Engineering Yarrowia lipolytica for the sustainable production of ß-farnesene from waste oil feedstock.

BACKGROUND: ß-Farnesene is a sesquiterpene with versatile industrial applications. The production of ß-farnesene from waste lipid feedstock is an attractive method for sustainable production and recycling waste oil. Yarrowia lipolytica is an unconventional oleaginous yeast, which can use lipid feedstock and has great potential to synthesize acetyl-CoA-derived chemicals. RESULTS: In this study, we engineered Y. lipolytica to produce ß-farnesene from lipid feedstock. To direct the flux of acetyl-CoA, which is generated from lipid ß-oxidation, to ß-farnesene synthesis, the mevalonate synthesis pathway was compartmentalized into peroxisomes. ß-Farnesene production was then engineered by the protein engineering of ß-farnesene synthase and pathway engineering. The regulation of lipid metabolism by enhancing ß-oxidation and eliminating intracellular lipid synthesis was further performed to improve the ß-farnesene synthesis. As a result, the final ß-farnesene production with bio-engineering reached 35.2 g/L and 31.9 g/L using oleic acid and waste cooking oil, respectively, which are the highest ß-farnesene titers reported in Y. lipolytica. CONCLUSIONS: This study demonstrates that engineered Y. lipolytica could realize the sustainable production of value-added acetyl-CoA-derived chemicals from waste lipid feedstock.

The regulatory role of the Aspergillus flavus core retromer complex in aflatoxin metabolism.

Aflatoxins are a series of highly toxic and carcinogenic secondary metabolites that are synthesized by Aspergillus species. The degradation of aflatoxin enzymes is an important regulatory mechanism which modulates mycotoxin producing. The retromer complex is responsible for the retrograde transport of specific biomolecules and the vacuolar fusion in the intracellular transport. Late endosomal-associated GTPase (Rab7) has been shown to be a downstream effector protein of the retromer complex. A deficiency in the retromer complex or Rab7 results in several cellular trafficking problems in yeast and humans, like protein abnormal accumulation. However, whether retromer dysfunction is involved in aflatoxin synthesis remains unclear. Here, we report that the core retromer complex, which comprises three vacuolar protein sorting-associated proteins (AflVps26-AflVps29-AflVps35), is essential for the development of dormant and resistant fungal forms such as conidia (asexual reproductive spore) and sclerotia (hardened fungal mycelium), as well as aflatoxin production and pathogenicity, in Aspergillus flavus. In particular, we show the AflVps26-AflVps29-AflVps35 complex is negatively correlated with aflatoxin exportation. Structural simulation, site-specific mutagenesis, and coimmunoprecipitation experiments showed that interactions among AflVps26, AflVps29, and AflVps35 played crucial roles in the retromer complex executing its core functions. We further found an intrinsic connection between AflRab7 and the retromer involved in vesicle-vacuole fusion, which in turn affected the accumulation of aflatoxin synthesis-associated enzymes, suggesting that they work together to regulate the production of toxins. Overall, these results provide mechanistic insights that contribute to our understanding of the regulatory role of the core retromer complex in aflatoxin metabolism.

Regulator of G Protein Signaling Contributes to the Development and Aflatoxin Biosynthesis in Aspergillus flavus through the Regulation of Gα Activity.

Heterotrimeric G-proteins play crucial roles in growth, asexual development, and pathogenicity of fungi. The regulator of G-protein signaling (RGS) proteins function as negative regulators of the G proteins to control the activities of GTPase in Gα subunits. In this study, we functionally characterized the six RGS proteins (i.e., RgsA, RgsB, RgsC, RgsD, RgsE, and FlbA) in the pathogenic fungus Aspergillus flavus. All the aforementioned RGS proteins were also found to be functionally different in conidiation, aflatoxin (AF) biosynthesis, and pathogenicity in A. flavus. Apart from FlbA, all other RGS proteins play a negative role in regulating both the synthesis of cyclic AMP (cAMP) and the activation of protein kinase A (PKA). Additionally, we also found that although RgsA and RgsE play a negative role in regulating the FadA-cAMP/PKA pathway, they function distinctly in aflatoxin biosynthesis. Similarly, RgsC is important for aflatoxin biosynthesis by negatively regulating the GanA-cAMP/PKA pathway. PkaA, which is the cAMP-dependent protein kinase catalytic subunit, also showed crucial influences on A. flavus phenotypes. Overall, our results demonstrated that RGS proteins play multiple roles in the development, pathogenicity, and AF biosynthesis in A. flavus through the regulation of Gα subunits and cAMP-PKA signals. IMPORTANCE RGS proteins, as crucial regulators of the G protein signaling pathway, are widely distributed in fungi, while little is known about their roles in Aspergillus flavus development and aflatoxin. In this study, we identified six RGS proteins in A. flavus and revealed that these proteins have important functions in the regulation of conidia, sclerotia, and aflatoxin formation. Our findings provide evidence that the RGS proteins function upstream of cAMP-PKA signaling by interacting with the Gα subunits (GanA and FadA). This study provides valuable information for controlling the contamination of A. flavus and mycotoxins produced by this fungus in pre- and postharvest of agricultural crops.

Progress and perspectives for microbial production of farnesene.

Farnesene is increasingly used in industry, agriculture, and other fields due to its unique and excellent properties, necessitating its efficient synthesis. Microbial synthesis is an ideal farnesene production method. Recently, researchers have used several strategies to optimize the production performance of microorganisms. This review summarized these strategies, including regulation of farnesene synthesis pathways, and proposed some emerging tools and methods in stain engineering. Meanwhile, new farnesene biosynthetic pathways and effective farnesene production from cheap or waste substrates were emphatically introduced. Finally, future farnesene biosynthesis challenges were discussed.

Characterization of the Bifunctional Enzyme BioDA Involved in Biotin Synthesis and Pathogenicity in Aspergillus flavus.

Biotin is an important enzyme cofactor that plays a key role in all three domains. The classical bifunctional enzyme BioDA in eukaryotes (such as Aspergillus flavus and Arabidopsis thaliana) is involved in the antepenultimate and penultimate steps of biotin biosynthesis. In this study, we identified a A. flavus bifunctional gene bioDA which could complement both Escherichia coli ΔEcbioD and ΔEcbioA mutants. Interestingly, the separated domain of AfBioD and AfBioA could, respectively, fuse with EcBioA and EcBioD well and work together. What is more, we found that BioDA was almost localized to the mitochondria in A. flavus, as shown by N-terminal red fluorescent protein tag fusion. Noteworthy, the subcellular localization of AfBioDA is never affected by common environmental stresses (such as hyperosmotic stress or oxidative stress). The knockout strategy demonstrated that the deletion of AfbioDA gene from the chromosome impaired the biotin de novo synthesis pathway in A. flavus. Importantly, this A. flavus mutant blocked biotin production and decreased its pathogenicity to infect peanuts. Based on the structural comparison, we found that two inhibitors (amiclenomycin and gemcitabine) could be candidates for antifungal drugs. Taken together, our findings identified the bifunctional AfbioDA gene and shed light on biotin biosynthesis in A. flavus.

Engineering of Yarrowia lipolytica transporters for high-efficient production of biobased succinic acid from glucose.

BACKGROUND: Succinic acid (SA) is a crucial metabolic intermediate and platform chemical. Development of biobased processes to achieve sustainable SA production has attracted more and more attention in biotechnology industry. Yarrowia lipolytica has a strong tricarboxylic acid cycle and tolerates low pH conditions, thus making it a potential platform for SA production. However, its SA titers in glucose media remain low. RESULTS: In this study, we screened mitochondrial carriers and C4-dicarboxylic acid transporters to enhance SA secretion in Y. lipolytica. PGC62-SYF-Mae strain with efficient growth and SA production was constructed by optimizing SA biosynthetic pathways and expressing the transporter SpMae1. In fed-batch fermentation, this strain produced 101.4 g/L SA with a productivity of 0.70 g/L/h and a yield of 0.37 g/g glucose, which is the highest SA titer achieved using yeast, with glucose as the sole carbon resource. CONCLUSION: Our results indicated that transporter engineering is a powerful strategy to achieve the efficient secretion of SA in Y. lipolytica, which will promote the industrial production of bio-based SA.

α-Farnesene production from lipid by engineered Yarrowia lipolytica.

Producing high value-added products from waste lipid feedstock by microbial cell factory has great advantages to minimize the pollution as well as improve the economic value of wasted oils and fats. Yarrowia lipolytica is a non-conventional oleaginous yeast and can grow on a variety of hydrophobic substrates. In this study, we explored its ability to synthesize α-farnesene, an important sesquiterpene, using lipid feedstock. Based on the α-farnesene production strain, we constructed previously, we identified that Erg12 was the key limiting factor to further increase the α-farnesene production. The α-farnesene production was improved by 35.8% through increasing the copy number of ERG12 and FSERG20 on oleic acid substrate. Expression of heterologous VHb further improved α-farnesene production by 12.7%. Combining metabolic engineering with the optimization of fermentation conditions, the α-farnesene titer and yield reached 10.2 g/L and 0.1 g/g oleic acid, respectively, in fed-batch cultivation. The α-farnesene synthesis ability on waste cooking oil and other edible oils were also explored. Compared with using glucose as carbon source, using lipid substrates obtained higher α-farnesene yield and titer, but lower by-products accumulation, demonstrating the advantage of Y. lipolytica to synthesize high value-added products using lipid feedstock.

Cyclase-Associated Protein Cap with Multiple Domains Contributes to Mycotoxin Biosynthesis and Fungal Virulence in Aspergillus flavus.

In Aspergillus, the cyclic adenosine monophosphate (cAMP) signaling modulates asexual development and mycotoxin biosynthesis. Here, we characterize the cyclase-associated protein Cap in the pathogenic fungus Aspergillus flauvs. The cap disruption mutant exhibited dramatic reduction in hyphal growth, conidiation, and spore germination, while an enhanced production of the sclerotia was observed in this mutant. Importantly, the cap gene was found to be important for mycotoxin biosynthesis and virulence. The domain deletion study demonstrated that each domain played an important role for the Cap protein in regulating cAMP/protein kinase A (PKA) signaling, while only P1 and CARP domains were essential for the full function of Cap. The phosphorylation of Cap at S35 was identified in A. flavus, which was found to play a negligible role for the function of Cap. Overall, our results indicated that Cap with multiple domains engages in mycotoxin production and fungal pathogenicity, which could be designed as potential control targets for preventing this fungal pathogen.

Engineering the oleaginous yeast Yarrowia lipolytica for production of α-farnesene.

BACKGROUND: Yarrowia lipolytica, a non-traditional oil yeast, has been widely used as a platform for lipid production. However, the production of other chemicals such as terpenoids in engineered Y. lipolytica is still low. α-Farnesene, a sesquiterpene, can be used in medicine, bioenergy and other fields, and has very high economic value. Here, we used α-farnesene as an example to explore the potential of Y. lipolytica for terpenoid production. RESULTS: We constructed libraries of strains overexpressing mevalonate pathway and α-farnesene synthase genes by non-homologous end-joining (NHEJ) mediated integration into the Y. lipolytica chromosome. First, a mevalonate overproduction strain was selected by overexpressing relevant genes and changing the cofactor specificity. Based on this strain, the downstream α-farnesene synthesis pathway was overexpressed by iterative integration. Culture conditions were also optimized. A strain that produced 25.55 g/L α-farnesene was obtained. This is the highest terpenoid titer reported in Y. lipolytica. CONCLUSIONS: Yarrowia lipolytica is a potentially valuable species for terpenoid production, and NHEJ-mediated modular integration is effective for expression library construction and screening of high-producer strains.

G Protein α Subunit GpaB is Required for Asexual Development, Aflatoxin Biosynthesis and Pathogenicity by Regulating cAMP Signaling in Aspergillus flavus.

The heterotrimeric G proteins are critical for signal transduction and function in numerous biological processes including vegetative growth, asexual development and fungal virulence in fungi. Here, we identified four G protein alpha subunits (GanA, GpaB, FadA and GaoC) in the notorious Aflatoxin-producing fungus Aspergillus flavus. GanA, GpaB and FadA have homologues in other fungal species, while GaoC is a novel one. Here, we showed that the loss function of gpaB displayed a defect in conidiophore formation and considerably reduced expression levels of conidia-specific genes brlA and abaA. A decreased viability of cell wall integrity stress and oxidative stress were also found in the ∆gpaB mutant. More importantly, aflatoxin (AF) biosynthesis and infection on crop seeds were severely impaired in the gpaB-deficient mutant. Further analyses demonstrated that the intracellular cAMP levels significantly reduced in the gpaB-deficient mutant compared to wildtype strains. Additionally, an alteration of PKA activities in the ∆gpaB mutant was also found. Overall, our results indicated that GpaB played diverse roles in asexual sporulation, AF biosynthesis and virulence by regulating cAMP signaling in Aspergillus flavus.

Investigation of Aspergillus flavus in animal virulence.

Aspergillus flavus is a common fungal pathogen of plants, animals and humans. Recently, many genes of A. flavus have been reported involving in regulation of pathogenesis in crops, but whether these genes are involved in animal virulence is still unknown. Here, we used a previous easy-to-use infection model for A. flavus based on mouse model by intravenous inoculation of A. flavus conidia. The outcome of infections in mice model showed that A. flavus NRRL3357 and laboratory strain CA14 PTS were both in dose dependent manner and highly reproducible. The progress of disease could be monitored by mice survival and histology analysis. Fungal burden analysis indicated it was gradually decreased within 7 days after infection. Moreover, aspergillosis caused by A. flavus significantly up-regulated gene expression levels of immune response mediators, including INF-γ, TNF-α, Dectin-1 and TLR2. Furthermore, the defined deletion A. flavus strains that previously displayed virulence in crop infection were also determined in this mouse model, and the results showed comparable degrees of infection in mice. Our results suggested that intravenous inoculation of conidia could be a suitable model for testing different A. flavus mutants in animal virulence. We hope to use this model to determine distinct A. flavus strains virulence in animals and study novel therapeutic methods to help control fungus diseases in the future.

The Putative Histone Methyltransferase DOT1 Regulates Aflatoxin and Pathogenicity Attributes in Aspergillus flavus.

Lysine methyltransferases transfer methyl groups in specific lysine sites, which regulates a variety of important biological processes in eukaryotes. In this study, we characterized a novel homolog of the yeast methyltransferase DOT1 in A. flavus, and observed the roles of dot1 in A. flavus. Deletion of dot1 showed a significant decrease in conidiation, but an increase in sclerotia formation. A change in viability to multiple stresses was also found in the Δdot1 mutant. Additionally, aflatoxin (AF) production was found severely impaired in the Δdot1 mutant. Further analysis by qRT-PCR revealed that the transcription of AF structural genes and their regulator gene aflS were prominently suppressed in the Δdot1 mutant. Furthermore, our data revealed that Dot1 is important for colonizing maize seeds in A. flavus. Our research indicates that Dot1 is involved in fungal development, aflatoxin biosynthesis and fungal virulence in A. flavus, which might provide a potential target for controlling A. flavus with new strategies.

The high-affinity phosphodiesterase PdeH regulates development and aflatoxin biosynthesis in Aspergillus flavus.

Cyclic AMP signaling controls a range of physiological processes in response to extracellular stimuli in organisms. Among the signaling cascades, cAMP, as a second messenger, is orchestrated by adenylate cyclase (biosynthesis) and cAMP phosphodiesterases (PDEs) (hydrolysis). In this study, we investigated the function of the high-affinity (PdeH) and low-affinity (PdeL) cAMP phosphodiesterase from the carcinogenic aflatoxin producing fungus Aspergillus flavus, and found that instead of PdeL, inactivation of PdeH exhibited a reduction in conidiation and sclerotia formation. However, the ΔpdeL/ΔpdeH mutant exhibited an enhanced phenotype defects, a similar phenotype defects to wild-type strain treated with exogenous cAMP. The activation of PKA activity was inhibited in the ΔpdeH or ΔpdeL/ΔpdeH mutant, both of whom exhibited increasing AF production. Further analysis by qRT-PCR revealed that pdeH had a high transcriptional level compared to pdeL in wild-type strain, and affected pdeL transcription. Green fluorescent protein tagging at the C-terminus of PDEs showed that PdeH-GFP is broadly compartmentalized in the cytosol, while PdeL-GFP localized mainly to the nucleus. Overall, our results indicated that PdeH plays a major role, but has overlapping function with PdeL, in vegetative growth, development and AF biosynthesis in A. flavus.

The DmtA methyltransferase contributes to Aspergillus flavus conidiation, sclerotial production, aflatoxin biosynthesis and virulence.

DNA methylation is essential for epigenetic regulation of gene transcription and development in many animals, plants and fungi. We investigated whether DNA methylation plays a role in the development and secondary metabolism of Aspergillus flavus, identified the DmtA methyltransferase from A. flavus, and produced a dmtA knock-out mutant by replacing the dmtA coding sequence with the pyrG selectable marker. The A. flavus dmtA null mutant lines produced white fluffy mycelium in liquid medium, and displayed a slightly flavescent conidial pigmentation compared with the normal yellow of the wild-type strain when grown on agar. The ΔdmtA lines exhibited decreased conidiation and aflatoxin (AF) biosynthesis, compared with the wild-type line, suggesting that the DmtA knock-out affected the transcriptional level of genes in the AF cluster. In particular, sclerotia development and host colonization were altered in the dmtA null mutants. Green fluorescent protein tagging at the C-terminus of DmtA showed that DmtA localized to the nucleus and cytoplasm. DNA methylation content measurements in the dmtA mutants revealed no widespread DNA methylation in the mutants or wild-type lines. Thus, our findings suggest that DmtA, apart from being a C-5 cytosine methyltransferase in A. flavus, contributes to asexual development, aflatoxin biosynthesis, sclerotial production and virulence.

Adenylate Cyclase AcyA Regulates Development, Aflatoxin Biosynthesis and Fungal Virulence in Aspergillus flavus.

Aspergillus flavus is one of the most important opportunistic pathogens of crops and animals. The carcinogenic mycotoxin, aflatoxins produced by this pathogen cause a health problem to human and animals. Since cyclic AMP signaling controls a range of physiological processes, like fungal development and infection when responding to extracellular stimuli in fungal pathogens, in this study, we investigated the function of adenylate cyclase, a core component of cAMP signaling, in aflatoxins biosynthesis and virulence on plant seeds in A. flavus. A gene replacement strategy was used to generate the deletion mutant of acyA that encodes the adenylate cyclase. Severe defects in fungal growth, sporulation and sclerotia formation were observed in the acyA deletion mutant. The defect in radical growth could be partially rescued by exogenous cAMP analog. The acyA mutant was also significantly reduced in aflatoxins production and virulence. Similar to the former studies in other fungi, The acyA mutant showed enhancing tolerance to oxidative stress, but more sensitive to heat stress. Overall, the pleiotropic defects of the acyA deletion mutant indicates that the cAMP-PKA pathway is involved in fungal development, aflatoxins biosynthesis and plant seed invasion in A. flavus.

[Current prevalence of Clonorchis sinensis infection in Jiangmen, Guangdong Province].

OBJECTIVE: To investigate the prevalence of clonorchiasis in Jiangmen City. METHODS: From May to December 2011, each town was randomly chosen from east, south, west, north and central area of 7 cities/districts of Jiangmen City. Four or five villages were randomly selected from each town. In each village, the residents above 3-year-old in 10% randomly sampled families were treated as research objects. Total of 14,000 fecal boxes were issued and 12,661 ones back. Eggs in stool were examined by modified Kato-Katz thick smear method (three slides per specimen). RESULTS: A total of 1316 clonorchiasis cases were found from 12,661 pepople in 140 villages with a prevalence of 10.39% (1316/12,661). The average egg density was 98.3 eggs per gram (EPG) feces. Among 7 cities/districts, the prevalence in Pengjiang District (26.68%, 402/1507) was the highest, and that of Taishan City (0.93%, 19/2049) was the lowest. The egg density in Heshan City was the highest (225.4 EPG) and the lowest one was found in Taishan City (5.13 EPG). The prevalence was negatively related with the distance to major rivers (r=-0.61, P<0.01). The prevalence and the egg density in males and females was 13.20% (807/6112) and 80.9 EPG, and 7.77% (509/6549) and 39.4 EPG, respectively. The prevalence and intensity of infection increased obviously in the groups of above 20-year-old. The people with a higher prevalence was the group of 60-69 year-old, and the people above 70 years showed heavier infection (153.8 EPG). Light, moderate and heavy infection occupied 99.91%, 0.09%, and 0. CONCLUSION: Clonorchiasis is endemic in seven districts of Jiangmen City with different epidemic degrees. There are significant differences in the prevalence and intensity of infection among different areas. The villages with higher prevalence distribute along the middle and lower sections of the two major rivers.