Enzyme and Microbial Technology 40 (2007) 1598–1603 Sequencing, cloning and expression of the dsz genes required for dibenzothiophene sulfone desulfurization from Gordonia alkanivorans strain 1B L. Alves a , M. Melo a , D. Mendonc ¸a a , F. Sim˜ oes a , J. Matos a , R. Tenreiro b , F.M. G´ ırio a,∗ a INETI, Departamento de Biotecnologia, Estrada do Pa¸ co do Lumiar, 22, 1649-038 Lisboa, Portugal b FCUL, Departamento de Biologia Vegetal, Centro de Gen´ etica e Biologia Molecular, Campo Grande, 1749-016 Lisboa, Portugal Received 4 January 2006; received in revised form 11 November 2006; accepted 14 November 2006 Abstract Biological desulfurization of fossil fuels may offer an alternative process to reduce sulfur dioxide emissions that cause environmental pollution. Gordonia alkanivorans strain 1B is able to convert dibenzothiophene sulfone (DBTS) to 2-hydroxybiphenyl (2-HBP), the final product of 4S pathway. G. alkanivorans genes that code for the enzymes involved in this degrading pathway were PCR amplified using homologous primers based on known sequences from Rhodococcus erythropolis IGTS8. Amplified fragments were further cloned and sequenced. Strain 1B desulfurization genes (dsz) were identified and compared with previously described bacterial genes from other strains. Three open reading frames were identified (dszA, dszB and dszC) and have shown high similarity when compared to those from R. erythropolis IGTS8 (88% for dszA, 88% for dszB and 90% for dszC). G. alkanivorans dszAB genes were further expressed in Escherichia coli. This recombinant strain was able to grow in a culture medium containing dibenzothiophene sulfone (DBTS) as the only sulfur source desulfurizing the same amount of DBTS (0.2 mM) to 2-HBP but 4.5-fold faster than strain 1B. In addition, the recombinant strain could also desulfurize DBTS in LB medium containing other sulfur compounds such as sulfates, showing no sulfate repression of the dszAB genes expression. © 2006 Elsevier Inc. All rights reserved. Keywords: Biodesulfurization; Dibenzothiophene sulfone; dsz genes; Gordonia alkanivorans; Escherichia coli; Recombinant bacteria 1. Introduction The combustion of petroleum distillates produces sulfur oxides that contribute to air pollution. In addition, the decreased availability of low sulfur crude oils has resulted in a need to uti- lize higher sulfur crude oil [1]. Regulatory agencies throughout the world have recognized these problems producing regula- tions limiting both the sulfur emissions from power plants and the level of sulfur allowed in transportation fuels [2]. Nowadays the petroleum industry treats the crude oil by hydrodesulfurization using extremely high temperature and pressure conditions. Microbiological desulfurization offers the potential of a complementary method for lowering the sulfur content of petroleum products [3]. There are many desulfuriz- ing bacteria which has shown the ability to desulfurize DBT producing 2-hydroxybiphenyl (2-HBP) using the specific sulfur ∗ Corresponding author. Tel.: +351 21 7165141; fax: +351 21 7163636. E-mail address: francisco.girio@ineti.pt (F.M. G´ ırio). pathway known as “4S pathway”. Several model compounds such as DBT, DBTS, and simple mononuclear thiophene deriva- tives can be used to characterize organic sulfur in coal, coal tars, and crude oils [4]. The dsz genes of Rhodococcus erythropolis strain IGTS8 were the first to be characterized [5]. These genes are orga- nized in an operon that comprises three open reading frames, transcribed in the same direction coding for two monooxyge- nases (dszC and dszA) and a desulfinase (dszB). The dsz operon of Mycobacterium phlei GTIS10 [6] and Arthrobacter sp. DS7 [7] were cloned and sequenced and found to be identical to that of R. erythropolis IGTS8. However, this homology of the dsz nucleotide sequence is not observed for other bacterial species [8–11]. The dsz promoter has also been characterized and it was found that the dsz genes expression is strongly repressed by sulfate or other sulfur compounds even in the presence of DBT [12,13]. Gallardo et al. [14] reported one of the first studies of engineering R. erythropolis IGTS8 dsz genes under the control of heterologous broad-host-range regulatory control to alleviate 0141-0229/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2006.11.008