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.

Physiological Traits of Dihomo-γ-Linolenic Acid Production of the Engineered Aspergillus oryzae by Comparing Mathematical Models.

Dihomo-γ-linolenic acid (DGLA; C20:3 n-6) is expected to dominate the functional ingredients market for its role in anti-inflammation and anti-proliferation. The DGLA production by the engineered strain of Aspergillus oryzae with overexpressing Pythium Δ6-desaturase and Δ6-elongase genes was investigated by manipulating the nutrient and fermentation regimes. Of the nitrogen sources tested, the maximum biomass and DGLA titers were obtained in the cultures using NaNO3 grown at pH 6.0. For establishing economically feasible process of DGLA production, the cost-effective medium was developed by using cassava starch hydrolysate (CSH) and NaNO3 as carbon and nitrogen sources, respectively. The supplementation with 1% (v/v) mother liquor (ML) into the CSH medium promoted the specific yield of DGLA production (Y DGLA / X ) comparable with the culture grown in the defined NaNO3 medium, and the DGLA proportion was over 22% in total fatty acid (TFA). Besides, the GLA was also generated at a similar proportion (about 25% in TFA). The mathematical models of the cultures grown in the defined NaNO3 and CSH/ML media were generated, describing that the lipid and DGLA were growth-associated metabolites corresponding to the relevant kinetic parameters of fermentations. The controlled mode of submerged fermentation of the engineered strain was explored for governing the PUFA biosynthesis and lipid-accumulating process in relation to the biomass production. This study provides an informative perspective in the n-6 fatty acid production through physiological manipulation, thus leading to a prospect in viable production of the DGLA-enriched oil by the engineered strain.

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.

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.