LipFam1 on the total bacterial DNA from U. lactuca surface and on the B. cepacia and P. aeruginosa genomic DNA, resulted in ∼ 2.0 kb fragments (14 ng/µl -20 ng/µl) (Fig. 2B). However, no data was recovered after sequencing due to the low quality and short length of the reads. Despite this, some PCR products were effectively cloned and expressed in E. coli TOP10 (efficiency 1 -5 x 10 6 UFC/µg DNA). The clones tested on tributyrin-LB showed a clear zone around the colonies ( Fig. 3A and Fig. 3C) while on tween-20 agar, a white precipitate was observed below the colonies and widespread all over the plate (Fig. 3B). Likewise, clones tested on olive-oil agar (Fig. 4A) [39,42] with some suitable modifications for obtaining DNA isolated of epiphytic bacteria. Total DNA was used for PCR-based analysis and subsequent functional evaluation [58]. To isolate bacterial DNA, we selected the thallus base section of U. lactuca due to the high density of bacteria present there (around to 10 7 cells/cm 2 ), which decreases closer to the distal tips (around to 10 2 cells/cm 2 ) [2, 59]. Moreover, we attempted physical cell disruption to release the bacteria from the thallus surface as suggested by Longford et al. [60]. Various studies have shown that this technique allows the isolation of DNA from bacteria with complex cell walls (thicker peptidoglycan layers) such as Actinobacteria and Firmicutes, which occur at low densities [61] at the surface of Ulva species [62,63]. Additionally, the methodology used in this study allows DNA clean-up [64,65] throughout the filtration of possible inhibitors such as phenolic and humic compounds that bind to amine groups from the DNA and that negatively affect the PCR reactions [66]. Therefore, the total bacterial DNA isolated in this study served as a template for PCR-based analysis; and additives such as Bovine Serum Albumin (BSA) and Dimethyl-sulfoxide (DMSO) were necessary to enhance PCR reactions [67][68][69].

Identification of a Thiolase
Although no data was recovered for the abg3 gene reported by Burke [42], we identified a 1.0 kb fragment corresponding to a "thiolase gene". Thiolases are a group of enzymes that have important uses on cellular engineering for industrial processes [70]. These enzymes appear to be functionally closely related to lipases and esterases since they are involved in the same lipid-degrading pathways and show genetic proximity to thioesterases [71]. In consequence, we gather that the presence of this thiolase on the total bacterial DNA from the U. lactuca surfaces could strongly entail lipolytic functions in the epiphytic bacterial communities. Thiolases could be involved in biotic degradation of some lipids on the algal host surface, considering that these enzymes participate in the cleavage of mid and long-length fatty acids in β-oxidation catabolism [25,72]. Thiolases also catalyze reactions involved in lipid transport [73], biosynthetic metabolism [74], host stress-response [75], and bacterial virulence [76], which necessarily include the use of lipid backbones as different authors have pointed.
It is well known that thiolases are widely distributed among bacteria and eukaryotes and it has been also observed that they possess a promiscuous functionality by catalyzing different reactions [77]. Therefore, thiolases have different roles in cell engineering especially for the polyhydroxyalkanoates (PHAs) [78][79][80], organic acids and solvents [81,82], and biofuel overproduction, as well as for wax ester fermentation [83] and bioremediation [84]. In consequence, further studies on thiolases produced by the U. lactuca epiphytic communities are required to understand their features and thereby explore their potential applications.

Identification of a "True" Lipase
In this study we proposed the LipFam1 primers for the PCR-based identification of "true" lipases; however, no data of its sequence was recovered. A problem in genomics is to functionally classify DNA sequences derived from environmental sampling. Sometimes the query sequence does not have a close relative in the database; this could be the case of the fragment obtained with LipFam1 primers [85].
On the other hand, the bacteria transformed with the PCR products involving the LipFam1 primers showed hydrolytic activity on fatty acids of different chain lengths such as tributyrin (C: 4), tween 20 (C: 12), and olive oil (C: 18). In tributyrin media, the clear halos around the colonies were the result of the loss of emulsion that indicates the release of soluble glycerol and butyric acid after hydrolysis [12,86]. The white precipitate was the evidence of the salt formed between the anionic lauric acid from the tween 20 and the Ca 2+ ions in the medium after the hydrolase activity [52,87]. Furthermore, the fluorescent halos were visible upon UV irradiation due to the interaction between the Rhodamine B dye and the free long-chain fatty acids (such as oleic, linoleic and palmitic) after the hydrolysis of olive oil [88][89][90]. In consequence, here we infer that this fragment could correspond to a versatile true lipase since it appears to hydrolyze different length-chain fatty acids, in contrast to common true lipases which only are capable of hydrolyzing ester bonds from long-chain fatty acids (> C: 12) in triacylglycerides [17]. Various lipases show a broad substrate specificity and regiospecificity [18] because of a flexible active site that appears to change its conformation with the presence and binding of different substrates [91,92].
Our work is potentially the first report of a versatile "true" lipase identified on the total DNA of the epiphytic bacteria from U. lactuca. Marine "true" lipases have been only identified on the free-living bacteria such as Oceanobacillus sp. [93] and Pseudomonas sp. [94], the metagenomes of the marine sponges Ircinia sp. (LipA) [35] and Haliclona simulans (Lpc53E1) [95], and recently, in the epiphytic bacterium Shewanella algae from the brown macroalgae Ascophyllum nodosum [96]. Although, knowledge of lipases from green macroalgae epiphytic bacteria is limited [57,97], the efforts to describe and characterize marine lipases from this source have actually shown that cultivable epiphytic bacteria from U. lactuca are capable of producing lipases and other hydrolytic enzymes [1].
Future studies on this topic could reveal the features and properties of these "true" lipases from green macroalgae-associated bacteria. Lipases from marine sources have shown attractive characteristics such as thermostability  [98], high-salt tolerance [99], cold-adaptation [100], extreme pH tolerance [101], organic solvents tolerance [102], and enantioselectivity [103]. These important properties are largely required in industrial and biotechnological applications such as plastic degradation [104] and biodiesel synthesis [105], as well as anti-biofilm and biofouling additives [106].

Conclusions
According to the importance of the marine biocatalysts for industrial and biotechnological purposes, here we were able to describe a suitable method to obtain two possible candidate enzymes. These candidates appear to be synthesized by the Ulva lactuca-associated epiphytic bacteria and could play important roles in lipid metabolism and lipid degradation.
One candidate was sequence-identified as a thiolase and the other was functionally-described as a versatile "true" lipase. The presence of these candidates can be related to the lipolytic functions in the U. lactuca-associated bacterial communities, and therefore, could represent a potential source of these enzymes and their related.
Notwithstanding this functional evidence, only nucleotide and peptide sequence analyses of these fragments will confirm our assumptions, and further structural and functional characterizations will reveal the features and properties of these candidates. Finally, these approaches will allow the search of potential candidate enzymes from un-exploited sources, such as green algae-associated epiphytic bacteria.