The
            N
            ‐acetylglucosamine catabolic gene cluster in
            Trichoderma reesei
            is controlled by the Ndt80‐like transcription factor RON1

Polaromonas naphthalenivorans strain CJ2 metabolizes naphthalene via the gentisate pathway and has recently been shown to carry a third copy of gentisate 1,2-dioxygenase (GDO), encoded by nagI3, within a previously uncharacterized naphthalene catabolic gene cluster. The role of this cluster (especially nagI3) in naphthalene metabolism of strain CJ2 was investigated by documenting patterns in regulation, transcription and enzyme activity. Transcriptional analysis of wild-type cells showed the third cluster to be polycistronic and that nagI3 was expressed at a relatively high level. Individual knockout mutants of all three nagI genes were constructed and their influence on both GDO activity and cell growth was evaluated. Of the three knockout strains, CJ2ΔnagI3 showed severely diminished GDO activity and grew slowest on aromatic substrates. These observations are consistent with the hypothesis that nagI3 may prevent toxic intracellular levels of gentisate from accumulating in CJ2 cells. All three nagI genes from strain CJ2 were cloned into Escherichia coli: the nagI2 and nagI3 genes were successfully overexpressed. The subunit mass of the GDOs were ~36–39 kDa, and their structures were deduced to be dimeric. The K m values of NagI2 and NagI3 were 31 and 10 µM, respectively, indicating that the higher affinity of NagI3 for gentisate may protect the wild-type cells from gentisate toxicity. These results provide clues for explaining why the third gene cluster, particularly the nagI3 gene, is important in strain CJ2. The organization of genes in the third gene cluster matched that of clusters in Polaromonas sp. JS666 and Leptothrix cholodnii SP-6. While horizontal gene transfer (HGT) is one hypothesis for explaining this genetic motif, gene duplication within the ancestral lineage is equally valid. The HGT hypothesis was discounted by noting that the nagI3 allele of strain CJ2 did not share high sequence identity with its homologues in Polaromonas sp. JS666 and L. cholodnii SP-6.

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The SLIM1 transcription factor regulates arsenic sensitivity in Arabidopsis thaliana

10.22541/au.160917937.72198173/v1 ◽

2020 ◽

Author(s):

Timothy Jobe ◽

Qi Yu ◽

Felix Hauser ◽

Qingqing Xie ◽

Yuan Meng ◽

...

Keyword(s):

Gene Expression ◽

Transcription Factor ◽

Gene Cluster ◽

Arsenic Exposure ◽

Redox Status ◽

Sulfur Assimilation ◽

Thiol Compounds ◽

Arsenic Tolerance ◽

Arsenic Accumulation ◽

Arsenic Detoxification

The transcriptional regulators of arsenic-induced gene expression remain largely unknown; however, arsenic exposure rapidly depletes cellular glutathione levels increasing demand for thiol compounds from the sulfur assimilation pathway. Thus, sulfur assimilation is tightly linked with arsenic detoxification. To explore the hypothesis that the key transcriptional regulator of sulfur assimilation, SLIM1, is involved in arsenic-induced gene expression, we evaluated the response of slim1 mutants to arsenic treatments. We found that slim1 mutants were sensitive to arsenic in root growth assays. Furthermore, arsenic treatment caused high levels of oxidative stress in the slim1 mutants, and slim1 mutants were impaired in both thiol and sulfate accumulation. We also found enhanced arsenic accumulation in the roots of slim1 mutants. Furthermore, microarray analyses identified genes from a highly co-regulated gene cluster (the O-acetylserine gene cluster), as being significantly upregulated in the slim1-1 mutant background in response to arsenic exposure. The present study identified the SLIM1 transcription factor as an important component in arsenic-induced gene expression and arsenic tolerance. Our results suggest that the severe arsenic sensitivity of the slim1 mutants is a result of both altered redox status as well as mis-regulation of key genes.

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Synthetic control devices for gene regulation in Penicillium chrysogenum

Microbial Cell Factories ◽

10.1186/s12934-019-1253-3 ◽

2019 ◽

Vol 18 (1) ◽

Cited By ~ 1

Author(s):

László Mózsik ◽

Zsófia Büttel ◽

Roel A. L. Bovenberg ◽

Arnold J. M. Driessen ◽

Yvonne Nygård

Keyword(s):

Transcription Factor ◽

Dna Binding ◽

Gene Cluster ◽

Penicillium Chrysogenum ◽

Quinic Acid ◽

Binding Domain ◽

Cell Factory ◽

Dna Binding Domain ◽

Synthetic Control ◽

Control Devices

Abstract Background Orthogonal, synthetic control devices were developed for Penicillium chrysogenum, a model filamentous fungus and industrially relevant cell factory. In the synthetic transcription factor, the QF DNA-binding domain of the transcription factor of the quinic acid gene cluster of Neurospora crassa is fused to the VP16 activation domain. This synthetic transcription factor controls the expression of genes under a synthetic promoter containing quinic acid upstream activating sequence (QUAS) elements, where it binds. A gene cluster may demand an expression tuned individually for each gene, which is a great advantage provided by this system. Results The control devices were characterized with respect to three of their main components: expression of the synthetic transcription factors, upstream activating sequences, and the affinity of the DNA binding domain of the transcription factor to the upstream activating domain. This resulted in synthetic expression devices, with an expression ranging from hardly detectable to a level similar to that of highest expressed native genes. The versatility of the control device was demonstrated by fluorescent reporters and its application was confirmed by synthetically controlling the production of penicillin. Conclusions The characterization of the control devices in microbioreactors, proved to give excellent indications for how the devices function in production strains and conditions. We anticipate that these well-characterized and robustly performing control devices can be widely applied for the production of secondary metabolites and other compounds in filamentous fungi.

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Identification of the Main Regulator Responsible for Synthesis of the Typical Yellow Pigment Produced by Trichoderma reesei

Applied and Environmental Microbiology ◽

10.1128/aem.01408-16 ◽

2016 ◽

Vol 82 (20) ◽

pp. 6247-6257 ◽

Cited By ~ 29

Author(s):

Christian Derntl ◽

Alice Rassinger ◽

Ewald Srebotnik ◽

Robert L. Mach ◽

Astrid R. Mach-Aigner

Keyword(s):

Transcription Factor ◽

Secondary Metabolism ◽

Trichoderma Reesei ◽

Penicillium Chrysogenum ◽

Yellow Pigment ◽

Mass Spectrometry Analysis ◽

Acremonium Chrysogenum ◽

Pigment Formation ◽

Content Type ◽

Cluster A

ABSTRACTThe industrially used ascomyceteTrichoderma reeseisecretes a typical yellow pigment during cultivation, while otherTrichodermaspecies do not. A comparative genomic analysis suggested that a putative secondary metabolism cluster, containing two polyketide-synthase encoding genes, is responsible for the yellow pigment synthesis. This cluster is conserved in a set of rather distantly related fungi, includingAcremonium chrysogenumandPenicillium chrysogenum. In an attempt to silence the cluster inT. reesei, two genes of the cluster encoding transcription factors were individually deleted. For a complete genetic proof-of-function, the genes were reinserted into the genomes of the respective deletion strains. The deletion of the first transcription factor (termed yellow pigment regulator 1 [Ypr1]) resulted in the full abolishment of the yellow pigment formation and the expression of most genes of this cluster. A comparative high-pressure liquid chromatography (HPLC) analysis of supernatants of theypr1deletion and its parent strain suggested the presence of several yellow compounds inT. reeseithat are all derived from the same cluster. A subsequent gas chromatography/mass spectrometry analysis strongly indicated the presence of sorbicillin in the major HPLC peak. The presence of the second transcription factor, termed yellow pigment regulator 2 (Ypr2), reduces the yellow pigment formation and the expression of most cluster genes, including the gene encoding the activator Ypr1.IMPORTANCETrichoderma reeseiis used for industry-scale production of carbohydrate-active enzymes. During growth, it secretes a typical yellow pigment. This is not favorable for industrial enzyme production because it makes the downstream process more complicated and thus increases operating costs. In this study, we demonstrate which regulators influence the synthesis of the yellow pigment. Based on these data, we also provide indication as to which genes are under the control of these regulators and are finally responsible for the biosynthesis of the yellow pigment. These genes are organized in a cluster that is also found in other industrially relevant fungi, such as the two antibiotic producersPenicillium chrysogenumandAcremonium chrysogenum. The targeted manipulation of a secondary metabolism cluster is an important option for any biotechnologically applied microorganism.

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