DNA marker utilization for the sustainable production of trehalose

Trehalose is a type of sugar that is known by its stability and resilience towards acid and low temperature. Furthermore, trehalose has numerous health benefits and has been used by several industries, including food, cosmetics, and pharmaceuticals. Even though trehalose could be easily produced using trehalose synthase (TreS) enzyme, a sustainable production of trehalose is still a problem. Our work aims to develop an approach to identify a novel trehalose synthase enzyme from various organisms, especially thermophilic bacteria, by implementing a deoxyribonucleic acid (DNA) marker technique. We first collected protein and DNA sequences from public biological databases and subsequently conducted sequence analysis. We then designed degenerate primers based on the conserved regions identified from the sequence analysis. The designed primers were subjected to primer characterization using Oligo Calc software. The primers were further validated via in-silico PCR amplification. In general, our designed primers possess the properties to work optimally. In addition, agarose gel electrophoresis that the primers successfully amplified nucleotides encoding TreS enzyme from all samples. Our findings may serve as a basis to discover the TreS enzyme variants which possess superior attributes, allowing the sustainable production of trehalose.


Introduction
Trehalose is a type of rare sugar, which is vastly distributed in many organisms, such as bacteria, fungi, insects, nematodes, and plants.Further, trehalose is also known as mushroom/mycose sugar as some fungi contain a high amount of it.Based on its structure, Trehalose contains two glucose units, which are connected with α, α−1, 1-glycosidic bond.In addition, the structure of trehalose is quite stable, as it has a high tolerance to acidity and freezing conditions [1].Until now, trehalose has been utilized in several industries.In the food industry, trehalose is commonly used as texturizer, preservative, and 1297 (2024) 012079 IOP Publishing doi:10.1088/1755-1315/1297/1/012079 2 stabilizer for various foods, such as instant noodles, fruits, and creams (for cakes and pastries) [2].
Meanwhile, in the pharmaceutical industry, trehalose has been used for preserving tissues/organs, for cryopreservation of cells and for eye treatment [3].In addition, in the cosmetic industry, trehalose is added into creams and lotions to preserve the moisture and enhance the stability of the products [4].
Furthermore, trehalose can also be used to reduce the human body odor as it prevents the degradation of unsaturated fatty acids [4].
Besides the utilization of trehalose in many industries, previous research has also shown the health benefits of trehalose.Yoshizane et al. studied the effect of daily consumption of trehalose to the blood glucose in humans [5].The result showed that consuming trehalose could reduce blood glucose levels, indicating that trehalose has the potential to reduce the development of metabolic disorders, for instance type 2 diabetes [6].Furthermore, a study by Portbury et al. displayed the efficacy of trehalose to improve traumatic brain injury [7].This suggests that trehalose may be proposed as a treatment for neurodegenerative diseases.In addition, Neta et al. showed that trehalose has a low-cariogenic properties, which possess a low potency in promoting dental caries [8].
The production of trehalose can be achieved either by chemically or biologically.The chemical route is performed through the reaction between 2,3,4,5-tetra-O-acetyl-D-glucose and 3,4,6-tri-O-acetyl-1,2anhydro-D-glucose [9].Meanwhile, the biological route of trehalose production is undertaken by utilizing enzymes via three different biosynthetic pathways [9].The first pathway uses two enzymes, namely trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase.The second pathway also involves two enzymes, maltooligosyl trehalose synthase and maltooligosyl trehalose trehalohydrolase.On the other hand, the third pathway only needs one enzyme, called trehalose synthase (TreS)  [10].Compared to the chemical route, producing trehalose via biological route is preferable as it is easier, more economical, and environmentally friendly.Furthermore, for the sustainable large-scale production of trehalose, employing the third pathway is more straightforward and economical approach due to the involvement of only one enzyme, the TreS [11].However, there are some issues in the sustainable large-scale production of trehalose, including the activity and specificity of the enzyme.In addition, a high-temperature condition is also required for the large-production, as this can boost the enzymatic activity and prevent microbial contamination [12,13].Therefore, utilizing TreS enzyme with a high activity and specificity that could function in a high temperature is the optimal solution to tackle the issues.
One promising approach to produce trehalose in a large-scale setting involves the utilization of trehalose enzyme derived from extremophilic sources, such as thermophilic bacteria.This can be implemented either using the native bacteria or heterologous expression of recombinant protein.In addition, the large-scale production needs to be performed in a bioreactor.This needs an optimization in the processes, such as integrating the fermentation and biocatalyst processes or uses an integrated setting of biocatalyst and bio-removal processes [14,15].To achieve a sustainable large-scale production of trehalose, discovering TreS enzyme with the superior attributes becomes urgent.The discovery of the trehalose enzyme with such attributes could be facilitated by employing DNA marker/DNA barcoding technique [16].In this work, we have devised an innovative approach for the identification of novel TreS enzyme from different organisms.The approach incorporates the application of DNA marker technique, encompassing data mining of gene expressing TreS enzyme (treS gene), as the subsequent design and validation of degenerate primer.The outcome of our research has the potential to serve as a valuable reference to discover TreS enzyme with optimal characteristics, particularly those expressed by thermophilic bacteria.

Materials and methods
The proposed DNA marker technique comprises of five main steps (Figure 1): mining DNA and protein sequences, sequence analysis, generation of degenerate primer for DNA marker, primer characterization, testing and validation.

Data collection
The protein sequences of TreS enzyme, as well as DNA sequences (treS gene), were obtained from National Center for Biotechnology Information (NCBI) (https://www.ncbi.nlm.nih.gov).In addition, we also collected sequences from Kyoto Encyclopedia of Genes and Genomes (KEGG) (https://www.genome.jp/kegg/pathway.html).The TreS enzyme from various bacteria, including mesophilic bacteria, thermophilic bacteria, and cyanobacteria were retrieved and used in this study.

Sequence analysis
To acquire the relevant TreS sequences, we used the "Search" function in the NCBI webserver.Focusing on the gene database (gene ID treS), we then collected the sequences from various taxa, including Pseudomonas putida, Mycobacterium tuberculosis, Fischerella thermalis, Streptomyces scabiei, Rhizobium leguminosarum, Corynebacterium glutamicum, and Thermus thermophilus.All sequences were subjected to multiple sequence alignment using the MUltiple Sequence Comparison by Log-Expectation (MUSCLE) Algorithm in the European Molecular Biology Laboratory-European Bioinformatics Institute (EMBL-EBI) online software (accessed from https://www.ebi.ac.uk/).The alignment created a consensus sequence that acts as a reference for creating degenerate primer.

DNA marker/primer design
The DNA marker (degenerate primer) for TreS enzyme were designed using SnapGene Viewer software version 5.2.5, based on the multiple sequence alignment result.For the degenerate primer, several degenerate bases were incorporated, following the one-letter codes of International Union of Biochemistry (IUB).The designed primer was then characterized using Oligo Calc software (http://biotools.nubic.northwestern.edu/OligoCalc.html).This software measures several parameters, including melting temperature (Tm), GC content, and any potential hairpin or self-dimerization formation.

Primer testing and validation
After performing primer characterization, the primer was then tested and validated whether they are able to amplify the treS gene, which encodes TreS enzyme.Primer testing and validation was conducted via in-silico polymerase chain reaction (PCR) amplification, using SnapGene® software version 6.1.The result was subsequently displayed using Simulate Agarose Gel Tools (SnapGene® software); 1 % agarose and 1 kb plus DNA ladder from New England Biolabs were selected for the gel electrophoresis simulation.

Sequence analysis of TreS enzyme
First, we performed data mining to retrieve TreS enzyme protein sequence, as well as the DNA sequence of treS gene, based on the data that are available in the NCBI.We successfully retrieved both protein and DNA sequences from 17 organisms, including thermophilic bacteria, mesophilic bacteria, actinomycete, and cyanobacteria (Table 1).Then, MUSCLE Algorithm was used to observe the differences between the sequences and discover the conserved regions that are required for DNA marker.Sequence analysis result displayed the aligned TreS protein sequence from all samples (Figure 2).The alignment of all sequences was achieved successfully.Subsequently, we explored for regions demonstrating a higher degree of conservation.We successfully discovered two conserved regions, demarcated within a blue box, which could be used for primer/DNA marker design.In conclusion, the sequence analysis revealed the presence of two prospective regions to design the primer suitable for TreS enzyme.

DNA marker/primer characterization
The sequence analysis discovered two conserved regions that can be utilized, as the DNA marker approach requires a set of primers that could recognize the TreS sequence.Using SnapGene Viewer software, a set of degenerate primers were designed, resulting in primers namely TreS_1 and TreS_2.Then, Oligo Calc software was used to characterize both primers.The result of primer characterization (primer properties) was shown in Table 2.Both primer (TreS_1 and TreS_2) had similar length, 19 base pairs and 20 base pairs, respectively.Furthermore, they also had a similar melting temperature (Tm) and GC content (Table 2).In addition, there was no indication for formation of either self-complementary or hairpin for both primers.Overall, the properties of both primers are comparable.To obtain a primer that work optimally, several criteria should be addressed: melting temperature (Tm), sequence length, GC concentration, and any potential self-complementary and hairpin formations [17].Ideally, a primer should have a sequence length between 18 and 30 nucleotides long.If the primer is too short, the specificity will decrease.On the other hand, if the primer is too long, it will be difficult to utilize it as a DNA marker.In addition, a primer should have a Tm within the range of 52⁰C and 58⁰C, as this is the best range for the primer to optimally interact with the DNA region [17].GC concentration IOP Publishing doi:10.1088/1755-1315/1297/1/0120796 is also affect the interaction between a primer and the DNA region.To have an optimum interaction, a GC concentration of 40-60% is essential [18].Meanwhile, any self-complementary and hairpin formations should be avoided as this could reduce the effectivity of the binding to the DNA region.For both self-complementary and hairpin, it is best to have the value closest to zero [18].Overall, primer characterization results displayed that the designed primers (TreS_1 and TreS_2) met the criteria to work optimally.These primers should be tested and validated to find out whether it could work as a DNA marker.

TreS primer testing and validation
Next, we conducted primer testing and validation by performing in-silico PCR amplification.All organisms that were listed in Table 1 were chosen as the sample.If both primers (TreS_1 and TreS_2) work optimally, there will only produce one DNA fragment.The result of the in-silico PCR was then displayed using agarose gel electrophoresis.The gel electrophoresis result showed only one amplicon of the treS gene (encoding Tres enzyme) from all samples (Figure 3), which confirms the specificity of the TreS primers.Our findings indicate that the designed primers exhibit potential utility as DNA marker to isolate TreS enzyme across diverse bacterial species.These primers may be harnessed for the identification of the TreS enzyme variants that display optimal attributes, which can be further applied for large-scale trehalose production.Prospective research will involve the application of protein engineering techniques to enhance the TreS characteristics, such as genetic modification and chemical modification.This may well promote a sustainable large-scale trehalose production, which holds several advantages for human consumption and applications.

Conclusions
Our work employed a DNA marker technique to facilitate the identification of the TreS enzyme across various bacterial species.Using protein and DNA sequences collected from publicly available biological databases, we performed sequence analysis that led to the generation of TreS primers.Extensive characterization and validation were further conducted to confirm the primers' functionality.Overall, the results showed that both primers (TreS_1 and TreS_2) had successfully amplified TreS enzyme from all samples.Consequently, the designed primers may serve as a valuable tool for the discovery of TreS

Figure 1 .
Figure 1.The workflow used in this study.2.1.Data collectionThe protein sequences of TreS enzyme, as well as DNA sequences (treS gene), were obtained from National Center for Biotechnology Information (NCBI) (https://www.ncbi.nlm.nih.gov).In addition, we also collected sequences from Kyoto Encyclopedia of Genes and Genomes (KEGG) (https://www.genome.jp/kegg/pathway.html).The TreS enzyme from various bacteria, including mesophilic bacteria, thermophilic bacteria, and cyanobacteria were retrieved and used in this study.2.2.Sequence analysisTo acquire the relevant TreS sequences, we used the "Search" function in the NCBI webserver.Focusing on the gene database (gene ID treS), we then collected the sequences from various taxa, including Pseudomonas putida, Mycobacterium tuberculosis, Fischerella thermalis, Streptomyces scabiei, Rhizobium leguminosarum, Corynebacterium glutamicum, and Thermus thermophilus.All sequences were subjected to multiple sequence alignment using the MUltiple Sequence Comparison by Log-Expectation (MUSCLE) Algorithm in the European Molecular Biology Laboratory-European Bioinformatics Institute (EMBL-EBI) online software (accessed from https://www.ebi.ac.uk/).The alignment created a consensus sequence that acts as a reference for creating degenerate primer.2.3.DNA marker/primer designThe DNA marker (degenerate primer) for TreS enzyme were designed using SnapGene Viewer software version 5.2.5, based on the multiple sequence alignment result.For the degenerate primer, several degenerate bases were incorporated, following the one-letter codes of International Union of Biochemistry (IUB).The designed primer was then characterized using Oligo Calc software (http://biotools.nubic.northwestern.edu/OligoCalc.html).This software measures several parameters, including melting temperature (Tm), GC content, and any potential hairpin or self-dimerization formation.2.4.Primer testing and validationAfter performing primer characterization, the primer was then tested and validated whether they are able to amplify the treS gene, which encodes TreS enzyme.Primer testing and validation was conducted via

Figure 2 .
Figure 2. Sequence analysis of TreS protein sequence from various organisms.The blue box displays the conserved region of the protein sequence.

Figure 3 .
Figure 3. Gel electrophoresis of the samples amplified using TreS primer.

Table 1 .
List of organisms used in this study.

Table 2 .
The properties of primers for TreS enzyme