Molecular Determination of a Gren Algae Isolate to Detecting 1-Deoxy-D-Xylulose-5-phosphate Synthase (DXS) Gene in Improvement of Carotenoid Production

Abstract

Sintesis karotenoid alami belum pernah melebihi produk sintetik pada skala komersial. Kurangnya pemahaman mengenai aspek mikrobiologis dan ekofisiologis isolat penghasil karotenoid menyebabkan terjadinya kesalahan penamaan spesies. Satu isolat lokal alga hijau dari BBAP Jepara yang digunakan sebagai pakan alami sumber karotenoid hewan-hewan perikanan, pada mulanya dianggap sebagai Dunaliella. Namun pengembangan produksi karotenoid menggunakan teknologi rekayasa genetik dan rekayasa metabolit terhadap isolat alga hijau lebih lanjut memperlihatkan ketidaksesuaian hasil dengan penamaan yang ada. Akumulasi karotenoid jalur non-MVA pada alga hijau ditentukan oleh enzim D-1-Deoksixilulosa 5-fosfat Sintase, yang disandi oleh gen D-1-deoksixilulosa 5-fosfat sintase (DXS). Determinasi spesies secara molekuler menjadi penting dilakukan untuk menentukan spesies isolat dan jalur biosintesis karotenoid yang digunakan. Hasil determinasi digunakan untuk analisis keserupaan putative partial fragment gen DXS Isolat alga hijau yang telah berhasil diperoleh pada penelitian sebelumnya. Tujuan utama penelitian ini adalah menentukan spesies satu isolat lokal alga hijau secara molekuler menggunakan 23S rRNA untuk mendeteksi keberadaan gen DXS penyandi biosintesis karotenoid. Hasil penelitian memperlihatkan bahwa Isolat alga hijau menunjukkan keserupaan yang tinggi dengan anggota-anggota Sianobakteria. Keserupaan tertinggi dimiliki dengan Cyanobacterium sp. MBIC 1021 sebesar 99 %, diikuti Synechocystis PCC6308 sebesar 95 %. Satu-satunya anggota Cyanobacteria yang memiliki gen DXS adalah Synechocystis. Hasil analisis keserupaan parsial gen DXS isolat alga hijau terhadap tujuh parsial gen DXS pada daerah lestari yang telah ditemukan, memperlihatkan bahwa putative partial fragment gen DXS Isolat lokal alga hijau juga memiliki keserupaan tertinggi dengan gen DXS Sianobakteria Synechocystis. Kata kunci : isolasi alga hijau, Dunaliella, gen DXS, 23S rRNA, Cyanobacteria, Synechocystis Carotenoids production levels are not yet competitive with carotenoid levels presently produced by fermentation, synthesis and isolation. It needs application of metabolic engineering and genetic engineering techniques in improving their production. An attempt to optimize carotenoid production from local isolate of green algae from BBAP Jepara has faced several problems, primarily related to the microbiological and ecophysiological characteristic which affecting growth that have not sufficiently been understood. A misnamed of species also have arisen due to wrong characterization. One local isolate of an algal species from BBAP Jepara was found potentially useful as source of carotenoids in food additives or as food supplement in fish farming. It was suspected as representing a strain of Dunaliella. Previous studies to improve carotenoid production using molecular approach on have shown unagreement. Therefore, the present study aimed to determinate the species of green algae isolate from Jepara waters based on molecular techniques using 23S rRNA approach for detecting DXS gene. Molecular analysis by 23S rRNA alignment showed the close relationship among isolate of green algae and most all of member of Cyanobacteria. Closest similarities was showed by Cyanobacterium sp. MBIC 1021 with 99 % similarity and Synechocystis PCC6308 with 95 % similarity. Synechocystis was the only member of Cyanobacteria which have DXS gene. Multiples aligment sequences of partial DXS gene on the conserve region among seven species confirmed this result. The DXS gene analysis also showed closest relationship between partial DXS gene of Cyanobacteria Synechocystis anda green algae isolate. The result of this analysis proven as valuable parameter for the interpretation of the relation among DXS gene of a green algae isolate and Cyanobacteria and increase the possibility in getting the complete DXS gene from local isolate of green algae by designing primers from DXS gene of Synechocystis as a member of Cyanobcteria. Key words : a green algae isolate, Dunaliella, DXS gene, 23S rRNA, Cyanobacteria, Synechocystis