Production of squalene by squalene synthases and their truncated mutants in Escherichia coli.

Squalene is a precursor of thousands of bioactive triterpenoids and also has industrial value as a lubricant, health-promoting agent, and/or drop-in biofuel. To establish an efficient Escherichia coli-based system for squalene production, we tested two different squalene synthases and their mutants in combination with precursor pathways. By co-expressing a chimeric mevalonate pathway with human or Thermosynechococcus squalene synthase, E.

Directed evolution of squalene synthase for dehydrosqualene biosynthesis.

Squalene synthase (SQS) catalyzes the first step of sterol/hopanoid biosynthesis in various organisms. It has been long recognized that SQSs share a common ancestor with carotenoid synthases, but it is not known how these enzymes selectively produce their own product. In this study, SQSs from yeast, human, and bacteria were independently subjected to directed evolution for the production of the C30 carotenoid backbone, dehydrosqualene.

Construction of carotenoid biosynthetic pathways using squalene synthase.

The first committed steps of steroid/hopanoid pathways involve squalene synthase (SQS). Here, we report the Escherichia coli production of diaponeurosporene and diapolycopene, yellow C30 carotenoid pigments, by expressing human SQS and Staphylococcus aureus dehydrosqualene (C30 carotenoid) desaturase (CrtN). We suggest that the carotenoid pigments are synthesized mainly via the desaturation of squalene rather than the direct synthesis of dehydrosqualene through the non-reductive condensation of prenyl diphosphate precursors, indicating the possible existence of a "squalene route" and a "lycopersene route" for C30 and C40 carotenoids, respectively.

A system for the rapid determination of the mutation spectrum in Escherichia coli.

The first step toward elucidating the mutagenic effects of chemicals and pathways is to determine the specificity of the mutations generated spontaneously or in response to treatment with mutagens. We constructed a set of plasmid-encoded probes for the specific detection of each type of base substitution mutation. Using these probes, we were able to quickly determine both the mutation rate and the specificity of the mutations caused by different types of mutagens and mutagenic conditions.