Cloning, nucleotide sequence, and overexpression of smoS, a component of a novel operon encoding an ABC transporter and polyol dehydrogenases of Rhodobacter sphaeroides Si4.

Abstract:

:The gene coding for sorbitol dehydrogenase (SDH) of Rhodobacter sphaeroides Si4 was located 55 nucleotides upstream of the mannitol dehydrogenase gene (mtlK) within a previously unrecognized polyol operon. This operon probably consists of all the proteins necessary for transport and metabolization of various polyols. The gene encoding SDH (smoS) was cloned and sequenced. Analysis of the deduced amino acid sequence revealed homology to enzymes of the short-chain dehydrogenase/reductase protein family. For structure analysis of this unique bacterial enzyme, smoS was subcloned into the overexpression vector pET-24a(+) and then overproduced in Escherichia coli BL21(DE3), which yielded a specific activity of 24.8 U/mg of protein and a volumetric yield of 38,000 U/liter. Compared to values derived with the native host, R. sphaeroides, these values reflected a 270-fold increase in expression of SDH and a 971-fold increase in the volumetric yield. SDH was purified to homogeneity, with a recovery of 49%, on the basis of a three-step procedure. Upstream from smoS, another gene (smoK), which encoded a putative ATP-binding protein of an ABC transporter, was identified.

journal_name

J Bacteriol

journal_title

Journal of bacteriology

authors

Stein MA,Schäfer A,Giffhorn F

doi

10.1128/jb.179.20.6335-6340.1997

subject

Has Abstract

pub_date

1997-10-01 00:00:00

pages

6335-40

issue

20

eissn

0021-9193

issn

1098-5530

journal_volume

179

pub_type

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