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Amino acids and related targets are typically produced by well-characterized heterotrophs including Corynebacterium glutamicum and Escherichia coli. Cyanobacteria offer an opportunity to supplant these sugar-intensive processes by instead directly utilizing atmospheric CO2 and sunlight. Synechococcus elongatus UTEX 2973 (hereafter UTEX 2973) is a particularly promising photoautotrophic platform due to its fast growth rate. Here, we first engineered UTEX 2973 to overproduce l-lysine (hereafter lysine), after which both cadaverine and glutarate production were achieved through further pathway engineering. To facilitate metabolic engineering, the relative activities of a subset of previously uncharacterized promoters were investigated, in each case, while also comparing the effects of both chromosomal (from neutral site NS3) and episomal (from pAM4788) expressions. Using these parts, lysine overproduction in UTEX 2973 was engineered by introducing a feedback-resistant copy of aspartate kinase (encoded by lysCfbr) and a lysine exporter (encoded by ybjE), both from E. coli. While chromosomal expression resulted in lysine production up to just 325.3 ± 14.8 mg/L after 120 h, this was then increased to 556.3 ± 62.3 mg/L via plasmid-based expression, also surpassing prior reports of photoautotrophic lysine bioproduction. Lastly, additional products of interest were then targeted by modularly extending the lysine pathway to glutarate and cadaverine, two 5-carbon, bioplastic monomers. By this approach, glutarate has so far been produced at final titers reaching 67.5 ± 2.2 mg/L by 96 h, whereas cadaverine has been produced at up to 55.3 ± 6.7 mg/L. Overcoming pathway and/or transport bottlenecks, meanwhile, will be important to improving upon these initial outputs.

Colanic acid has promising applications in food, cosmetic, and healthcare fields. In this study, a recombinant WQM003/pRAU was constructed by deleting genes lon and hns and overexpressing genes rcsA and galU in E. coli MG1655Δ(L-Q). After systematic optimization of fermentation conditions, colanic acid yield in WQM003/pRAU reached 19.79 g/L, the highest yield reported so far. The colanic acid produced by WQM003/pRAU was purified and its structure and physical properties were determined. This colanic acid shows a triple-helical structure and is stable up to 102 °C, and its melting temperature is 253.9 °C. This colanic acid shows a sphere-like chain conformation in aqueous solution. The viscosity of this colanic acid solution is related to concentration, shear rate, salt, temperature, and pH. At high concentrations, this colanic acid shows both viscous and elastic behaviors. These results suggest that the colanic acid produced by WQM003/pRAU has broad application prospects.

Bacillus subtilis CH3-5 isolated from cheonggukjang secretes a 28 kDa protease with a strong fibrinolytic activity. Its gene, aprE3-5, was cloned and expressed in a heterologous host (Jeong et al., 2007). In this study, the promoter of aprE3-5 was replaced with other stronger promoters (Pcry3A, P10, PSG1, PsrfA) of Bacillus spp. using PCR. The constructed chimeric genes were cloned into pHY300PLK vector, and then introduced into B. subtilis WB600. The P10 promoter conferred the highest fibrinolytic activity, i.e., 1.7-fold higher than that conferred by the original promoter. Overproduction of the 28 kDa protease was confirmed using SDS-PAGE and fibrin zymography. RT-qPCR analysis showed that aprE3-5 expression was 2.0-fold higher with the P10 promoter than with the original promoter. Change of the initiation codon from GTG to ATG further increased the fibrinolytic activity. The highest aprE3-5 expression was observed when two copies of the P10 promoter were placed in tandem upstream of the ATG initiation codon. The construct with P10 promoter and ATG and the construct with two copies of P10 promoter in tandem and ATG exhibited 117% and 148% higher fibrinolytic activity, respectively, than that exhibited by the construct containing P10 promoter and GTG. These results confirmed that significant overproduction of a fibrinolytic enzyme can be achieved by suitable promoter modification, and this approach may have applications in the industrial production of AprE3-5 and related fibrinolytic enzymes.

L-tryptophan (L-trp) production in Escherichia coli has been developed by employing random mutagenesis and selection for a long time, but this approach produces an unclear genetic background. Here, we generated the L-trp overproducer TPD5 by combining an intracellular L-trp biosensor and fluorescence-activated cell sorting (FACS) in E. coli, and succeeded in elucidating the genetic basis for L-trp overproduction. The most significant identified positive mutations affected TnaA (deletion), AroG (S211F), TrpE (A63V), and RpoS (nonsense mutation Q33*). The underlying structure–function relationships of the feedback-resistant AroG (S211F) and TrpE (A63V) mutants were uncovered based on protein structure modeling and molecular dynamics simulations, respectively. According to transcriptomic analysis, the global regulator RpoS not only has a great influence on cell growth and morphology, but also on carbon utilization and the direction of carbon flow. Finally, by balancing the concentrations of the L-trp precursors’ serine and glutamine based on the above analysis, we further increased the titer of L-trp to 3.18 g/L with a yield of 0.18 g/g. The analysis of the genetic characteristics of an L-trp overproducing E. coli provides valuable information on L-trp synthesis and elucidates the phenotype and complex cellular properties in a high-yielding strain, which opens the possibility to transfer beneficial mutations and reconstruct an overproducer with a clean genetic background.

The objective of this study was to characterize Polish penicillin-resistant, ampicillin-susceptible Enterococcus faecalis (PRASEF), increasingly reported to the National Reference Centre for Susceptibility Testing, Poland, to elucidate the path of emergence of such strains. A total of 136 isolates were examined by antimicrobial susceptibility testing and for the β-lactamase production (cefinase test). The clonality of isolates was established by multilocus sequence typing (MLST) and the penicillin-binding protein pbp4 gene was sequenced to search for putative mutation(s). The presence of pheromone-responsive plasmids was investigated by clumping test and PCR detection of plasmid-specific genes. All Polish PRASEF were multidrug resistant and β-lactamase-negative. MLST assigned isolates mostly to high-risk enterococcal clonal complexes (HIRECCs) 6 (57.4%) and 87 (30.1%), in addition to to CC88 (12.5%). The sequencing of pbp4 revealed mutations upstream of a putative promoter region and amino acid alterations in PBP4, affecting 24 positions and resulting in 30 variants. While production of aggregation substance was observed for 17.6% of isolates, genes of pheromone plasmids were much more commonly detected. However, no conjugal transfer of penicillin resistance was observed. Penicillin resistance in E. faecalis emerges mostly in HiRECCs due to PBP4 overproduction and/or mutations. The acquisition of penicillin resistance by HiRECCs may represent the next step in the evolution of E. faecalis as human nosocomial pathogen.

SQSTM1/p62, also known as sequestosome 1 (SQSTM1) or p62, is an intracellular protein induced by stress and functions as an adaptor molecule in diverse cellular processes. Oxidative damage induced by overproduction of amyloid-β (Aβ) and the impairment of endogenous antioxidant Nrf2 signaling have been documented in the brains of Alzheimer’s disease (AD) patients. The causes of the inactivation of Nrf2 signaling under Aβ-induced oxidative stress are unclear, and p62 might be involved in this process. In this study, APP/PS1 transgenic mice, Aβ intrahippocampal injection rat model, and SH-SY5Y cells were used to reveal that the alterations in the oligomeric state of p62 participated in the regulation of Nrf2 signaling under Aβ insult. The present in vivo and in vitro studies revealed that short-term treatment of Aβ activated Nrf2 signaling, while long-term Aβ treatment inhibited it through either canonical or noncanonical Nrf2 activation pathway. p62 oligomerization was largely attenuated under long-term Aβ treatment. The reduction of p62 oligomerization weakened p62 sequestration to Keap1, leading to Nrf2 signaling inhibition. Our findings provide a better understanding of p62-mediated modulation on Nrf2 activity and highlight a potential therapeutic target of p62 in AD.

Integral membrane proteins (IMPs) constitute ~30% of all proteins encoded by the genome of any organism and Escherichia coli remains the first-choice host for recombinant production of prokaryotic IMPs. However, the expression levels of prokaryotic IMPs delivered by this bacterium are often low and overproduced targets often accumulate in inclusion bodies. The targets are therefore often discarded to avoid an additional and inconvenient refolding step in the purification protocol. Here we compared expression of five prokaryotic (bacterial and archaeal) IMP families in E. coli and Saccharomyces cerevisiae. We demonstrate that our S. cerevisiae-based production platform is superior in expression of four investigated IMPs, overall being able to deliver high quantities of active target proteins. Surprisingly, in case of the family of zinc transporters (Zrt/Irt-like proteins, ZIPs), S. cerevisiae rescued protein expression that was undetectable in E. coli. We also demonstrate the effect of localization of the fusion tag on expression yield and sample quality in detergent micelles. Lastly, we present a road map to achieve the most efficient expression of prokaryotic IMPs in our yeast platform. Our findings demonstrate the great potential of S. cerevisiae as host for high-throughput recombinant overproduction of bacterial and archaeal IMPs for downstream biophysical characterization., Graphical abstract Image 1, Highlights • S. cerevisiae is superior to E. coli in expressing correctly folded and active IMPs. • S. cerevisiae completely rescues the expression of the family of zinc transporters. • Localization of the fusion tag affects expression yields and protein quality. • We provide a roadmap to efficient expression of prokaryotic IMPs in S. cerevisiae.

A high efficiency and low toxicity radiosensitizer, OsN(PhenOH)Cl3, was designed and synthesized through substituent regulation. To the best of our knowledge, this is the first osmium-based coordination complex radiosensitizer. The experimental results shown that this radiosensitizer induced G2/M cell cycle arrest mainly through induction of intracellular ROS overproduction.

l-Cysteine is a ubiquitous and unique sulfur-containing amino acid with numerous applications in agricultural and food industries. The efficient production of l-cysteine via microbial fermentation has received a great deal of attention. In this study, the fitness of different Escherichia coli K-12 strains harboring plasmid pLH03 was investigated. The enhancement of the precursor synthetic pathway and thiosulfate assimilation pathway resulted in the good performance of the E. coli BW25113 strain. The expression levels of synthetic pathway genes were optimized by two constitutive promoters to assess their effects on cysteine production. In conjunction, the main degradation pathway genes were also deleted for more efficient production of cysteine. l-Cysteine production was further increased through the manipulation of the sulfur transcription regulator cysB and sulfur supplementation. After process optimization in a 1.5 L bioreactor, LH2A1M0BΔYTS-pLH03 [BW25113 Ptrc2-serA Ptrc1-cysMPtrc-cysBΔyhaMΔtnaAΔsdaA-(pLH03)] accumulated 8.34 g/L cysteine, laying a foundation for application in the cysteine fermentation industry.

Tumor necrosis factor-alpha (TNF-α) is an inflammatory cytokine that plays a major role in immune regulation, homeostatic function, and cellular organization. The present study was undertaken to overproduce recombinant human TNF-α (rhTNF-α) in Escherichia coli (E.coli) in high cell density culture. The use of a codon-optimized gene and strong promoter-based (T7) expression system, choice of Terrific Broth (TB) as medium, and subsequent optimization of culture conditions in shake flasks resulted in production of 0.95 g/L insoluble rhTNF-α comprising upto 50% of total cellular protein (TCP) The protein yield further increased upto 1.26 g/L in 1 L TB medium batch culture in bioreactor with the controlled temperature, pH, and dissolved oxygen. In a series of chemostats operated at dilution rates of 0.2 h-1, 0.3 h-1, 0.4 h-1 and 0.5 h-1 the specific growth rate (μ) positively correlated with specific yield (Yp/x) and a maximum yield of 164 mg/g DCW was obtained at μ = 0.4 h-1 within 4 h post-induction. A fed-batch cultivation in TB with an exponential feeding profile (μ = ∼0.4 h-1) of concentrated feed resulted in an accumulation of 5.5 g/L of rhTNF-α within 14 h of cultivation which accounted for ∼29% of TCP.

Synthetic methylotrophy aims to engineer methane and methanol utilization pathways in platform hosts like Escherichia coli for industrial bioprocessing of natural gas and biogas. While recent attempts to engineer synthetic methylotrophs have proved successful, autonomous methylotrophy, i.e. the ability to utilize methane or methanol as sole carbon and energy substrates, has not yet been realized. Here, we address an important limitation of autonomous methylotrophy in E. coli: the inability of the organism to synthesize several amino acids when grown on methanol. By activating the stringent/stress response via ppGpp overproduction, or DksA and RpoS overexpression, we demonstrate improved biosynthesis of proteinogenic amino acids via endogenous upregulation of amino acid synthesis pathway genes. Thus, we were able to achieve biosynthesis of several limiting amino acids from methanol-derived carbon, in contrast to the control methylotrophic E. coli strain. This study addresses a key limitation currently preventing autonomous methylotrophy in E. coli and possibly other synthetic methylotrophs and provides insight as to how this limitation can be alleviated via stringent/stress response activation.

Aims: To determine whether the use of an octreotide suppression test will reliably distinguish pituitary from ectopic ACTH overproduction. Somatostatin receptors are expressed in NETs, but are downgraded in the pituitary as the result of hypercortisolaemia. Octreotide should therefore lower ACTH and cortisol levels in patients with NETs but not in patients with Cushing’s disease and pituitary tumors. Methodology: A cross sectional study was performed in 13 patents with ACTH dependent Cushing’s (8 women, 5 men) with ages ranging between 21 to 40 years were studied. Serum cortisol concentrations were measured at 0800 hrs before and during the administration of. Octreotide at a dosage of 100 mcg subcutaneously every 8 hours for 72 hours. Results: The serum cortisol concentrations returned to normal in 4 patients who were later documented to have ectopic disease, two with typical bronchial carcinoids and two with pancreatic NETs and metastatic disease. The other 9 patients had no suppression in serum cortisol concentrations and were documented later to have pituitary tumours. Conclusion: These results indicate that a short trial of octreotide will identify patients with ectopic disease as evidenced by a fall in serum cortisol levels whereas in those with Cushing’s disease and pituitary tumours serum cortisol levels remains unchanged. Recommendation: We recommend all patients with ACTH dependent Cushing’s syndrome have an octreotide suppression test, even if the MRI shows an adenoma, so as to exclude the possibility of a pituitary incidentaloma in a patient with ectopic disease, or false localization from IPSS to the pituitary gland due to ectopic CRH secretion.

The compatible solutes ectoine and hydroxyectoine are synthesized by many microorganisms as potent osmostress and desiccation protectants. Besides their successful implementation into various skincare products, they are of increasing biotechnological interest due to new applications in the healthcare sector. To meet this growing demand, efficient heterologous overproduction solutions for ectoines need to be found. This study is the first report on the utilization of the non-halophilic biosynthesis enzymes from Acidiphilium cryptum DSM 2389T for efficient heterologous production of ectoines in Escherichia coli. When grown at low salt conditions (≤ 0.5% NaCl) and utilizing the cheap carbon source glycerol, the production was characterized by the highest specific production of ectoine [2.9 g/g dry cell weight (dcw)] and hydroxyectoine (2.2 g/g dcw) reported so far and occurred at rapid specific production rates of up to 345 mg/(g dcw × h). This efficiency in production was related to an unprecedented carbon source conversion rate of approx. 60% of the theoretical maximum. These findings confirm the unique potential of the here implemented non-halophilic enzymes for ectoine production processes in E. coli and demonstrate the first efficient heterologous solution for hydroxyectoine production, as well as an extraordinary efficient low-salt ectoine production. Electronic supplementary material The online version of this article (10.1007/s00792-020-01188-8) contains supplementary material, which is available to authorized users.

Controlling metabolism of engineered microbes is important to modulate cell growth and production during a bioprocess. For example, external parameters such as light, chemical inducers, or temperature can act on metabolism of production strains by changing the abundance or activity of enzymes. Here, we created temperature-sensitive variants of an essential enzyme in arginine biosynthesis of Escherichia coli (argininosuccinate synthetase, ArgG) and used them to dynamically control citrulline overproduction and growth of E. coli. We show a method for high-throughput enrichment of temperature-sensitive ArgG variants with a fluorescent TIMER protein and flow cytometry. With 90 of the thus derived ArgG variants, we complemented an ArgG deletion strain showing that 90% of the strains exhibit temperature-sensitive growth and 69% of the strains are auxotrophic for arginine at 42 °C and prototrophic at 30 °C. The best temperature-sensitive ArgG variant enabled precise and tunable control of cell growth by temperature changes. Expressing this variant in a feedback-dysregulated E. coli strain allowed us to realize a two-stage bioprocess: a 33 °C growth-phase for biomass accumulation and a 39 °C stationary-phase for citrulline production. With this two-stage strategy, we produced 3 g/L citrulline during 45 h cultivation in a 1-L bioreactor. These results show that temperature-sensitive enzymes can be created en masse and that they may function as metabolic valves in engineered bacteria., Graphical abstract Image 1, Highlights • Method to enrich temperature-sensitive enzymes en masse. • Temperature-sensitive enzymes function as metabolic valve. • Temperature controlled two-stage production of citrulline.

Rationale: Aspirin-exacerbated respiratory disease is characterized by severe asthma, nonsteroidal antiinflammatory drug hypersensitivity, nasal polyposis, and leukotriene overproduction. Systemic ...

microRNAs (miRNAs) participate in the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). However, few reports have discussed the effect of miRNAs on the magnesium chloride (MgCl2)-induced promotion of osteogenic differentiation of BMSCs, a process involved in the healing of bone tissue. As determined in the present investigation, MgCl2 decreased miR-16 levels; increased levels of fibroblast growth factor 2 (FGF2), p-p38, and p-ERK; and promoted the osteogenic differentiation of BMSCs. Enhancement of miR-16 levels by an miR-16 mimic blocked these MgCl2-induced changes. Moreover, luciferase reporter assays confirmed that miR-16 binds to the 3′UTR region of FGF2 mRNA. Down-regulation of FGF2 blocked the MgCl2-induced increases of p-p38 and p-ERK and the promotion of the osteogenic differentiation of BMSCs. Furthermore, over-expression of miR-16 attenuated the MgCl2-induced overproduction of p-p38 and p-ERK1/2 and the high levels of osteogenic differentiation, effects that were reversed by elevated expression of FGF2. In summary, the present findings provide a mechanism by which miR-16 regulates MgCl2-induced promotion of osteogenic differentiation by targeting FGF2-mediated activation of the ERK/MAPK pathway.

The lincosamide family antibiotic lincomycin is a widely used antibacterial pharmaceutical generated by Streptomyces lincolnensis, and the high-yield strain B48 produces 2.5 g/L lincomycin, approximately 30-fold as the wild-type strain NRRL 2936. Here, the genome of S. lincolnensis B48 was completely sequenced, revealing a ~10.0 Mb single chromosome with 71.03% G + C content. Based on the genomic information, lincomycin-related primary metabolism network was constructed and the secondary metabolic potential was analyzed. In order to dissect the overproduction mechanism, a comparative genomic analysis with NRRL 2936 was performed. Three large deletions (LDI-III), one large inverted duplication (LID), one long inversion and 80 small variations (including 50 single nucleotide variations, 13 insertions and 17 deletions) were found in B48 genome. Then several crucial mutants contributing to higher production phenotype were validated. Deleting of a MarR-type regulator-encoding gene slinc377 from LDI, and the whole 24.7 kb LDII in NRRL 2936 enhanced lincomycin titer by 244% and 284%, respectively. Besides, lincomycin production of NRRL 2936 was increased to 7.7-fold when a 71 kb supercluster BGC33 from LDIII was eliminated. As for the duplication region, overexpression of the cluster situated genes lmbB2 and lmbU, as well as two novel transcriptional regulator-encoding genes (slinc191 and slinc348) elevated lincomycin titer by 77%, 75%, 114% and 702%, respectively. Furthermore, three negative correlation genes (slinc6156, slinc4481 and slinc6011) on lincomycin biosynthesis, participating in regulation were found out. And surprisingly, inactivation of RNase J-encoding gene slinc6156 and TPR (tetratricopeptide repeat) domain-containing protein-encoding gene slinc4481 achieved lincomycin titer equivalent to 83% and 68% of B48, respectively, to 22.4 and 18.4-fold compared to NRRL 2936. Therefore, the comparative genomics approach combined with confirmatory experiments identified that large fragment deletion, long sequence duplication, along with several mutations of genes, especially regulator genes, are crucial for lincomycin overproduction.

Colistin resistant Acinetobacter baumannii strains are of great concern worldwide. However, the role of efflux pumps in colistin resistance needs to be elucidated. We investigated the changes in colistin MICs of 29 colistin resistant A. baumannii isolates in response to resistance-nodulation-division (RND)-type efflux pump inhibitor (EPI) and the alterations in AdeR and AdeS two-component regulatory proteins previously associated with the overproduction of AdeAB. The EPI, 1-(1-naphthylmethyl)-piperazine (NMP), led to significant reductions in colistin MICs. At least one of the following amino acid substitutions was found in AdeS proteins from 18 of the isolates: L172P, A94V, V27I, V32I, G186V, and G164A. Besides, A136V and V120I alterations were identified in AdeR from five isolates. Therefore, EPI-responsive colistin resistance in our isolates is most likely due to the action of an RND-type efflux system. The underlying mechanism of resistance might be the result of certain AdeRS alterations, leading to AdeAB overexpression.

The creation of microbial cell factories for sustainable production of natural products is important for medical and industrial applications. This requires stable expression of biosynthetic pathways in a host organism with favorable fermentation properties such as Bacillus subtilis. The aim of this study is to construct B. subtilis strains that produce valuable terpenoid compounds by overexpressing the innate methylerythritol phosphate (MEP) pathway. A synthetic operon allowing the concerted and regulated expression of multiple genes was developed. Up to 8 genes have been combined in this operon and a stably inherited plasmid-based vector was constructed resulting in a high production of C30 carotenoids. For this, two vectors were examined, one with rolling circle replication and another with theta replication. Theta-replication constructs were clearly superior in structural and segregational stability compared to rolling circle constructs. A strain overexpressing all eight genes of the MEP pathway on a theta-replicating plasmid clearly produced the highest level of carotenoids. The level of transcription for each gene in the operon was similar as RT-qPCR analysis indicated. Hence, that corresponding strain can be used as a stable cell factory for production of terpenoids. This is the first report of merging and stably expressing this large-size operon (eight genes) from a plasmid-based system in B. subtilis enabling high C30 carotenoid production.

Heavy infection of the virus leads to overproduction of cytokines. The overproduction of cytokine (cytokines storms) is responsible for the critical cases and deaths of COVID-19. The nuclear factor kappa-B stimulates the expression of the genes, which is responsible for cytokines storm and RNA transcription. The COVID-19 virus can be controlled by inhibition of nuclear factor kappa-B. Nuclear factor kappa-B is controlled by inhibition of hydrogen peroxide and inhibitor kappa-B kinase enzyme.

Fermentative production of L-cysteine has been established using Escherichia coli. In that procedure, thiosulfate is a beneficial sulfur source, whereas repressing sulfate utilization. We first found that thiosulfate decreased transcript levels of genes related to sulfur assimilation, particularly whose expression is controlled by the transcription factor CysB. Therefore, a novel approach, i.e. increment of expression of genes involved in sulfur-assimilation, was attempted for further improvement of L-cysteine overproduction. Disruption of the rppH gene significantly augmented transcript levels of the cysD, cysJ, cysM and yeeE genes (≥1.5-times) in medium containing sulfate as a sole sulfur source, probably because the rppH gene encodes mRNA pyrophosphohydrolase that triggers degradation of certain mRNAs. In addition, the ΔrppH strain appeared to preferentially uptake thiosulfate rather than sulfate, though thiosulfate dramatically reduced expression of the known sulfate/thiosulfate transporter complexes in both ΔrppH and wild-type cells. We also found that both YeeE and YeeD are required for the strain without the transporters to grow in the presence of thiosulfate as a sole sulfur source. Therefore, yeeE and yeeD are assigned as genes responsible for thiosulfate uptake (tsuA and tsuB, respectively). In final, we applied the ΔrppH strain to the fermentative production of L-cysteine. Disruption of the rppH gene enhanced L-cysteine biosynthesis, as a result, a strain producing approximately twice as much L-cysteine as the control strain was obtained.

Hydrogen peroxide (H2O2), an important marker for oxidative stress, plays a vital role in cellular biological functions. Overproduction of H2O2 causes oxidative damage to cellular functions and pro...

Coenzyme Q10 (CoQ10) is an important component of the respiratory chain in humans and some bacteria. As a high-value-added nutraceutical antioxidant, CoQ10 has excellent capacity to prevent cardiovascular disease. The content of CoQ10 in the industrial Rhodobacter sphaeroides HY01 is hundreds of folds higher than normal physiological levels. In this study, we found that overexpression or optimization of the synthetic pathway failed CoQ10 overproduction in the HY01 strain. Moreover, under phosphate- limited conditions (decreased phosphate or in the absence of inorganic phosphate addition), CoQ10 production increased significantly by 12% to220 mg/L, biomass decreased by 12%, and the CoQ10 productivity of unit cells increased by 27%. In subsequent fed-batch fermentation, CoQ10 production reached 272 mg/L in the shake-flask fermentation and 1.95 g/L in a 100-L bioreactor under phosphate limitation. Furthermore, to understand the mechanism associated with CoQ10 overproduction under phosphate- limited conditions, the comparatve transcriptome analysis was performed. These results indicated that phosphate limitation combined with glucose fed-batch fermentation represented an effective strategy for CoQ10 production in the HY01. Phosphate limitation induced a pleiotropic effect on cell metabolism, and that improved CoQ10 biosynthesis efficiency was possibly related to the disturbance of energy metabolism and redox potential. Keywords: R sphaeroides, CoQ10, Phosphate limitation, Overproduction, Scale-up, Transcriptome

The wild-type transcription factors are sensitive to their corresponding signal molecules. Using wild-type transcription factors as biosensors to screen industrial overproducers are generally impractical because of their narrow detection ranges. This study took transcription factor BmoR as an example and aimed to expand the detection range of BmoR for screening alcohols overproducers. Firstly, a BmoR mutation library was established, and the mutations distributed randomly in all predicted functional domains of BmoR. Structure of BmoR-isobutanol complex were modelled, and isobutanol binding sites were confirmed by site-directed mutagenesis. Subsequently, the effects of the mutations on the detection range or output were confirmed in the BmoR mutants. Four combinatorial mutants containing one increased-detection-range mutation and one enhanced-output mutation were constructed. Compared with wild-type BmoR, F276A/E627N BmoR and D333N/E627N BmoR have wider detection ranges (0–100 mM) and relatively high outputs to the isobutanol added quantitatively or produced intracellularly, demonstrating they have potential for screening isobutanol overproduction strains. This work presented an example of engineering the wild-type transcription factors with physiological significance for industrial utilization.

Hydroxytyrosol (HT) is one of the most powerful dietary antioxidants with numerous applications in different areas, including cosmetics, nutraceuticals and food. In the present work, heterologous hydroxylase complex HpaBC from Escherichia coli was integrated into the Saccharomyces cerevisiae genome in multiple copies. HT productivity was increased by redirecting the metabolic flux towards tyrosol synthesis to avoid exogenous tyrosol or tyrosine supplementation. After evaluating the potential of our selected strain as an HT producer from glucose, we adjusted the medium composition for HT production. The combination of the selected modifications in our engineered strain, combined with culture conditions optimization, resulted in a titre of approximately 375 mg l-1 of HT obtained from shake-flask fermentation using a minimal synthetic-defined medium with 160 g l-1 glucose as the sole carbon source. To the best of our knowledge, this is the highest HT concentration produced by an engineered S. cerevisiae strain., This work was supported by the Spanish Ministry of Science, Innovation and Universities through the grants AGL2016-77505-C3-1-R and PID2019-108722RB-C31, awarded to JMG.

Background The phospholipase domain-containing 3 gene (PNPLA3)-148M variant is associated with liver steatosis but its influence on the metabolism of triglyceride-rich lipoproteins remains unclear. Here, we investigated the kinetics of large, triglyceride-rich very-low-density lipoprotein (VLDL), (VLDL1), and smaller VLDL2 in homozygotes for the PNPLA3-148M variant. Methods and results The kinetics of apolipoprotein (apo) B100 (apoB100) and triglyceride in VLDL subfractions were analysed in nine subjects homozygous for PNPLA3-148M and nine subjects homozygous for PNPLA3-148I (controls). Liver fat was >3-fold higher in the 148M subjects. Production rates for apoB100 and triglyceride in VLDL1 did not differ significantly between the two groups. Likewise, production rates for VLDL2-apoB100 and -triglyceride, and fractional clearance rates for both apoB100 and triglyceride in VLDL1 and VLDL2, were not significantly different. Conclusions Despite the higher liver fat content in PNPLA3 148M homozygotes, there was no increase in VLDL production. Equally, VLDL production was maintained at normal levels despite the putative impairment in cytosolic lipid hydrolysis in these subjects.

Itaconic acid is an unsaturated organic acid with two carboxyls and one methylene group. The presence of these functional groups, along with a conjugated double bond, makes itaconic acid a versatile molecule with a vast number of applications. Itaconic acid can be produced through chemical as well as biological routes. Aspergillus terreus is the most prevalent microbial cell factory for the biological production of itaconic acid, reaching titers of >100 g/L. However, it suffers from low yield and volumetric productivities leading to high manufacturing costs. The wider applications of itaconic acid can be enabled with a low-cost production process, which can be achieved with cheaper feedstocks and robust cell factories accumulating itaconic acid efficiently. The current review summarizes the recent advances in the biological production of itaconic acid with a focus on the metabolic engineering of prokaryotic and eukaryotic systems for the overproduction of itaconic acid. It comprehensively describes various microbial cell factories with an insight into the pathway leading to itaconic acid production in natural producers like A. terreus and U. maydis. It also discusses the metabolic engineering approaches to improve strain performance in terms of high itaconic acid productivity, less by-product generation, and the ability to utilize unconventional cheap substrates. Moreover, the alternative strategies for the development of non-native producers through genome engineering and the hurdles related to itaconic acid production have been elaborated.

Pseudomonas aeruginosa is a ubiquitous environmental bacterium and an opportunistic pathogen that represents an important health hazard. The quorum-sensing response regulates the expression of several virulence factors and involves three regulons: Las, Rhl, and Pqs. The P. aeruginosa ATCC 9027 strain, which belongs to the genetically diverse PA7 clade, contains a frame-shift mutation in the pqsR gene that encodes a transcriptional activator necessary for pyocyanin (PYO) synthesis in type strains PAO1 and PA14. Here we characterize the PqsE-dependent production of PYO in strain ATCC 9027. We show that this strain expresses pqsE independently of PqsR and in the absence of quinolone production, and that PqsE promotes the RhlR-dependent production of PYO, yet this production is not strictly dependent on PqsE. In addition, we show that in both strains ATCC 9027 and PAO1, PqsE overexpression causes an increased concentration of RhlR and enhances PYO production but does not affect rhamnolipids (RL) production in the same way. These results suggest that PqsE interaction with RhlR preferentially modifies its ability to activate transcription of genes involved in PYO production and provide new evidence about PqsE-dependent RhlR activation, highlighting the variability of the QS response among different P. aeruginosa clades and strains. HIGHLIGHTS: Pseudomonas aeruginosa ATCC 9027 is able to produce pyocyanin in phosphate limiting conditions, even in the absence of a functional PqsR. This strain does not produce alkyl quinolones like PQS and HHQ, but expresses pqsE. Synthesis of pyocyanin by ATCC 9027 is only partially dependent on pqsE. The overexpression of pqsE in the ATCC 9027 and PAO1 strains causes pyocyanin overproduction. The overexpression of pqsE in these strains causes an increased RhlR concentration without affecting rhlR transcription or translation. Rhamnolipids production is not affected to the same extent as pyocyanin by overexpression of pqsE in these strains.

The ability of chaperonins to buffer mutations that affect protein folding pathways suggests that their abundance should be evolutionarily advantageous. Here, we investigate the effect of chaperonin overproduction on cellular fitness in Escherichia coli. We demonstrate that chaperonin abundance confers 1) an ability to tolerate higher temperatures, 2) improved cellular fitness, and 3) enhanced folding of metabolic enzymes, which is expected to lead to enhanced energy harvesting potential.

Neuroblastoma (NB) is a solid tumor in the nervous system and has a high mortality rate in children. Curcumin has well-characterized anticancer properties, while there is no effective method in clinical treatment. MTT assays revealed that curcumin dramatically inhibited the proliferation of SK-N-SH cells. Compared with the control group, curcumin markedly restrained the migration of SK-N-SH cells. Curcumin induced SK-N-SH cell apoptosis by G2/M cycle arrest and activated caspase-3 activity. Furthermore, curcumin promoted the overproduction of intracellular ROS and apoptosis induced by activating p53 and Bcl-2 signal pathways. This finding demonstrated the application of curcumin is an effective strategy for the therapeutics of NB.

Research on microbial fatty acid metabolism started in the late 1960s, and till date, various developments have aided in elucidating the fatty acid metabolism in great depth. Over the years, synthesis of microbial fatty acid has drawn industrial attention due to its diverse applications. However, fatty acid overproduction imparts various stresses on its metabolic pathways causing a bottleneck to further increase the fatty acid yields. Numerous strategies to increase fatty acid titres in Escherichia coli by pathway modulation have already been published, but the stress generated during fatty acid overproduction is relatively less studied. Stresses like pH, osmolarity and oxidative stress, not only lower fatty acid titres, but also alter the cell membrane composition, protein expression and membrane fluidity. This review discusses an overview of fatty acid synthesis pathway and presents a panoramic view of various stresses caused due to fatty acid overproduction in E. coli. It also addresses how certain stresses like high temperature and nitrogen limitation can boost fatty acid production. This review paper also highlights the interconnections that exist between these stresses.

Acidithiobacillus ferrooxidans are well-studied iron- and sulfur-oxidizing acidophilic chemolithoautotrophs that are exploited for their ability to participate in the bioleaching of metal sulfides. Here, we overexpressed the endogenous glutamate--cysteine ligase and glutathione synthetase genes in separate strains and found that glutathione synthetase overexpression increased intracellular glutathione levels. We explored the impact of pH on the halotolerance of iron oxidation in wild type and engineered cultures. The increase in glutathione allowed the modified cells to grow under salt concentrations and pH conditions that are fully inhibitory to wild type cells. These results indicate that glutathione overexpression can be used to increase halotolerance in A. ferrooxidans and would likely be a useful strategy on other acidophilic bacteria.ImportanceThe use of acidophilic bacteria in the hydrometallurgical processing of sulfide ores can enable many benefits including the potential reduction of environmental impacts. The cells involved in bioleaching tend to have limited halotolerance, and increased halotolerance could enable several benefits, including a reduction in the need for fresh water resources. We show that the genetic modification of A. ferrooxidans for the overproduction of glutathione is a promising strategy to enable cells to resist the oxidative stress that can occur during growth in the presence of salt.

Hypercalcemia is a common electrolyte abnormality in malignancy and is largely caused by activation of parathyroid hormone (PTH) pathways. We report the case of a 76-year-old man with hypercalcemia primarily owing to 1,25-dihydroxyvitamin D3 overproduction from a high-grade fetal lung adenocarcinoma. Histologically, the tumor itself and tumor-adjacent macrophages were positive for the CYP27B1 protein, a key enzyme that generates 1,25-dihydroxyvitamin D3. Suppression was observed in serum PTH and PTH-related hormone levels, suggesting hypercalcemia is independent of the PTH pathway. Serum calcium level returned to normal after surgical resection of the lung cancer, supporting extrarenal overproduction of 1,25-dihydroxyvitamin D3 elicited by the tumors is the cause of hypercalcemia in this patient.

Aims: This study attempted to evaluate the five host strains, including BL21 (DE3), Rosetta (DE3), DH5α, XL1-BLUE, and SHuffle, in terms of arginine deiminase (ADI) production and enzyme activity. Background: Escherichia coli is one of the most preferred host microorganisms for the production of recombinant proteins due to its well-characterized genome, availability of various expression vectors, and host strains. Choosing a proper host strain for the overproduction of a desired recombinant protein is very important because of the diversity of genetically modified expression strains. Various E. coli cells have been examined in different patent applications. Method: ADI was chosen as a bacterial enzyme that degrades L-arginine. It is effective in the treatment of some types of human cancers like melanoma and hepatocellular carcinoma (HCC), which are arginine-auxotrophic. Five mentioned E. coli strains were cultivated. The pET-3a was used as the expression vector. The competent E. coli cells were obtained through the CaCl2 method. It was then transformed with the construct of pET3a-ADI using the heat shock strategy. The ADI production levels were examined by 10% SDS-PAGE analysis. The ability of host strains for the expression of the requested recombinant protein was compared. The enzymatic activity of the obtained recombinant ADI from each studied strain was assessed by a colorimetric 96-well microtiter plate assay. Result: All the five strains exhibited a significant band at 46 kDa. BL21 (DE3) produced the highest amount of ADI protein, followed by Rosetta (DE3). The following activity assay showed that ADI from BL21 (DE3) and Rosetta (DE3) had the most activity. Conclusion: There are some genetic and metabolic differences among the various E. coli strains, leading to differences in the amount of recombinant protein production. The results of this study can be used for the efficacy evaluation of the five studied strains for the production of similar pharmaceutical enzymes. The strains also could be analyzed in terms of proteomics.

International audience; Catalases (CAT) are antioxidant metalloenzymes necessary for life in oxygen-metabolizing cells to regulate H 2 O 2 concentration by accelerating its dismutation. Many physio-pathological situations are associated with oxidative stress resulting from H 2 O 2 overproduction during which antioxidant defenses are overwhelmed. We have used a combinatorial approach associated with an activity-based screening to discover a first peptidyl di copper complex mimicking CAT. The complex was studied in detail and characterized for its CAT activity both in solution and in cells using different analytical methods. The complex exhibited CAT activity in solution and, more interestingly, on HyPer HeLa cells which possess a genetically encoded ratiometric fluorescent sensors of H 2 O 2. These results highlight the efficiency of a combinatorial approach for the discovery of peptidyl complexes that exhibit catalytic activity.

With the flexibility to fold into complex structures, RNA is well-suited to act as a cellular sensor to recognize environmental fluctuations and respond to changes by regulating the corresponding genes. In this study, we established a high-throughput screening platform to screen tryptophan high-producing strains from a large repertoire of candidate strains. This platform consists of a tryptophan-specific aptamer-based biosensor and fluorescence-activated droplet sorting technology. One mutant strain, with a 165.9% increase in Trp titer compared with the parental strain, was successfully screened from a random mutagenesis library. Sequencing results revealed that a total of 10 single-nucleotide polymorphisms were discovered in the genome of the mutant strain, among which CRP(T29K) was proven to significantly increase Trp production through improving the strain's tolerance of the harsh environment during the stationary phase of the fermentation process. Our results indicate that this strategy has great potential for improving the production of other amino acids in Escherichia coli.

Legume plants are able to establish nitrogen-fixing symbiotic relations with Rhizobium bacteria. This symbiosis is, however, affected by a number of abiotic constraints, particularly drought. One of the consequences of drought stress is the overproduction of reactive oxygen (ROS) and nitrogen species (RNS), leading to cellular damage and, ultimately, cell death. Ascorbic acid (AsA), also known as vitamin C, is one of the antioxidant compounds that plants synthesize to counteract this oxidative damage. One promising strategy for the improvement of plant growth and symbiotic performance under drought stress is the overproduction of AsA via the overexpression of enzymes in the Smirnoff-Wheeler biosynthesis pathway. In the current work, we generated Medicago truncatula plants with increased AsA biosynthesis by overexpressing MtVTC2, a gene coding for GDP-L-galactose phosphorylase. We characterized the growth and physiological responses of symbiotic plants both under well-watered conditions and during a progressive water deficit. Results show that increased AsA availability did not provide an advantage in terms of plant growth or symbiotic performance either under well-watered conditions or in response to drought. This work had been funded by the Spanish Ministry of Science and Innovation-European Regional Development Fund (grant RTI2018-094623-B-C22) and the Government of Navarra (project PC112-113 LEGUSI). EL was a Ramón y Cajal fellow (RYC2018-023867-I) and LC-P and MR were Formación de Personal Investigador fellows from the Spanish Ministry of Economy and Competitiveness (BES-2015-074411 and BES-2012-059972, respectively).

Violacein is a naturally occurring purple pigment, widely used in cosmetics and has potent antibacterial and antiviral properties. Violacein can be produced from tryptophan, consequently sufficient tryptophan biosynthesis is the key to violacein production. However, the complicated biosynthetic pathways and regulatory mechanisms often make the tryptophan overproduction challenging in Escherichia coli. In this study, we used the adaptive laboratory evolution (ALE) strategy to improve violacein production using galactose as a carbon source. During the ALE, a tryptophan-responsive biosensor was employed to provide selection pressure to enrich tryptophan-producing cells. From the biosensor-assisted ALE, we obtained an evolved population of cells capable of effectively catabolizing galactose to tryptophan and subsequently used the population to obtain the best violacein producer. In addition, whole-genome sequencing of the evolved strain identified point mutations beneficial to the overproduction. Overall, we demonstrated that the biosensor-assisted ALE strategy could be used to rapidly and selectively evolve the producers to yield high violacein production.

Some macrolides such as 14- and 15-membered macrolides have immunomodulatory effects such as suppression of mucin overproduction. Because a novel macrolide, solithromycin, was developed, we examined whether it suppresses the overexpression of mucin in vitro. A human airway epithelial cell line NCI–H292 was stimulated by Pseudomonas aeruginosa lipopolysaccharides to induce the overproduction of a major mucin, MUC5AC. Treatment with 10 μg/mL of solithromycin significantly inhibited LPS-induced MUC5AC in both mRNA and protein levels as well as a 15-membered macrolide, azithromycin. These findings support that solithromycin has a potential immunomodulatory effect.

The pressure injury environment is characterized by overproduction of reactive oxygen species and exacerbated inflammation, which impair the healing of these lesions. Mediterranean-like diet may be a good intervention to improve the healing of pressure injury owing to its anti-inflammatory and antioxidant components. Thus, this study evaluated the hypothesis that olive oil, as a main source of lipid in Mediterranean diet, could improve cutaneous wound healing of pressure injury in mice. Male Swiss mice were randomly divided into standard, olive oil, or soybean oil plus olive oil groups and fat represented 10% of total calories in all groups. Four weeks after the beginning of diet administration, 2 cycles of ischemia–reperfusion (IR) by external application of 2 magnets disks were performed in the dorsal skin to induce pressure injury formation. Fourteen days after the end of the second IR cycle, olive oil-based diet reduced neutrophils cells and cyclooxygenase-2 protein expression and increased nitric oxide synthase-2 and protein and lipid oxidation. Olive oil based-diet also increased nuclear factor erythroid 2-related factor 2 protein expression and collagen type I precursor protein expression. In addition, administration of olive oil-based diet promoted wound closure at 7, 10, and 14 days after the end of the second IR cycle. These findings support the hypothesis that olive oil-based diet improves cutaneous wound healing of pressure injury in mice through the reduction of inflammation and stimulation of redox equilibrium.

Microbial production of steroid drugs exhibits great potentials in much greener and more sustainable manners, in which engineering multiple cytochrome P450s is the prerequisite requirement. The pairing of multicomponents of P450 systems is a tremendous challenge. Herein, biosynthesis of pregnenolone (a common precursor of steroid drugs) in Yarrowia lipolytica was taken as a typical instance to explore the engineering strategy of the cytochrome P450 side-chain cleavage enzyme (P450scc) system. The mature forms of the components belonging to P450scc system, CYP11A1, adrenodoxin (Adx), and adrenodoxin reductase (AdR), were coexpressed in a former constructed campesterol producing strain. To maximize pregnenolone production, an integrative components pairing strategy was proposed for pairing the component sources and balancing the expression levels of CYP11A1, Adx, and AdR. Led by the above approaches, a 2344-fold improvement of pregnenolone titer was achieved at the shake flask level. Consequently, a highest reported pregnenolone titer of 78.0 mg/L in microbes was obtained in a 5 L bioreactor. Our study not only highlights the integrative components pairing of cytochrome P450scc as a general strategy for engineering other cytochrome P450s, but also provides a feasible and efficient platform of Y. lipolytica for other steroids production.

α-Pinene is an important monoterpene, which is widely used as a flavoring agent and in fragrances, pharmaceuticals and biofuels. Although an evolved strain Escherichia coli YZFP, which had higher tolerance to pinene and titer, has been successfully used to produce high levels of pinene, the pinene titer is much lower than that of hemiterpene (isoprene) and sesquiterpenes (farnesene) to date. Moreover, the overall cellular physiological and metabolic changes caused by higher tolerance to pinene and overproduction of pinene remains unclear. To reveal the mechanism of Escherichia coli YZFP with the higher tolerance to pinene and titer, a comparative genomics and transcriptional level analyses combining with CRISPR activation (CRISPRa) and interference (CRISPRi) were carried out. The results show that the tolerance to pinene and the overproduction of pinene in E. coli may be associated with: 1) the mutations of the DXP pathway genes, the rpoA and some membrane protein genes, and their upregulations of transcription levels; and 2) the mutations of some genes and their downregulation of transcriptional levels. These comparative omics analyses provided some genetic modification strategies to further improve pinene production. Overexpression of the mutated cbpA, tabA, pitA, rpoA, sufBCDS, mutS, ispH, oppF, dusB, dnaK, dxs, dxr and flgFGH genes further improved pinene production. This study also demonstrated that combining comparative omics analysis with CRISPRa and CRISPRi is an efficient technology to quickly find a new metabolic engineering strategy., Highlights • A genomics and transcriptional level analyses combining with CRISPRa and CRISPRi was carried out. • The mechanism of the tolerance to pinene and overproduction of pinene was obtained. • Some target genes difficultly found by rational analysis were identified. • Combining comparative omics analysis with CRISPRa/i is an efficient technology for metabolic engineering.

As an opportunistic bacterial pathogen, Pseudomonas aeruginosa PAO1 contains two phenazineproducing gene operons, phzA1B1C1D1E1F1G1 (phz1) and phzA2B2C2D2E2F2G2 (phz2), each of which is independently capable of encoding all enzymes for biosynthesizing phenazines, including phenazine-1-carboxylic acid and its derivatives. Other previous study reported that the RpoS-deficient mutant SS24 overproduced pyocyanin, a derivative of phenazine-1- carboxylic acid. However, it is not known how RpoS mediates the expression of two phz operons and regulates pyocyanin biosynthesis in detail. In this study, with deletion of the rpoS gene in the PAΔphz1 mutant and the PAΔphz2 mutant respectively, we demonstrated that RpoS exerted opposite regulatory roles on the expression of the phz1and phz2 operons. We also confirmed that the phz1 operon played a critical role and especially biosynthesized much more phenazines than the phz2 operon when the rpoS gene was knocked out in P. aeruginosa. By constructing the translational reporter fusion vector lasR'-'lacZ and the chromosomal fusion mutant PAΔlasR::lacZ, we verified that RpoS deficiency caused increased expression of lasR, a transcription regulator gene in a first quorum sensing system (las) that activates overexpression of the phz1 operon, suggesting that in the absence of RpoS, LasR might act as an intermediate in overproduction of phenazine biosynthesis mediated by the phz1 operon in P. aeruginosa.

Removing transcriptional feedback regulation of metabolic pathways is a classical approach to enhance overproduction of chemicals in microbes. However, disrupting transcriptional regulation can have broad physiological consequences that decrease cellular growth and productivity. Here, we compared downregulation and deletion of the transcriptional repressor ArgR in arginine overproducing Escherichia coli. Different levels of ArgR downregulation were achieved with CRISPR interference (CRISPRi) and resulted in 2-times higher growth rates compared to deletion of ArgR, while specific arginine production was similar (∼2 mmol gDW-1 h-1). Metabolomics and proteomics data revealed that poor growth of the ArgR deletion strain was caused by a limitation of pyrimidine nucleotide biosynthesis, because a 17-fold overexpression of ornithine carbamoyltransferase (ArgI) perturbed the arginine-pyrimidine branch point. These results demonstrate that overexpression of enzymes in an engineered pathway can impair metabolism of the host, especially in the case of branch-point enzymes. Thus, balancing enzyme levels is important to optimize industrial microbes, and CRISPRi of a transcription factor is a versatile tool for this purpose.

Genomic analysis of the clavulanic acid (CA)-high-producing Streptomyces clavuligerus strains, OL13 and OR, developed through random mutagenesis revealed a frameshift mutation in the cas1 gene-encoding clavaminate synthase 1. Overexpression of the intact cas1 in S. clavuligerus OR enhanced the CA titer by approximately 25%, producing ~ 4.95 g/L of CA, over the OR strain in the flask culture. Moreover, overexpression of the pathway-specific positive regulatory genes, ccaR and claR, in the OR strain improved CA yield by approximately 43% (~ 5.66 g/L) in the flask. However, co-expression of the intact cas1 with ccaR-claR did not further improve CA production. In the 7 L fermenter culture, maximum CA production by the OR strain expressing the wild-type cas1 and ccaR-claR reached approximately 5.52 g/L and 6.01 g/L, respectively, demonstrating that reverse engineering or simple rational metabolic engineering is an efficient method for further improvement of industrial strains.

リファンピシンが粘液活性を減少させ、高病原性肺炎桿菌の病原性を低下させることを見出した。, A recent increase in the incidence of hypervirulent Klebsiella pneumoniae (hvKP) infections, especially those caused by a sublineage of the clonal group CG23 (CG23-I), is raising serious health concerns worldwide. The high virulence of hvKP is, at least in part, attributed to the overproduction of capsular polysaccharide (CPS), which is triggered by a positive regulator of capsular polysaccharide synthesis (cps) genes, named RmpA. Although extensive research has been conducted on the mechanisms ofhvKP virulence, no study has focused on the development of anti-virulence therapeutics....

Streptomyces is currently the main producer of microbial pharmaceuticals from its secondary metabolites as natural products. It will be more beneficial if the promoters, which are particularly strong during the secondary metabolism of Streptomyces, are used to drive the efficient production of desired natural products with the coordination of bacterial growth. Here, in an industrial natamycin producer Streptomyces chattanoogensis L10, a strong promoter groESp was identified for this purpose based on the comparative proteomic analysis of the primary and secondary metabolism. With a constitutive promoter ermEp* as a control, the activity of groESp was weak in the primary metabolism, but about sixfold higher than ermEp* in the secondary metabolism, when the representative antibiotic natamycin was highly produced. Furthermore, when ScnRII, a pathway-specific positive regulator in natamycin biosynthesis, was expressed under groESp, the productivity of natamycin was about 20% higher in the secondary metabolism than that from ermEp*, but had no discrimination in the early 2 days. Thus, we showed that proteomics is an effective alternative way to identify promoters for the high yield of natamycin in S. chattanoogensis, and this strategy can be widely adaptable to other Streptomyces species for the full development of secondary metabolites with promising bioactivities.