Sequence Analysis for Metabolic Evolution of Archaeoglobus fulgidus Geranylgeranyl Diphosphate Synthase in Escherichia coli for Carotenoid Biosynthesis
Chia-Wei Wang；Tae H. Han；James C. Liao
Sequence analysis ； Directed evolution ； geranylgeranyl diphosphate ； Escherichia coli ； Carotenoid
Journal of the Chinese Institute of Chemical Engineers
|Volume or Term/Year and Month of Publication||
34卷1期（2003 / 01 / 01）
47 - 56
To investigate possible ways for improving the metabolic output after horizontal gene transfer, we mutated a DNA fragment from the hyperthermophile Archaeoglobus fulgidus containing geranylgeranyl pyrophosphate synthase (GPS), and screened for higher metabolic output in an Escherichia coli strain that expressed the carotenoid biosynthesis gene cluster (crtBIYZW). A total of 16 independent, positive mutants were isolated, sequenced, and analyzed. A binomial statistical analysis identified a high mutation frequency region upstream of the protein coding region, which may be responsible for optimizing gene expression level in the new host. The same analysis also identified two low frequency regions within the protein coding region. These two regions represent the highly conserved regions during evolution and contain the presumed active site domains. Mutations in the protein coding region also showed a trend of amino acid substitutions that increase structural flexibility, a reversal of trend for increasing thermostability. This work provides a rapid and practical method to map important regions for function, and obtained mutants that are useful for biosynthesis of carotenoids.