Cloning of upstream region and cellulose synthase operon genes involved in bacterial cellulose biosynthesis by Gluconacetobacter hansenii ATCC23769

Abstract

Background Cellulose is a homopolysaccharide composed of extracellular D-glucose monomers connected by glycosidic linkages in b-1,4 conformation which can be synthesized by a variety of living organisms, possessing numerous applications in food, pharmaceutical, medical area, etc.[1][2]. In 1990, cellulose synthase operon encoding four proteins required for bacterial cellulose (BC) biosynthesis by Gluconacetobacter hansenii was isolated. It was demonstrated that these genes bcsA, bcsB, bcsC and bcsD were, together, required for maximum production of celulose [3]. Later, researches identified other relevant regions for bacterial cellulose biosynthesis that occur upstream (cmcax and ccpAx genes) and downstream of the operon (bglx gene) [4]. In this study, we aimed to clone the most important genes related to the BC synthesis. With the purpose of cloning those genes, the genomic DNA of the mentioned microorganism was extracted and proceeded with the amplification of cmcax, ccpAx and cellulose synthase operon genes. Later, the genes were ligated to a cloning vector, transformed in Escherichia coli and the identity of the clones was confirmed by sequencing and comparing with the GenBank using Blastn tool. The cloning achievement of the operon and upstream genes from G. hansenii and their overexpression will enable studies on BC synthesis improvement.