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Evolution of aromatic ?-glucoside utilization by successive mutational steps in Escherichia coli.

ABSTRACT: The bglA gene of Escherichia coli encodes phospho-?-glucosidase A capable of hydrolyzing the plant-derived aromatic ?-glucoside arbutin. We report that the sequential accumulation of mutations in bglA can confer the ability to hydrolyze the related aromatic ?-glucosides esculin and salicin in two steps. In the first step, esculin hydrolysis is achieved through the acquisition of a four-nucleotide insertion within the promoter of the bglA gene, resulting in enhanced steady-state levels of the bglA transcript. In the second step, hydrolysis of salicin is achieved through the acquisition of a point mutation within the bglA structural gene close to the active site without the loss of the original catabolic activity against arbutin. These studies underscore the ability of microorganisms to evolve additional metabolic capabilities by mutational modification of preexisting genetic systems under selection pressure, thereby expanding their repertoire of utilizable substrates.

SUBMITTER: Zangoui P

PROVIDER: S-EPMC4334193 | biostudies-literature | 2015 Feb

REPOSITORIES: biostudies-literature

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Evolution of aromatic β-glucoside utilization by successive mutational steps in Escherichia coli.

Zangoui Parisa P Vashishtha Kartika K Mahadevan Subramony S

Journal of bacteriology 20141201 4

The bglA gene of Escherichia coli encodes phospho-β-glucosidase A capable of hydrolyzing the plant-derived aromatic β-glucoside arbutin. We report that the sequential accumulation of mutations in bglA can confer the ability to hydrolyze the related aromatic β-glucosides esculin and salicin in two steps. In the first step, esculin hydrolysis is achieved through the acquisition of a four-nucleotide insertion within the promoter of the bglA gene, resulting in enhanced steady-state levels of the bgl ...[more]

PMID: 25448815

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Project description:BackgroundMulti-monocistronic and multi-variate vectors were designed, built, and tested for the improved production of cyanidin 3-O-glucoside (C3G) in Escherichia coli BL21 (DE3). The synthetic bio-parts were designed in such a way that multiple genes can be assembled using the bio-brick system, and expressed under different promoters in a single vector. The vectors harbor compatible cloning sites, so that the genes can be shuffled from one vector to another in a single step, and assembled into a single vector. The two required genes: anthocyanidin synthase (PhANS) from Petunia hybrida, and cyanidin 3-O-glucosyltransferase (At3GT) from Arabidopsis thaliana, were individually cloned under PT7, Ptrc, and PlacUV5 promoters. Both PhANS and At3GT were shuffled back and forth, so as to generate a combinatorial system for C3G production. The constructed systems were further coupled with the genes for UDP-D-glucose synthesis, all cloned in a multi-monocistronic fashion under PT7. Finally, the production of C3G was checked and confirmed using the modified M9 media, and analyzed through various chromatography and spectrometric analyses.ResultsThe engineered strains endowed with newly generated vectors and the genes for C3G biosynthesis and UDP-D-glucose synthesis were fed with 2 mM (+)-catechin and D-glucose for the production of cyanidin, and its subsequent conversion to C3G. One of the engineered strains harboring At3GT and PhANS under Ptrc promoter and UDP-D-glucose biosynthesis genes under PT7 promoter led to the production of ~?439 mg/L of C3G within 36 h of incubation, when the system was exogenously fed with 5% (w/v) D-glucose. This system did not require exogenous supplementation of UDP-D-glucose.ConclusionA synthetic vector system using different promoters has been developed and used for the synthesis of C3G in E. coli BL21 (DE3) by directing the metabolic flux towards the UDP-D-glucose. This system has the potential of generating better strains for the synthesis of valuable natural products.

Project description:The isolation of aerobic citrate-utilizing Escherichia coli (Cit(+)) in long-term evolution experiments (LTEE) has been termed a rare, innovative, presumptive speciation event. We hypothesized that direct selection would rapidly yield the same class of E. coli Cit(+) mutants and follow the same genetic trajectory: potentiation, actualization, and refinement. This hypothesis was tested with wild-type E. coli strain B and with K-12 and three K-12 derivatives: an E. coli ?rpoS::kan mutant (impaired for stationary-phase survival), an E. coli ?citT::kan mutant (deleted for the anaerobic citrate/succinate antiporter), and an E. coli ?dctA::kan mutant (deleted for the aerobic succinate transporter). E. coli underwent adaptation to aerobic citrate metabolism that was readily and repeatedly achieved using minimal medium supplemented with citrate (M9C), M9C with 0.005% glycerol, or M9C with 0.0025% glucose. Forty-six independent E. coli Cit(+) mutants were isolated from all E. coli derivatives except the E. coli ?citT::kan mutant. Potentiation/actualization mutations occurred within as few as 12 generations, and refinement mutations occurred within 100 generations. Citrate utilization was confirmed using Simmons, Christensen, and LeMaster Richards citrate media and quantified by mass spectrometry. E. coli Cit(+) mutants grew in clumps and in long incompletely divided chains, a phenotype that was reversible in rich media. Genomic DNA sequencing of four E. coli Cit(+) mutants revealed the required sequence of mutational events leading to a refined Cit(+) mutant. These events showed amplified citT and dctA loci followed by DNA rearrangements consistent with promoter capture events for citT. These mutations were equivalent to the amplification and promoter capture CitT-activating mutations identified in the LTEE.IMPORTANCE E. coli cannot use citrate aerobically. Long-term evolution experiments (LTEE) performed by Blount et al. (Z. D. Blount, J. E. Barrick, C. J. Davidson, and R. E. Lenski, Nature 489:513-518, 2012, http://dx.doi.org/10.1038/nature11514 ) found a single aerobic, citrate-utilizing E. coli strain after 33,000 generations (15 years). This was interpreted as a speciation event. Here we show why it probably was not a speciation event. Using similar media, 46 independent citrate-utilizing mutants were isolated in as few as 12 to 100 generations. Genomic DNA sequencing revealed an amplification of the citT and dctA loci and DNA rearrangements to capture a promoter to express CitT, aerobically. These are members of the same class of mutations identified by the LTEE. We conclude that the rarity of the LTEE mutant was an artifact of the experimental conditions and not a unique evolutionary event. No new genetic information (novel gene function) evolved.

Dataset Information

Evolution of aromatic ?-glucoside utilization by successive mutational steps in Escherichia coli.

Publications

Evolution of aromatic β-glucoside utilization by successive mutational steps in Escherichia coli.

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