Hierarchical mutational events compensate for glutamate auxotrophy of a <scp><i>B</i></scp><i>acillus subtilis gltC</i> mutant

Dormeyer, Miriam; Lübke, Anastasia L.; Müller, Peter; Lentes, Sabine; Reuß, Daniel R.; Thürmer, Andrea; Stülke, Jörg; Daniel, Rolf; Brantl, Sabine; Commichau, Fabian M.

doi:10.1111/1758-2229.12531

Cited by 13 publications

(16 citation statements)

References 56 publications

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“…One of these arose by spontaneous gene amplification encompassing gcoAB, a result that further emphasizes the importance of gene amplification. An appreciation for this process continues to grow as evolutionary outcomes are documented in diverse contexts (6,8,42,43). The EASy methodology both accelerates evolution in the laboratory and expands options for phenotypic selection.…”

Section: Discussionmentioning

confidence: 99%

Accelerating pathway evolution by increasing the gene dosage of chromosomal segments

Tumen-Velasquez

Johnson

Ahmed

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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Experimental evolution is a critical tool in many disciplines, including metabolic engineering and synthetic biology. However, current methods rely on the chance occurrence of a key step that can dramatically accelerate evolution in natural systems, namely increased gene dosage. Our studies sought to induce the targeted amplification of chromosomal segments to facilitate rapid evolution. Since increased gene dosage confers novel phenotypes and genetic redundancy, we developed a method, Evolution by Amplification and Synthetic Biology (EASy), to create tandem arrays of chromosomal regions. In Acinetobacter baylyi, EASy was demonstrated on an important bioenergy problem, the catabolism of lignin-derived aromatic compounds. The initial focus on guaiacol (2-methoxyphenol), a common lignin degradation product, led to the discovery of Amycolatopsis genes (gcoAB) encoding a cytochrome P450 enzyme that converts guaiacol to catechol. However, chromosomal integration of gcoAB in Pseudomonas putida or A. baylyi did not enable guaiacol to be used as the sole carbon source despite catechol being a growth substrate. In ∼1,000 generations, EASy yielded alleles that in single chromosomal copy confer growth on guaiacol. Different variants emerged, including fusions between GcoA and CatA (catechol 1,2-dioxygenase). This study illustrates the power of harnessing chromosomal gene amplification to accelerate the evolution of desirable traits.

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Section: Discussionmentioning

confidence: 99%

Accelerating pathway evolution by increasing the gene dosage of chromosomal segments

Tumen-Velasquez

Johnson

Ahmed

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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“…Bypassing essential metabolic pathways is not unusual in bacteria. For example, glutamate and proline auxotrophic mutants of B. subtilis can revert to prototrophy by suppressor mutations or amplification of specific genomic loci ( Zaprasis et al, 2014 ; Dormeyer et al, 2017 ).…”

Section: Discussionmentioning

confidence: 99%

Genetic Adaptation of a Mevalonate Pathway Deficient Mutant in Staphylococcus aureus

et al. 2018

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In this study we addressed the question how a mevalonate (MVA)-auxotrophic Staphylococcus aureusΔmvaS mutant can revert to prototrophy. This mutant couldn’t grow in the absence of MVA. However, after a long lag-phase of 4–6 days the mutant adapted from auxotrophic to prototrophic phenotype. During that time, it acquired two point mutations: One mutation in the coding region of the regulator gene spx, which resulted in an amino acid exchange that decreased Spx function. The other mutation in the upstream-element within the core-promoter of the mevalonolactone lactonase gene drp35. This mutation led to an increased expression of drp35. In repeated experiments the mutations always occurred in spx and drp35 and in the same order. The first detectable mutation appeared in spx and allowed slight growth; the second mutation, in drp35, increased growth further. Phenotypical characterizations of the mutant showed that small amounts of the lipid-carrier undecaprenol are synthesized, despite the lack of mvaS. The growth of the adapted clone, ΔmvaSad, indicates that the mutations reawake a rescue bypass. We think that this bypass enters the MVA pathway at the stage of MVA, because blocking the pathway downstream of MVA led to growth arrest of the mutant. In addition, the lactonase Drp35 is able to convert mevalonolactone to MVA. Summarized, we describe here a mutation-based two-step adaptation process that allows resuscitation of growth of the ΔmvaS mutant.

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“…Therefore, it is likely to assume that the slow growth of the parent strain BP978 (Δ pdxST Δ bshC aprE::pdxH amyE::pdxJ ) can be relieved either by mutations increasing the activity of the P ytoQ promoter or by enhancing the ytoQ gene dosage via gene amplification (see below). Similarly, other studies uncovered that promoter‐up mutations and gene amplifications cause the same phenotypic outcome (Kershner et al ., ; Dormeyer et al ., ).…”

Section: Resultsmentioning

confidence: 97%

A two‐step evolutionary process establishes a non‐native vitamin B6 pathway in Bacillus subtilis

Rosenberg

Yeak

Commichau

2017

Environmental Microbiology

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Summary Pyridoxal 5′‐phosphate (PLP), the most important form of vitamin B6 serves as a cofactor for many proteins. Two alternative pathways for de novo PLP biosynthesis are known: the short deoxy‐xylulose‐5‐phosphate (DXP)‐independent pathway, which is present in the Gram‐positive model bacterium Bacillus subtilis and the longer DXP‐dependent pathway, which has been intensively studied in the Gram‐negative model bacterium Escherichia coli. Previous studies revealed that bacteria contain many promiscuous enzymes causing a so‐called ‘underground metabolism’, which can be important for the evolution of novel pathways. Here, we evaluated the potential of B. subtilis to use a truncated non‐native DXP‐dependent PLP pathway from E. coli for PLP synthesis. Adaptive laboratory evolution experiments revealed that two non‐native enzymes catalysing the last steps of the DXP‐dependent PLP pathway and two genomic alterations are sufficient to allow growth of vitamin B6 auxotrophic bacteria as rapid as the wild type. Thus, the existence of an underground metabolism in B. subtilis facilitates the generation of a pathway for synthesis of PLP using parts of a non‐native vitamin B6 pathway. The introduction of non‐native enzymes into a metabolic network and rewiring of native metabolism could be helpful to generate pathways that might be optimized for producing valuable substances.

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Hierarchical mutational events compensate for glutamate auxotrophy of a Bacillus subtilis gltC mutant

Cited by 13 publications

References 56 publications

Accelerating pathway evolution by increasing the gene dosage of chromosomal segments

Accelerating pathway evolution by increasing the gene dosage of chromosomal segments

Genetic Adaptation of a Mevalonate Pathway Deficient Mutant in Staphylococcus aureus

A two‐step evolutionary process establishes a non‐native vitamin B6 pathway in Bacillus subtilis

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