Potensi Genetik Limosilactobacillus fermentum 5.1.2 dalam Menghasilkan Senyawa Metabolit dengan Aktivitas Antioksidan

Abstract

Bakteri asam laktat (BAL) memiliki manfaat yang sangat luas sehingga penelitian yang mengkaji isolate BAL multifungsional menarik untuk dikembangkan. Isolat Limosilactobacillus fermentum 5.1.2 yang diisolasi dari tempe memiliki sifat probiotik dan mampu menyintesis metabolit esensial, yaitu vitamin B12. Eksplorasi lebih lanjut diperlukan untuk mengkarakterisasi senyawa metabolit yang dihasilkan oleh isolat L. fermentum 5.1.2. Beberapa penelitian telah mengkaji gen-gen pada genom BAL yang terlibat dalam biosintesis senyawa metabolit, namun penelitian yang mengkaji gen-gen terkait biosintesis senyawa metabolit pada genom L. fermentum 5.1.2 belum dilakukan. Penelitian ini bertujuan mengevaluasi potensi L. fermentum 5.1.2 secara genetik dalam menghasilkan senyawa metabolit melalui analisis sekuen genom total, mengidentifikasi senyawa metabolit pada ekstrak L. fermentum 5.1.2, melakukan uji aktivitas antioksidan ekstrak L. fermentum 5.1.2 dan mengevaluasi efek perlindungannya pada organisme model Schizosaccharomyces pombe. Ekstrak DNA genom didapatkan dengan menggunakan kit Quick-DNA Magbead Plus (D4082 – Zymo Research). Analisis sekuens genom total L. fermentum 5.1.2 dilakukan oleh PT. Genetika Science Indonesia. Data genom total dianalisis dengan menggunakan antiSMASH 7.0 digunakan untuk identifikasi dan analisis Biosynthetic Gene Cluster for Secondary Metabolites (BGC-SC). Orthovenn3 digunakan untuk mendapatkan klaster gen ortolog. Anotasi gen fungsional terkait biosintesis senyawa metabolit berdasarkan basis data KEGG dilakukan menggunakan program GhostKOALA. Supernatan bebas sel L. fermentum 5.1.2 diekstraksi dengan pelarut etil asetat 1:1 (v/v). Senyawa metabolit ekstrak diidentifikasi menggunakan LC-QTOF-MS. Aktivitas antioksidan ekstrak L. fermentum 5.1.2 diuji dengan metode DPPH dan ABTS. Efek perlindungan ekstrak tersebut diamati pada S. pombe dengan metode titik. Klaster gen biosintesis prekursor terpen terdeteksi pada genom L. fermentum 5.1.2. Keberadaan klaster gen biosintesis ini mengindikasikan kemampuan isolat 5.1.2 menghasilkan prekursor terpen. Berdasarkan KEGG, klaster gen tersebut teridentifikasi ke dalam modul biosintesis isoprenoid dan terlibat dalam jalur biosintesis terpenoid dan poliketida. Beberapa gen potensial yang diduga terlibat dalam biosintesis senyawa metabolit, teridentifikasi sebagai gen terkait biosintesis terpenoid backbone dan gen terkait ubikuinon dan terpenoid-kuinon lainnya. Beberapa senyawa turunan terpenoid dan terpen-kuinon terdeteksi pada ekstrak L. fermentum 5.1.2 antara lain linalil propionat, geranilbenzokuinon, geranilhidrokuinon, momordol, dan 4,4'-diapofitofluen. Aktivitas antioksidan ekstrak L. fermentum 5.1.2 tergolong rendah dengan IC50 660,56 ± 35,67 µg/mL berdasarkan metode DPPH, dan 333,886 µg/mL berdasarkan metode ABTS. Ekstrak dengan konsentrasi 334,65 µg/mL dan 669,3 µg/mL mampu memberi perlindungan kepada S. pombe pada kondisi cekaman oksidatif yang dipicu oleh H2O2 3mM. Berdasarkan hasil tersebut, L. fermentum 5.1.2 berpotensi menghasilkan senyawa terpenoid dan terpen-kuinon secara genetik, didukung dengan keberadaan senyawa turunan terpenoid dan terpen-kuinon pada ekstrak L. fermentum 5.1.2. Aktivitas antioksidan ekstrak tersebut juga menunjukkan efek perlindungan pada S. pombe pada cekaman oksidatif.

Kata kunci: Anotasi gen, Cekaman oksidatif, Limosilactobacillus fermentum, Schizosaccharomyces pombe

Lactic acid bacteria (LAB) are known for their broad range of beneficial properties, making studies on multifunctional LAB isolates an attractive area for further development. The isolate Limosilactobacillus fermentum 5.1.2, obtained from tempeh, has been reported to exhibit probiotic characteristics and the ability to synthesize essential metabolites, particularly vitamin B12. However, further investigation is necessary to characterize the metabolic compounds produced by this isolate. Although several studies have examined genes in LAB genomes that are associated with metabolite biosynthesis, research specifically addressing genes related to metabolite production in the genome of L. fermentum 5.1.2 remains limited. Therefore, this study aimed to evaluate the genetic potential of L. fermentum 5.1.2 to produce metabolic compounds through whole-genome sequence analysis, to identify metabolites present in the extract of L. fermentum 5.1.2, to determine the antioxidant activity of the extract, and to assess its protective effects using the model organism Schizosaccharomyces pombe. Genomic DNA was extracted using the Quick-DNA Magbead Plus kit (D4082; Zymo Research). Whole-genome sequencing of L. fermentum 5.1.2 was performed by PT Genetika Science Indonesia. The resulting genomic data were analyzed using antiSMASH 7.0 to identify and examine biosynthetic gene clusters associated with secondary metabolite production (BGC-SC). Orthologous gene clusters were identified using Orthovenn3. Functional annotation of genes related to metabolic compound biosynthesis, based on the KEGG database, was carried out using the GhostKOALA program. The cell-free supernatant of L. fermentum 5.1.2 was extracted using ethyl acetate at a ratio of 1:1 (v/v). Metabolites present in the extract were identified using LC-QTOF-MS. Antioxidant activity of the extract was evaluated using DPPH and ABTS radical scavenging assays. The protective effect of the extract was assessed in S. pombe using a spot assay. Genome analysis revealed the presence of biosynthetic gene clusters associated with terpene precursor production in the genome of L. fermentum 5.1.2. The detection of these clusters suggests that this isolate has the capability to produce terpene precursors. According to KEGG classification, these clusters belong to the isoprenoid biosynthesis module and are involved in the biosynthesis of terpenoids and polyketides pathway. Several candidate genes potentially involved in metabolite biosynthesis were identified, including genes related to terpenoid backbone biosynthesis as well as those associated with ubiquinone and other terpenoid-quinone biosynthesis. Metabolite detection of the L. fermentum 5.1.2 extract revealed several terpenoid and terpene-quinone derivatives, including linalyl propionate, geranylbenzoquinone, geranylhydroquinone, momordol, and 4,4'-diapophytofluene. The antioxidant activity of the extract was relatively low, with an IC50 value of 660.56 ± 35.67 µg/mL in the DPPH assay and 333.886 µg/mL in the ABTS assay. Nevertheless, extract concentrations of 334.65 µg/mL and 669.3 µg/mL were able to protect S. pombe cells under oxidative stress induced by 3 mM H2O2. These findings indicate that L. fermentum 5.1.2 possesses genetic potential to produce terpenoid and terpene-quinone compounds, which is supported by the detection of these derivatives in the extract. Furthermore, the extract exhibited a protective effect against oxidative stress in S. pombe

Keywords: Gene annotation, Limosilactobacillus fermentum, Oxidative stress, Schizosaccharomyces pombe