Ligand recognition and allosteric regulation of DRD1-Gs signaling complexes.

Dopamine receptors, including D1- and D2-like receptors, are important therapeutic targets in a variety of neurological syndromes, as well as cardiovascular and kidney diseases. Here, we present five cryoelectron microscopy (cryo-EM) structures of the dopamine D1 receptor (DRD1) coupled to Gs heterotrimer in complex with three catechol-based agonists, a non-catechol agonist, and a positive allosteric modulator for endogenous dopamine. These structures revealed that a polar interaction network is essential for catecholamine-like agonist recognition, whereas specific motifs in the extended binding pocket were responsible for discriminating D1- from D2-like receptors. Moreover, allosteric binding at a distinct inner surface pocket improved the activity of DRD1 by stabilizing endogenous dopamine interaction at the orthosteric site. DRD1-Gs interface revealed key features that serve as determinants for G protein coupling. Together, our study provides a structural understanding of the ligand recognition, allosteric regulation, and G protein coupling mechanisms of DRD1.

CD1a on Langerhans cells controls inflammatory skin disease.

CD1a is a lipid-presenting molecule that is abundantly expressed on Langerhans cells. However, the in vivo role of CD1a has remained unclear, principally because CD1a is lacking in mice. Through the use of mice with transgenic expression of CD1a, we found that the plant-derived lipid urushiol triggered CD1a-dependent skin inflammation driven by CD4(+) helper T cells that produced the cytokines IL-17 and IL-22 (TH17 cells). Human subjects with poison-ivy dermatitis had a similar cytokine signature following CD1a-mediated recognition of urushiol. Among various urushiol congeners, we identified diunsaturated pentadecylcatechol (C15:2) as the dominant antigen for CD1a-restricted T cells. We determined the crystal structure of the CD1a-urushiol (C15:2) complex, demonstrating the molecular basis of urushiol interaction with the antigen-binding cleft of CD1a. In a mouse model and in patients with psoriasis, CD1a amplified inflammatory responses that were mediated by TH17 cells that reacted to self lipid antigens. Treatment with blocking antibodies to CD1a alleviated skin inflammation. Thus, we propose CD1a as a potential therapeutic target in inflammatory skin diseases.

A genome assembly of ginger (Zingiber officinale Roscoe) provides insights into genome evolution and 6-gingerol biosynthesis.

Ginger is cultivated in tropical and subtropical regions and is one of the most crucial spices worldwide owing to its special taste and scent. Here, we present a high-quality genome assembly for 'Small Laiwu Ginger', a famous cultivated ginger in northern China. The ginger genome was phased into two haplotypes, haplotype A (1.55Gb), and haplotype B (1.44Gb). Analysis of Ty1/Copia and Ty3/Gypsy LTR retrotransposon families revealed that both have undergone multiple retrotransposon bursts about 0-1 million years ago. In addition to a recent whole-genome duplication event, there has been a lineage-specific expansion of genes involved in stilbenoid, diarylheptanoid, and gingerol biosynthesis, thereby enhancing 6-gingerol biosynthesis. Furthermore, we focused on the biosynthesis of 6-gingerol, the most important gingerol, and screened key transcription factors ZoMYB106 and ZobHLH148 that regulate 6-gingerol synthesis by transcriptomic and metabolomic analysis in the ginger rhizome at four growth stages. The results of yeast one-hybrid, electrophoretic mobility shift, and dual-luciferase reporter gene assays showed that both ZoMYB106 and ZobHLH148 bind to the promoters of the key rate-limiting enzyme genes ZoCCOMT1 and ZoCCOMT2 in the 6-gingerol synthesis pathway and promote their transcriptional activities. The reference genome, transcriptome, and metabolome data pave the way for further research on the molecular mechanism underlying the biosynthesis of 6-gingerol. Furthermore, it provides precious new resources for the study on the biology and molecular breeding of ginger.

Comparing in planta accumulation with microbial routes to set targets for a cost-competitive bioeconomy.

Plants and microbes share common metabolic pathways for producing a range of bioproducts that are potentially foundational to the future bioeconomy. However, in planta accumulation and microbial production of bioproducts have never been systematically compared on an economic basis to identify optimal routes of production. A detailed technoeconomic analysis of four exemplar compounds (4-hydroxybenzoic acid [4-HBA], catechol, muconic acid, and 2-pyrone-4,6-dicarboxylic acid [PDC]) is conducted with the highest reported yields and accumulation rates to identify economically advantaged platforms and breakeven targets for plants and microbes. The results indicate that in planta mass accumulation ranging from 0.1 to 0.3 dry weight % (dwt%) can achieve costs comparable to microbial routes operating at 40 to 55% of maximum theoretical yields. These yields and accumulation rates are sufficient to be cost competitive if the products are sold at market prices consistent with specialty chemicals ($20 to $50/kg). Prices consistent with commodity chemicals will require an order-of-magnitude-greater accumulation rate for plants and/or yields nearing theoretical maxima for microbial production platforms. This comparative analysis revealed that the demonstrated accumulation rates of 4-HBA (3.2 dwt%) and PDC (3.0 dwt%) in engineered plants vastly outperform microbial routes, even if microbial platforms were to reach theoretical maximum yields. Their recovery and sale as part of a lignocellulosic biorefinery could enable biofuel prices to be competitive with petroleum. Muconic acid and catechol, in contrast, are currently more attractive when produced microbially using a sugar feedstock. Ultimately, both platforms can play an important role in replacing fossil-derived products.

Biodegradation of chloroethene compounds under microoxic conditions.

The biodegradation of chloroethene compounds under oxic and anoxic conditions is well established. However, the biological reactions that take place under microoxic conditions are unknown. Here, we report the biostimulated (BIOST: addition of lactate) and natural attenuated (NAT) degradation of chloroethene compounds under microoxic conditions by bacterial communities from chloroethene compounds-contaminated groundwater. The degradation of tetrachloroethene was significantly higher in NAT (15.14% on average) than in BIOST (10.13% on average) conditions at the end of the experiment (90 days). Sporomusa, Paracoccus, Sedimentibacter, Pseudomonas, and Desulfosporosinus were overrepresented in NAT and BIOST compared to the source groundwater. The NAT metagenome contains phenol hydrolase P1 oxygenase (dmpL), catechol-1,2-dioxygenase (catA), catechol-2,3-dioxygenases (dmpB, todE, and xylE) genes, which could be involved in the cometabolic degradation of chloroethene compounds; and chlorate reductase (clrA), that could be associated with partial reductive dechlorination of chloroethene compounds. Our data provide a better understanding of the bacterial communities, genes, and pathways potentially implicated in the reductive and cometabolic degradation of chloroethene compounds under microoxic conditions.

Oxidation of dopamine and related catechols in dopaminergic brain regions in Parkinson's disease and during ageing in non-Parkinsonian subjects.

The present study was performed to examine if catechol oxidation is higher in brains from patients with Parkinson's disease compared to age-matched controls, and if catechol oxidation increases with age. Brain tissue from Parkinson patients and age-matched controls was examined for oxidation of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC) and 3,4-dihydroxyphenylalanine (DOPA) to corresponding quinones, by measurement of 5-S-cysteinyl-dopamine, 5-S-cysteinyl-DOPAC and 5-S-cysteinyl-DOPA. The cysteinyl catechols are assumed to be biomarkers for DA, DOPAC and DOPA autoxidation and part of the biosynthetic pathway of neuromelanin. The concentrations of the 5-S-cysteinyl catechols were lower, whereas the 5-S-cysteinyl-DA/DA and 5-S-cysteinyl-DOPAC/DOPAC ratios tended to be higher in the Parkinson group compared to controls, which was interpreted as a higher degree of oxidation. High 5-S-cysteinyl-DA/DA ratios were found in the substantia nigra of a sub-population of the Parkinson group. Based on 5-S-cysteinyl-DA/DA ratios, dopamine oxidation was found to increase statistically significantly with age in the caudate nucleus, and non-significantly in the substantia nigra. In conclusion, the occurrence of 5-S-cysteinyl-DA, 5-S-cysteinyl-DOPAC and 5-S-cysteinyl-DOPA was demonstrated in dopaminergic brain areas of humans, a tendency for higher oxidation of DA in the Parkinson group compared to controls was observed as well as a statistically significant increase in DA oxidation with age. Possibly, autoxidation of DA and other catechols are involved in both normal and pathological ageing of the brain. This study confirms one earlier but small study, as well as complements one study on non-PD cases and one study on both PD cases and controls on NM bound or integrated markers or catechols.

SifR is an Rrf2-family quinone sensor associated with catechol iron uptake in Streptococcus pneumoniae D39.

Streptococcus pneumoniae (pneumococcus) is a Gram-positive commensal and human respiratory pathogen. How this bacterium satisfies its nutritional iron (Fe) requirement in the context of endogenously produced hydrogen peroxide is not well understood. Here, we characterize a novel virulence-associated Rrf2-family transcriptional repressor that we term SifR (streptococcal IscR-like family transcriptional repressor) encoded by spd_1448 and conserved in Streptococci. Global transcriptomic analysis of a ΔsifR strain defines the SifR regulon as genes encoding a candidate catechol dioxygenase CatE, an uncharacterized oxidoreductase YwnB, a candidate flavin-dependent ferric reductase YhdA, a candidate heme-based ferric reductase domain-containing protein and the Piu (pneumococcus iron uptake) Fe transporter (piuBCDA). Previous work established that membrane-anchored PiuA binds FeIII-bis-catechol or monocatechol complexes with high affinity, including the human catecholamine stress hormone, norepinephrine. We demonstrate that SifR senses quinone via a single conserved cysteine that represses its regulon when in the reduced form. Upon reaction with catechol-derived quinones, we show that SifR dissociates from the DNA leading to regulon derepression, allowing the pneumococcus to access a catechol-derived source of Fe while minimizing reactive electrophile stress induced by quinones. Consistent with this model, we show that CatE is an FeII-dependent 2,3-catechol dioxygenase with broad substrate specificity, YwnB is an NAD(P)H-dependent quinone reductase capable of reducing the oxidized and cyclized norepinephrine, adrenochrome, and YhdA is capable of reducing a number of FeIII complexes, including PiuA-binding transport substrates. These findings are consistent with a model where FeIII-catechol complexes serve as significant nutritional Fe sources in the host.

Systems metabolic engineering upgrades Corynebacterium glutamicum to high-efficiency cis, cis-muconic acid production from lignin-based aromatics.

Lignin displays a highly challenging renewable. To date, massive amounts of lignin, generated in lignocellulosic processing facilities, are for the most part merely burned due to lacking value-added alternatives. Aromatic lignin monomers of recognized relevance are in particular vanillin, and to a lesser extent vanillate, because they are accessible at high yield from softwood-lignin using industrially operated alkaline oxidative depolymerization. Here, we metabolically engineered C. glutamicum towards cis, cis-muconate (MA) production from these key aromatics. Starting from the previously created catechol-based producer C. glutamicum MA-2, systems metabolic engineering first discovered an unspecific aromatic aldehyde reductase that formed aromatic alcohols from vanillin, protocatechualdehyde, and p- hydroxybenzaldehyde, and was responsible for the conversion up to 57% of vanillin into vanillyl alcohol. The alcohol was not re-consumed by the microbe later, posing a strong drawback on the producer. The identification and subsequent elimination of the encoding fudC gene completely abolished vanillyl alcohol formation. Second, the initially weak flux through the native vanillin and vanillate metabolism was enhanced up to 2.9-fold by implementing synthetic pathway modules. Third, the most efficient protocatechuate decarboxylase AroY for conversion of the midstream pathway intermediate protocatechuate into catechol was identified out of several variants in native and codon optimized form and expressed together with the respective helper proteins. Fourth, the streamlined modules were all genomically combined which yielded the final strain MA-9. MA-9 produced bio-based MA from vanillin, vanillate, and seven structurally related aromatics at maximum selectivity. In addition, MA production from softwood-based vanillin, obtained through alkaline depolymerization, was demonstrated.

High-efficient production of mushroom polyketide compounds in a platform host Aspergillus oryzae.

BACKGROUND: Orsellinic acid (2,4-dihydroxy-6-methylbenzoic acid, OA) and its structural analog o-Orsellinaldehyde, have become widely used intermediates in clinical drugs synthesis. Although the research on the biosynthesis of such compounds has made significant progress, due to the lack of suitable hosts, there is still far from the industrial production of such compounds based on synthetic biology. RESULTS: With the help of genome mining, we found a polyketide synthase (PKS, HerA) in the genome of the Hericium erinaceus, which shares 60% amino acid sequence homology with ArmB from Armillaria mellea, an identified PKS capable of synthesizing OA. To characterize the function of HerA, we cloned herA and heterologously expressed it in Aspergillus oryzae, and successfully detected the production of OA. Subsequently, the introduction of an incomplete PKS (Pks5) from Ustilago maydis containing only three domains (AMP-ACP-R), which was into herA-containing A. oryzae, the resulted in the production of o-Orsellinaldehyde. Considering the economic value of OA and o-Orsellinaldehyde, we then optimized the yield of these compounds in A. oryzae. The screening showed that when maltose was used as carbon source, the yields of OA and o-Orsellinaldehyde were 57.68 mg/L and 15.71 mg/L respectively, while the yields were 340.41 mg/Kg and 84.79 mg/Kg respectively in rice medium for 10 days. CONCLUSIONS: Herein, we successfully expressed the genes of basidiomycetes using A. oryzae heterologous host. As a fungus of ascomycetes, which not only correctly splices genes of basidiomycetes containing multiple introns, but also efficiently produces their metabolites. This study highlights that A. oryzae is an excellent host for the heterologous production of fungal natural products, and has the potential to become an efficient chassis for the production of basidiomycete secondary metabolites in synthetic biology.

Compartmentalized processing of catechols during mussel byssus fabrication determines the destiny of DOPA.

Inspired largely by the role of the posttranslationally modified amino acid dopa (DOPA) in mussel adhesion, catechol functional groups have become commonplace in medical adhesives, tissue scaffolds, and advanced smart polymers. Yet, the complex redox chemistry of catechol groups complicates cross-link regulation, hampering fabrication and the long-term stability/performance of mussel-inspired polymers. Here, we investigated the various fates of DOPA residues in proteins comprising mussel byssus fibers before, during, and after protein secretion. Utilizing a combination of histological staining and confocal Raman spectroscopy on native tissues, as well as peptide-based cross-linking studies, we have identified at least two distinct DOPA-based cross-linking pathways during byssus fabrication, achieved by oxidative covalent cross-linking or formation of metal coordination interactions under reducing conditions, respectively. We suggest that these end states are spatiotemporally regulated by the microenvironments in which the proteins are stored prior to secretion, which are retained after formation-in particular, due to the presence of reducing moieties. These findings provide physicochemical pathways toward greater control over properties of synthetic catechol-based polymers and adhesives.

Transcriptomic profiling reveals the molecular responses of Rhodococcus aetherivorans DMU1 to skatole stress.

Skatole is a typical malodor compound in animal wastes. Several skatole-degrading bacterial strains have been obtained, whereas the molecular response of strains to skatole stress has not been well elucidated. Herein, the skatole degradation by a Gram-positive strain Rhodococcus aetherivorans DMU1 was investigated. Strain DMU1 showed high efficiency in skatole degradation under the conditions of 25-40 °C and pH 7.0-10.0. It could utilize various aromatics, including cresols, phenol, and methylindoles, as the sole carbon source for growth, implying its potential in the bioremediation application of animal wastes. Transcriptomic sequencing revealed that 328 genes were up-regulated and 640 genes were down-regulated in strain DMU1 when grown in the skatole-containing medium. Skatole increased the gene expression levels of antioxidant defense systems and heat shock proteins. The expression of ribosome-related genes was significantly inhibited which implied the growth inhibition of skatole. A rich set of oxidoreductases were changed, and a novel gene cluster containing the flavoprotein monooxygenase and ring-hydroxylating oxygenase genes was highly up-regulated, which was probably involved in skatole upstream degradation. The upregulation pattern of this gene cluster was further verified by qRT-PCR assay. Furthermore, skatole should be mainly degraded via the catechol ortho-cleavage pathway with cat25170 as the functional gene. The gene cat25170 was cloned and expressed in E. coli BL21(DE3). Pure enzyme assays showed that Cat25170 could catalyze catechol with Km 9.96 µmol/L and kcat 12.36 s-1.

Polycatechols with Robust Efficiency in Cytosolic Peptide Delivery via Catechol-Boronate Chemistry.

Cytosolic delivery of peptides remains a challenging task because of the limited binding sites on peptides and the existence of multiple intracellular barriers. Here, we proposed the use of polycatechols with a high cell permeability to deliver peptides of different physicochemical properties using the catechol-boronate chemistry. Peptides were decorated with boronate moieties via three strategies, and the introduced boronate groups greatly increased the binding affinity of cargo peptides with polycatechols. The loading peptides could be released under the endolysosomal acidity. When the cargo peptide was modified with boronate moiety via a p-hydroxybenzylcarbamate self-immolative spacer, it could be loaded by polycatechols and released in a traceless manner triggered by reactive oxygen species. The proposed strategies greatly promote the cytosolic delivery efficiency of different peptides into various cell lines and restored their biofunctions after intracellular delivery and release. This study provides a general and robust platform for the intracellular delivery of membrane-impermeable peptides.

Conjunctive Analyses of BSA-Seq and BSR-Seq to Identify Candidate Genes Controlling the Black Lemma and Pericarp Trait in Barley.

Black barley seeds are a health-beneficial diet resource because of their special chemical composition and antioxidant properties. The black lemma and pericarp (BLP) locus was mapped in a genetic interval of 0.807 Mb on chromosome 1H, but its genetic basis remains unknown. In this study, targeted metabolomics and conjunctive analyses of BSA-seq and BSR-seq were used to identify candidate genes of BLP and the precursors of black pigments. The results revealed that five candidate genes, purple acid phosphatase, 3-ketoacyl-CoA synthase 11, coiled-coil domain-containing protein 167, subtilisin-like protease, and caffeic acid-O-methyltransferase, of the BLP locus were identified in the 10.12 Mb location region on the 1H chromosome after differential expression analysis, and 17 differential metabolites, including the precursor and repeating unit of allomelanin, were accumulated in the late mike stage of black barley. Phenol nitrogen-free precursors such as catechol (protocatechuic aldehyde) or catecholic acids (caffeic, protocatechuic, and gallic acids) may promote black pigmentation. BLP can manipulate the accumulation of benzoic acid derivatives (salicylic acid, 2,4-dihydroxybenzoic acid, gallic acid, gentisic acid, protocatechuic acid, syringic acid, vanillic acid, protocatechuic aldehyde, and syringaldehyde) through the shikimate/chorismite pathway other than the phenylalanine pathway and alter the metabolism of the phenylpropanoid-monolignol branch. Collectively, it is reasonable to infer that black pigmentation in barley is due to allomelanin biosynthesis in the lemma and pericarp, and BLP regulates melanogenesis by manipulating the biosynthesis of its precursors.

Unraveling the skatole biodegradation process in an enrichment consortium using integrated omics and culture-dependent strategies.

3-Methylindole (skatole) is regarded as one of the most offensive compounds in odor emission. Biodegradation is feasible for skatole removal but the functional species and genes responsible for skatole degradation remain enigmatic. In this study, an efficient aerobic skatole-degrading consortium was obtained. Rhodococcus and Pseudomonas were identified as the two major and active populations by integrated metagenomic and metatranscriptomic analyses. Bioinformatic analyses indicated that the skatole downstream degradation was mainly via the catechol pathway, and upstream degradation was likely catalyzed by the aromatic ring-hydroxylating oxygenase and flavin monooxygenase. Genome binning and gene analyses indicated that Pseudomonas, Pseudoclavibacter, and Raineyella should cooperate with Rhodococcus for the skatole degradation process. Moreover, a pure strain Rhodococcus sp. DMU1 was successfully obtained which could utilize skatole as the sole carbon source. Complete genome sequencing showed that strain DMU1 was the predominant population in the consortium. Further crude enzyme and RT-qPCR assays indicated that strain DMU1 degraded skatole through the catechol ortho-cleavage pathway. Collectively, our results suggested that synergistic degradation of skatole in the consortium should be performed by diverse bacteria with Rhodococcus as the primary degrader, and the degradation mainly proceeded via the catechol pathway.

Biobased PET from lignin using an engineered cis, cis-muconate-producing Pseudomonas putida strain with superior robustness, energy and redox properties.

Polyethylene terephthalate (PET), the most common synthetic polyester today, is largely produced from fossil resources, contributing to global warming. Consequently, sustainable sources must be developed to meet the increasing demand for this useful polymer. Here, we demonstrate a cascaded value chain that provides green PET from lignin, the world's most underutilized renewable, via fermentative production of cis, cis-muconate (MA) from lignin-based aromatics as a central step. Catechol, industrially the most relevant but apparently also a highly toxic lignin-related aromatic, strongly inhibited MA-producing Pseudomonas putida MA-1. Assessed by 13C metabolic flux analysis, the microbe substantially redirected its carbon core fluxes, resulting in enhanced NADPH supply for stress defense but causing additional ATP costs. The reconstruction of MA production in a genome-reduced P. putida chassis yielded novel producers with superior pathway fluxes and enhanced robustness to catechol and a wide range of other aromatics. Using the advanced producer P. putida MA-10 catechol, MA could be produced in a fed-batch process from catechol (plus glucose as additional growth substrate) up to an attractive titer of 74 g L-1 and a space-time-yield of 1.4 g L-1 h-1. In terms of co-consumed sugar, the further streamlined strain MA-11 achieved the highest yield of 1.4 mol MA (mol glucose)-1, providing a striking economic advantage. Following fermentative production, bio-based MA was purified and used to chemically synthetize the PET monomer terephthalic acid and the comonomer diethylene glycol terephthalic acid through five steps, which finally enabled the first green PET from lignin.

Degradation of salicylic acid to catechol in Solanaceae by SA 1-hydroxylase.

The hormone salicylic acid (SA) plays crucial roles in plant defense, stress responses, and in the regulation of plant growth and development. Whereas the biosynthetic pathways and biological functions of SA have been extensively studied, SA catabolism is less well understood. In this study, we report the identification and functional characterization of an FAD/NADH-dependent SA 1-hydroxylase from tomato (Solanum lycopersicum; SlSA1H), which catalyzes the oxidative decarboxylation of SA to catechol. Transcript levels of SlSA1H were highest in stems and its expression was correlated with the formation of the methylated catechol derivatives guaiacol and veratrole. Consistent with a role in SA catabolism, SlSA1H RNAi plants accumulated lower amounts of guaiacol and failed to produce any veratrole. Two O-methyltransferases involved in the conversion of catechol to guaiacol and guaiacol to veratrole were also functionally characterized. Subcellular localization analyses revealed the cytosolic localization of this degradation pathway. Phylogenetic analysis and functional characterization of SA1H homologs from other species indicated that this type of FAD/NADH-dependent SA 1-hydroxylases evolved recently within the Solanaceae family.

Characterization of two 1,2,4-trihydroxybenzene 1,2-dioxygenases from Phanerochaete chrysosporium.

Lignin is the most abundant aromatic compound in nature, and it plays an important role in the carbon cycle. White-rot fungi are microbes that are capable of efficiently degrading lignin. Enzymes from these fungi possess exceptional oxidative potential and have gained increasing importance for improving bioprocesses, such as the degradation of organic pollutants. The aim of this study was to identify the enzymes involved in the ring cleavage of the lignin-derived aromatic 1,2,4-trihydroxybenzene (THB) in Phanerochaete chrysosporium, a lignin-degrading basidiomycete. Two intradiol dioxygenases (IDDs), PcIDD1 and PcIDD2, were identified and produced as recombinant proteins in Escherichia coli. In the presence of O2, PcIDD1 and PcIDD2 acted on eight and two THB derivatives, respectively, as substrates. PcIDD1 and PcIDD2 catalyze the ring cleavage of lignin-derived fragments, such as 6-methoxy-1,2,4-trihydroxybenzene (6-MeOTHB) and 3-methoxy-1,2-catechol. The current study also revealed that syringic acid (SA) was converted to 5-hydroxyvanillic acid, 2,6-dimethoxyhydroquinone, and 6-MeOTHB by fungal cells, suggesting that PcIDD1 and PcIDD2 may be involved in aromatic ring fission of 6-MeOTHB for SA degradation. This is the first study to show 6-MeOTHB dioxygenase activity of an IDD superfamily member. These findings highlight the unique and broad substrate spectra of PcIDDs, rendering it an attractive candidate for biotechnological application. KEY POINTS: • Novel intradiol dioxygenases (IDD) in lignin degradation were characterized. • PcIDDs acted on lignin-derived fragments and catechol derivatives. • Dioxygenase activity on 6-MeOTHB was identified in IDD superfamily enzymes.

Phenotypic Adaption of Pseudomonas aeruginosa by Hacking Siderophores Produced by Other Microorganisms.

Bacteria secrete siderophores to access iron, a key nutrient poorly bioavailable and the source of strong competition between microorganisms in most biotopes. Many bacteria also use siderophores produced by other microorganisms (exosiderophores) in a piracy strategy. Pseudomonas aeruginosa, an opportunistic pathogen, produces two siderophores, pyoverdine and pyochelin, and is also able to use a panel of exosiderophores. We first investigated expression of the various iron-uptake pathways of P. aeruginosa in three different growth media using proteomic and RT-qPCR approaches and observed three different phenotypic patterns, indicating complex phenotypic plasticity in the expression of the various iron-uptake pathways. We then investigated the phenotypic plasticity of iron-uptake pathway expression in the presence of various exosiderophores (present individually or as a mixture) under planktonic growth conditions, as well as in an epithelial cell infection assay. In all growth conditions tested, catechol-type exosiderophores were clearly more efficient in inducing the expression of their corresponding transporters than the others, showing that bacteria opt for the use of catechol siderophores to access iron when they are present in the environment. In parallel, expression of the proteins of the pyochelin pathway was significantly repressed under most conditions tested, as well as that of proteins of the pyoverdine pathway, but to a lesser extent. There was no effect on the expression of the heme and ferrous uptake pathways. Overall, these data provide precise insights on how P. aeruginosa adjusts the expression of its various iron-uptake pathways (phenotypic plasticity and switching) to match varying levels of iron and competition.

Engineered Pseudomonas putida for biosynthesis of catechol from lignin-derived model compounds and biomass hydrolysate.

Catechol is an industrially relevant chemical with myriad applications. Its production via chemical route suffers from several drawbacks the major being a non-green and nonselective route. Currently, bio-based products using biocatalyst are gaining attention due to the growing environmental and health hazards concerns over the use of petroleum-derived feedstock. Lignocellulosic biomass serves as a promising feedstock. Lignin valorization is the demand of the current scenario which is complicated task by its complexity, heterogeneity and diversity of lignin structures posing limitations toward lignin valorization via chemical routes. There are several microorganisms that possess the ability to metabolize lignin monomers via their central metabolic pathways and this paves the way to the synthesis of a number of products. Pseudomonas putida KT2440 is one such organism and was chosen for genetic manipulations for catechol biosynthesis using lignin-derived model compounds and biomass hydrolysate stream comprising of various lignin monomers. Catechol production was engineered by diverting various lignin monomers and addressing the identified metabolic bottlenecks particularly vanillic acid accumulation toward catechol biosynthesis. The engineered strain could convert the model lignin monomers as well as monomers in the biomass hydrolysates to catechol and vanillic acid in more than 60% and 90% molar yields, respectively.

Metabolic Engineering of Shikimic Acid Biosynthesis Pathway for the Production of Shikimic Acid and Its Branched Products in Microorganisms: Advances and Prospects.

The shikimate pathway is a necessary pathway for the synthesis of aromatic compounds. The intermediate products of the shikimate pathway and its branching pathway have promising properties in many fields, especially in the pharmaceutical industry. Many important compounds, such as shikimic acid, quinic acid, chlorogenic acid, gallic acid, pyrogallol, catechol and so on, can be synthesized by the shikimate pathway. Among them, shikimic acid is the key raw material for the synthesis of GS4104 (Tamiflu®), an inhibitor of neuraminidase against avian influenza virus. Quininic acid is an important intermediate for synthesis of a variety of raw chemical materials and drugs. Gallic acid and catechol receive widespread attention as pharmaceutical intermediates. It is one of the hotspots to accumulate many kinds of target products by rationally modifying the shikimate pathway and its branches in recombinant strains by means of metabolic engineering. This review considers the effects of classical metabolic engineering methods, such as central carbon metabolism (CCM) pathway modification, key enzyme gene modification, blocking the downstream pathway on the shikimate pathway, as well as several expansion pathways and metabolic engineering strategies of the shikimate pathway, and expounds the synthetic biology in recent years in the application of the shikimate pathway and the future development direction.