Novel pentose-regulated promoter of Aspergillus oryzae with application in controlling heterologous gene expression.
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ABSTRACT:
SUBMITTER: Jeennor S
PROVIDER: S-EPMC8718821 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:In the genome of Aspergillus oryzae, 12 genes have been predicted to encode serine-type carboxypeptidases. However, the carboxypeptidase activities of the proteins encoded by these genes have not yet been confirmed experimentally. In this study, we have constructed three of these 12 genes overexpressing strains using Aspergillus nidulans and characterized their overproduced recombinant proteins. Of these three genes, one was previously named cpI; the other two have not been reported yet, and hence, we named them ocpA and ocpB. The recombinant proteins released amino acid residues from the C terminus of peptides, and the activity of the enzymes was inhibited by phenylmethylsulfonyl fluoride, indicating the enzymes to be serine-type carboxypeptidases. Recombinant OcpA, OcpB, and CpI were stable at 45 degrees C, 55 degrees C, and 55 degrees C, respectively, at a low pH. The enzymatic properties of recombinant OcpB were different from those of any reported serine-type carboxypeptidase. On the other hand, recombinant OcpA had similar enzymatic properties to A. oryzae carboxypeptidases O1 and O2. The DNA and N-terminal amino acid sequences of carboxypeptidases O1 and O2 from A. oryzae IAM2640 were similar to those of OcpA. Result of transcriptional analysis of ocpA, ocpB, and cpI suggest differences in transcriptional regulation between these genes.
| S-EPMC2773364 | biostudies-literature
Project description:A novel cyclic peptide compound, KK-1, was originally isolated from the plant-pathogenic fungus Curvularia clavata. It consists of 10 amino acid residues, including five N-methylated amino acid residues, and has potent antifungal activity. Recently, the genome-sequencing analysis of C. clavata was completed, and the biosynthetic genes involved in KK-1 production were predicted by using a novel gene cluster mining tool, MIDDAS-M. These genes form an approximately 75-kb cluster, which includes nine open reading frames, containing a non-ribosomal peptide synthetase (NRPS) gene. To determine whether the predicted genes were responsible for the biosynthesis of KK-1, we performed heterologous production of KK-1 in Aspergillus oryzae by introduction of the cluster genes into the genome of A. oryzae. The NRPS gene was split in two fragments and then reconstructed in the A. oryzae genome, because the gene was quite large (approximately 40 kb). The remaining seven genes in the cluster, excluding the regulatory gene kkR, were simultaneously introduced into the strain of A. oryzae in which NRPS had already been incorporated. To evaluate the heterologous production of KK-1 in A. oryzae, gene expression was analyzed by RT-PCR and KK-1 productivity was quantified by HPLC. KK-1 was produced in variable quantities by a number of transformed strains, along with expression of the cluster genes. The amount of KK-1 produced by the strain with the greatest expression of all genes was lower than that produced by the original producer, C. clavata. Therefore, expression of the cluster genes is necessary and sufficient for the heterologous production of KK-1 in A. oryzae, although there may be unknown factors limiting productivity in this species.
| S-EPMC5900794 | biostudies-literature
Project description:Microbial natural products have had phenomenal success in drug discovery and development yet form distinct classes based on the origin of their native producer. Methods that enable metabolic engineers to combine the most useful features of the different classes of natural products may lead to molecules with enhanced biological activities. In this study, we modified the metabolism of the fungus Aspergillus oryzae to enable the synthesis of triketide lactone (TKL), the product of the modular polyketide synthase DEBS1-TE engineered from bacteria. We established (2S)-methylmalonyl-CoA biosynthesis via introducing a propionyl-CoA carboxylase complex (PCC); reassembled the 11.2 kb DEBS1-TE coding region from synthetic codon-optimized gene fragments using yeast recombination; introduced bacterial phosphopantetheinyltransferase SePptII; investigated propionyl-CoA synthesis and degradation pathways; and developed improved delivery of exogenous propionate. Depending on the conditions used titers of TKL ranged from <0.01-7.4 mg/L. In conclusion, we have demonstrated that A. oryzae can be used as an alternative host for the synthesis of polyketides from bacteria, even those that require toxic or non-native substrates. Our metabolically engineered A. oryzae may offer advantages over current heterologous platforms for producing valuable and complex natural products.
| S-EPMC8708903 | biostudies-literature
Project description:We investigated the effect of codon optimization on the expression levels of heterologous proteins in Aspergillus oryzae, using the mite allergen Der f 7 as a model protein. A codon-optimized Der f 7 gene was synthesized according to the frequency of codon usage in A. oryzae by recursive PCR. Both native and optimized Der f 7 genes were expressed under the control of a high-level-expression promoter with their own signal peptides or in a fusion construct with A. oryzae glucoamylase (GlaA). Codon optimization markedly increased protein and mRNA production levels in both nonfused and GlaA-fused Der f 7 constructs. For constructs with native codons, analysis by 3' rapid amplification of cDNA ends revealed that poly(A) tracts tended to be added within the coding region, producing aberrant mRNAs that lack a termination codon. Insertion of a termination codon between the carrier GlaA and native Der f 7 proteins in the GlaA fusion construct resulted in increases in mRNA and secreted-carrier-GlaA levels. These results suggested that mRNAs without a termination codon as a result of premature polyadenylation are degraded, possibly through the nonstop mRNA decay pathway. We suggest that codon optimization in A. oryzae results in elimination of cryptic polyadenylation signals in native Der f 7, thereby circumventing the production of truncated transcripts and resulting in an increase in steady-state mRNA levels.
| S-EPMC2576710 | biostudies-literature
Project description:The yeast Kluyveromyces lactis has been a successful host for the production of heterologous proteins for over 30 years. Currently, the galactose-/lactose-inducible and glucose-repressible LAC4 promoter (P LAC4 ) is the most widely used promoter to drive recombinant protein expression in K. lactis However, P LAC4 is not fully repressed in the presence of glucose and significant protein expression still occurs. Thus, P LAC4 is not suitable in processes where tight regulation of heterologous gene expression is required. In this study, we devised a novel K. lactis promoter system that is both strong and tightly controllable. We first tested several different endogenous K. lactis promoters for their ability to express recombinant proteins. A novel hybrid promoter (termed P350) was created by combining segments of two K. lactis promoters, namely, the strong constitutive P GAP1 promoter and the carbon source-sensitive P ICL1 promoter. We demonstrate that P350 is tightly repressed in the presence of glucose or glycerol and becomes derepressed upon depletion of these compounds by the growing cells. We further illustrate the utility of P350-controlled protein expression in shake flask and high-cell-density bioreactor cultivation strategies. The P350 hybrid promoter is a strong derepressible promoter for use in autoinduction of one-step fermentation processes for the production of heterologous proteins in K. lactisIMPORTANCE The yeast Kluyveromyces lactis is an important host for the expression of recombinant proteins at both laboratory and industrial scales. However, the system lacks a tightly regulated promoter that permits controlled expression of heterologous proteins. In this study, we report the engineering of a highly regulated strong hybrid promoter (termed P350) for use in K. lactis P350 is tightly repressed by glucose or glycerol in the medium but strongly promotes gene expression once the carbon source has been consumed by the cells. This feature permits heterologous protein expression to be "autoinduced" at any scale without the addition of a gratuitous inducer molecule or changing feed solutions.
| S-EPMC6606884 | biostudies-literature
Project description:Oxysterol-binding proteins (OSBPs) comprise a family of sterol-binding proteins. In this study, we focused on AoOSBP1, one of the five OSBP proteins identified from the industrial fungus Aspergillus oryzae. The temporal expression pattern analysis showed that the expression of AoOSBP1, in both gene and protein levels, was stably expressed throughout the developmental stages, while was upregulated during the accelerated growth stage. The immunofluorescence observation revealed that AoOSBP1 protein was mainly distributed in the conidiophore, indicating its underlying role in spore formation. The ligand-binding domain of AoOSBP1, namely OSBP-related domain (ORD), was heterologously expressed in Escherichia coli and purified. The binding assay carried out using microscale thermophoresis showed that the recombinant AoORD protein exhibited binding affinity for ergosterol, and exhibited much higher affinity to oxysterols (25-hydroxycholesterol and 7-ketocholesterol) and phytosterols (?-sitosterol and stigmasterol). By contrast, MBP tag as the negative control showed no binding affinity for sterols. The present work demonstrates that AoORD domain in AoOSBP1 is capable of binding sterols, plays an underlying role in sterols transportation, and may participate in spore formation.
| S-EPMC6863284 | biostudies-literature
Project description:BackgroundCyclic dipeptides are an important class of natural products owing to their structural diversity and biological activities. In fungi, the cyclo-ring system is formed through the condensation of two α-amino acids via non-ribosomal peptide synthetase (NRPS). However, there are few investigations on the functional identification of this enzyme. Additionally, information on how to increase the production of cyclic dipeptide molecules is relatively scarce.ResultsWe isolated the Eurotium cristatum NWAFU-1 fungus from Jing-Wei Fu brick tea, whose fermentation metabolites contain echinulin-related cyclic dipeptide molecules. We cloned the cirC gene, encoding an NRPS, from E. Cristatum NWAFU-1 and transferred it into the heterologous host Aspergillus oryzae. This transformant produced a novel metabolite possessing an L-tryptophan-L-alanine cyclic dipeptide backbone (Cyclo-TA). Based on the results of heterologous expression and microsomal catalysis, CriC is the first NRPS characterized in fungi that catalyzes the formation of a cyclic dipeptide from L-tryptophan and L-alanine. After substrate feeding, the final yield reached 34 mg/L. In this study, we have characterized a novel NRPS and developed a new method for cyclic dipeptide production.ConclusionsIn this study we successfully expressed the E. Cristatum NWAFU-1 criC gene in A. oryzae to efficiently produce cyclic dipeptide compounds. Our findings indicate that the A. oryzae heterologous expression system constitutes an efficient method for the biosynthesis of fungal Cyclic dipeptides.
| S-EPMC9290255 | biostudies-literature
Project description:Efficient expression of the dye-decolorizing peroxidase, DyP, from Geotrichum candidum Dec 1 in Aspergillus oryzae M-2-3 was achieved by fusing mature cDNA encoding dyp with the A. oryzae alpha-amylase promoter (amyB). The activity yield of the purified recombinant DyP (rDyP) was 42-fold compared with that of the purified native DyP from Dec 1. No exogenous heme was necessary for the expression of rDyP in A. oryzae. From the N-terminal amino acid sequence analyses of native DyP and rDyP, the absence of a histidine residue in both DyPs, which was considered to be important for heme binding of DyP, was confirmed. These results suggest that rDyP without a typical heme-binding region produced by A. oryzae exhibits a function similar to that of native DyP.
| S-EPMC92058 | biostudies-literature
Project description:Xylanases (EC 3.2.1.8) hydrolyze the hemicellulose of plant cell walls. Xylanases are used in the food and paper industries and for bioconversion of lignocellulose to biofuel. In this work, the producer-strain with four copies of the xAor xylanase gene was organized in two tandem copies for optimal expression in Komagataella phaffii T07 yeast. The secreted 35 kDa xylanase was purified from culture medium by gel filtration on Sephadex G-25 and anion exchange chromatography on DEAE-Sepharose 6HF. Tryptic peptides of the recombinant enzyme were analyzed by liquid chromatography-tandem mass spectrometry where the amino acid sequence corresponded to Protein Accession # O94163 for Endo-1,4-beta-xylanase from Aspergillus oryzae RIB40. The recombinant xylanase was produced in a bioreactor where the secreted enzyme hydrolyzed oat xylane with an activity of 258240 IU/mL. High activity in the culture medium suggested xylanase could be used for industrial applications without being purified or concentrated. The pH optimum for xylanase xAor was 7.5, though the enzyme was active from pH 2.5 to pH 10. Xylanase was active at temperatures from 35 °C to 85 °C with a maximum at 60 °C. In conclusion, this protocol yields soluble, secreted xylanase suitable for industrial scale production.
| S-EPMC9369408 | biostudies-literature
Project description:BackgroundLactic acid is the building block of poly-lactic acid (PLA), a biopolymer that could be set to replace petroleum-based plastics. To make lactic acid production cost-effective, the production process should be carried out at low pH, in low-nutrient media, and with a low-cost carbon source. Yeasts have been engineered to produce high levels of lactic acid at low pH from glucose but not from carbohydrate polymers (e.g. cellulose, hemicellulose, starch). Aspergilli are versatile microbial cell factories able to naturally produce large amounts of organic acids at low pH and to metabolize cheap abundant carbon sources such as plant biomass. However, they have never been used for lactic acid production.ResultsTo investigate the feasibility of lactic acid production with Aspergillus, the NAD-dependent lactate dehydrogenase (LDH) responsible for lactic acid production by Rhizopus oryzae was produced in Aspergillus brasiliensis BRFM103. Among transformants, the best lactic acid producer, A. brasiliensis BRFM1877, integrated 6 ldhA gene copies, and intracellular LDH activity was 9.2 × 10(-2) U/mg. At a final pH of 1.6, lactic acid titer reached 13.1 g/L (conversion yield: 26%, w/w) at 138 h in glucose-ammonium medium. This extreme pH drop was subsequently prevented by switching nitrogen source from ammonium sulfate to Na-nitrate, leading to a final pH of 3 and a lactic acid titer of 17.7 g/L (conversion yield: 47%, w/w) at 90 h of culture. Final titer was further improved to 32.2 g/L of lactic acid (conversion yield: 44%, w/w) by adding 20 g/L glucose to the culture medium at 96 h. This strain was ultimately able to produce lactic acid from xylose, arabinose, starch and xylan.ConclusionWe obtained the first Aspergillus strains able to produce large amounts of lactic acid by inserting recombinant ldhA genes from R. oryzae into a wild-type A. brasiliensis strain. pH regulation failed to significantly increase lactic acid production, but switching nitrogen source and changing culture feed enabled a 1.8-fold increase in conversion yields. The strain produced lactic acid from plant biomass. Our findings make A. brasiliensis a strong contender microorganism for low-pH acid production from various complex substrates, especially hemicellulose.
| S-EPMC4425913 | biostudies-literature