RESUMO
The cellular activities of gymnosperms monoterpene synthases are largely compromised due to their requirement for manganese, which is deficient in microbial cells. Through site-saturation mutagenesis of the residue adjacent to metal-binding glutamate, we found that pinene synthase is highly mutable at this position yet drastically alter their metal binding preference, thereby quickly improving the cellular performance in heterologous hosts.
Assuntos
Liases Intramoleculares/metabolismo , Cloretos/química , Liases Intramoleculares/genética , Compostos de Manganês/química , Mutagênese , Engenharia de ProteínasRESUMO
To achieve an efficient production of geraniol and its derivatives in Escherichia coli, we aimed to improve the activity of geraniol synthase (GES) through a single round of mutagenesis and screening for higher substrate consumption. We isolated GES variants that outperform their parent in geraniol production. The analysis of GES variants indicated that the expression level of GES was the bottleneck for geraniol synthesis. Over-expression of the mutant GESM53 with a 5'-untranslated sequence designed for high translational efficiency, along with the additional expression of mevalonate pathway enzymes, isopentenyl pyrophosphate isomerase, and geranyl pyrophosphate synthase, yielded 300 mg/L/12 h geraniol and its derivatives (>1000 mg/L/42 h in total) in a shaking flask.
Assuntos
Evolução Molecular Direcionada , Proteínas de Escherichia coli , Escherichia coli/enzimologia , Escherichia coli/genética , Geraniltranstransferase , Terpenos/metabolismo , Monoterpenos Acíclicos , Isomerases de Ligação Dupla Carbono-Carbono/genética , Carotenoides/metabolismo , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/biossíntese , Proteínas de Escherichia coli/genética , Geraniltranstransferase/biossíntese , Geraniltranstransferase/genética , Hemiterpenos , Ensaios de Triagem em Larga Escala , Engenharia Metabólica , Mutagênese , Iniciação Traducional da Cadeia Peptídica/genética , Proteínas de Plantas/biossíntese , Proteínas de Plantas/genética , Fosfatos de Poli-Isoprenil , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Especificidade por SubstratoRESUMO
Successful feeding of the substrate geranylpyrophosphate (GPP) to monoterpene synthase is critical to the efficient microbial production of monoterpenes. Overexpression of GPP synthases, metabolic channeling from GPP synthase to terpene synthases, and down-tuning of endogenous competitors have been successfully used to increase the production of monoterpene. Nevertheless, the production of monoterpenes has remained considerably lower than that of hemi-/sesqui-terpenoids. We tested whether it is effective to improve the cellular activity of monoterpene synthases. To this end, we developed a high-throughput screening system to monitor for elevated GPP consumption. Through a single round of mutagenesis and screening, we isolated a pinene synthase variant that outperformed the wild-type (parent) enzyme in multiple contexts in Escherichia coli and cyanobacteria. The purified variant exhibited drastically altered metal dependency, enabling to keep the activity in the cytosol that is manganese-deficient. Coexpression of this variant with mevalonate pathway enzymes, isopentenylpyrophosphate isomerase, and GPP synthase yielded 140 mg/L pinene in a flask culture.
