Development of plasmid vectors for zymomonas organisms

Abstract

For the ultimate purpose of introducing genes of cellulase or amylase into $\underline{Zymomonas}$ cells in order to make them able to produce ethanol from cellulose or starch, a suitable plasmid vector was attempted to develope. Plasmids were isolated from $\underline{Z}$. $\underline{anaerobia}$ and $\underline{Z}$. $\underline{mobilis}$. Among them a small plasmid isolated from $\underline{Z}$. $\underline{anaerobia}$ and designated as pZA2 was found to be most suitable. The size of plasmid pZA2 was estimated as 1.74 kilobase. It has one each cleavage site for Hind III and Bal I. Plasmids, pBR322 and pRK2501, have also been known to have one cleavage site each for Hind III. A hybrid plasmid was constructed using pZA2 and pBR322. The hybrid plasmid could be maintained in $\underline{E}$. $\underline{coli}$and be amplified by chloramphenicol. The hybrid plasmid could still be maintained in $\underline{E}$. $\underline{coli}$ even after the replicon portion of pBR322 was destroyed by treating with Hae II. The fact suggests that the replicon of pZA2 was not attacted by Hind III and was used for replication of the hybrid plasmid in E. coli. For the purpose of inserting selective markers another hybrid plasmid between pZA2 and pRK2501 was formed. Transformation of $\underline{Zymomonas}$ cells with this hybrid plasmid is now being tried.