Multiomics Analysis Revealed Colorectal Cancer Pathogenesis.

Gut microbiota-derived microbial compounds may link to the pathogenesis of colorectal cancer (CRC). However, the role of the host-microbiome in the incidence and progression of CRC remains elusive. We performed 16S rRNA sequencing, metabolomics, and proteomic studies on samples from 85 CRC patients who underwent colonoscopy examination and found two distinct changed patterns of microbiome in CRC patients. The relative abundances of Catabacter and Mogibacterium continuously increased from intramucosal carcinoma to advanced stages, whereas Clostridium, Anaerostipes, Vibrio, Flavonifractor, Holdemanella, and Hungatella were significantly altered only in intermediate lesions. Fecal metabolomics analysis exhibited consistent increases in bile acids, indoles, and urobilin as well as a decrease in heme. Serum metabolomics uncovered the highest levels of bilin, glycerides, and nucleosides together with the lowest levels of bile acids and amino acids in the stage of intermediate lesions. Three fecal and one serum dipeptides were elevated in the intermediate lesions. Proteomics analysis of colorectal tissues showed that oxidation and autophagy through the PI3K/Akt-mTOR signaling pathway contribute to the development of CRC. Diagnostic analysis showed multiomics features have good predictive capability, with AUC greater than 0.85. Our overall findings revealed new candidate biomarkers for CRC, with potentially significant diagnostic and prognostic capabilities.

SpecLipIDA: a pseudotargeted lipidomics approach for polyunsaturated fatty acids in milk.

Polyunsaturated fatty acids (PUFAs), such as arachidonic acid (ARA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), play an important role in the nutritional value of milk lipids. However, a comprehensive analysis of PUFAs and their esters in milk is still scarce. In this study, we developed a novel pseudotargeted lipidomics approach, named SpecLipIDA, for determining PUFA lipids in milk. Triglycerides (TGs) and phospholipids (PLs) were separated using NH2 cartridges, and mass spectrometry data in the information-dependent acquisition (IDA) mode were preprocessed by MS-DIAL, leading to improved identification in subsequent targeted analysis. The target matching algorithm, based on specific lipid cleavage patterns, demonstrated enhanced identification of PUFA lipids compared to the lipid annotations provided by MS-DIAL and GNPS. The approach was applied to identify PUFA lipids in various milk samples, resulting in the detection of a total of 115 PUFA lipids. The results revealed distinct differences in PUFA lipids among different samples, with 44 PUFA lipids significantly contributing to these differences. Our study indicated that SpecLipIDA is an efficient method for rapidly and specifically screening PUFA lipids.

Non-targeted mass spectrometry and feature-based molecular networking for determination of branched fatty acid esters of hydroxy fatty acids in milk.

Branched fatty acid esters of hydroxy fatty acids (FAHFAs) represent trace lipids with significant natural biological functions. While exogenous FAHFAs have been extensively studied, research on FAHFAs in milk remains limited, constraining our grasp of their nutritional roles. This study introduces a non-targeted mass spectrometry approach combined with chemical networking of spectral fragmentation patterns to uncover FAHFAs. Through meticulous sample handling and comparisons of various data acquisition and processing modes, we validate the method's superiority, identifying twice as many FAHFAs compared to alternative techniques. This validated method was then applied to different milk samples, revealing 45 chemical signals associated with known and potential FAHFAs, alongside findings of 66 ceramide/hexosylceramide (Cer/HexCer), 48 phosphatidyl ethanolamine/lyso phosphatidyl ethanolamine (PE/LPE), 21 phosphatidylcholine/lysophosphatidylcholine (PC/LPC), 16 phosphatidylinositol (PI), 7 phosphatidylserine (PS), and 11 sphingomyelin (SM) compounds. This study expands our understanding of the FAHFA family in milk and provides a fast and convenient method for identifying FAHFAs.

Advances in encapsulation systems of Antarctic krill oil: From extraction to encapsulation, and future direction.

Antarctic krill oil (AKO) is highly sought after by consumers and the food industry due to its richness in a variety of nutrients and physiological activities. However, current extraction methods are not sufficient to better extract AKO and its nutrients, and AKO is susceptible to lipid oxidation during processing and storage, leading to nutrient loss and the formation of off-flavors and toxic compounds. The development of various extraction methods and encapsulation systems for AKO to improve oil yield, nutritional value, antioxidant capacity, and bioavailability has become a research hotspot. This review summarizes the research progress of AKO from extraction to encapsulation system construction. The AKO extraction mechanism, technical parameters, oil yield and composition of solvent extraction, aqueous enzymatic extraction, supercritical/subcritical extraction, and three-liquid-phase salting-out extraction system are described in detail. The principles, choice of emulsifier/wall materials, preparation methods, advantages and disadvantages of four common encapsulation systems for AKO, namely micro/nanoemulsions, microcapsules, liposomes and nanostructured lipid carriers, are summarized. These four encapsulation systems are characterized by high encapsulation efficiency, low production cost, high bioavailability and high antioxidant capacity. Depending on the unique advantages and conditions of different encapsulation methods, as well as consumer demand for health and nutrition, different products can be developed. However, existing AKO encapsulation systems lack relevant studies on digestive absorption and targeted release, and the single product category of commercially available products limits consumer choice. In conjunction with clinical studies of AKO encapsulation systems, the development of encapsulation systems for special populations should be a future research direction.

Windows Scanning Multiomics: Integrated Metabolomics and Proteomics.

Metabolomics and proteomics offer significant advantages in understanding biological mechanisms at two hierarchical levels. However, conventional single omics analysis faces challenges due to the high demand for specimens and the complexity of intrinsic associations. To obtain comprehensive and accurate system biological information, we developed a multiomics analytical method called Windows Scanning Multiomics (WSM). In this method, we performed simultaneous extraction of metabolites and proteins from the same sample, resulting in a 10% increase in the coverage of the identified biomolecules. Both metabolomics and proteomics analyses were conducted by using ultrahigh-performance liquid chromatography mass spectrometry (UPLC-MS), eliminating the need for instrument conversions. Additionally, we designed an R-based program (WSM.R) to integrate mathematical and biological correlations between metabolites and proteins into a correlation network. The network created from simultaneously extracted biomolecules was more focused and comprehensive compared to those from separate extractions. Notably, we excluded six pairs of false-positive relationships between metabolites and proteins in the network established using simultaneously extracted biomolecules. In conclusion, this study introduces a novel approach for multiomics analysis and data processing that greatly aids in bioinformation mining from multiomics results. This method is poised to play an indispensable role in systems biology research.

Monomethyl branched-chain fatty acids-a pearl dropped in the ocean.

As an emerging group of bioactive fatty acids, monomethyl branched-chain fatty acids (mmBCFAs) have sparked the interest of many researchers both domestically and internationally. In addition to documenting the importance of mmBCFAs for growth and development, there is increasing evidence that mmBCFAs are highly correlated with obesity and insulin resistance. According to previous pharmacological investigations, mmBCFAs also exhibit anti-inflammatory effects and anticancer properties. This review summarized the distribution of mmBCFAs, which are widely found in dairy products, ruminants, fish, and fermented foods. Besides, we discuss the biosynthesis pathway in different species and detection methods of mmBCFAs. With the hope to unveil their mechanisms of action, we recapitulated detailed the nutrition and health benefits of mmBCFAs. Furthermore, this study provides a thorough, critical overview of the current state of the art, upcoming difficulties, and trends in mmBCFAs.

Metabolomics identify landscape of food sensory properties.

Sensory evaluation is a key component of food production strategy. The classical food sensory evaluation method is time-consuming, laborious, costly, and highly subjective. Since flavor (taste and smell), texture, and mouthfeel are all related to the chemical properties of food, there has been a growing interest in how they affect the senses of food. In the past decades, emerging metabolomics has received much attention in the field of sensory evaluation, because it not only offers a broad picture of chemical composition for sensory properties but also revealed their changes and functions in food proceeding. This article reviewed food chemicals regarding the flavor, smell, and texture of foods, and discussed the advantages and limitations of applying metabolomics approaches to sensory evaluation, including GC-MS, LC-MS, and NMR. Taken together, this review gives a comprehensive, critical overview of the current state, future challenges, and trends in metabolomics on food sensory properties.

Comparative evaluation of static and dynamic simulated digestion models.

BACKGROUND: The digestion behavior of lipids plays a crucial role in their nutritional values. Currently, the complex dynamic variations of human gastrointestinal conditions are considered in simulated digestion models. The present study compared the digestion behavior of glycerol trilaurate (GTL), glycerol tripalmitate (GTP) and glycerol tristearate (GTS) in a static in vitro digestion model and a dynamic in vitro digestion model. In the dynamic digestion model, the parameters of gastric juice secretion, the rate of gastric emptying, the secretion of intestinal juice and the pH variations were estimated. RESULTS: The dynamic digestion model showed a certain extent of gastric lipase hydrolysis, while almost no lipolysis happened in the gastric phase of the static digestion model. A smoother digestive behavior was observed in the dynamic model than that in the static model. In the static model, the particle size distribution in gastric and intestinal phase changed rapidly in all triacylglycerol (TAG) groups. The change of particle size during the whole digestion period in GTL is more moderate than GTP and GTS. In addition, the final free fatty acids release degree was 58.558%, 54.36%, and 52.97% for GTL, GTP, and GTS, respectively. CONCLUSION: This study illustrated the different digestion profiles of TAGs in two digestion models and the results will contribute to a better understanding of different in vitro digestion models in lipid digestion. © 2023 Society of Chemical Industry.

Fasting and metabolic syndrome: A systematic review and Meta-analyses.

Objective: Fasting is considered to be a food structure that can improve body health. Several randomized clinical trials (RCTs) have investigated the effects of fasting in patients with metabolic syndrome (MS). In this review, we performed a meta-analysis to assess the effects of fasting on patients with MS. Methods: Following PRISMA guidelines, a systematic literature search was conducted in PubMed, Embase, and Cochrane Central updated to September 2021. The quality evaluation and heterogeneity detection of the included research literature were carried out by Revman and Stata software through a random-effects model. Results: A total of 268 subjects were included. The pooled results revealed that fasting significantly reduced body weight (WMD: -2.48 kg, 95% CI: -3.22, -1.74), BMI (WMD = -2.72 cm; 95%CI: -4.04, -1.40 cm), body fat percent (WMD: -1.57%, 95%CI: -2.47, -0.68), insulin level (WMD: -2.45 mmol/L; 95%CI: -4.40, -0.49 mmol/L) and HOMA-IR (WMD:-0.65 mmol/L; 95%CI: -0.90, -0.41 mmol/L) in patients with MS, whereas had no effect on glucose, blood pressure and lipids profile. Conclusions: Our findings provide insights into the effect of fasting on the anthropometric outcomes, insulin resistance, and gut microbiota in MS.

Blueberry Anthocyanins from Commercial Products: Structure Identification and Potential for Diabetic Retinopathy Amelioration.

The aim of the present study was to determine the major anthocyanins of blueberry extracts from northeast China and explore their vision health improvement effects. HPLC-Q-TOF-MS/MS results suggested that six different anthocyanins were accurately identified, among which the Cy-3-glu (C3G) was the most abundant, ranging from 376.91 ± 7.91 to 763.70 ± 4.99 µM. The blueberry extract contained a higher purity of anthocyanins, and the anthocyanosides reached 342.98 mg/kg. The anti-oxidative stress function of C3G on HG-treated ARPE-19 cells were evaluated, and showed that the GSSG level of HG-cells pretreated with 10 µM C3G was significantly decreased, while the Nrf2 and NQO1 gene expression levels were increased. Further molecular docking (MD) results indicated that the C3G displayed favorable binding affinity towards REDD1, and only the B-ring of the C3G molecule displayed binding interactions with the CYS-140 amino acids within the REDD1 protein. It implied that the oxidative stress amelioration effects of C3G on the ARPE-19 cells were related to the REDD1 protein, which was probably via the Nrf2 pathways, although further studies are needed to provide mechanism evidence. The present study provides novel insights into understanding the roles of blueberry anthocyanins in ameliorating oxidative stress-induced BRB damage in the retina.

Different typical dietary lipid consumption affects the bile acid metabolism and the gut microbiota structure: an animal trial using Sprague-Dawley rats.

BACKGROUND: The palm oil (PO), leaf lard oil (LO), rapeseed oil (RO), sunflower oil (SO) and linseed oil (LN) are five of the most typical dietary lipids in most Asian countries. However, their influences on gut health, and the connections between the fatty acid composition, the gut microbiota, and the bile acid metabolism are not fully understood. RESULTS: In the present study, results showed that compared with polyunsaturated fatty acid (PUFA)-rich SO and LN, the saturated fatty acid (SFA)-rich and monounsaturated fatty acid (MUFA)-rich PO, LO and RO were more likely to decrease the re-absorption of bile acid in the colon, which was probably caused by their different role in modulating the gut microbiota structure. LO consumption significantly up-regulated the Cyp27a1, FXR and TGR5 gene expression level (P < 0.05). The correlation results suggested that the C18:0 was significantly positive correlated with these three genes, indicating that intake of SFA-rich dietary lipids, especially for the C18:0, could specifically increase the bile acid production by stimulating the bile acid alternative synthesis pathway. Although the bile acid receptor expression in the colon was increased, the re-absorption of bile acid did not show a significant increase (P > 0.05) as compared with other dietary lipids. Moreover, the C18:2-rich SO maintained the bile acid metabolic balance probably by decreasing the Romboutsia, while increasing the Bifidobacterium abundance in the colon. CONCLUSIONS: The different dietary lipids showed different effects on the bile acid metabolism, which was probably connected with the alterations in the gut microbiota structure. The present study could provide basic understandings about the influences of the different dietary lipids consumption on gut homeostasis and bile acid metabolism. © 2021 Society of Chemical Industry.

Deep-frying oil induces cytotoxicity, inflammation and apoptosis on intestinal epithelial cells.

BACKGROUND: Deep-frying oil has been found to cause inflammatory bowel disease (IBD). However, the molecular mechanism of the effect of deep-frying palm oil on IBD still remains undetermined. RESULTS: In the present study, bioinformatics and cell biology were used to investigate the functions and signal pathway enrichments of differentially expressed genes. The bioinformatics analysis of three original microarray datasets (GSE73661, GSE75214 and GSE126124) in the NCBI-Gene Expression Omnibus database showed 17 down-regulated genes (logFC < 0) and 2 up-regulated genes (logFC > 0) existed in the enteritis tissue. Meanwhile, pathway enrichment and protein-protein interaction network analysis suggested that IBD is relevant to cytotoxicity, inflammation and apoptosis. Furthermore, Caco-2 cells were treated with the main oxidation products of deep-frying oil-total polar compounds (TPC) and its components (polymerized triglyceride, oxidized triglycerides and triglyceride degradation products) isolated from deep-frying oil. The flow cytometry experiment revealed that TPC and its components could induce apoptosis, especially for oxidized triglyceride. A quantitative polymerase chain reaction analysis demonstrated that TPC and its component could induce Caco-2 cell apoptosis through AQP8/CXCL1/TNIP3/IL-1. CONCLUSION: The present study provides fundamental knowledge for understanding the effects of deep-frying oils on the cytotoxic and inflammatory of Caco-2 cells, in addition to clarifying the molecular function mechanism of deep-frying oil in IBD. © 2021 Society of Chemical Industry.

Bioanalytical insights into the association between eicosanoids and pathogenesis of hepatocellular carcinoma.

It has been noted that inflammatory were intimately associated with the development and progression of hepatocellular carcinoma (HCC). Eicosanoids derived from arachidonic acid play crucial roles in chronic inflammation. Accordingly, there is an intricate relationship between eicosanoids and HCC, being supported by the epidemiological, clinical, and basic science studies. Herein, we intend to provide bioanalytical insights into the role of eicosanoids in HCC progression, from cell proliferation, angiogenesis migration, to apoptosis. Also, the analytical methods and biochemistry of eicosanoids are described.

Choline metabolite, trimethylamine N-oxide (TMAO), is associated with inflammation in psoriatic arthritis.

OBJECTIVES: Dietary intake of choline has been linked to systemic inflammation through the microbial production of two metabolites, trimethylamine (TMA) and trimethylamine-N-oxide (TMAO). Herein we explore the association between choline metabolites and inflammation in psoriatic arthritis (PsA) patients. METHODS: Thirty-eight patients with PsA, all of whom satisfied the CASPAR classification criteria for PsA, were studied. Outcomes reflecting the activity of peripheral arthritis as well as skin psoriasis, Disease Activity Score (DAS)28, Clinical Disease Index (CDAI) and Body Surface Area (BSA) were assessed. Serum concentration of choline metabolites (choline, TMA, TMAO, betaine and carnitine) were determined by LC-MS, and metabolite levels associated with disease scores. RESULTS: Among the 38 PsA patients included, the mean DAS28PCR was 2.74±1.29. Twenty-seven patients had active skin disease, with an average BSA of 7.2±16.22. TMAO, but not TMA or choline, significantly correlated with measures of disease activity for both skin and peripheral joints. CONCLUSIONS: In our cohort, only TMAO, but not TMA, choline, betaine or carnitine, was associated with inflammation in PsA patients, establishing a mechanistic link between TMAO and PsA phenotypes. Future studies will explore the modulation of TMAO and disease severity in PsA.

Influence of total polar compounds on lipid metabolism, oxidative stress and cytotoxicity in HepG2 cells.

BACKGROUND: Recently, the harmful effects of frying oil on health have been gradually realized. However, as main components of frying oils, biochemical effects of total polar compounds (TPC) on a cellular level were underestimated. METHODS: The effects of total polar compounds (TPC) in the frying oil on the lipid metabolism, oxidative stress and cytotoxicity of HepG2 cells were investigated through a series of biochemical methods, such as oil red staining, real-time polymerase chain reaction (RT-PCR), cell apoptosis and cell arrest. RESULTS: Herein, we found that the survival rate of HepG2 cells treated with TPC decreased in a time and dose dependent manner, and thereby presented significant lipid deposition over the concentration of 0.5 mg/mL. TPC were also found to suppress the expression levels of PPARα, CPT1 and ACOX, elevate the expression level of MTP and cause the disorder of lipid metabolism. TPC ranged from 0 to 2 mg/mL could significantly elevate the amounts of reactive oxygen species (ROS) in HepG2 cells, and simultaneously increase the malondialdehyde (MDA) content from 21.21 ± 2.62 to 65.71 ± 4.20 µmol/mg of protein (p < 0.05) at 24 h. On the contrary, antioxidant enzymes superoxide dismutase (SOD), glutathione (GSH), and catalase (CAT) respectively decreased by 0.52-, 0.56- and 0.28-fold, when HepG2 cells were exposed to 2 mg/mL TPC for 24 h. In addition, TPC could at least partially induce the apoptosis of HepG2 cells, and the transition from G0/G1 to G2 phase in HepG2 cells was impeded. CONCLUSIONS: TPC could progressively cause lipid deposition, oxidative stress and cytotoxicity, providing the theoretical support for the detrimental health effects of TPC.

Krüppel-Like Factor 4 Regulation of Cholesterol-25-Hydroxylase and Liver X Receptor Mitigates Atherosclerosis Susceptibility.

BACKGROUND: Atherosclerosis is a multifaceted inflammatory disease involving cells in the vascular wall (eg, endothelial cells [ECs]), as well as circulating and resident immunogenic cells (eg, monocytes/macrophages). Acting as a ligand for liver X receptor (LXR), but an inhibitor of SREBP2 (sterol regulatory element-binding protein 2), 25-hydroxycholesterol, and its catalyzing enzyme cholesterol-25-hydroxylase (Ch25h) are important in regulating cellular inflammatory status and cholesterol biosynthesis in both ECs and monocytes/macrophages. METHODS: Bioinformatic analyses were used to investigate RNA-sequencing data to identify cholesterol oxidation and efflux genes regulated by Krüppel-like factor 4 (KLF4). In vitro experiments involving cultured ECs and macrophages and in vivo methods involving mice with Ch25h ablation were then used to explore the atheroprotective role of KLF4-Ch25h/LXR. RESULTS: Vasoprotective stimuli increased the expression of Ch25h and LXR via KLF4. The KLF4-Ch25h/LXR homeostatic axis functions through suppressing inflammation, evidenced by the reduction of inflammasome activity in ECs and the promotion of M1 to M2 phenotypic transition in macrophages. The increased atherosclerosis in apolipoprotein E-/-/Ch25h-/- mice further demonstrates the beneficial role of the KLF4-Ch25h/LXR axis in vascular function and disease. CONCLUSIONS: KLF4 transactivates Ch25h and LXR, thereby promoting the synergistic effects between ECs and macrophages to protect against atherosclerosis susceptibility.

Measurement of Serum and Hepatic Eicosanoids by Liquid Chromatography Tandem-Mass Spectrometry (LC-MS/MS) in a Mouse Model of Hepatocellular Carcinoma (HCC) with Delivery of c-Met and Activated ß-Catenin by Hepatocyte Hydrodynamic Injection.

BACKGROUND Most forms of cancer, including hepatocellular carcinoma (HCC), are associated with varying degrees of chronic inflammation. The association between the expression of eicosanoids, which are bioactive lipid mediators of inflammation, and HCC remains unknown. The aim of this study was to measure serum and hepatic eicosanoids in a mouse model of HCC with the delivery of c-Met and activated b-catenin by hepatocyte hydrodynamic injection. MATERIAL AND METHODS The HCC mouse model, and normal control mice, were used in this study with co-delivery of human c-Met combined with activated ß-catenin into hepatocytes through hydrodynamic injection. Liquid chromatography tandem-mass spectrometry (LC-MS/MS) analysis was used to measure serum and hepatic eicosanoid levels. RESULTS The combined activation of c-Met and ß-catenin was induced in the HCC mouse model. LC-MS/MS showed that a total of 13 eicosanoids in serum and 12 eicosanoids in liver tissue were significantly increased in the HCC mice, when compared with control mice. CONCLUSIONS In a mouse model of HCC, co-activation of the c-Met and ß-catenin signaling pathway resulted in increased levels of serum and hepatic eicosanoids.

Metabolomics Reveals that Momordica charantia Attenuates Metabolic Changes in Experimental Obesity.

Momordica charantia L., also known as bitter melon, has been shown to ameliorate obesity and insulin resistance. However, metabolic changes regulated by M. charantia in obesity are not clearly understood. In this study, serums obtained from obese and M. charantia-treated mice were analyzed by using gas and liquid chromatography-mass spectrometry, and multivariate statistical analysis was performed by Orthogonal partial least squares discriminant analysis. The results from this study indicated that body weight fat and insulin levels of obese mice are dramatically suppressed by 8 weeks of dietary supplementation of M. charantia. Metabolomic data revealed that overproductions of energy and nutrient metabolism in obese mice were restored by M. charantia treatment. The antiinflammatory and inhibition of insulin resistance effect of M. charantia in obesity was illustrated with the restoration of free fatty acids and eicosanoids. The findings achieved in this study further strengthen the therapeutic value of using M. charantia to treat obesity. Copyright © 2016 John Wiley & Sons, Ltd.

Phospholipid homeostasis maintains cell polarity, development and virulence in metarhizium robertsii.

The final product of the glycerol phosphate (GP) pathway is triacylglycerol (TAG) that regulates the homeostasis of energy, fatty acids and phospholipids in cells. The enzymes involved in this pathway have been characterized in many model organisms; however, their contributions to fungal infection are largely unclear. In this study, we performed serial deletion of genes in the GP pathway in the insect pathogenic fungus Metarhizium robertsii. The results indicated that a lysophosphatidate acyltransferase mrLPAAT1 was required for fungal growth, cell differentiation, maintenance of cell polarity and virulence. Lipidomic analysis indicated that deletion of mrLPAAT1 resulted in significant increases in TAG, fatty acids and phosphatidylcholine (PC) but decreased phosphatidic acid (PA), phosphatidylethanolamine (PE) and other species of phospholipids when compared to the wild type. Disruption of the isozymatic gene mrLPAAT2, however, resulted in a reduction in PC but not PA in the mutant cells. There were no changes in development and virulence in ΔmrLPAAT2. Phospholipid feeding assays verified that a PE supplement could rescue the cell differentiation defect in ΔmrLPAAT1. The results of this study reveal that cellular phospholipid homeostasis mediated by the GP pathway regulates fungal growth, cell polarity, differentiation and virulence.

Biosynthesis of non-melanin pigment by a divergent polyketide synthase in Metarhizium robertsii.

Fungal polyketide synthases (PKSs) and their related gene clusters are highly diversified at both inter- and intra-specific levels. The most well characterized PKS enzymes include those responsible for the biosynthesis of polyketide pigments such as melanins. The genome of the insect pathogenic fungus Metarhizium robertsii contains 20 type I PKSs but none has been functionally characterized. In this study, two PKS genes (designated as MrPks1 and MrPKs2) showing homologies to those counterparts for the biosynthesis of heptaketide pigments and dihydroxynaphthalene (DHN)-melanins, respectively, were deleted in two different strains of M. robertsii. The results indicated that disruption of MrPks1 but not MrPks2 impaired fungal culture pigmentation and cell wall structure. In addition to the negative effect of the DHN-melanin pathway inhibitor, it was postulated that DHN-melanin would not be produced by M. robertsii. Various assays revealed that the stress resistance abilities against ultraviolet radiation, heat shock and oxidants, as well as virulence against insects were not impaired in ΔMrPks1 and ΔMrPks2 isolates when compared with the wild-type strain. Thus, the non-melanin pigment(s) produced by the fungus do not contribute to cell damage protection and pathogenicity in M. robertsii. Physiological differences were evident in the two examined wild-type strains. The results from this study advance the understanding of functional divergence of fungal PKSs.