Amplification of D-xylose and D-glucose isomerase activities in Escherichia coli by gene cloning

Abstract

A recombinant plasmid, designated pUC1002, was constructed by ligation of a HindIlI restriction endonuclease fragment of Escherichia coli chromosomal DNA to vector plasmid pMB9. Strains carrying this plasmid were selected by transformation of an E. coli strain bearing the xyl-7 mutation to a xylose-positive (Xyl+) phenotype. Strains containing pUC1002 produced coordinately elevated levels of D-xylose isomerase and D-xylulose kinase. Under appropriate conditions, the isomerase also efficiently catalyzed the conversion of D-glucose to D-fructose. A number of bacteria, including Escherichia coli, contain D-xylose isomerases which function physiologically to catalyze the first reaction in the catabolism of D-xylose but which also convert D-glucose to D-fructose under certain conditions (24). The latter reaction is the basis for industrial processes currently used to produce very large quantities of high-fructose syrups (4). This use, as well as interest in the use of the enzyme to promote efficient fermentation of D-xylose to ethanol (5, 12, 21, 22; T. W. Jeffries and S. Choi, Abstr. Annu. Meet. Am. Soc. Microbiol. 1981, 034, p. 193), led us to try to increase the yield of the enzyme by gene cloning. This report describes the construction and selection of a recombinant plasmid coding for both E. coli D-xylose isomerase and E. coli Dxylulose kinase, the second enzyme in the pathway for D-xylose utilization by E. coli. We also describe the production of the enzymes and the D-glucose isomerase activity in strains bearing the plasmid. The recipient strain used in the cloning was JC1553 (6), an E. coli K-12 derivative obtained from the E.

Molecular and industrial aspects of glucose isomerase.

“…Chromosomal fragments with the ability to complement all three different classes of xyl-negative mutants were cloned on a plasmid. Localization of the genes indicated that the putative xylulose kinase gene resides near the glucose isomerase gene, which is consistent with the organization of the locus in Salmonella typhimurium (73), E. coli (186), and Bacillus subtilis (183) (Fig. 4).…”

“…The first report on the isolation of the GI gene was from E. coli by Ho et al (84). D-Xylose isomerase and xylulokinase activities were amplified by transformation of a GIdeficient E. coli strain with plasmid pMB9 bearing a HindIII restriction fragment of E. coli chromosomal DNA (186). The GI gene from E. coli was sequenced and was shown to code for GI by purification of the cloned gene product (142).…”

Molecular cloning and functional expression of d-arabitol dehydrogenase gene fromGluconobacter oxydansinEscherichia coli

“…This indicated that TG1, DH5a, JM109 and BL21(DE3) strains utilize xylose via the pentose phosphate pathway, whereas HB101 could not. E. coli strains which could utilize xylose also could utilize D-xylulose via xylose isomerase (EC.5.3.1.5) [11]. Considering that strain TG1 grew faster than DH5a, JM109 and BL21 in a minimal medium containing xylose as the sole carbon source (data not shown), we selected TG1 as the host for cloning the ArDH gene from G. oxydans CGMCC 1.110.…”

Hyperexpression of Escherichia coli Xylose Isomerase

“…Wovcha et uf. [21] reported the amplification of xylose isomerase activity by cloning of a 13 kb Hind111 E. coli chromosomal DNA fragment that carried the x y l A gene. E. coli JC1553 (xy17) with cloned x y l A carried on pMB9 exhibited a fivefold increase in xylose isomerase activity over the wild type strain.…”