LIP4 gene expression from Candida viswanathii strain

Abstract


Background
Lipases (E.C. 3.1.1.3)belong to a class of hydrolases and water-soluble enzyme which catalyses the hydrolysis of insoluble triacylglycerols to generate free fatty acids, diacylglycerols, monoacylglycerols and glycerol.They catalyze a variety of reactions including hydrolysis, transesterification and interesterification of other esters as well as the synthesis of esters, and exhibit a range of regio, enantio and stereoselective transformation properties [1].Lipases are the most popular biocatalysts that have remarkable applications in promoting various biochemical processes in the industry.They are found to be successful in catalyzing numerous processes relevant to the food, pharmaceutical, leather, cosmetic, detergent, medical diagnostics, dairy, beverage, fatty acid, and paper industries [2].
Candida genera can produce several lipases isoform with different catalytic properties.The genes responsible for encoding the lipases comprise a family called LIP gene, being composed of several members and with different characteristics, such as the LIP1 to LIP8 genes of C. rugosa, as well as other sequences of these genes deposited in the GenBank [3,4].This gene family comprise a different catalytic behavior in terms of substrate specificity, thermal and pH stabilities.The differences in substrate specificity may be related to specific amino acid variations in the different parts that compose the catalytic machinery.The commercial CRL is actually a mixture of three Lip isoenzymes (Lip1, Lip2, Lip3) present in different proportions (e.g., the CRL mixture of Sigma is composed of 73% Lip1, 8% Lip2 and 19% Lip3) with Lip1 being the major component [5].
Candida viswanathii was firstly isolated from wastewater of a Brazilian oil refinery Replan/Petrobras, Paulínia, São Paulo, Brazil) and used for biodiesel and hydrocarbon bioremediation [6].Almeida et al. [7] described the C. viswanathii strain as a potential lipase producer using hydrophobic carbons sources in which produced high level of enzyme under submerged cultivation using olive oil as carbon sources.
Biochemical properties of crude lipase showed optimum pH and temperature activities at 4.0 and 40 o C, high thermal stability and activity in organic solvent [8].The purified lipase presented high solvent stability and hydrolytic activity on natural triacylglycerols and soy lecithin [9].C. viswanathii was also able to produce high level of lipase using vegetable oils from Brazilian Amazonian with different fatty acids compositions [10].The aim of this work was to evaluate at molecular level the effect of carbon sources on LIP4 isoform expression of C. viswanathii by polymerase chain reaction under submerged cultivation conditions.

Maintenance lineage
C. viswanathii strain is maintained in Laboratory of Biotechnology, Food Analysis and Products (LABAP), Habite -Biotechnology Incubator Companies, Federal University of Tocantins -UFT.C. viswanathii was routinely cultivated on potato dextrose agar (PDA) for 3 days at 28 °C and then stored at 4 °C.

Culture conditions
Cultures were also performed in PDA slants at the same conditions for inoculum preparation.Submerged cultivations were carried out in Erlenmeyer flasks (250 mL) containing 60 mL of modified Vogel liquid medium [11] supplemented with 0.2% (w/v) yeast extract and 1.0% (w/v) carbon sources (glucose, tributyrin, triolein or olive oil), pH 6.0.Flasks containing the liquid cultures were autoclaved at 121°C for 20 min and inoculated with 3 mL of cells suspension (10 7 cells/mL).Cultivation was carried out at 28 °C, 180 rpm for 25 h.After cultivations, biomasses were centrifugated at 4,000 rpm, 4 o C for 5 min.
One hundred of biomass was macerated using liquid nitrogen and stores at -80 o C.

In silico analyzes
C. viswanathii strain does not have its genome sequenced and are not available in annotated form through the GenBank nucleotide sequence database.A survey of known coding sequences of LIP4 gene from Candida species using GenBank database (www.ncbi.nlm.nih.gov/genbank/).Analysis of the sequence's alignment of Candida tropicalis (XM_002548709.1),Candida albicans (AF191317.1)and Candida orthopsilosis (XM_003871089.1) were performed with the ClustalW program [12], followed by standard parameters.For the construction of the phylogenetic tree, the MEGA 7.0 program [13] was adopted using the Neighbor-joining comparison model [14], with distance model p and pair-wise suppression.The statistical bootstrap method was used for validation of the phylogenetic tree with 10,000 replicates [15].

Design of degenerate primers
The nucleotide sequences of several species of the Candida genus were aligned in the same clade of the phylogenetic tree, using the Multiple Sequence Alignment by CLUSTALW program.With the result of the alignment an image was made using the GeneDoc program, where the regions of greater similarity were evaluated.The degenerate primers were designed in the most conserved regions for the LIP4 gene, where the lowest degree of degeneration was obtained using the Primer Express v2.0 program of Applied Biosystems.For the use of degenerate bases was followed the information of the own manufacturer (https://www.thermofisher.com/br).

Conventional PCR conditions
Deoxyribonucleic acid (DNA) extraction was carried out using Ilustra Nucleon PhytoPure for small samples kit (GE Helthcare), following manufacturer´s protocol.Samples purity were analyzed in spectrophotometer Nanodrop One (Thermo Fischer Scientific) and stored at -

Design of primers CvLIP4
The primers design LIP4 gene from C. viswanathii (named CvLIP4) was carried out by analysis of sequencing results obtained previously compared to the sequences used to design the degenerate primers (Table 1).From the sequence of CvLIP4 to C. viswanathii obtained in the sequencing, the primers were designed using the Primer Express v2.0 program of Applied Biosystems.Table 1 Sequencing Amplified products were eluted with Illustra™ GFX™ PCR DNA and Gel Band Purification (GE Healthcare) according manufacturer´s protocol.Purified samples 2.0 µL (50 ng/ µL) were transferred to Eppendorf containing 1.0 µL of primer forward [10µM], and 4.5 µL of nuclease free water.These samples were submitted to Myleus Biotechnology (Belo Horizonte, Brazil) for sequencing by capillary electrophoresis, using POP7 polymer and BigDye v3.1.

RNA extraction, DNase treatment and cDNA synthesis
For ribonucleic acid (RNA) total extraction, Trizol TM was used according manufacturer´s protocol.
Turbo DNA-Free TM (Applied Biosystems) was also used to eliminate possible trace amounts of contaminating genomic DNA according do kit instructions.The absence of DNA was also verified through PCR, using as template the total RNA.Nucleic acid quantification was assessed spectrophotometrically by measuring the absorbance at 260 nm using a NanoDrop One spectrophotometer (Thermo Fisher Scientific).
RNA purity of samples was evaluated through the A260nm/A280nm and A260nm/A230nm ratio.This samples were stored at -80 o C. Samples of treated RNA (1,000 ng/µL) with high-degree purity and integrity were used to cDNA synthesis using High-Capacity cDNA Revere Transcription (Applied Biosystems).

Amplification of CvLIP4 by conventional PCR
CvLIP4 amplification was performed using GoTaq® Hot Start Green Master Mix 2X (Promega) by conventional PCR according manufacturer´s protocol.For each reaction with 12.5 µL final volume were added 6.25 µL GoTaq Hot Start reen Master Mix 2X, 0.5 µL primer forward (10 µM), 0.5 µL primer reverse (10 µM), 0.75 µL cDNA (100 ng/µL) and 4.5 µL nuclease free water.PCR was run (T100 Thermal Cycler, Bio-Rad) with an initial step at 95 °C for 5 min and 40 cycles with denaturation at 95 °C for 45 s, annealing at 59 °C for 45 s, extension at 72 °C for 30 s, and final extension at 72 °C for 5 min.Samples were stored at -20 o C.

Results
C. viswanathii not have its genome sequenced and are not available in annotated form through the GenBank nucleotide sequence database for lipase production.For this study, in silico analysis was carried out with LIP4 gene from Candida species (Figure 1A).Namely CvLIP4 gene product was used to align the degenerate prime designed for C. viswanathii strain with LIP4 gene from C. orthopsilosis, C. albicans and C. tropicalis (Figure 1B), where the similarity between the sequences in black indicates similarity between the bases of all species, gray indicates that similarity occurs in at least two species and white means that there is no similarity.Candida species showed little differences in conserved regions from LIP4 gene.CvLIP4 gene was amplified from C. viswanathii using degenerated primers (Figure 2).In this experiment, it was observed one band of 1,000 bp (amplicon expected) and one unspecific product; however, the annealing temperature of 51.4 o C was more specific than 48.3 o C to produce the interest product.The amplification and expression of CvLIP4 gene was evaluated by cultivation of C. viswanathii under minimal salt medium containing sole carbon sources e.g.glucose (G), tributyrin (TB), triolein (TO) and olive oil (O) (Figure 3).In these conditions, CvLIP4 gene expression is directly associated to the carbon source once the glucose was a strong repressor source, tributyrin a weak inducer source and triolein and olive oil were a strong inducer to C. viswanathii lipase production.Triolein and olive oil presented amplification bands with higher intensity when compared to tributyrin and glucose.Triolein and olive oil present oleic acid (C18:1Δ 9 ) in your fatty acid composition.Figure 3.

Discussion
Candida species can express eight LIP gene family with a high amino acid identity and similarity among them [5].In these work, CvLIP4 from C. viswanathii was identify among homologous sequence from Candida species and degenerated primers were designed and analyzed in different carbon sources.The highly similarity in sequence but partially different in regions among the yeast species studied in this work can offer distinct biochemical properties such as substrates molecules interactions, sugar content, hydrophobicity and isoelectric point.Brocca et al. [16] extends the family of known Lip genes from Candida rugosa to seven highly homologous sequences.However, five-lipase genes comprising lipase gene family of C. rugosa with 80-88% pair wise identity, in nucleotide sequence.All genes encoding these isozymes were located on the same chromosome, which suggests their origin through gene duplication.
The effect of culture conditions on the microbial growth e.g., carbon and nitrogen sources, not only influence the lipase production but also changed the pattern formation of multiple forms of lipase.C. viswanathii has strong repression of lipase expression compared to olive oil and triolein.The yeast follows a complex pattern of lipase production depending on the presence of multiple lipase encoding genes whose expression is modulate by carbon sources that can act as repressor (e.g.glucose), inducer sources (e.g.oleic acid) or neutral substrates which is employed in two-step fermentation, where the first step of biomass growth is followed by the induction of lipase gene expression [17].In the first report about lipase production by C. viswanathii, Almeida et al. [7] related that this yeast can growth in several pure carbon sources such as glucose, galactose, mannitol sorbitol and glycerol but was not observed lipase production; on the other hand, pure fatty acids and triacylglycerols were able to promote the microbial growth and lipase secretion.
On the other hand, the complex triacylglycerols were also evaluated according to presence of high percentage of long chain fatty acids which observed that presence of oleic acid in the total composition of fatty acids was the most important inducer to lipase production.Oleic acid in widely described as main inducer source to true lipase production for many microorganisms.Olive oil contains approximately 80% oleic acid and it is considered a good inducer for lipase synthesis by many microorganisms what could be related with the fact of the lipases hydrolyze preferentially fatty acids residues at positions 1 and 3 of the glycerides, and some extracellular lipase requires oleic acid as stabilizer/activator [18]; and also to the presence of several tocopherols and other liposoluble vitamins that are important for microbial growth.
The weak intensity band formation using tributyrin source observed to CvLIP4 gene expression by C. viswanathii can be explained due the specificity of true lipase to long-chain fatty acids ester.Lipases are activated on interfaces of insoluble lipid substrates oil-water, where the catalytic reaction is combined with various interfacial phenomena/processes.The catalytic mechanisms involved for lipase activity depend strongly on the mode of organization of the lipid substrates present in interfacial structures such as membrane bilayers, monolayers, micelles, vesicles, and oil-in-water emulsions [19].A recombinant LIP4 isoform from C. rugosa (formerly Candida cylindracea) was previously related to have higher esterase activity toward long acyl-chain ester and lower lipase activity toward triglycerides [20].Tributyrin present short-chain fatty acid ester in triacylglycerol which are soluble in water and does not provide an interface oil-water formation.True lipases display a hydrophobic lid structure on active-site that regulate the activity mechanism in the presence of interface oil-water, which rearranges its position leaving an open gate to the active center.Then, the position of the lid marks the difference between the open (active) or closed (inactive) forms of these proteins [21,22].On the other hand, Fikers et al. [23] and Lotti et al. [24] reported the repression of lipase production caused by glucose from culture both.

Conclusion
This is the first report on gene expression from lipase family using C. viswanathii strain under cultivation growth conditions.These results will contribute to further studies about regulation of the lipase genes and heterologous expression of this enzyme and to understand and improve the catalytic conditions in industries processes.
20 o C. Conventional PCR were carried out using kit GoTaq® Hot Start Green Master Mix, 2X (Promega), according manufacturer´s protocol.PCR reactions (T100 Thermal Cycler -Bio-Rad) were performed in reaction mixture in a total volume of 25 μL in an Eppendorf as follow: 12.5 µL GoTaq Hot Start Green Master Mix, 2X, 1.0 µL of primer forward [10 µM], 1.0 µL of primer reverse [10 µM], 1.5 µL of DNA [100.0ng/µL] and 9.0 µL of nuclease free water.PCR cycles conditions were carried out as follow: 95 o C for 5 min and 40 cycles with denaturation at 95 o C for 30 s, annealing at 51.4 o C for 45 s, and final extension at 72 °C for 1 min.

Figure 1 .
Figure 1.Phylogenetic tree of Candida species that Lip4 gene encoded (A).Alignment of LIP4 gene

Figures
Figures

Table 1 .
Degenerate and design primer for LIP4 gene expression in C. viswanathii.