@misc{SchwedtSchoeneEckertetal., author = {Schwedt, Inge and Sch{\"o}ne, Kerstin and Eckert, Maike and Pizzinato, Manon and Winkler, Laura and Knotkova, Barbora and Richts, Bj{\"o}rn and Hau, Jann-Louis and Steuber, Julia and Mireles, Raul and Noda-Garcia, Lianet and Fritz, G{\"u}nter and Mittelst{\"a}dt, Carolin and Hertel, Robert and Commichau, Fabian M.}, title = {The low mutational flexibility of the EPSP synthase in Bacillus subtilis is due to a higher demand for shikimate pathway intermediates}, series = {Environmental Microbiology}, volume = {25}, journal = {Environmental Microbiology}, number = {12}, issn = {1462-2912}, doi = {10.1111/1462-2920.16518}, pages = {3604 -- 3622}, abstract = {Glyphosate (GS) inhibits the 5-enolpyruvyl-shikimate-3-phosphate (EPSP) synthase that is required for aromatic amino acid, folate and quinone biosynthesis in Bacillus subtilis and Escherichia coli. The inhibition of the EPSP synthase by GS depletes the cell of these metabolites, resulting in cell death. Here, we show that like the laboratory B. subtilis strains also environmental and undomesticated isolates adapt to GS by reducing herbicide uptake. Although B. subtilis possesses a GS-insensitive EPSP synthase, the enzyme is strongly inhibited by GS in the native environment. Moreover, the B. subtilis EPSP synthase mutant was only viable in rich medium containing menaquinone, indicating that the bacteria require a catalytically efficient EPSP synthase under nutrient-poor conditions. The dependency of B. subtilis on the EPSP synthase probably limits its evolvability. In contrast, E. coli rapidly acquires GS resistance by target modification. However, the evolution of a GS-resistant EPSP synthase under non-selective growth conditions indicates that GS resistance causes fitness costs. Therefore, in both model organisms, the proper function of the EPSP synthase is critical for the cellular viability. This study also revealed that the uptake systems for folate precursors, phenylalanine and tyrosine need to be identified and characterized in B. subtilis.}, language = {en} } @misc{RichtsCommichau, author = {Richts, Bj{\"o}rn and Commichau, Fabian M.}, title = {Underground metabolism facilitates the evolution of novel pathways for vitamin B6 biosynthesis}, series = {Applied Microbiology and Biotechnology}, volume = {105}, journal = {Applied Microbiology and Biotechnology}, number = {6}, issn = {1432-0614}, doi = {10.1007/s00253-021-11199-w}, pages = {2297 -- 2305}, abstract = {The term vitamin B6 is a designation for the vitamers pyridoxal, pyridoxamine, pyridoxine and the respective phosphate esters pyridoxal-5′-phosphate (PLP), pyridoxamine-5′-phosphate and pyridoxine-5′-phosphate. Animals and humans are unable to synthesise vitamin B6. These organisms have to take up vitamin B6 with their diet. Therefore, vitamin B6 is of commercial interest as a food additive and for applications in the pharmaceutical industry. As yet, two naturally occurring routes for de novo synthesis of PLP are known. Both routes have been genetically engineered to obtain bacteria overproducing vitamin B6. Still, major genetic engineering efforts using the existing pathways are required for developing fermentation processes that could outcompete the chemical synthesis of vitamin B6. Recent suppressor screens using mutants of the Gram-negative and Gram-positive model bacteria Escherichia coli and Bacillus subtilis, respectively, carrying mutations in the native pathways or heterologous genes uncovered novel routes for PLP biosynthesis. These pathways consist of promiscuous enzymes and enzymes that are already involved in vitamin B6 biosynthesis. Thus, E. coli and B. subtilis contain multiple promiscuous enzymes causing a so-called underground metabolism allowing the bacteria to bypass disrupted vitamin B6 biosynthetic pathways. The suppressor screens also show the genomic plasticity of the bacteria to suppress a genetic lesion. We discuss the potential of the serendipitous pathways to serve as a starting point for the development of bacteria overproducing vitamin B6.}, language = {en} } @misc{RichtsLentesPoehleinetal., author = {Richts, Bj{\"o}rn and Lentes, Sabine and Poehlein, Anja and Daniel, Rolf and Commichau, Fabian M.}, title = {A Bacillus subtilis ΔpdxT mutant suppresses vitamin B6 limitation by acquiring mutations enhancing pdxS gene dosage and ammonium assimilation}, series = {Environmental Microbiology Reports}, volume = {13}, journal = {Environmental Microbiology Reports}, number = {2}, issn = {1758-2229}, doi = {10.1111/1758-2229.12936}, pages = {218 -- 233}, abstract = {Pyridoxal-5'-phosphate (PLP), the biologically active form of vitamin B6, serves as a cofactor for many enzymes. The Gram-positive model bacterium Bacillus subtilis synthesizes PLP via the PdxST enzyme complex, consisting of the PdxT glutaminase and the PdxS PLP synthase subunits, respectively. PdxT converts glutamine to glutamate and ammonia of which the latter is channelled to PdxS. At high extracellular ammonium concentrations, the PdxS PLP synthase subunit does not depend on PdxT. Here, we assessed the potential of a B. subtilis ΔpdxT mutant to adapt to PLP limitation at the genome level. The majority of ΔpdxT suppressors had amplified a genomic region containing the pdxS gene. We also identified mutants having acquired as yet undescribed mutations in ammonium assimilation genes, indicating that the overproduction of PdxS and the NrgA ammonium transporter partially relieve vitamin B6 limitation in a ΔpdxT mutant when extracellular ammonium is scarce. Furthermore, we found that PdxS positively affects complex colony formation in B. subtilis. The catalytic mechanism of the PdxS PLP synthase subunit could be the reason for the limited evolution of the enzyme and why we could not identify a PdxS variant producing PLP independently of PdxT at low ammonium concentrations.}, language = {en} } @misc{MichalikRederRichtsetal., author = {Michalik, Stephan and Reder, Alexander and Richts, Bj{\"o}rn and Faßhauer, Patrick and M{\"a}der, Ulrike and Pedreira, Tiago and Poehlein, Anja and Heel, Auke van and Tilburg, Amanda van and Altenbuchner, Josef and Klewing, Anika and Reuß, Daniel and Daniel, Rolf and Commichau, Fabian M. and Kuipers, Oscar and Hamoen, Leendert and V{\"o}lker, Uwe and St{\"u}lke, J{\"o}rg}, title = {The Bacillus subtilis Minimal Genome Compendium}, series = {ACS Synthetic Biology}, volume = {10}, journal = {ACS Synthetic Biology}, number = {10}, issn = {2161-5063}, doi = {10.1021/acssynbio.1c00339}, pages = {2767 -- 2771}, abstract = {To better understand cellular life, it is essential to decipher the contribution of individual components and their interactions. Minimal genomes are an important tool to investigate these interactions. Here, we provide a database of 105 fully annotated genomes of a series of strains with sequential deletion steps of the industrially relevant model bacterium Bacillus subtilis starting with the laboratory wild type strain B. subtilis 168 and ending with B. subtilis PG38, which lacks approximately 40\% of the original genome. The annotation is supported by sequencing of key intermediate strains as well as integration of literature knowledge for the annotation of the deletion scars and their potential effects. The strain compendium presented here represents a comprehensive genome library of the entire MiniBacillus project. This resource will facilitate the more effective application of the different strains in basic science as well as in biotechnology.}, language = {en} } @misc{RichtsHertelPototetal., author = {Richts, Bj{\"o}rn and Hertel, Robert and Potot, S{\´e}bastien and Poehlein, Anja and Daniel, Rolf and Schyns, Ghislain and Pr{\´a}gai, Zolt{\´a}n and Commichau, Fabian M.}, title = {Complete Genome Sequence of the Prototrophic Bacillus subtilis subsp. subtilis Strain SP1}, series = {Microbiology Resource Announcements}, volume = {9}, journal = {Microbiology Resource Announcements}, number = {32}, issn = {2576-098X}, doi = {10.1128/MRA.00825-20}, pages = {3}, abstract = {Here, we present the complete genome sequence of the Bacillus subtilis strain SP1. This strain is a descendant of the laboratory strain 168. The strain is suit- able for biotechnological applications because the prototrophy for tryptophan has been restored. Due to laboratory cultivation, the strain has acquired 24 additional sequence variations.}, language = {en} } @incollection{RosenbergRichtsCommichau, author = {Rosenberg, Jonathan and Richts, Bj{\"o}rn and Commichau, Fabian M.}, title = {Fermentative Production of Vitamin B6}, series = {Pharmaceutical Biocatalysis : Drugs, Genetic Diseases, and Epigenetics}, booktitle = {Pharmaceutical Biocatalysis : Drugs, Genetic Diseases, and Epigenetics}, editor = {Grunwald, Peter}, publisher = {Jenny Stanford publishing}, address = {Singapur}, isbn = {978-981-4877-14-5}, abstract = {Volume 7 of the Jenny Stanford Series on Biocatalysis deals with several different aspects of pharmaceuticals, which include not only various applications of drugs and their metabolism but also natural resources for active pharmaceutical ingredients as well as the removal of pharmaceutical pollution. In detail, novel approaches for developing microbial fermentation processes to produce vitamin B6 using microorganisms are described together with novel routes for vitamin B6 biosynthesis. The other topics discussed are new approaches for producing the successful anticancer drug Taxol from naturally occurring precursors, molecular farming through plant engineering as a cost-effective means to produce therapeutic and prophylactic proteins, and successful screening of potent microorganisms producing L-asparaginase for various chemotherapeutic applications. Furthermore, microbial biotransformations in the production and degradation of fluorinated pharmaceuticals are described. The other chapters inform the reader about the biotransformation of xenobiotics/drugs in living systems, the degradation of pharmaceuticals by white-rot fungi and their ligninolytic enzymes, and the removal of pharmaceutical pollution from municipal sewage using laccase}, language = {en} }