Identification of natural inhibitors to inhibit C. acnes lipase through docking and simulation studies.

Acne vulgaris is a common skin disease affecting 80-90% of teenagers worldwide. C. acnes producing lipases are the main virulence factor that catalyzes sebum lipid into free fatty acid that is used for C. acnes growth. Recently, computational biology and bioinformatics play a significant role in drug discovery programs and the identification of novel lead(s). In this study, potential inhibitors against the C. acnes lipase have been identified via cost-effective computational investigations. Molecular docking, MD simulations, and binding affinity analysis have been performed between the active site of C. acnes lipase protein and selected natural plant constituents. First, C. acnes lipase protein was downloaded from PDB and defined the catalytically active site. Next, 16 active natural plant constituents were shortlisted from the PubChem library (based on their pharmacokinetics, pharmacodynamics, and antibacterial activity). Docking studies identified the best five active compounds that showed significantly strong binding affinity interacted through hydrogen bonding, hydrophobic interactions, and π-stacking with the active site residues of the target protein. Furthermore, a 100 ns MD simulation run showed a stable RMSD and less fluctuating RMSF graph for luteolin and neryl acetate. In silico investigation suggested that luteolin, neryl acetate, and isotretinoin were involved in stable interactions which were maintained throughout the MD run with the C. acnes lipase enzyme, virtually. The results advocated that these could potentially inhibit lipase activity and be used in the clinical management of acne.

Propionibacterium (Cutibacterium) granulosum Extracellular DNase BmdE Targeting Propionibacterium (Cutibacterium) acnes Biofilm Matrix, a Novel Inter-Species Competition Mechanism.

Acne vulgaris is the most common dermatological disorder worldwide affecting more than 80% of adolescents and young adults with a global prevalence of 231 million cases in 2019. The involvement of the skin microbiome disbalance in the pathophysiology of acne is recognized, especially regarding the relative abundance and diversity of Propionibacterium acnes a well-known dominant human skin commensal. Biofilms, where bacteria are embedded into a protective polymeric extracellular matrix, are the most prevalent life style for microorganisms. P. acnes and its biofilm-forming ability is believed to be a contributing factor in the development of acne vulgaris, the persistence of the opportunistic pathogen and antibiotic therapy failures. Degradation of the extracellular matrix is one of the strategies used by bacteria to disperse the biofilm of competitors. In this study, we report the identification of an endogenous extracellular nuclease, BmdE, secreted by Propionibacterium granulosum able to degrade P. acnes biofilm both in vivo and in vitro. This, to our knowledge, may represent a novel competitive mechanism between two closely related species in the skin. Antibiotics targeting P. acnes have been the mainstay in acne treatment. Extensive and long-term use of antibiotics has led to the selection and spread of resistant bacteria. The extracellular DNase BmdE may represent a new bio-therapeutical strategy to combat P. acnes biofilm in acne vulgaris.

Rhein inhibits the growth of Propionibacterium acnes by blocking NADH dehydrogenase-2 activity.

Introduction. Rhein (4, 5-dihydroxyanthraquinone-2-carboxylic acid) has various properties, including anti-inflammatory, antioxidant and anticancer activities. However, the mechanism underlying the role of rhein in antimicrobial activity remains largely unknown.Aim. This study aims to identify potential natural compounds of rhein that are capable of inhibiting Cutibacterium acnes and elucidate the effects of rhein on NADH dehydrogenase-2 activity in C. acnes.Methodology. The anti-C. acnes activity of compounds was analysed using minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), the paper disc diffusion test and the checkerboard dilution test. To check whether rhein was inhibitory, putative type II NADH dehydrogenase (NDH-2) of C. acnes was analysed, cloned and expressed in Escherichia coli, and then NDH-2 purification was assessed with Ni-NTA before rhein inhibition of NADH dehydrogenase-2 activity was checked with ferricyanide [K3Fe(CN)6] as a substrate.Results. The results showed that the MIC of rhein against C. acnes was 6.25 µg ml-1, while the MBC was 12.5 µg ml-1, and there was a 38 mm inhibition zone in the paper disc diffusion test. Rhein showed an additive two- to fourfold reduction of the MIC value with four antibiotics on the checkerboard dilution test. The purified NADH dehydrogenase gene product showed a size of approximately 51 kDa and had a V max of 23 µmol and a K m of 280 µm. The inhibitory effect of rhein against NADH dehydrogenase-2 activity was non-competitive with ferricyanide [K3Fe(CN)6] with a K i value of 3.5-4.5 µm.Conclusion. This study provided evidence of the inhibitory effects of rhein on the growth of C. acnes by blocking of NADH dehydrogenase-2 activity. This mechanism of inhibitory activity in the reduction of ROS formation and ATP productivity should be further tested in C. acnes and the question of whether rhein inhibits the natural growth of C. acnes should be investigated.

A single amino acid substitution in the FAD-binding domain causes the inactivation of Propionibacterium Acnes isomerase.

We previously demonstrated the efficient production of trans 10, cis 12-conjugated linoleic acid (t10c12-CLA) in Lactococcus lactis by ectopically expressing a Propionibacterium acnes isomerase (pai) gene and also mentioned that a recombinant strain was unable to accumulate t10c12-CLA product, despite the normal transcription. Here, the molecular analysis indicated that this mutated strain harbors a pai gene with a single-nucleotide mutation converting GC50A to GTA, leading to a corresponding change of Alanine residue into Valine. The expression of the reverse mutation resulted in the recovery for enzyme activity. Site-directed mutagenesis indicated that the codon usage of Val17 was not responsible for the enzyme inactivation in the Ala17Val mutation. Western blot analysis revealed that the recombinant PAI protein was not detectable in the His tag-marked Ala17Val mutant. It is, therefore, reasonable to assume that Ala17 residue is critical for PAI functionality.Abbreviations: pai: propionibacterium acnes isomerase; CLA: conjugated linoleic acid; t10c12-CLA: trans 10, cis 12-CLA; LA: linoleic acid (18:2n-6); FAD: flavin adenine dinucleotide.

Catalase expression of Propionibacterium acnes may contribute to intracellular persistence of the bacterium in sinus macrophages of lymph nodes affected by sarcoidosis.

Bacterial catalase is important for intracellular survival of the bacteria. This protein of Propionibacterium acnes, one of possible causes of sarcoidosis, induces hypersensitive Th1 immune responses in sarcoidosis patients. We examined catalase expression in cultured P. acnes isolated from 19 sarcoid and 18 control lymph nodes and immunohistochemical localization of the protein in lymph nodes from 43 sarcoidosis and 102 control patients using a novel P. acnes-specific antibody (PAC) that reacts with the catalase protein, together with the previously reported P. acnes-specific PAB and TIG antibodies. High catalase expression of P. acnes cells was found during stationary phase in more isolates from sarcoid than from non-sarcoid lymph nodes and was associated with bacterial survival under H2O2-induced oxidative stress. In many sarcoid and some control lymph nodes, catalase expression was detected at the outer margins of PAB-reactive Hamazaki-Wesenberg (HW) bodies in sinus macrophages, the same location as catalase expression on the surface of cultured P. acnes and the same distribution as bacterial cell membrane-bound lipoteichoic acid in HW bodies. Some or no catalase expression was detected in sarcoid granulomas with PAB reactivity or in clustered paracortical macrophages packed with many PAB-reactive small-round bodies. HW bodies expressing catalase may be persistent P. acnes in sinus macrophages whereas PAB-reactive small-round bodies with undetectable catalase may be activated P. acnes proliferating in paracortical macrophages. Intracellular proliferation of P. acnes in paracortical macrophages may lead to granuloma formation by this commensal bacterium in sarcoidosis patients with Th1 hypersensitivity to certain P. acnes antigens, including catalase.

Target Proteins of Phloretin for Its Anti-Inflammatory and Antibacterial Activities Against Propionibacterium acnes-Induced Skin Infection.

Phloretin is a natural chalcone with antibacterial and anti-inflammatory effects. This study investigated the anti-acne activity of phloretin against Propionibacterium acnes-induced skin infection and the potential target proteins of its anti-inflammatory and antibacterial effects. Phloretin potently inhibited the growth of P. acnes and P. acnes-induced Toll-like receptor (TLR) 2-mediated inflammatory signaling in human keratinocytes. Secreted embryonic alkaline phosphatase assay confirmed that the anti-inflammatory activity of phloretin is associated with the P. acnes-stimulated TLR2-mediated NF-κB signaling pathway. Phloretin significantly decreased the level of phosphorylated c-Jun N-terminal kinase (JNK), showing a binding affinity of 1.184 × 10-5 M-1. We also found that phloretin binds with micromolar affinity to P. acnes ß-ketoacyl acyl carrier protein (ACP) synthase III (KAS III), an enzyme involved in fatty acid synthesis. Conformation-sensitive native polyacrylamide gel electrophoresis showed that phloretin reduced KAS III-mediated 3-ketoacyl ACP production by over 66%. A docking study revealed that phloretin interacts with the active sites of JNK1 and KAS III, suggesting their involvement in P. acnes-induced inflammation and their potential as targets for the antibacterial activity of phloretin. These results demonstrate that phloretin may be useful in the prevention or treatment of P. acnes infection.

Characterization of the housekeeping sortase from the human pathogen Propionibacterium acnes: first investigation of a class F sortase.

Sortase enzymes play an important role in Gram-positive bacteria. They are responsible for the covalent attachment of proteins to the surface of the bacteria and perform this task via a highly sequence-specific transpeptidation reaction. Since these immobilized proteins are often involved in pathogenicity of Gram-positive bacteria, characterization of this type of enzyme is also of medical relevance. Different classes of sortases (A-F) have been found, which recognize characteristic recognition sequences present in substrate proteins. Up to date, sortase A from Staphylococcus aureus, a housekeeping class A sortase, is the most thoroughly studied representative of the sortase family of enzymes. Here we report the in-depth characterization of the class F sortase from Propionibacterium acnes, a class of sortases that has not been investigated before. As Sortase F is the only transpeptidase found in the P. acnes genome, it is the housekeeping sortase of this organism. Sortase F from P. acnes shows a behavior similar to sortases from class A in terms of pH dependence, recognition sequence and catalytic activity; furthermore, its activity is independent of bivalent ions, which contrasts to sortase A from S. aureus We demonstrate that sortase F is useful for protein engineering applications, by producing a site-specifically conjugated homogenous antibody-drug conjugate with a potency similar to that of a conjugate prepared with sortase A. Thus, the detailed characterization presented here will not only enable the development of anti-virulence agents targeting P. acnes but also provides a powerful alternative to sortase A for protein engineering applications.

Structural basis of branched-chain fatty acid synthesis by Propionibacterium acnes ß-ketoacyl acyl Carrier protein synthase.

Propionibacterium acnes is an anaerobic gram-positive bacterium found in the niche of the sebaceous glands in the human skin, and is a causal pathogen of inflammatory skin diseases as well as periprosthetic joint infection. To gain effective control of P. acnes, a deeper understanding of the cellular metabolism mechanism involved in its ability to reside in this unique environment is needed. P. acnes exhibits typical cell membrane features of gram-positive bacteria, such as control of membrane fluidity by branched-chain fatty acids (BCFAs). Branching at the iso- or anteiso-position is achieved by incorporation of isobutyryl- or 2-methyl-butyryl-CoA via ß-ketoacyl acyl carrier protein synthase (KAS III) from fatty acid synthesis. Here, we determined the crystal structure of P. acnes KAS III (PaKAS III) at the resolution of 1.9 Šfor the first time. Conformation-sensitive urea polyacrylamide gel electrophoresis and tryptophan fluorescence quenching experiments confirmed that PaKAS III prefers isobutyryl-CoA as the acetyl-CoA, and the unique shape of the active site cavity complies with incorporation of branched-short chain CoAs. The determined structure clearly illustrates how BCFA synthesis is achieved in P. acnes. Moreover, the unique shape of the cavity required for the branched-chain primer can be invaluable in designing novel inhibitors of PaKAS III and developing new specifically targeted antibiotics.

Alpha- and gamma-mangostins exhibit anti-acne activities via multiple mechanisms.

Objective: Acne is a chronic skin disease that involves four key pathogenic factors: excess sebum production, ductal epidermal hyperproliferation, Propionibacterium acnes (P. acnes) colonization, and skin inflammation. Mangostins are well-known for their anti-bacterial and anti-inflammatory effects, suggesting that mangostins may have therapeutic potential for acne. The present study aimed to explore the anti-acne effects of mangostins from the perspective of multiple pathogenic mechanisms of acne. Methods: The effects of α- and γ-mangostins on the growth of P. acnes and lipase activity were analyzed. Their effects on P. acnes-induced keratinocyte proliferation were examined by CCK-8. The expression of inflammatory genes and activation of NF-κB and MAPK signaling pathways were detected by quantitative real-time PCR and western blotting, respectively. Results: Alpha- and γ-mangostins not only inhibited the growth of P. acnes, but also reduced the proliferation of keratinocytes induced by heat-killed P. acnes. Furthermore, α- and γ-mangostins were able to suppress P. acnes-induced expression of pro-inflammatory cytokines, including TNF-α, IL-1ß, and IL-6 in keratinocytes by inhibiting the activation of NF-κB and MAPK signaling pathways. Discussion and conclusions: Mangostins appeared to possess multiple anti-acne activities, including the inhibition of P. acnes growth, regulation of keratinocytes proliferation, and attenuation of skin inflammatory reaction. Hence, mangostins might be developed into a potential therapeutic agent for the treatment of acne.

Combined photodynamic and antibiotic therapy for skin disorder via lipase-sensitive liposomes with enhanced antimicrobial performance.

A lipase-sensitive singlet oxygen-producible and erythromycin-loaded liposome (LSSPL) was developed for combination antibacterial therapy for skin disorder. The LSSPL was synthesized by coating pullulan-pheophorbide a (PU-Pheo A) conjugates onto erythromycin-loaded liposomes composed of 1,2-dipalmitoyl-sn-phosphatidylcholine (DPPC) and cholesterol. Lipase activity was chosen as the environmental-stimulus for the controlled release of erythromycin and Pheo A from LSSPL because skin inflammation-inducing Propionibacterium acnes (P. acnes) secrete extracellular lipases. The presence of P. acnes lipases disrupted LSSPLs by selective cleavage of their ester linkages, liberating erythromycin and Pheo A. Along with the antibacterial effect of erythromycin, additional laser irradiation onto Pheo A further achieved the inhibition of P. acnes growth and treatment of P. acnes-infected inflammation in nude mice back skin. Therefore, antimicrobial therapy, using a stimulus-responsiveness moiety, presents a feasible way to treat bacteria-induced skin disorders.

Green Synthesis of Conjugated Linoleic Acids from Plant Oils Using a Novel Synergistic Catalytic System.

A novel and efficient method has been developed for converting plant oil into a specific conjugated linoleic acid (CLA) using a synergistic biocatalytic system based on immobilized Propionibacterium acnes isomerase (PAI) and Rhizopus oryzae lipase (ROL). PAI exhibited the greatest catalytic activity when immobilized on D301R anion-exchange resin under optimal conditions (PAI dosage of 12 410 U of PAI/g of D301R, glutaraldehyde concentration of 0.4%, and reaction conditions of pH 7.0, 25 °C, and 60 min). Up to 109 g/L trans-10,cis-12-CLA was obtained after incubation of 200 g/L sunflower oil with PAI (1659 U/g of oil) and ROL (625 mU/g of oil) at pH 7.0 and 35 °C for 36 h; the corresponding conversion ratio of linoleic acid (LA) to CLA was 90.5%. This method exhibited the highest proportion of trans-10,cis-12-CLA yet reported and is a promising method for large-scale production.

A metabolic engineering strategy for producing conjugated linoleic acids using the oleaginous yeast Yarrowia lipolytica.

Conjugated linoleic acids (CLAs) have been found to have beneficial effects on human health when used as dietary supplements. However, their availability is limited because pure, chemistry-based production is expensive, and biology-based fermentation methods can only create small quantities. In an effort to enhance microbial production of CLAs, four genetically modified strains of the oleaginous yeast Yarrowia lipolytica were generated. These mutants presented various genetic modifications, including the elimination of ß-oxidation (pox1-6∆), the inability to store lipids as triglycerides (dga1∆ dga2∆ are1∆ lro1∆), and the overexpression of the Y. lipolytica ∆12-desaturase gene (YlFAD2) under the control of the constitutive pTEF promoter. All strains received two copies of the pTEF-oPAI or pPOX-oPAI expression cassettes; PAI encodes linoleic acid isomerase in Propionibacterium acnes. The strains were cultured in neosynthesis or bioconversion medium in flasks or a bioreactor. The strain combining the three modifications mentioned above showed the best results: when it was grown in neosynthesis medium in a flask, CLAs represented 6.5% of total fatty acids and in bioconversion medium in a bioreactor, and CLA content reached 302 mg/L. In a previous study, a CLA degradation rate of 117 mg/L/h was observed in bioconversion medium. Here, by eliminating ß-oxidation, we achieved a much lower rate of 1.8 mg/L/h.

Inhibition of Propionibacterium acnes lipase activity by the antifungal agent ketoconazole.

The common skin disease acne vulgaris is caused by Propionibacterium acnes. A lipase secreted by this microorganism metabolizes sebum and the resulting metabolites evoke inflammation in human skin. The antifungal drug ketoconazole inhibits P. acnes lipase activity. We previously showed that the drug also inhibits the growth of P. acnes. Thus, ketoconazole may serve as an alternative treatment for acne vulgaris, which is important because the number of antibiotic-resistant P. acnes strains has been increasing.

Rhodomyrtone inhibits lipase production, biofilm formation, and disorganizes established biofilm in Propionibacterium acnes.

Virulence enzymes and biofilm a play crucial role in the pathogenesis of Propionibacterium acnes, a major causative agent of acne vulgaris. In the present study, the effects of rhodomyrtone, a pure compound identified from Rhodomyrtus tomentosa (Aiton) Hassk. leaves extract against enzyme production and biofilm formation production by 5 clinical isolates and a reference strain were evaluated. The degree of hydrolysis by both lipase and protease enzymes significantly decreased upon treatment with the compound at 0.125-0.25 µg/mL (p < 0.05). Lipolytic zones significantly reduced in all isolates while decrease in proteolytic activities was found only in 50% of the isolates. Rhodomyrtone at 1/16MIC and 1/8MIC caused significant reduction in biofilm formation of the clinical isolates (p < 0.05). Percentage viability of P. acnes within mature biofilm upon treated with the compound at 4MIC and 8MIC ranged between 40% and 85%. Pronounced properties of rhodomyrtone suggest a path towards developing a novel anti-acne agent.

Propionibacterium acnes Recovered from Atherosclerotic Human Carotid Arteries Undergoes Biofilm Dispersion and Releases Lipolytic and Proteolytic Enzymes in Response to Norepinephrine Challenge In Vitro.

In the present study, human atherosclerotic carotid arteries were examined following endarterectomy for the presence of the Gram-positive bacterium Propionibacterium acnes and its potential association with biofilm structures within the arterial wall. The P. acnes 16S rRNA gene was detectable in 4 of 15 carotid artery samples, and viable P. acnes was one among 10 different bacterial species recoverable in culture. Fluorescence in situ hybridization analysis of 5 additional atherosclerotic carotid arteries demonstrated biofilm bacteria within all samples, with P. acnes detectable in 4 samples. We also demonstrated that laboratory-grown cultures of P. acnes biofilms were susceptible to induction of a biofilm dispersion response when challenged with physiologically relevant levels of norepinephrine in the presence of iron-bound transferrin or with free iron. The production and release of lipolytic and proteolytic extracellular enzymes by P. acnes were shown to increase in iron-induced dispersed biofilms, and these dispersion-induced P. acnes VP1 biofilms showed increased expression of mRNAs for the triacylglycerol lipases PPA2105 and PPA1796 and the hyaluronate lyase PPA380 compared to that in untreated biofilms. These results demonstrate that P. acnes can infect the carotid arteries of humans with atherosclerosis as a component of multispecies biofilms and that dispersion is inducible for this organism, at least in vitro, with physiologically relevant levels of norepinephrine resulting in the production and release of degradative enzymes.

Propionibacterium acnes catalase induces increased Th1 immune response in sarcoidosis patients.

BACKGROUND: Propionibacterium acnes is one of the most commonly implicated etiologic agents of sarcoidosis. We screened antigenic proteins from this indigenous bacterium that increase Th1 responses in sarcoidosis patients. METHODS: Antigenic bacterial proteins were screened by probing western blots of P. acnes whole cell lysates with blood plasma samples from 52 sarcoidosis patients and 34 healthy volunteers. Soluble protein antigens from the bands most frequently detected on blotting membranes were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS). Recombinant proteins were prepared from DNA sequences of the proteins identified by MALDI-TOF/MS and analyzed by immunologic assays. RESULTS: MALDI-TOF/MS analysis identified propionyl-CoA carboxylase subunit beta, arginine deiminase (ADI), catalase (KAT), and UDP-N-acetylglucosamine pyrophosphorylase (UAP). Successfully prepared recombinant proteins from ADI, KAT, and UAP provoked humoral and cellular immune responses in mice immunized with P. acnes when measured by enzyme-linked immunosorbent assay for serum antibodies and enzyme-linked immunospot assay for interferon (IFN)-γ-secreting cells (ELISPOT IFN-γ assay) with lymph node cells. Plasma IgG and IgA titers to KAT and UAP were significantly higher in sarcoidosis patients than in healthy volunteers. When Th1 immune responses to ADI, KAT, and UAP were measured by ELISPOT IFN-γ assay with peripheral blood mononuclear cells from 12 sarcoidosis patients, 13 other pneumonitis patients, and 11 healthy volunteers, only the KAT protein provoked a significantly higher response in sarcoidosis patients (p=0.0032). CONCLUSION: These results suggest that P. acnes KAT is an antigen that provokes allergic Th1 immune responses in sarcoidosis patients.

Production of conjugated linoleic acid by heterologous expression of linoleic acid isomerase in oleaginous fungus Mortierella alpina.

OBJECTIVE: To increase the commercial value of oleaginous fungus Mortierella alpina by incorporation of trans-10,cis-12 conjugated linoleic acid (CLA) into the polyunsaturated fatty acids (PUFAs) of M. alpina via Propionibacterium acnes isomerase (PAI) conversion. RESULTS: The PAI gene and the codon optimization version were heterologously expressed in M. alpina via Agrobacterium tumefaciens-mediated transformation (ATMT). Coding usage modification significantly improved the translation of PAI transcripts and trans-10,cis-12 CLA was produced up to 1.2 mg l(-1), which corresponds to approx. 0.05% of the total fatty acid (TFA). Since PAI prefers free linoleic acid as a substrate rather than any other forms, 5 µM long-chain acyl CoA synthetase inhibitor was added and the trans-10,cis-12 CLA content increased approx. 24-fold to 29 mg l(-1), reaching up to 1.2% (w/w) of the TFA in M. alpina. CONCLUSION: Heterologous expression of PAI in M. alpina by ATMT methods is a practicable way in biosynthesis of CLA and this system may be a feasible platform for industrial production of CLA.

Yeast cell surface display of linoleic acid isomerase from Propionibacterium acnes and its application for the production of trans-10, cis-12 conjugated linoleic acid.

Conjugated linoleic acid (CLA), a family of geometric and positional isomers of linoleic acid, has many health-promoting properties. Different isomers of CLA may have very different physiological effects. In the current work, we explore the possibility to produce single isomer of CLA by using biocatalysis based on displayed biocatalysts on the yeast cell surfaces. A reporter system used to assess gene expression and protein distribution was established by combining the egfp gene to the N-terminus of Propionibacterium acnes pai gene encoding the linoleic isomerase onto vector pYD1. After induction of the yeast strains containing pYD1::egfp::pai with galactose, strong green fluorescence was observed on the surface of cells, demonstrating that the fusion protein was successfully displayed. Using the engineered strains as whole-cell biocatalyst, trans-10, cis-12 CLA was detected in the reaction mixture. To improve the biocatalytic potential of this system, the first 20 amino codons of pai were modified, and the catalytic reaction conditions were optimized. Optimization of the codon usage resulted in 35% increase of CLA production, and the maximum yield of CLA was observed within 20 H in the optimal conditions: pH 7.0, 4 mg/mL linoleic acid, 37 °C. The system established in the present work can guide the development of biocatalytic strategies to produce trans-10, cis-12 CLA single isomer.

Inflammation and acne: putting the pieces together.

Acne vulgaris is a common skin disease in which abnormal desquamation, excess sebum production, proliferation of Propionibacterium acnes, and production of proinflammatory mediators all contribute to the pathogenesis of the disease. A review of the literature shows that our current understanding of acne pathogenesis continues to evolve. Recent data suggests that inflammatory mediators may play a more important role than previously realized; however, how these mediators work independently as well as together in acne lesion progression is not well understood. Several cell types and mediators involved in the pathology of acne are responsible for producing or exacerbating an inflammatory response. Here, we present an updated theoretical model of acne lesion progression that highlights the role inflammatory mediators may play throughout acne lesion development.

Genetic engineering of Yarrowia lipolytica for enhanced production of trans-10, cis-12 conjugated linoleic acid.

BACKGROUND: Conjugated linoleic acid (CLA) has been extensively studied for decades because of its health benefits including cancer prevention, anti-atherogenic and anti-obesity effects, and modulation of the immune system. We previously described the production of trans-10, cis-12 CLA in Yarrowia lipolytica by expressing the gene coding for linoleic acid isomerase from Propionibacterium acnes (pai). However the stable strain produced CLA at about 0.08% of dry cell weight (DCW), a level of production which was not high enough for practical applications. The goal of the present study was to enhance production of CLA by genetic engineering of Y. lipolytica strains. RESULTS: We have now co-expressed the delta 12-desaturase gene (FADS12, d12) from Mortierella alpina together with the codon-optimized linoleic acid isomerase (opai) gene in Y. lipolytica, expressed under the control of promoter hp16d modified by fusing 12 copies of UAS1B to the original promoter hp4d. A multi-copy integration plasmid was used to further enhance the expression of both genes. Using glucose as the sole carbon source, the genetically-modified Y. lipolytica produced trans-10, cis-12-CLA at a level of up to 10% of total fatty acids and 0.4% of DCW. Furthermore, when the recombinant yeast was grown with soybean oil, trans-10, cis-12-CLA now accumulated at a level of up to 44% of total fatty acids, which represented 30% of DCW after 38.5 h of cultivation. In addition, trans-10, cis-12-CLA was also detected in the growth medium up to 0.9 g/l. CONCLUSIONS: We have successfully produced trans-10, cis-12-CLA with a titre of 4 g/l of culture (3.1 g/l in cells and 0.9 g/l in culture medium). Our results demonstrate the potential use of Y. lipolytica as a promising microbial cell factory for trans-10, cis-12-CLA production.