RESUMEN
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. coli accumulated squalene up to 230 mg/L or 55 mg/g-DCW in flask culture. We also determined that a significant truncation of squalene synthase at the C-terminus retains partial cellular activity. The squalene-producing strain described herein represents a convenient platform for gene discovery and the construction of the pathway toward natural and non-natural hopanoids/steroids.
Asunto(s)
Escherichia coli/metabolismo , Farnesil Difosfato Farnesil Transferasa/genética , Farnesil Difosfato Farnesil Transferasa/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutación/genética , Escualeno/metabolismo , Cromatografía Líquida de Alta Presión , Cianobacterias/enzimología , Cianobacterias/genética , Escherichia coli/genética , Farnesil Difosfato Farnesil Transferasa/química , Humanos , Ácido Mevalónico/metabolismo , Proteínas Mutantes/genética , TemperaturaRESUMEN
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. This was accomplished via high-throughput screening with Pantoea ananatis phytoene desaturase, which can selectively convert dehydrosqualene into yellow carotenoid pigments. Genetic analysis of the resultant mutants revealed various mutations that could effectively convert SQS into a "dehydrosqualene synthase." All of these mutations are clustered around the residues that have been proposed to be important for NADPH binding.
Asunto(s)
Proteínas Bacterianas/genética , Farnesil Difosfato Farnesil Transferasa/genética , Proteínas de Saccharomyces cerevisiae/genética , Escualeno/análogos & derivados , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Proteínas Bacterianas/química , Biocatálisis , Evolución Molecular Dirigida , Farnesil Difosfato Farnesil Transferasa/química , Humanos , Modelos Moleculares , Proteínas de Saccharomyces cerevisiae/química , Escualeno/síntesis químicaRESUMEN
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. Additionally, this finding yields a new method of colorimetric screening for the cellular activity of squalene synthases, which are major targets for cholesterol-lowering drugs.
Asunto(s)
Vías Biosintéticas , Carotenoides/biosíntesis , Farnesil Difosfato Farnesil Transferasa/metabolismo , Proteínas Bacterianas , Carotenoides/química , Carotenoides/genética , Escherichia coli/enzimología , Farnesil Difosfato Farnesil Transferasa/química , Farnesil Difosfato Farnesil Transferasa/genética , Humanos , Oxidorreductasas , Staphylococcus aureus/enzimologíaRESUMEN
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. We also developed a PCR-based method to rapidly and robustly determine the mutation spectrum in response to various mutagenic samples in parallel. This system allows one to not only analyze the mutation specificity of various chemicals, but also to search for novel genetic elements that promote the specific mutation events.