Rhodoferax potami sp. nov. and Rhodoferax mekongensis sp. nov., isolated from the Mekong River in Thailand.

Four newly discovered Gram-stain-negative bacteria, designated as BL00010T, BL00058, D8-11T and BL00200, were isolated from water samples collected at three hydrological monitoring stations (namely Chiang Saen, Chiang Khan and Nong Khai) located along the Mekong River in Thailand. An investigation encompassing phenotypic, chemotaxonomic and genomic traits was conducted. The results of phylogenetic analysis based on 16S rRNA gene sequences indicated that all four isolates represented members of the genus Rhodoferax. These isolates were closely related to Rhodoferax bucti KCTC 62564T with a similarity of 99.59%. The major fatty acids of the four novel isolates included C16:0 and C16:1ω7c and/or C16 : 1ω6c, whereas the major respiratory quinone was identified as ubiquinone Q-8. In addition, phosphatidylethanolamine was identified as a major polar lipid in these bacteria. The genomes of BL00010T, BL00058, D8-11T and BL00200 were similar in size (3.88-4.01 Mbp) and DNA G+C contents (59.5, 59.3, 59.5 and 59.3 mol%, respectively). In contrast to R. bucti KCTC 62564T and Rhodoferax aquaticus KCTC 32394T, the newly discovered species possessed several genes involved in nitrite and nitrile metabolism, which may be related to their unique adaptation to nitrile-rich environments. From the results of the pairwise analysis of average nucleotide identity of the whole genome and digital DNA-DNA hybridisation, it was evident that BL00010T and D8-11T represented two novel species, for which we propose the nomenclature Rhodoferax potami sp. nov., with the type strain BL00010T (TBRC 17198T = NBRC 116413T), and Rhodoferax mekongensis sp. nov., with the type strain D8-11T (TBRC 17307T = NBRC 116415T).

Efficient de novo production of bioactive cordycepin by Aspergillus oryzae using a food-grade expression platform.

BACKGROUND: Cordycepin (3'-deoxyadenosine) is an important bioactive compound in medical and healthcare markets. The drawbacks of commercial cordycepin production using Cordyceps spp. include long cultivation periods and low cordycepin yields. To overcome these limitations and meet the increasing market demand, the efficient production of cordycepin by the GRAS-status Aspergillus oryzae strain using a synthetic biology approach was developed in this study. RESULTS: An engineered strain of A. oryzae capable of cordycepin production was successfully constructed by overexpressing two metabolic genes (cns1 and cns2) involved in cordycepin biosynthesis under the control of constitutive promoters. Investigation of the flexibility of carbon utilization for cordycepin production by the engineered A. oryzae strain revealed that it was able to utilize C6-, C5-, and C12-sugars as carbon sources, with glucose being the best carbon source for cordycepin production. High cordycepin productivity (564.64 ± 9.59 mg/L/d) was acquired by optimizing the submerged fermentation conditions. CONCLUSIONS: This study demonstrates a powerful production platform for bioactive cordycepin production by A. oryzae using a synthetic biology approach. An efficient and cost-effective fermentation process for cordycepin production using an engineered strain was established, offering a powerful alternative source for further upscaling.

Transcriptome-based Mining of the Constitutive Promoters for Tuning Gene Expression in Aspergillus oryzae.

Transcriptional regulation has been adopted for developing metabolic engineering tools. The regulatory promoter is a crucial genetic element for strain optimization. In this study, a gene set of Aspergillus oryzae with highly constitutive expression across different growth stages was identified through transcriptome data analysis. The candidate promoters were functionally characterized in A. oryzae by transcriptional control of ß-glucuronidase (GUS) as a reporter. The results showed that the glyceraldehyde triphosphate dehydrogenase promoter (PgpdA1) of A. oryzae with a unique structure displayed the most robust strength in constitutively controlling the expression compared to the PgpdA2 and other putative promoters tested. In addition, the ubiquitin promoter (Pubi) of A. oryzae exhibited a moderate expression strength. The deletion analysis revealed that the 5' untranslated regions of gpdA1 and ubi with the length of 1028 and 811 nucleotides, counted from the putative translation start site (ATG), respectively, could efficiently drive the GUS expression. Interestingly, both promoters could function on various carbon sources for cell growth. Glucose was the best fermentable carbon source for allocating high constitutive expressions during cell growth, and the high concentrations (6-8% glucose, w/v) did not repress their functions. It was also demonstrated that the secondary metabolite gene coding for indigoidine could express under the control of PgpdA1 or Pubi promoter. These strong and moderate promoters of A. oryzae provided beneficial options in tuning the transcriptional expression for leveraging the metabolic control towards the targeted products.

The Exploring Functional Role of Ammonium Transporters of Aspergillus oryzae in Nitrogen Metabolism: Challenges towards Cell Biomass Production.

Ammonium is a source of fermentable inorganic nitrogen essential for the growth and development of filamentous fungi. It is involved in several cellular metabolic pathways underlying nitrogen transport and assimilation. Ammonium can be transferred into the cell by an ammonium transporter. This study explored the role of ammonium transporters in nitrogen metabolism and cell biomass production in Aspergillus oryzae strain BCC 7051. Specific sequences encoding ammonium transporters (Amts) in A. oryzae were identified using genomic analysis. Four of the identified ammonium transporter genes, aoamt1-aoamt4, showed similarity in deduced amino acid sequences to the proteins in the ammonium transporter/methylammonium permease (AMT/MEP) family. Transcriptional analysis showed that the expression of aoamt2 and aoamt3 was ammonium-dependent, and was highly upregulated under ammonium-limited conditions. Their functional roles are characterized by genetic perturbations. The gene disruption and overexpression of aoamt3 indicated that the protein encoded by it was a crucial ammonium transporter associated with nitrogen metabolism and was required for filamentous growth. Compared with the wild type, the aoamt3-overexpressing strain showed superior growth performance, high biomass yield, and low glucose consumption. These results shed light on further improvements in the production of potent bioproducts by A. oryzae by manipulating the ammonium uptake capacity and nitrogen metabolism.

Potential of Aspergillus oryzae as a biosynthetic platform for indigoidine, a non-ribosomal peptide pigment with antioxidant activity.

The growing demand for natural pigments in the industrial sector is a significant driving force in the development of production processes. The production of natural blue pigments, which have wide industrial applications, using microbial systems has been gaining significant attention. In this study, we used Aspergillus oryzae as a platform cell factory to produce the blue pigment indigoidine (InK), by genetic manipulation of its non-ribosomal peptide synthetase system to overexpress the indigoidine synthetase gene (AoinK). Phenotypic analysis showed that InK production from the engineered strain was growth associated, owing to the constitutive control of gene expression. Furthermore, the initial pH, temperature, and glutamine and MgSO4 concentrations were key factors affecting InK production by the engineered strain. The pigment secretion was enhanced by addition of 1% Tween 80 solution to the culture medium. The maximum titer of total InK was 1409.22 ± 95.33 mg/L, and the maximum productivity was 265.09 ± 14.74 mg/L·d. Moreover, the recombinant InK produced by the engineered strain exhibited antioxidant activity. These results indicate that A. oryzae has the potential to be used as a fungal platform for overproduction of extracellular non-ribosomal peptide pigments.

Novel pentose-regulated promoter of Aspergillus oryzae with application in controlling heterologous gene expression.

The potent promoter and its transcriptional control make a significant contribution to strain optimization. Using transcriptome-based approach, a novel pentose-regulated promoter of the xylose reductase gene (PxyrA) of Aspergillus oryzae was identified. The promoter analysis showed that the PxyrA was tightly regulated by pentose sugars, which xylose and xylan were favorable inducers. The PxyrA function was highly efficient as compared with the maltose-inducible promoters of A. oryzae. It also exhibited the efficient transcription induction even though certain amounts of glucose and sucrose existed in the cultures. The expression control of PxyrA was dependent on xylose consumption capacity for fungal growth. The control mode of PxyrA offers a simple operation in simultaneous gene expression and cultivation optimization in Aspergilli. This study provides a prospective development of fungal production platform using cellulosic sugars by the xylose-utilizing strains for sustainable growing in circular economy.

Promoter exchange of the cryptic nonribosomal peptide synthetase gene for oligopeptide production in Aspergillus oryzae.

Oligopeptides with functional activities are of current interest in the nutraceutical and medical sectors. The development of the biosynthetic process of oligopeptides through a nonribosomal peptide synthetase (NRPS) system has become more challenging. To develop a production platform for nonribosomal peptides (NRPs), reprogramming of transcriptional regulation of the acv gene encoded ACV synthetase (ACVS) was implemented in Aspergillus oryzae using the CRISPR-Cas9 system. Awakening silent acv expression was successfully achieved by promoter substitution. Among the three exchanged promoters, AoPgpdA, AoPtef1, and PtPtoxA, the replacement of the native promoter with AoPgpdA led to the highest ACV production in A. oryzae. However, the ACV production of the AoPGpdA strain was also dependent on the medium composition, in which urea was the best nitrogen source, and a C:N ratio of 20:1 was optimal for tripeptide production. In addition to cell growth, magnesium ions are an essential element for ACV production and might participate in ACVS activity. It was also found that ACV was the growth-associated product of the engineered strain that might be a result of constitutive transcriptional control by the AoPgpdA promoter. This study offers a potential strategy for nonribosomal ACV production using the fungal system, which is applicable for redesigning bioactive oligopeptides with industrial relevance.

Significance of two intracellular triacylglycerol lipases of Aspergillus oryzae in lipid mobilization: A perspective in industrial implication for microbial lipid production.

Microbial lipid production of oleaginous strains involves in a complex cellular metabolism controlling lipid biosynthesis, accumulation and degradation. Particular storage lipid, triacylglycerol (TAG), contributes to dynamic traits of intracellular lipids and cell growth. To explore a basis of TAG degradation in the oleaginous strain of Aspergillus oryzae, the functional role of two intracellular triacylglycerol lipases, AoTgla and AoTglb, were investigated by targeted gene disruption using CRISPR/Cas9 system. Comparative lipid profiling of different cultivation stages between the control, single and double disruptant strains (ΔAotgla, ΔAotglb and ΔAotglaΔAotglb strains) showed that the inactivation of either AoTgla or AoTglb led to the increase of total lipid contents, particularly in the TAG fraction. Moreover, the prolonged lipid-accumulating stage of all disruptant strains was obtained as indicated by a reduction in specific rate of lipid turnover, in which a holding capacity in maximal lipid and TAG levels was achieved. The involvement of AoTgls in spore production of A. oryzae was also discovered. In addition to the significance in lipid physiology of the oleaginous fungi, this study provides an impact on industrial practice by overcoming the limitation in short lipid-accumulating stage of the fungal strain, which facilitate the cell harvesting step at the maximum lipid production yield.

Exploring differential traits of lipid-producing stages of the wild type and morphologically engineered strain of Aspergillus oryzae by comparative kinetic modeling.

Comparative profilings of cell growth and lipid production in the morphologically engineered strain (Δags1) and the wild type (WT) of Aspergillus oryzae BCC7051 were implemented. Using various nitrogen sources, a discrimination in cell morphology between the two strains was found, of which the Δags1 culture exhibited mycelial growth as small pellets in contrast to the WT. Of them, sodium nitrate and potassium nitrate were optimal for lipid production of the WT and Δags1 strains, respectively, which the highest lipid concentrations of 7.2 and 7.9 g L-1 were obtained in the respective cultures. The mathematical models of the growth kinetics and lipid phenotypes of both fungal strains were developed, enabling to distinguish three lipid-producing stages, including low lipid-producing, lipid accumulation, and lipid turnover stages. The model validation showed good performances in all nitrogen sources tested for the WT, but only NaNO3 and mixed yeast extract/NH4Cl were fitted well for the Δags1. The difference in the period of lipid-producing stages between the WT and Δags1 indicated the metabolic alterations of A. oryzae by the defect of a gene involved in the cell wall biosynthesis, which exhibited benefits for bioprocessing practices in addition to the high productivities of biomass and lipid. These findings would further permit the manipulation in the metabolic hub of the fungal production platform for other industrial purposes.

Functional Characterization of Novel U6 RNA Polymerase III Promoters: Their Implication for CRISPR-Cas9-Mediated Gene Editing in Aspergillus oryzae.

U6 RNA polymerase III promoter (PU6), which is a key element in controlling the generation of single-guide RNA (sgRNA) for gene editing through CRISPR-Cas9 system, was investigated in this work. Using bioinformatics approach, two novel U6 ribonucleic acid (U6 RNA) sequences of Aspergillus niger were identified, showing that they had conserved motifs similar to other U6 RNAs. The putative PU6 located at the upstream sequence of A. niger U6 RNA exhibited the consensus motif, CCAATYA, and the TATA box which shared highly conserved characteristics across Aspergilli, whereas the A- and B-boxes were found at the intragenic and downstream of the structural genes, respectively. Using Aspergillus oryzae as a workhorse system, the function of A. niger PU6s for controlling the transcripts of sgRNA was verified, in which the orotidine-5'-phosphate decarboxylase (pyrG) sequence was used as a target for gene disruption through CRISPR-Cas9 system. Quantitative reverse transcription-polymerase chain reaction (RT-qPCR) analysis of the selected pyrG auxotrophic strains showed the expression of sgRNA, indicating that the non-native promoters could efficiently drive sgRNA expression in A. oryzae. These identified promoters are useful genetic tools for precise engineering of metabolic pathways in the industrially important fungus through the empowered CRISPR-Cas9-associated gene-editing system.

Morphologically engineered strain of Aspergillus oryzae as a cell chassis for production development of functional lipids.

Cell morphology of the oleaginous fungus, Aspergillus oryzae BCC7051, was genetically engineered by disruption of non-essential genes involved in cell wall biosynthesis. Comparative phenotypic analysis of two disruptant strains defective either in α-1,3-glucan synthase 1 (ΔAoAgs1) or chitin synthase B (ΔAoChsB), and the wild type showed that the ΔAoAgs1 strain had no alterations in colonial growth and sporulation when grown on agar medium whereas the ΔAoChsB disruptant showed growth retardation and lower sporulation. However, tiny and loose pellets were found in the ΔAoAgs1 culture grown in liquid medium, where fungal pellet size was decreased by 35-50% of the wild type size. Further investigation of the ΔAoAgs1 mutant grown under stress-induced conditions, including high salt concentration, ionic strength and osmolarity, showed that its growth and development remained similar to that of the wild type. When cultivating the ΔAoAgs1 strain in a stirred-tank bioreactor, lipid production in terms of titer and productivity was significantly improved. As compared to the wild type, an increase of triacylglycerol and ergosterol contents with a proportional decrease in steryl ester content was observed in the ΔAoAgs1 strain. These results suggest that the morphologically engineered strain of A. oryzae is a robust cell chassis useful for exploitation in further production development of functional lipids with industrial significance.

Reengineering lipid biosynthetic pathways of Aspergillus oryzae for enhanced production of γ-linolenic acid and dihomo-γ-linolenic acid.

Biological significance of 18-carbon polyunsaturated fatty acids, γ-linolenic acid (GLA; C18:3 n-6) and dihomo-γ-linolenic acid (DGLA; C20:3 n-6) has gained much attention in the systematic development of optimized strains for industrial applications. In this work, a n-6 PUFAs-producing strain of Aspergillus oryzae was generated by manipulating metabolic reactions in fatty acid modification and triacylglycerol biosynthesis. The codon-optimized genes coding for Δ6-desaturase and Δ6-elongase of Pythium sp., and diacylglycerol acyltransferase 2 (mMaDGAT2) of Mortierella alpina were co-transformed in a single vector into A. oryzae BCC14614, yielding strain TD6E6-DGAT2. Comparative phenotypic analysis showed that a 70% increase of lipid titer was found in the engineered strain, which was a result of a significant increase in triacylglycerol (TAG) content (52.0 ±â€¯1.8% of total lipids), and corresponded to the increased size of lipid particles observed in the fungal cells. Interestingly, the proportions of GLA and DGLA in neutral lipids of the engineered strain were similar, with the highest titers obtained in the high C:N culture (29:0; 6% glucose) during the lipid-accumulating stage of growth. Time-course expression analysis of the engineered strain revealed transcriptional control of TAG biosynthesis through a co-operation between the native DGAT2 of A. oryzae and the transformed mMaDGAT2.

Genome Characterization of Oleaginous Aspergillus oryzae BCC7051: A Potential Fungal-Based Platform for Lipid Production.

The selected robust fungus, Aspergillus oryzae strain BCC7051 is of interest for biotechnological production of lipid-derived products due to its capability to accumulate high amount of intracellular lipids using various sugars and agro-industrial substrates. Here, we report the genome sequence of the oleaginous A. oryzae BCC7051. The obtained reads were de novo assembled into 25 scaffolds spanning of 38,550,958 bps with predicted 11,456 protein-coding genes. By synteny mapping, a large rearrangement was found in two scaffolds of A. oryzae BCC7051 as compared to the reference RIB40 strain. The genetic relationship between BCC7051 and other strains of A. oryzae in terms of aflatoxin production was investigated, indicating that the A. oryzae BCC7051 was categorized into group 2 nonaflatoxin-producing strain. Moreover, a comparative analysis of the structural genes focusing on the involvement in lipid metabolism among oleaginous yeast and fungi revealed the presence of multiple isoforms of metabolic enzymes responsible for fatty acid synthesis in BCC7051. The alternative routes of acetyl-CoA generation as oleaginous features and malate/citrate/pyruvate shuttle were also identified in this A. oryzae strain. The genome sequence generated in this work is a dedicated resource for expanding genome-wide study of microbial lipids at systems level, and developing the fungal-based platform for production of diversified lipids with commercial relevance.

Diacylglycerol acyltransferase 2 of Mortierella alpina with specificity on long-chain polyunsaturated fatty acids: A potential tool for reconstituting lipids with nutritional value.

Based on available genome sequences and bioinformatics tools, we searched for an uncharacterized open reading frame of Mortierella alpina (MaDGAT2) using diacylglycerol acyltransferase sequence (fungal DGAT type 2B) as a query. Functional characterization of the identified native and codon-optimized M. alpina genes were then performed by heterologous expression in Saccharomyces cerevisiae strain defective in synthesis of neutral lipid (NL). Lipid analysis of the yeast tranformant carrying MaDGAT2 showed that the NL biosynthesis and lipid particle formation were restored by the gene complementation. Substrate specificity study of the fungal enzyme by fatty acid supplementation in the transformant cultures showed that it had a broad specificity on saturated and unsaturated fatty acid substrates for esterification into triacylglycerol (TAG). The n-6 polyunsaturated fatty acids (PUFAs) with 18 and 20 carbon atoms, including linoleic acid, γ-linolenic acid, dihomo γ-linolenic and arachidonic acid could be incorporated into TAG fraction in the yeast cells. Interestingly, among n-3 PUFAs tested, the MaDGAT2 enzyme preferred eicosapentaenoic acid (EPA) substrate as its highly proportional constituent found in TAG fraction. This study provides a potential genetic tool for reconstituting oils rich in long-chain PUFAs with nutritional value.

Metabolic engineering of long chain-polyunsaturated fatty acid biosynthetic pathway in oleaginous fungus for dihomo-gamma linolenic acid production.

Microbial lipids are promising alternative sources of long chain-polyunsaturated fatty acids (LC-PUFAs) for food, feed, nutraceutical and pharmaceutical sectors. Dihomo-γ-linolenic acid (C20:3Δ(8,11,14); DGLA) is an important LC-PUFAs with anti-inflammatory and anti-proliferative effects. To generate a DGLA-producing strain, fatty acid reconstitution in Aspergillus oryzae was performed by metabolic engineering through co-expression of codon-optimized Pythium Δ(6)-desaturase and Δ(6)-elongase, which had high conversion rates of substrates to respective products as compared to the native enzymes. The Δ(6)-desaturated and Δ(6)-elongated products, γ-linolenic acid (C18:3Δ(6,9,12); GLA) and DGLA, were accumulated in phospholipids rather than triacylglycerol. Interestingly, the manipulation of lipid quality in the oleaginous fungus did not affect growth and lipid phenotypes. This strategy might expand to development of the oleaginous fungal strain for producing other tailor-made oils with industrial applications.

Systematic development of biomass overproducing Scheffersomyces stipitis for high-cell-density fermentations.

The development of economically feasible bio-based process requires efficient cell factories capable of producing the desired product at high titer under high-cell-density fermentation. Herein we present a combinatorial approach based on systems metabolic engineering and metabolic evolution for the development of efficient biomass-producing strain. Systems metabolic engineering guided by flux balance analysis (FBA) was first employed to rationally design mutant strains of Scheffersomyces stipitis with high biomass yield. By experimentally implementing these mutations, the biomass yield was improved by 30% in GPD1, 25% in TKL1, 30% in CIT1, and 44% in ZWF1 overexpressed mutants compared to wild-type. These designed mutants were further fine-tuned through metabolic evolution resulting in the maximal biomass yield of 0.49 g-cdw/g-glucose, which matches well with predicted yield phenotype. The constructed mutants are beneficial for biotechnology applications dealing with high cell titer cultivations. This work demonstrates a solid confirmation of systems metabolic engineering in combination with metabolic evolution approach for efficient strain development, which could assist in rapid optimization of cell factory for an economically viable and sustainable bio-based process.

The codon-optimized Δ(6)-desaturase gene of Pythium sp. as an empowering tool for engineering n3/n6 polyunsaturated fatty acid biosynthesis.

BACKGROUND: The ∆(6)-desaturase gene, encoding a key enzyme in the biosynthesis of polyunsaturated fatty acids, has potential in pharmaceutical and nutraceutical applications. RESULTS: The ∆(6)-desaturase gene has been isolated from a selected strain of Oomycetes, Pythium sp. BCC53698. The cloned gene (PyDes6) contained an open reading frame (ORF) of 1401 bp encoding 466 amino acid residues. The deduced amino acid sequence shared a high similarity to those of other ∆(6)-desaturases that contained the signature features of a membrane-bound ∆(6)-desaturase, including a cytochrome b 5 and three histidine-rich motifs and membrane-spanning regions. Heterologous expression in Saccharomyces cerevisiae showed that monoene, diene and triene fatty acids having ∆(9)-double bond were substrates for PyDes6. No distinct preference between the n-3 and n-6 polyunsaturated fatty acyl substrates was found. The ∆(6)-desaturated products were markedly increased by codon optimization of PyDes6. CONCLUSION: The codon-optimized ∆(6)-desaturase gene generated in this study is a promising tool for further reconstitution of the fatty acid profile, in a host system of choice, for the production of economically important fatty acids, particularly the n-3 and n-6 polyunsaturated fatty acids.

Novel elongase of Pythium sp. with high specificity on Δ(6)-18C desaturated fatty acids.

We identified a novel elongase gene from a selected strain of the Oomycete, Pythium sp. BCC53698. Using a PCR approach, the cloned gene (PyElo) possessed an open reading frame (ORF) of 834 bp encoding 277 amino acid residues. A similarity search showed that it had homology with the PUFA elongases of several organisms. In addition, the signature characteristics, including four conserved motifs, a histidine-rich catalytic motif and membrane-associated feature were present in the Pythium gene. Heterologous expression in Saccharomyces cerevisiae showed that it was specific for fatty acid substrates, having a double bond at Δ(6)-position, which included γ-linolenic acid (GLA) and stearidonic acid (STA), and preferentially elongated the n3-18C PUFA. This is an elongase in Oomycete fungi, which displays very high specificity on Δ(6)-18C desaturated fatty acids. This will be a powerful tool to engineer PUFA biosynthesis in organisms of interest through the n-6 series pathway for producing value-added fatty acids.

Comparative fatty acid profiling of Mucor rouxii under different stress conditions.

To understand the relationship between fatty acid metabolism and the growth morphology of Mucor rouxii, fatty acid profiling was studied comparatively in cells grown under conditions which included different atmospheric conditions or the addition of phenethyl alcohol (PEA). The significant difference in fatty acid profiles from M. rouxii grown under aerobic or anaerobic conditions was not found to be directly related to morphological growth. Oxygen limitation, which induced the formation of pure multipolar budding yeasts, led to a decrease in long-chain fatty acids-- particularly unsaturated fatty acids-- and an increase in medium-chain saturated fatty acids, a finding which contrasted with the aerobic cultures, including mycelia and PEA-induced bipolar budding cells. High levels of C18 : 1Delta(9) were found in aerobic yeast cultures with additional PEA when compared to that in the aerobically grown mycelia. The identification of unusual fatty acids in Mucor in response to alcoholic and hypoxic stresses - including odd-numbered fatty acids and 7-hydroxy dodecanoic acid (7-OH C12 : 0) in addition to the more common fatty acids - implied that an important role existed for these unusual fatty acids.