Rapid Identification of Ectoine-Producing Halophiles Using Newly Designed Degenerate Primers and LC-MS/MS

Öz

Ectoine is a valuable compatible solute widely used in pharmaceutical, cosmetic, and biotechnological applications due to its protective and stabilizing properties. This study aimed to identify ectoine-producing bacterial strains isolated from diverse saline environments, including the İzmir Çamaltı Saltern, İzmir Bird Paradise, and commercial Himalayan salt. A PCR-based screening approach was applied using in silico primer design. Newly designed degenerate primers targeting the ectB-ectC gene region was used in this study. Primer design was performed for each strain by analyzing conserved genomic regions between the adjacent ectB and ectC genes using representative type strains retrieved from NCBI. Gradient PCR conditions, particularly annealing temperature and extension time, were optimized individually for each isolate to improve amplification efficiency and specificity. For confirmation of genetic screening, ectoine production was assessed in 15% salinity medium and quantified using LC–MS/MS analysis and ectoine standard. A total of eight bacterial isolates were purified and screened for the presence of the ectB-ectC region. Among them, three isolates -Kocuria sp. CT-6, Oceanobacillus sp. HT-9, and Halomonas sp. CS-5- were found positive for the target region. A representative ectB-ectC positive Halomonas sp. CS-5 strain was selected for further analysis as a study-specific model and cultivated under 15% salinity conditions. Ectoine production was confirmed as 0.395 g/L (mean±SD, n=3, SD=0) in the culture reached the stationary phase at 48.5 h, total dry cell weight was 1.77 g/L corresponding to 22.3% (w/w) of dry cell weight by LC-MS/MS analysis. The study demonstrates that the combined application of PCR-based screening and analytical confirmation provides a potentially useful preliminary screening approach for identifying ectoine-producing microorganisms. This approach supports the selection of promising strains for further biotechnological evaluation.

Anahtar Kelimeler

• Ectoine
• In silico primer design
• Degenerate primers
• Halophilic bacteria
• LC-MS/MS

Destekleyen Kurum

Ege University Scientific Research Projects Coordination Unit

Proje Numarası

2011-FEN-010

Etik Beyan

No specific ethical approval was necessary for the study.

Teşekkür

Portions of this article are based on the Ebru Tekin’s PhD thesis. This study was supported by the Ege University Scientific Research Projects Coordination Unit (PhD Project number: 2011-FEN-010). The authors also thank the İzmir Institute of Technology, Biotechnology and Bioengineering Research and Application Center for DNA sequencing (BIYOMER). LC-MS/MS analyses were performed at Argefar, İzmir, Turkey by Barış Gümüştaş.

Dejenere Primerler ve LC-MS/MS Kullanılarak Ektoin Üreten Halofillerin Hızlı Tanısı

Öz

Ektoin, koruyucu ve stabilize edici özellikleri nedeniyle farmasötik, kozmetik ve biyoteknolojik uygulamalarda yaygın olarak kullanılan önemli bir biyouyumlu moleküldür. Bu çalışmanın amacı, İzmir Çamaltı Tuzlası, İzmir Kuş Cenneti ve ticari Himalaya tuzu gibi farklı tuzlu ortamlardan izole edilen ektoin üreten bakteriyel suşları belirlemektir. Bu amaçla, in silico primer tasarımına dayalı PCR temelli bir tarama yaklaşımı uygulanmıştır. Çalışmada, ectB-ectC gen bölgesini hedefleyen yeni tasarlanmış dejenere primerler kullanılmıştır. Primer tasarımı, NCBI veri tabanından elde edilen temsilci tip suşlar kullanılarak, komşu ectB ve ectC genleri arasındaki korunmuş genomik bölgelerin analiz edilmesiyle gerçekleştirilmiştir. Amplifikasyon verimini ve özgüllüğünü artırmak amacıyla, özellikle bağlanma sıcaklığı ve uzama süresi gibi gradient PCR koşulları her izolat için ayrı ayrı optimize edilmiştir. Genetik tarama sonuçlarının doğrulanması amacıyla, ektoin üretimi %15 tuzluluk içeren besiyerinde değerlendirilmiş ve ektoin standardı kullanılarak LC-MS/MS analizi ile miktar tayini yapılmıştır. Toplam sekiz bakteriyel izolat saflaştırılmış ve ectB-ectC bölgesinin varlığı açısından taranmıştır. Bunlardan üç izolat (Kocuria sp. CT-6, Oceanobacillus sp. HT-9 ve Halomonas sp. CS-5) hedef gen bölgesi açısından pozitif bulunmuştur. ectB-ectC gen bölgesi pozitif olan temsili olarak Halomonas sp. CS-5 suşu, çalışmaya özgü bir model olarak seçilmiş ve %15 tuzluluk koşullarında kültive edilmiştir. LC-MS/MS analizi sonucunda, kültürün 48,5 saatte durağan faza ulaşmasıyla ektoin üretimi 0,395 g/L (ortalama±SD, n=3, SD=0) olarak doğrulanmıştır. Aynı zamanda toplam kuru hücre ağırlığı 1,77 g/L olarak belirlenmiş ve bu değer kuru hücre ağırlığının %22,3’üne (w/w) karşılık gelmiştir. Bu çalışma, PCR tabanlı genetik tarama ile analitik doğrulamanın birlikte uygulanmasının, ektoin üreten mikroorganizmaların belirlenmesinde potansiyel olarak yararlı bir ön tarama yaklaşımı sunduğunu göstermektedir. Bu yaklaşım, ileri biyoteknolojik değerlendirmeler için umut verici suşların seçimini desteklemektedir.

Anahtar Kelimeler

• Ektoin
• In silico primer dizaynı
• Dejenere primerler
• Halofilik bakteri
• LC-MS/MS

Destekleyen Kurum

Ege Üniversitesi Bilimsel Araştırma Proje Koordinatörlüğü

Proje Numarası

2011-FEN-010

Etik Beyan

Çalışma için etik kurul onayına gerek yoktur.

Teşekkür

Bu makalenin bazı bölümleri Ebru Tekin'in doktora tezine dayanmaktadır. Bu çalışma Ege Üniversitesi Bilimsel Araştırma Proje Koordinatörlüğü tarafından desteklenmiştir (Doktora Projesi numarası: 2011-FEN-010). İzmir Teknoloji Enstitüsü, Biyoteknoloji ve Biyomühendislik DNA Dizileme Araştırma ve Uygulama Merkezi'ne (BIYOMER) sekans analizi için hizmet alımı yapılmıştır. LC-MS/MS analizleri, Argefar (İzmir, Türkiye)'da, Barış Gümüştaş tarafından gerçekleştirilmiştir.

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