Enzyme evolution in Rhodobacter sphaeroides: selection of a mutant expressing a new galactitol dehydrogenase and biochemical characterization of the enzyme

Abstract

A gain of function mutant of Rhodobacter sphaeroides Si4, capable of growing on galactitol, was isolated from a chemostat culture. Continuous cultivation was performed for 54 d with a limiting concentration (1 mM) of the substrate D-glucitol and an excess (20 mM) of the non-metabolizable galactitol. The mutant strain, R. sphaeroides D, grew in galactitol minimal medium with a growth rate of 0-11 h-1 (t d = 6-3 h). In crude extracts of R. sphaeroides D, a specific galactitol dehydrogenase (GDH) activity of 380 mU mg-1 was found, while the wild-type strain exhibited GDH activities lower than 50 mU mg-1 when grown on different polyols. Unlike mannitol, sorbitol or ribitol dehydrogenase from the wild-type strain, the new GDH was expressed constitutively. To study whether it was a newly evolved enzyme or an improved side activity of one of the pre-existing polyol dehydrogenases, GDH was purified to apparent homogeneity by ammonium sulfate precipitation and chromatography on Phenyl-Sepharose, Q-Sepharose, Matrex Gel Red-A and Mono-Q. The relative molecular mass (M r) of the native GDH was 110000. SDS-PAGE resulted in one single band that represented a polypeptide with a M r of 28000, indicating that the native protein is a tetramer. The isoelectric point of GDH was determined to be pH 4-2. The enzyme was specific for NAD+ but catalysed the oxidation of different sugar alcohols as well as different diols and secondary alcohols. The apparent K m values were: galactitol, 240 mM; D-threitol, 85 mM; 1,2-hexandiol, 0-2 mM; NAD+, 12μM; L-erythrulose, 144 mM; acetoin, 62 mM; dihydroxyacetone, 48 mM; and NADH, 4μM. GDH activity was strictly dependent on the presence of divalent cations. The properties of GDH are different to any of the three polyol dehydrogenases from R. sphaeroides Si4. In addition, comparison of the N-terminal amino acid sequence of the isolated GDH with the N-terminal sequence of the other three polyol dehydrogenases clearly demonstrates that GDH is an additional enzyme, so far unrecognized in the wild-type strain.

Prolonged Maltose-Limited Cultivation of Saccharomyces cerevisiae Selects for Cells with Improved Maltose Affinity and Hypersensitivity

“…The term evolutionary engineering has recently been coined to describe the selection of microbial strains with specific characteristics via selection under rationally designed culture conditions (18,20,31,40,53,55). The use of chemostat cultures as "evolutionary devices" for selection of mutant microorganisms was already described in the 1950s and has since been successfully applied to improve numerous physiological traits (42,64).…”

Polyol metabolism of Rhodobacter sphaeroides: biochemical characterization of a short-chain sorbitol dehydrogenase

“…S23835). Although only a relatively short amino acid sequence of the R. sphaeroides SDH is known, the comparative N-terminal amino acid sequences of SDH and MDH show a significant identity between the enzymes (Schneider et al, 1995), and it will be the subject of further studies to elucidate whether SDH and MDH are evolutionarily related. There is also a high degree of homology between the N-terminal parts of SDH and those of other short-chain dehydrogenases.…”

“…Since it is intended to use R. spbaeroides Si4 as a model for experimental enzyme evolution of polyol dehydrogenases with biotechnomannitol, D-glucitol (sorbitol) and D-arabitol, when logical significance (Schneider et a!., 1995), the origin of grown in the presence of any of these substrates the SDH activity in the mutant M22 has to be elucidated. (Schneider & G i h o r n , 1989).…”

Chemostat Approach for the Directed Evolution of Biodesulfurization Gain-of-Function Mutants

“…Chemostats have been designed to select for mutants that produce enzymes with increased activity rates (10,32,33) or with altered substrate specificity (29). The latter type have been termed gain-of-function mutants.…”