Global regulator IrrE on stress tolerance: a review.

Stress tolerance is a vital attribute for all living beings to cope with environmental adversities. IrrE (also named PprI) from Deinococcus radiodurans enhances resistance to extreme radiation stress by functioning as a global regulator, mediating the transcription of genes involved in deoxyribonucleic acid (DNA) damage response (DDR). The expression of IrrE augmented the resilience of various species to heat, radiation, oxidation, osmotic stresses and inhibitors, encompassing bacterial, fungal, plant, and mammalian cells. Moreover, IrrE was employed in a global regulator engineering strategy to broaden its applications in stress tolerance. The regulatory impacts of heterologously expressed IrrE have been investigated at the molecular and systems level, including the regulation of genes, proteins, modules, or pathways involved in DNA repair, detoxification proteins, protective molecules, native regulators and other aspects. In this review, we discuss the regulatory role and mechanism of IrrE in the antiradiation response of D. radiodurans. Furthermore, the applications and regulatory effects of heterologous expression of IrrE to enhance abiotic stress tolerance are summarized in particular.

A new peucemycin derivative and impacts of peuR and bldA on peucemycin biosynthesis in Streptomyces peucetius.

Streptomyces peucetius ATCC 27952 is known to produce a variety of secondary metabolites, including two important antitumor anthracyclines: daunorubicin and doxorubicin. Identification of peucemycin and 25-hydroxy peucemycin (peucemycin A), as well as their biosynthetic pathway, has expanded its biosynthetic potential. In this study, we isolated a new peucemycin derivative and identified it as 19-hydroxy peucemycin (peucemycin B). Its antibacterial activity was lower than those of peucemycin and peucemycin A. On the other hand, this newly identified peucemycin derivative had higher anticancer activity than the other two compounds for MKN45, NCI-H1650, and MDA-MB-231 cancer cell lines with IC50 values of 76.97 µM, 99.68 µM, and 135.2 µM, respectively. Peucemycin biosynthetic gene cluster revealed the presence of a SARP regulator named PeuR whose role was unknown. The presence of the TTA codon in the peuR and the absence of global regulator BldA in S. peucetius reduced its ability to regulate the peucemycin biosynthetic gene cluster. Hence, different mutants harboring these genes were prepared. S. peucetius bldA25 harboring bldA produced 1.75 times and 1.77 times more peucemycin A (11.8 mg/L) and peucemycin B (21.2 mg/L), respectively, than the wild type. On the other hand, S. peucetius R25 harboring peuR produced 1.86 and 1.79 times more peucemycin A (12.5 mg/L) and peucemycin B (21.5 mg/L), respectively, than the wild type. Finally, strain S. peucetius bldAR25 carrying bldA and peuR produced roughly 3.52 and 2.63 times more peucemycin A (23.8 mg/L) and peucemycin B (31.5 mg/L), respectively, than the wild type. KEY POINTS: • This study identifies a new peucemycin derivative, 19-hydroxy peucemycin (peucemycin B). • The SARP regulator (PeuR) acts as a positive regulator of the peucemycin biosynthetic gene cluster. • The overexpression of peuR and heterologous expression of bldA increase the production of peucemycin derivatives.

AaLaeA targets AaFla1 to mediate the production of antitumor compound in Alternaria alstroemeria.

Endophytic fungi are an important source of novel antitumor substances. Previously, we isolated an endophytic fungus, Alternaria alstroemeria, from the medicinal plant Artemisia artemisia, whose crude extracts strongly inhibited A549 tumor cells. We obtained a transformant, namely AaLaeAOE26 , which completely loses its antitumor activity due to overexpression of the global regulator AaLaeA. Re-sequencing analysis of the genome revealed that the insertion site was in the noncoding region and did not destroy any other genes. Metabolomics analysis revealed that the level of secondary antitumor metabolic substances was significantly lower in AaLaeAOE26 compared with the wild strain, in particular flavonoids were more downregulated according to the metabolomics analysis. A further comparative transcriptome analysis revealed that a gene encoding FAD-binding domain protein (Fla1) was significantly downregulated. On the other hand, overexpression of AaFla1 led to significant enhancement of antitumor activity against A549 with a sevenfold higher inhibition ratio than the wild strain. At the same time, we also found a significant increase in the accumulation of antitumor metabolites including quercetin, gitogenin, rhodioloside, liensinine, ginsenoside Rg2 and cinobufagin. Our data suggest that the global regulator AaLaeA negatively affects the production of antitumor compounds via controlling the transcription of AaFla1 in endophytic A. alstroemeria.

A Rare Mono-Rhamnolipid Congener Efficiently Produced by Recombinant Pseudomonas aeruginosa YM4 via the Expression of Global Transcriptional Regulator irrE.

Rhamnolipids (RLs) are widely used biosurfactants produced mainly by Pseudomonas aeruginosa and Burkholderia spp. in the form of mixtures of diverse congeners. The global transcriptional regulator gene irrE from radiation-tolerant extremophiles has been widely used as a stress-resistant element to construct robust producer strains and improve their production performance. A PrhlA-irrE cassette was constructed to express irrE genes in the Pseudomonas aeruginosa YM4 of the rhamnolipids producer strain. We found that the expression of irrE of Deinococcus radiodurans in the YM4 strain not only enhanced rhamnolipid production and the strain's tolerance to environmental stresses, but also changed the composition of the rhamnolipid products. The synthesized rhamnolipids reached a maximum titer of 26 g/L, about 17.9% higher than the original, at 48 h. The rhamnolipid production of the recombinant strain was determined to be mono-rhamnolipids congener Rha-C10-C12, accounting for 94.1% of total products. The critical micelle concentration (CMC) value of the Rha-C10-C12 products was 62.5 mg/L and the air-water surface tension decreased to 25.5 mN/m. The Rha-C10-C12 products showed better emulsifying activity on diesel oil than the original products. This is the first report on the efficient production of the rare mono-rhamnolipids congener Rha-C10-C12 and the first report that the global regulator irrE can change the components of rhamnolipid products in Pseudomonas aeruginosa.

Insights into the complementation potential of the extreme acidophile's orthologue in replacing Escherichia coli hfq gene-particularly in bacterial resistance to environmental stress.

Acidithiobacillus caldus is a typical extreme acidophile widely used in the biohydrometallurgical industry, which often experiences extreme environmental stress in its natural habitat. Hfq, an RNA-binding protein, typically functions as a global regulator involved in various cellular physiological processes. Yet, the biological functions of Hfq derived from such extreme acidophile have not been extensively investigated. In this study, the recombinant strain Δhfq/Achfq, constructed by CRISPR/Cas9-mediated chromosome integration, fully or partially restored the phenotypic defects caused by hfq deletion in Escherichia coli, including impaired growth performance, abnormal cell morphology, impaired swarming motility, decreased stress resistance, decreased intracellular ATP and free amino acid levels, and attenuated biofilm formation. Particularly noteworthy, the intracellular ATP level and biofilm production of the recombinant strain were increased by 12.2% and 7.0%, respectively, compared to the Δhfq mutant. Transcriptomic analysis revealed that even under heterologous expression, AcHfq exerted global regulatory effects on multiple cellular processes, including metabolism, environmental signal processing, and motility. Finally, we established a potential working model to illustrate the regulatory mechanism of AcHfq in bacterial resistance to environmental stress.

The leucine-responsive regulatory proteins/feast-famine regulatory proteins: an ancient and complex class of transcriptional regulators in bacteria and archaea.

Since the discovery of the Escherichia coli leucine-responsive regulatory protein (Lrp) almost 50 years ago, hundreds of Lrp homologs have been discovered, occurring in 45% of sequenced bacteria and almost all sequenced archaea. Lrp-like proteins are often referred to as the feast/famine regulatory proteins (FFRPs), reflecting their common regulatory roles. Acting as either global or local transcriptional regulators, FFRPs detect the environmental nutritional status by sensing small effector molecules (usually amino acids) and regulate the expression of genes involved in metabolism, virulence, motility, nutrient transport, stress tolerance, and antibiotic resistance to implement appropriate behaviors for the specific ecological niche of each organism. Despite FFRPs' complexity, a significant role in gene regulation, and prevalence throughout prokaryotes, the last comprehensive review on this family of proteins was published about a decade ago. In this review, we integrate recent notable findings regarding E. coli Lrp and other FFRPs across bacteria and archaea with previous observations to synthesize a more complete view on the mechanistic details and biological roles of this ancient class of transcription factors.

Overexpression of Global Regulator SCrp Leads to the Discovery of New Angucyclines in Streptomyces sp. XS-16.

Six angucyclines including three unreported compounds (1-3) were isolated from Streptomyces sp. XS-16 by overexpressing the native global regulator of SCrp (cyclic AMP receptor). The structures were characterized based on nuclear magnetic resonance (NMR) and spectrometry analysis and assisted by electronic circular dichroism (ECD) calculations. All compounds were tested for their antitumor and antimicrobial activities, and compound 1 showed different inhibitory activities against various tumor cell lines with IC50 values ranging from 0.32 to 5.33 µM.

Overexpression of the global regulator FnVeA upregulates antitumor substances in endophytic Fusarium nematophilum.

The special niche of endophytic fungi promotes their potential to produce antitumor compounds with novel structure and significant bioactivity for screening of new antitumor drugs. In our previous studies, we isolated a Fusarium strain from the roots of the medicinal plant Nothapodytes pittosporoides and identified it as Fusarium nematophilum. We found that the crude extract of F. nematophilum had significant antitumor activity on A549 cancer cells, and overexpressing the global regulatory factor FnVeA (the VeA gene of the fungus F. nematophilum) resulted in a significant increase in the antitumor activity, which was approximately fivefold higher than wild strain for relative inhibition rate. In FnVeAOE, the accumulation of indole, alkene, alkaloid, steroid, and flavonoid metabolites with potential antitumor activity was significantly upregulated compared with wild type via metabolomic analysis. Moreover, the transcriptome analysis showed that 134 differential genes were considered to be closely related to the biosynthesis of antitumor substances, of which 59 differential genes were considered as candidate key genes, and related to tryptophan dimethylallyltransferase, cytochrome P450 monooxygenase, polyketide synthases, and transcription factors. Taken together, we suggest that FnVeA may regulate the biosynthesis of antitumor substances by mediating the expression of genes related to secondary metabolic pathways in F. nematophilum.

Dissection of Functional Domains of Orc1-2, the Archaeal Global DNA Damage-Responsive Regulator.

Orc1-2 is a non-initiator ortholog of archaeal/eukaryotic Orc1 proteins, which functions as a global regulator in DNA damage-responsive (DDR) expression. As for Orc1 initiators, the DDR regulator harbors an AAA+ ATPase domain, an Initiator-Specific Motif (ISM) and a winged-helix (wH) DNA-binding domain, which are also organized in a similar fashion. To investigate how Orc1-2 mediates the DDR regulation, the orc1-2 mutants inactivating each of these functional domains were constructed with Saccharolobus islandicus and genetically characterized. We found that disruption of each functional domain completely abolished the DDR regulation in these orc1-2 mutants. Strikingly, inactivation of ATP hydrolysis of Orc1-2 rendered an inviable mutant. However, the cell lethality can be suppressed by the deficiency of the DNA binding in the same protein, and it occurs independent of any DNA damage signal. Mutant Orc1-2 proteins were then obtained and investigated for DNA-binding in vitro. This revealed that both the AAA+ ATPase and the wH domains are involved in DNA-binding, where ISM and R381R383 in wH are responsible for specific DNA binding. We further show that Orc1-2 regulation occurs in two distinct steps: (a) eliciting cell division inhibition at a low Orc1-2 content, and this regulation is switched on by ATP binding and turned off by ATP hydrolysis; any failure in turning off the regulation leads to growth inhibition and cell death; (b) activation of the expression of DDR gene encoding DNA repair proteins at an elevated level of Orc1-2.

Recent advances in metabolic regulation and bioengineering of gibberellic acid biosynthesis in Fusarium fujikuroi.

The plant growth hormone gibberellic acid (GA3), as one of the representative secondary metabolites, is widely used in agriculture, horticulture and brewing industry. GA3 is detected in both plants and several fungi with the ability to stimulate plant growth. Currently, the main mode of industrial production of GA3 is depended on the microbial fermentation via long-period submerged fermentation using Fusarium fujikuroi as the only producing strain, qualified for its natural productivity. However, the demand of large-sale industrialization of GA3 was still restricted by the low productivity. The biosynthetic route of GA3 in F. fujikuroi is now well-defined. Furthermore, the multi-level regulation mechanisms involved in the whole network of GA3 production have also been gradually unveiled by the past two decades based on the identification and characterization of several global regulators and their mutual functions. Combined with the quick development of genetic manipulation techniques, the rational modification of producing strain F. fujikuroi development become practical for higher productivity achievement. Herein, we review the latest advances in the molecular regulation of GA3 biosynthesis in F. fujikuroi and conclude a comprehensive network involving nitrogen depression, global regulator, histone modification and G protein signaling pathway. Correspondingly, the bioengineering strategies covering conventional random mutation, genetic manipulating platform development, metabolic edition and fermentation optimization were also systematically proposed.

Engineering of the Small Noncoding RNA (sRNA) DsrA Together with the sRNA Chaperone Hfq Enhances the Acid Tolerance of Escherichia coli.

Acid tolerance of microorganisms is a desirable phenotype for many industrial fermentation applications. In Escherichia coli, the stress response sigma factor RpoS is a promising target for engineering acid-tolerant phenotypes. However, the simple overexpression of RpoS alone is insufficient to confer these phenotypes. In this study, we show that the simultaneous overexpression of the noncoding small RNA (sRNA) DsrA and the sRNA chaperone Hfq, which act as RpoS activators, significantly increased acid tolerance in terms of cell growth under modest acidic pH, as well as cell survival upon extreme acid shock. Directed evolution of the DsrA-Hfq module further improved the acid tolerance, with the best mutants showing a 51 to 72% increase in growth performance at pH 4.5 compared with the starting strain, MG1655. Further analyses found that the improved acid tolerance of these DsrA-Hfq strains coincided with activation of genes associated with proton-consuming acid resistance system 2 (AR2), protein chaperone HdeB, and reactive oxygen species (ROS) removal in the exponential phase. This study illustrated that the fine-tuning of sRNAs and their chaperones can be a novel strategy for improving the acid tolerance of E. coliIMPORTANCE Many of the traditional studies on bacterial acid tolerance generally focused on improving cell survival under extreme-pH conditions, but cell growth under less harsh acidic conditions is more relevant to industrial applications. Under normal conditions, the general stress response sigma factor RpoS is maintained at low levels in the growth phase through a number of mechanisms. This study showed that RpoS can be activated prior to the stationary phase via engineering its activators, the sRNA DsrA and the sRNA chaperone Hfq, resulting in significantly improved cell growth at modest acidic pH. This work suggests that the sigma factors and likely other transcription factors can be retuned or retimed by manipulating the respective regulatory sRNAs along with the sufficient supply of the respective sRNA chaperones (i.e., Hfq). This provides a novel avenue for strain engineering of microbes.

Global Regulator of Rubber Degradation in Gordonia polyisoprenivorans VH2: Identification and Involvement in the Regulation Network.

A cAMP receptor protein (CRPVH2) was detected as a global regulator in Gordonia polyisoprenivorans VH2 and was proposed to participate in the network regulating poly(cis-1,4-isoprene) degradation as a novel key regulator. CRPVH2 shares a sequence identity of 79% with GlxR, a well-studied global regulator of Corynebacterium glutamicum Furthermore, CRPVH2 and GlxR have a common oligomerization state and similar binding motifs, and thus most likely have similar functions as global regulators. Size exclusion chromatography of purified CRPVH2 confirmed the existence as a homodimer with a native molecular weight of 44.1 kDa in the presence of cAMP. CRPVH2 bound to the TGTGAN6TCACT motif within the 131-bp intergenic region of divergently oriented lcp1VH2 and lcpRVH2, encoding a latex clearing protein and its putative repressor, respectively. DNase I footprinting assays revealed the exact operator size of CRPVH2 in the intergenic region (25 bp), which partly overlapped with the proposed promoters of lcpRVH2 and lcp1VH2 Our findings indicate that CRPVH2 represses the expression of lcpRVH2 while simultaneously directly or indirectly activating the expression of lcp1VH2 by binding the competing promoter regions. Furthermore, binding of CRPVH2 to upstream regions of additional putative enzymes of poly(cis-1,4-isoprene) degradation was verified in vitro. In silico analyses predicted 206 CRPVH2 binding sites comprising 244 genes associated with several functional categories, including carbon and peptide metabolism, stress response, etc. The gene expression regulation of several subordinated regulators substantiated the function of CRPVH2 as a global regulator. Moreover, we anticipate that the novel lcpR regulation mechanism by CRPs is widespread in other rubber-degrading actinomycetes.IMPORTANCE In order to develop efficient microbial recycling strategies for rubber waste materials, it is required that we understand the degradation pathway of the polymer and how it is regulated. However, only little is known about the transcriptional regulation of the rubber degradation pathway, which seems to be upregulated in the presence of the polymer. We identified a novel key regulator of rubber degradation (CRPVH2) that regulates several parts of the pathway in the potent rubber-degrader G. polyisoprenivorans VH2. Furthermore, we provide evidence for a widespread involvement of CRP regulators in the degradation of rubber in various other rubber-degrading actinomycetes. Thus, these novel insights into the regulation of rubber degradation are essential for developing efficient microbial degradation strategies for rubber waste materials by this group of actinomycetes.

The two-component system, BasSR, is involved in the regulation of biofilm and virulence in avian pathogenic Escherichia coli.

Avian pathogenic Escherichia coli (APEC) is a subgroup of extra-intestinal pathogenic E. coli (ExPEC) strains that cause avian colibacillosis, resulting in significant economic losses to the poultry industry worldwide. It has been reported that a few two-component signal transduction systems (TCS) participate in the regulation of the virulence factors of APEC infection. In this study, a basSR-deficient mutant strain was constructed from its parent strain APECX40 (WT), and high-throughput sequencing (RNA-seq) was performed to analyse the transcriptional profile of WT and its mutant strain XY1. Results showed that the deletion of basSR down-regulated the transcript levels of a series of biofilm- and virulence-related genes. Results of biofilm formation assays and bird model experiments indicated that the deletion of basSR inhibited biofilm formation in vitro and decreased bacterial virulence and colonization in vivo. In addition, electrophoretic mobility shift assays confirmed that the BasR protein could bind to the promoter regions of several biofilm- and virulence-related genes, including ais, opgC and fepA. This study suggests that the BasSR TCS might be a global regulator in the pathogenesis of APEC infection. RESEARCH HIGHLIGHTS Transcriptional profiling showed that BasSR might be a global regulator in APEC. BasSR increases APEC pathogenicity in vivo. BasSR positively regulates biofilm- and the virulence-associated genes. BasSR can bind to the promoter regions of virulence-associated genes ais, opgC and fepA.

Function and Regulation of the Pyruvate Transporter CstA in Escherichia coli.

Pyruvate is a central metabolite that connects many metabolic pathways in living organisms. To meet the cellular pyruvate requirements, the enterobacterium Escherichia coli has at least three pyruvate uptake systems-the H+/pyruvate symporter BtsT, and two thus far less well-characterized transporters, YhjX and CstA. BtsT and CstA belong to the putative carbon starvation (CstA) family (transporter classification TC# 2.A.114). We have created an E. coli mutant that cannot grow on pyruvate as the sole carbon source and used it to characterize CstA as a pyruvate transporter. Transport studies in intact cells confirmed that CstA is a highly specific pyruvate transporter with moderate affinity and is energized by a proton gradient. When cells of a reporter strain were cultured in complex medium, cstA expression was maximal only in stationary phase. A DNA affinity-capture assay combined with mass spectrometry and an in-vivo reporter assay identified Fis as a repressor of cstA expression, in addition to the known activator cAMP-CRP. The functional characterization and regulation of this second pyruvate uptake system provides valuable information for understanding the complexity of pyruvate sensing and uptake in E. coli.

Development of 3-hydroxypropionic-acid-tolerant strain of Escherichia coli W and role of minor global regulator yieP.

3-Hydroxypropionic acid (3-HP) is an important platform chemical, but its toxic effect at high concentrations (> 200 mM) is a serious challenge for commercial production. In this study, a highly 3-HP-tolerant strain of Escherichia coli W (tolerance concentration: 400 mM in M9 minimal medium and 800 mM when yeast extract was added) was developed by adaptive laboratory evolution (ALE) with glycerol as the carbon source. Genome analysis of the adapted strain (designated as E. coli WA) indicated the presence of mutations in 13 genes, including glpK (glycerol kinase) and yieP (a less-studied global regulator). The mutant GlpK (K67T) exhibited a higher activity than the wild-type enzyme, but it was not beneficial for 3-HP production due to its causing carbon overflow metabolism. Interestingly, among the other 12 genes, the mutation in yieP alone was almost fully responsible for the improved tolerance to 3-HP. When the mutant yieP was substituted with the wild-type counterpart, the adapted E. coli WA strain completely lost its tolerance to 3-HP, showing a tolerance similar to the wild-type W strain. Deletion of yieP conferred 3-HP tolerance to several other E. coli strains including K-12 W3110, K-12 MG1655, and B except BL21 (DE3). The E. coli WA with wild-type glpK showed, as compared with its parental strain, better 3-HP production, indicating that improved tolerance is beneficial for 3-HP production.

Transcription of the Subtilase Cytotoxin Gene subAB1 in Shiga Toxin-Producing Escherichia coli Is Dependent on hfq and hns.

Certain foodborne Shiga toxin-producing Escherichia coli (STEC) strains carry genes encoding the subtilase cytotoxin (SubAB). Although the mode of action of SubAB is under intensive investigation, information about the regulation of subAB gene expression is currently not available. In this study, we investigated the regulation of the chromosomal subAB1 gene in laboratory E. coli strain DH5α and STEC O113:H21 strain TS18/08 using a luciferase reporter gene assay. Special emphasis was given to the role of the global regulatory protein genes hfq and hns in subAB1 promoter activity. Subsequently, quantitative real-time PCR was performed to analyze the expression of Shiga toxin 2a (Stx2a), SubAB1, and cytolethal distending toxin V (Cdt-V) genes in STEC strain TS18/08 and its isogenic hfq and hns deletion mutants. The deletion of hfq led to a significant increase of up to 2-fold in subAB1 expression, especially in the late growth phase, in both strains. However, deletion of hns showed different effects on the promoter activity during the early and late exponential growth phases in both strains. Furthermore, upregulation of stx2a and cdt-V was demonstrated in hfq and hns deletion mutants in TS18/08. These data showed that the expression of subAB1, stx2a, and cdt-V is integrated in the regulatory network of global regulators Hfq and H-NS in Escherichia coliIMPORTANCE Shiga toxin-producing Escherichia coli (STEC) strains are responsible for outbreaks of foodborne diseases, such as hemorrhagic colitis and the hemolytic uremic syndrome. The pathogenicity of those strains can be attributed to, among other factors, the production of toxins. Recently, the subtilase cytotoxin was detected in locus of enterocyte effacement (LEE)-negative STEC, and it was confirmed that it contributes to the cytotoxicity of those STEC strains. Although the mode of action of SubAB1 is under intensive investigation, the regulation of gene expression is currently not known. The global regulatory proteins H-NS and Hfq have impact on many cellular processes and have been described to regulate virulence factors as well. Here, we investigate the role of hns and hfq in expression of subAB1 as well as stx2a and cdt-V in an E. coli laboratory strain as well as in wild-type STEC strain TS18/08.

Genetic variants of the oppA gene are involved in metabolic regulation of surfactin in Bacillus subtilis.

BACKGROUND: Bacillus subtilis 916 has been identified as an effective biocontrol agent against Rhizoctonia solani, the causal pathogen of rice sheath blight, under greenhouse and field conditions. HPLC analysis showed that surfactin, a member of the lipopeptide family produced by B. subtilis, was the major antimicrobial substance. RESULTS: Previously, we obtained a mutant strain of B. subtilis 916, Bs-H74, which produced significantly more surfactin than the wild type and presented 10% stronger inhibitory activity against R. solani. To explore the molecular mechanism underlying the higher surfactin productivity in the mutant, high-throughput proteomic analysis was carried out to analyze the differential protein expression. Our results showed that several differentially expressed proteins are involved in OppA, DegU and Carbon Catabolite Repression (CCR) regulatory pathways, which could be positively or negatively associated with surfactin biosynthesis. At both transcriptional and translational levels, we suggested that OppA may play a key role in surfactin synthesis regulation. Based on the above findings, we proposed the hypothesis that a point mutation in the oppA gene may lead to changes in oligopeptides acquisition in B. subtilis, and then the changed oligopeptides may activate or suppress the global regulatory protein, CcpA in the CCR pathway, and ComA and DegU may indirectly regulate surfactin synthesis in Bs-H74. To further explore the regulatory mechanisms in Bs-H74, metabolomics analysis was performed in this study. Interestingly, only 16 metabolites showed changes in abundance in Bs-H74 compared to Bs-916. Neohesperidin, a type of natural flavanone glycosides from citrus with a range of biological activities, increased by 18 times over the wild type Bs-916. This result implied exciting findings in regulatory mechanisms by OppA protein. CONCLUSIONS: In summary, this study has revealed the mechanisms underlying the improved antagonistic property with increased surfactin production in Bs-H74 at the gene, protein and metabolic levels, which may help to comprehend the map of the regulatory networks in B. subtilis. Findings from our work have provided a solid physical and theoretical basis for practically applying metabolic and genetic engineering to achieve improved and high-yielding biocontrol strains.

Engineering of Corynebacterium glutamicum as a prototrophic pyruvate-producing strain: Characterization of a ramA-deficient mutant and its application for metabolic engineering.

To construct a prototrophic Corynebacterium glutamicum strain that efficiently produces pyruvate from glucose, the effects of inactivating RamA, a global regulator responsible for activating the oxidative tricarboxylic acid (TCA) cycle, on glucose metabolism were investigated. ΔramA showed an increased specific glucose consumption rate, decreased growth, comparable pyruvate production, higher formation of lactate and acetate, and lower accumulation of succinate and 2-oxoglutarate compared to the wild type. A significant decrease in pyruvate dehydrogenase complex activity was observed for ΔramA, indicating reduced carbon flow to the TCA cycle in ΔramA. To create an efficient pyruvate producer, the ramA gene was deleted in a strain lacking the genes involved in all known lactate- and acetate-producing pathways. The resulting mutant produced 161 mM pyruvate from 222 mM glucose, which was significantly higher than that of the parent (89.3 mM; 1.80-fold).

Discovery of Two New Sorbicillinoids by Overexpression of the Global Regulator LaeA in a Marine-Derived Fungus Penicillium dipodomyis YJ-11.

Overexpression of the global regulator LaeA in a marine-derived fungal strain of Penicillium dipodomyis YJ-11 induced obvious morphological changes and metabolic variations. Further chemical investigation of the mutant strain afforded a series of sorbicillinoids including two new ones named 10,11-dihydrobislongiquinolide (1) and 10,11,16,17-tetrahydrobislongiquinolide (2), as well as four known analogues, bislongiquinolide (3), 16,17-dihydrobislongiquinolide (4), sohirnone A (5), and 2',3'-dihydrosorbicillin (6). The results support that the global regulator LaeA is a useful tool in activating silent gene clusters in Penicillium strains to obtain previously undiscovered compounds.

Thailandamide, a Fatty Acid Synthesis Antibiotic That Is Coexpressed with a Resistant Target Gene.

Microbes encode many uncharacterized gene clusters that may produce antibiotics and other bioactive small molecules. Methods for activating these genes are needed to explore their biosynthetic potential. A transposon containing an inducible promoter was randomly inserted into the genome of the soil bacterium Burkholderia thailandensis to induce antibiotic expression. This screen identified the polyketide/nonribosomal peptide thailandamide as an antibiotic and discovered its regulator, AtsR. Mutants of Salmonella resistant to thailandamide had mutations in the accA gene for acetyl coenzyme A (acetyl-CoA) carboxylase, which is one of the first enzymes in the fatty acid synthesis pathway. A second copy of accA in the thailandamide synthesis gene cluster keeps B. thailandensis resistant to its own antibiotic. These genetic techniques will likely be powerful tools for discovering other unusual antibiotics.