Screening of Bacillus subtilis transposon mutants with altered riboflavin production

Tännler, Simon; Zamboni, Nicola; Király, Csilla; Aymerich, Stéphane; Sauer, Uwe

doi:10.1016/j.ymben.2008.06.002

Cited by 52 publications

(23 citation statements)

References 40 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Overexpression of one of the identified genes, gapB (coding for gluconeogenic glyceraldehyde-3-phosphate dehydrogenase), due to knockout of its regulator ccpN, improved yield in the industrial RF producer. These data clearly show that insertion mutagenesis is a promising approach for the identification of novel genes involved in RF synthesis and their subsequent manipulation (493). Transcriptome analysis of a B. subtilis wild-type strain and the RF-overproducing mutant RH33 suggested that the potential bottleneck in RF overproduction is the low pool of phosphoribosyl pyrophosphate.…”

Section: B Subtilismentioning

confidence: 82%

“…Reduction of maintenance metabolism by knockout of the major cytochrome oxidase that catalyzes the less efficient branch of the respiratory chain significantly increased the yield in an RFoverproducing strain (548). Transposon mutagenesis of B. subtilis showed novel and unexpected genes involved in the regulation of RF synthesis: disruption of the fliP, yjaU, and yloN genes led to a 25 to 35% increase in RF yield, whereas transposon insertion in numerous genes (nearly 30) significantly decreased RF synthesis (493). The effect of many such insertions cannot yet be rationally explained.…”

Section: B Subtilismentioning

confidence: 99%

See 1 more Smart Citation

Genetic Control of Biosynthesis and Transport of Riboflavin and Flavin Nucleotides and Construction of Robust Biotechnological Producers

Abbas

Sibirny

2011

Microbiol Mol Biol Rev

316

272

View full text Add to dashboard Cite

SUMMARY Riboflavin [7,8-dimethyl-10-(1′- d -ribityl)isoalloxazine, vitamin B 2 ] is an obligatory component of human and animal diets, as it serves as the precursor of flavin coenzymes, flavin mononucleotide, and flavin adenine dinucleotide, which are involved in oxidative metabolism and other processes. Commercially produced riboflavin is used in agriculture, medicine, and the food industry. Riboflavin synthesis starts from GTP and ribulose-5-phosphate and proceeds through pyrimidine and pteridine intermediates. Flavin nucleotides are synthesized in two consecutive reactions from riboflavin. Some microorganisms and all animal cells are capable of riboflavin uptake, whereas many microorganisms have distinct systems for riboflavin excretion to the medium. Regulation of riboflavin synthesis in bacteria occurs by repression at the transcriptional level by flavin mononucleotide, which binds to nascent noncoding mRNA and blocks further transcription (named the riboswitch). In flavinogenic molds, riboflavin overproduction starts at the stationary phase and is accompanied by derepression of enzymes involved in riboflavin synthesis, sporulation, and mycelial lysis. In flavinogenic yeasts, transcriptional repression of riboflavin synthesis is exerted by iron ions and not by flavins. The putative transcription factor encoded by SEF1 is somehow involved in this regulation. Most commercial riboflavin is currently produced or was produced earlier by microbial synthesis using special selected strains of Bacillus subtilis , Ashbya gossypii , and Candida famata . Whereas earlier RF overproducers were isolated by classical selection, current producers of riboflavin and flavin nucleotides have been developed using modern approaches of metabolic engineering that involve overexpression of structural and regulatory genes of the RF biosynthetic pathway as well as genes involved in the overproduction of the purine precursor of riboflavin, GTP.

show abstract

Section: B Subtilismentioning

confidence: 82%

Section: B Subtilismentioning

confidence: 99%

Genetic Control of Biosynthesis and Transport of Riboflavin and Flavin Nucleotides and Construction of Robust Biotechnological Producers

Abbas

Sibirny

2011

Microbiol Mol Biol Rev

316

272

View full text Add to dashboard Cite

show abstract

“…Tannler et al (2008) devised a reverse engineering strategy for improving riboflavin production by expression of the gluconeogenic genes gapB and pckA through knockout of their genetic repressor ccpN. 13 C-based flux analysis revealed increased relative flux through the PPP for a ccpN mutant with improved riboflavin yield.…”

Section: Intracellular Metabolites Analysis In Different Genetic Modimentioning

confidence: 99%

“…In order to improve further riboflavin production, obvious additional targets for metabolic engineering have been employed. Examples include the over-expression of key riboflavin biosynthesis gene (ribA) (Humbelin et al, 1999), improvement of ATP formation through co-feeding experiments (Dauner et al, 2002) or reducing maintenance metabolism by metabolic engineering of respiration (Li et al, 2006;Zamboni et al, 2003), screening for improved mutant enzymes of riboflavin biosynthesis (ribA and ribH) (Fischer et al, 2003;Lehmann et al, 2009), enhancing precursor supply by up-regulation of purR controlled genes including the purine pathway genes (purE, purD) and the glycine biosynthesis pathway genes (glyA, folD) (Shi et al, 2009b) or by redirecting carbon flow through the pentose phosphate pathway (PPP) (Duan et al, 2010;Gershanovich et al, 2000;Tannler et al, 2008).…”

Section: Introductionmentioning

confidence: 99%

Enhancement of riboflavin production with Bacillus subtilis by expression and site-directed mutagenesis of zwf and gnd gene from Corynebacterium glutamicum

Wang

Chen

et al. 2011

Bioresource Technology

View full text Add to dashboard Cite

“…In addition, it has been shown that CcpN controls central carbon fluxes in the metabolism of B. subtilis and that the growth defect of CcpN knock-out mutants is caused by ATP dissipation via extensive futile cycling (8). It has been demonstrated that a CcpN knock-out is able to increase the industrial production of riboflavin in B. subtilis by a deregulation of the gapB gene (9). However, nothing is known about the actual repression mechanism of CcpN as yet.…”

mentioning

confidence: 99%

The Transcriptional Repressor CcpN from Bacillus subtilis Uses Different Repression Mechanisms at Different Promoters

Licht

Brantl

2009

Journal of Biological Chemistry

View full text Add to dashboard Cite

CcpN, a transcriptional repressor from Bacillus subtilis that is responsible for the carbon catabolite repression of three genes, has been characterized in detail in the past 4 years. However, nothing is known about the actual repression mechanism as yet. Here, we present a detailed study on how CcpN exerts its repression effect at its three known target promoters of the genes sr1, pckA, and gapB. Using gel shift assays under nonrepressive and repressive conditions, we showed that CcpN and RNA polymerase can bind simultaneously and that CcpN does not prevent RNA polymerase (RNAP) binding to the promoter. Furthermore, we investigated the effect of CcpN on open complex formation and demonstrate that CcpN also does not act at this step of transcription initiation at the sr1 and pckA and presumably at the gapB promoter. Investigation of abortive transcript synthesis revealed that CcpN acts differently at the three promoters: At the sr1 and pckA promoter, promoter clearance is impeded by CcpN, whereas synthesis of abortive transcripts is repressed at the gapB promoter. Eventually, we demonstrated with Far Western blots and co-elution experiments that CcpN is able to interact with the RNAP ␣-subunit, which completes the picture of the requirements for the repressive action of CcpN. On the basis of the presented results, we propose a new working model for CcpN action.CcpN, a transcriptional repressor from Bacillus subtilis, mediates CcpA-independent carbon catabolite repression of at least three genes: sr1, encoding a small RNA and pckA and gapB (1, 2), encoding two gluconeogenic enzymes (3, 4). Since its discovery in 2005, CcpN has been thoroughly investigated. Binding properties and binding motifs were examined, revealing that CcpN possesses two asymmetric binding sites that are bound cooperatively and positioned differently at the three regulated promoters (5): At the sr1 promoter, binding sites are located upstream of the Ϫ35 region and between the Ϫ35 and the Ϫ10 region, while binding sites cover the Ϫ35 as well as the Ϫ10 region at the pckA promoter. One operator at the gapB promoter overlaps the Ϫ10 region, the second one is located around ϩ20. ATP and low pH have been identified as signals required for CcpN-mediated repression (6) and the detailed biophysical properties of CcpN-DNA interaction have been reported (7). In addition, it has been shown that CcpN controls central carbon fluxes in the metabolism of B. subtilis and that the growth defect of CcpN knock-out mutants is caused by ATP dissipation via extensive futile cycling (8). It has been demonstrated that a CcpN knock-out is able to increase the industrial production of riboflavin in B. subtilis by a deregulation of the gapB gene (9). However, nothing is known about the actual repression mechanism of CcpN as yet.Initiation of transcription is a stepwise process (10), beginning with binding of RNA polymerase (RNAP) 2 to the promoter and formation of a loose closed complex, which is then rearranged into a tighter closed complex. This is followed by the ...

show abstract

Screening of Bacillus subtilis transposon mutants with altered riboflavin production

Cited by 52 publications

References 40 publications

Genetic Control of Biosynthesis and Transport of Riboflavin and Flavin Nucleotides and Construction of Robust Biotechnological Producers

Genetic Control of Biosynthesis and Transport of Riboflavin and Flavin Nucleotides and Construction of Robust Biotechnological Producers

Enhancement of riboflavin production with Bacillus subtilis by expression and site-directed mutagenesis of zwf and gnd gene from Corynebacterium glutamicum

The Transcriptional Repressor CcpN from Bacillus subtilis Uses Different Repression Mechanisms at Different Promoters

Contact Info

Product

Resources

About