Construction and Expression of Pet Operon using Shuttle Vector for Mesophilic and Thermophilic Bacteria Eny I. Riyanti 1 and Peter L. Rogers 2 1 Indonesian Center for Agricultural Biotechnology and Genetic Resources Research and Development, Jl. Tentara Pelajar 3A, Bogor 16111 2 School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, Australia ABSTRAK Konstruksi dan Ekspresi Pet Operon Menggunakan Shut- tle Vector untuk Bakteri Mesofilik dan Termofilik. Eny I. Riyanti dan Peter L. Rogers. Keuntungan fermentasi etanol pada suhu tinggi mendorong penelitian perakitan bakteri ter- mofilik etalogenik. Selain itu, kemampuan bakteri termofilik dalam penggunaan gula pentosa hasil degradasi biomasa memberi peluang untuk menekan biaya produksi bioetanol. Tujuan dari penelitian ini adalah untuk mengkonstruksi pet (production of ethanol) operon dengan menggunakan shuttle vector pMK18 dan melihat ekspresinya dalam bakteri mesofilik dan termofilik. Konstruksi dan ekspresi pet operon dengan menggunakan adhT dari bakteri termofilik dan pdc dari bakteri mesofilik, dan penggunaan mesofilik-termofilik shuttle vector sebagai backbone-nya baru pertama kali di- laporkan. Pet operon adalah suatu susunan gen penyandi produksi etanol yang terdiri dari gen pdc (pyruvate decarboxylase) dan adh (alcohol dehydrogenase). Konstruk- si pet operon menggunakan gen adhT dari bakteri termofilik Geobacillus thermoglucosidasius M10EXG dan pdc (pyruvate dehydrogenase) dari bakteri mesofilik Zymomonas mobilis ZM4 telah dilakukan dengan menggunakan mesofilik-termo- filik shuttle vector pMK18. Ekspresi pet operon pada bakteri mesofilik Eschericia coli dapat memproduksi 0,3 g/l etanol dengan aktivitas adhT sekitar 0,02 U/mg protein dan aktivitas pdc sekitar 0,004 U/mg protein. Perlu dilakukan penelitian lanjutan untuk perbaikan konstruksi pet operon untuk sis- tem termofik pada Thermus thermophilus HB27, karena konstruksi yang didapat belum optimum untuk sistem ter- mofilik ini. Hasil ini diharapkan akan mengawali pengem- bangan teknik manipulasi genetik pada bakteri termofilik yang masih sangat terbatas, khususnya pengembangan tek- nik manipulasi termofilik etanologenik. Kata kunci: Etanol, bakteri termofilik, bakteri mesofilik, pet operon, ekspresi gen. INTRODUCTION The natural ability for thermophiles to utilize a wide range of sugars, including pentose, at high tem- perature (Larsen et al. 1997) renders them as potential microorganisms for ethanol production from cheap lignocellulosic materials (Olsson and Hahn-Hagerdal 1996), which consist mainly of cellulose, hemicellulose and lignin. In addition, high temperature fermentation promises cost savings at an industrial scale, mainly Hak Cipta © 2009, BB-Biogen through the reduction of cooling costs following en- zymatic pre-treatment (Klapatch et al. 1994, Banat and Marchant 1995). Other advantages of fermentations carried out at evaluated temperatures include lower contamination risks (particularly by mesophilic con- taminants) and reduced energy costs for ethanol recovery (Edwards 1990, Lowe et al. 1993, Banat et al. 1998). The potential advantages associated with fermen- tation carried out at elevated temperatures have stimu- lated a significant interest in developing thermophilic ethanologens. However, naturally isolated ethanol producing thermophilic yield very low ethanol com- pared to mesophilic counterpart (Dien et al. 2003, Desai et al. 2004, Demain et al. 2005, Chinn et al. 2006, Keating et al. 2006, Stephanopoulos 2007). Current research on increasing ethanol yields in potential thermophilic ethanologens has been focused on optimization of fermentation conditions. Limited meta- bolic manipulations have also been reported in these thermophiles, including the development of high ethanol tolerant (Ljungdahl and Carreira 1983, Lovitt et al. 1988, Bryant et al. 1992) and LDH-negative mutant strains. So far, the metabolic/genetic manipulation of ethanol producing thermophiles was not revealed yet. In mesophilic bacteria such as Zymomonas mobilis, gene pdc and adh were reported to be involved in the production of ethanol (Conway et al. 1987a, 1987b). A number of thermostable adhT, have been isolated and characterized because of possible advantages associated with the use of thermophilic microorganisms and their enzymes in biotechnological processes (Coolbear et al. 1992, D'Auria et al. 1996). Thermostable variants of ADH from the obligately fermentative Z. mobilis have also been investigated (Rellos et al. 1998). However no adhT expression has been reported previously in T. thermophilus. There is no report of the isolation of pdc genes from thermophilic microorganisms or heterologous expression of thermostable pdc genes in thermophiles. It has been suggested that thermophilic ethanologens do not possess PDC enzymes (Payton 1984). However pdc genes have been isolated from a number of mesophiles with reports of at least four pdc genes Jurnal AgroBiogen 5(1):7-15