ELSEVIER FEMS Microbiology Letters 132 (1995) 23-26 Construction of an integrative shuttle vector for Zymomonas mobilis Osvaldo D. Delgado a, Carlos M. Abate a*b* * , Faustino Siaeriz ‘3’ a zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA PROIMI, Au. Belgrano Y Pje. Caseros, 4000- Tucumhn, Argentina h Ccitedras de Biologfa Celular y Biologia Molecular. Qca., y Fcia. UW , 4OOO-Tucunuin, Argentina ’ Critedras de Microbiologia Superior, Fat. de Bioqca., Qca.. y Fcia. W T. IOa)-Tucumh. Argentina Received 7 June 1995; revised 26 June 1995; accepted 12 July 1995 zyxwvutsrqponmlkjihgfedcbaZYXWV Abstract An integrative shuttle vector, pZMOCP1, was constructed by ligating EcoRV digests of the plasmid cloning vector pBluescript and pZMP1, a cryptic plasmid of Zymomomzs mobilis PROIMI Al. The 7.2-kb plasmid pZMOCP1 replicated in Escherichia coli and could also be transferred from this host by electroporation to Z. mobilis ATCC 29191. The transformants were selected by ampicillin resistance. The integrative characteristic was detected by hybridization in situ. The vector was stably maintained in Z mobilis after 200 generations without selective pressure. Keywords: Zymomonas mobilis; Plasmid; Integrative shuttle vector 1. Introduction Zymomonas mobilis is an anaerobic Gram-nega- tive bacterium capable of producing almost theoreti- cal yields of ethanol from glucose and has been shown to be a promising alternative to yeast for the industrial production of ethanol [l-3] reaching high ethanol concentrations (10%) [ 11.However, the range of substrate utilized by this microorganism is re- stricted to glucose, fructose and sucrose, and this severely limits its commercial use. It is possible to increase the substrate range of this microorganism by genetic manipulation [4]. Different studies have shown that plasmids having a broad host range can be conjugally transferred from Escherichia co/i to 2. mobilis, as in the case of * Corresponding author. Fax: + 54 (81) 330 087. RR1 plasmid containing a lactose transposon, Tn951, and be stably maintained [5,6]. Browne et al. [7] have reported that pNSW601 transformed Z. mobilis and was stably maintained in it. However, these plasmids are not suitable cloning vectors for the introduction of foreign genes into 2. mobilis, since they have high molecular masses [4]. The construc- tion of shuttle vectors using replicon sequences of indigenous plasmids of Zymomonas plus replicative and resistance marker sequences of E. coli plasmids such as pBR322 and pACYC184 was previously described [4,8]. However, although such vectors replicate stably in Zymomonus, there are reports that they can interact with indigenous plasmids or with host chromosome [9]. On the other hand, the avail- ability of shuttle vectors for use in E. coli and Z mobilis will aid the genetic investigations of Z. mobilis by means of genetic engineering [lo]. In this way the development of integrative vectors is con- 0378-1097/95/$09.50 0 1995 Federation of European Microbiological Societies. All rights reserved .SSDI 0378-1097(95)00275-8 Downloaded from https://academic.oup.com/femsle/article-abstract/132/1-2/23/903298 by guest on 05 June 2020