Microbial production of trans-aconitic acid.

Trans-aconitic acid (TAA) is a promising bio-based chemical with the structure of unsaturated tricarboxylic acid, and also has the potential to be a non-toxic nematicide as a potent inhibitor of aconitase. However, TAA has not been commercialized because the traditional production processes of plant extraction and chemical synthesis cannot achieve large-scale production at a low cost. The availability of TAA is a serious obstacle to its widespread application. In this study, we developed an efficient microbial synthesis and fermentation production process for TAA. An engineered Aspergillus terreus strain producing cis-aconitic acid and TAA was constructed by blocking itaconic acid biosynthesis in the industrial itaconic acid-producing strain. Through heterologous expression of exogenous aconitate isomerase, we further designed a more efficient cell factory to specifically produce TAA. Subsequently, the fermentation process was developed and scaled up step-by-step, achieving a TAA titer of 60 g L-1 at the demonstration scale of a 20 m3 fermenter. Finally, the field evaluation of the produced TAA for control of the root-knot nematodes was performed in a field trial, effectively reducing the damage of the root-knot nematode. Our work provides a commercially viable solution for the green manufacturing of TAA, which will significantly facilitate biopesticide development and promote its widespread application as a bio-based chemical.

Improving the production of the micafungin precursor FR901379 in an industrial production strain.

BACKGROUND: Micafungin is an echinocandin-type antifungal agent used for the clinical treatment of invasive fungal infections. It is semisynthesized from the sulfonated lipohexapeptide FR901379, a nonribosomal peptide produced by the filamentous fungus Coleophoma empetri. However, the low fermentation efficiency of FR901379 increases the cost of micafungin production and hinders its widespread clinical application. RESULTS: Here, a highly efficient FR901379-producing strain was constructed via systems metabolic engineering in C. empetri MEFC09. First, the biosynthesis pathway of FR901379 was optimized by overexpressing the rate-limiting enzymes cytochrome P450 McfF and McfH, which successfully eliminated the accumulation of unwanted byproducts and increased the production of FR901379. Then, the functions of putative self-resistance genes encoding ß-1,3-glucan synthase were evaluated in vivo. The deletion of CEfks1 affected growth and resulted in more spherical cells. Additionally, the transcriptional activator McfJ for the regulation of FR901379 biosynthesis was identified and applied in metabolic engineering. Overexpressing mcfJ markedly increased the production of FR901379 from 0.3 g/L to 1.3 g/L. Finally, the engineered strain coexpressing mcfJ, mcfF, and mcfH was constructed for additive effects, and the FR901379 titer reached 4.0 g/L under fed-batch conditions in a 5 L bioreactor. CONCLUSIONS: This study represents a significant improvement for the production of FR901379 and provides guidance for the establishment of efficient fungal cell factories for other echinocandins.

Discovery of a Unique Flavonoid Biosynthesis Mechanism in Fungi by Genome Mining.

Flavonoids are important plant natural products with variable structures and bioactivities. All known plant flavonoids are generated under the catalysis of a type III polyketide synthase (PKS) followed by a chalcone isomerase (CHI) and a flavone synthase (FNS). In this study, the biosynthetic gene cluster of chlorflavonin, a fungal flavonoid with acetolactate synthase inhibitory activity, was discovered using a self-resistance-gene-directed strategy. A novel flavonoid biosynthetic pathway in fungi was revealed. A core nonribosomal peptide synthetase-polyketide synthase (NRPS-PKS) is responsible for the generation of the key precursor chalcone. Then, a new type of CHI catalyzes the conversion of a chalcone into a flavanone by a histidine-mediated oxa-Michael addition mechanism. Finally, the desaturation of flavanone to flavone is catalyzed by a new type of FNS, a flavin mononucleotide (FMN)-dependent oxidoreductase.

Microbial production of the plant-derived fungicide physcion.

Physcion is a characteristic component of the traditional herb rhubarb with diverse pharmacological activities that has been commercially approved as an herbal fungicide. Nevertheless, its extremely low contents, costly purification procedure and geographically restricted planting severely hinder its application. Here, a cell factory was constructed in the filamentous fungus Aspergillus terreus for physcion production via microbial fermentation by integrating a pathway-modified emodin accumulation module and a position-selective emodin methylation module. Specifically, 1.71 g/L emodin accumulated when the transcriptional activator GedR and the emodin-1-OH-O-methyltransferase GedA in the geodin biosynthetic pathway were overexpressed and knocked out, respectively. Subsequently, potential emodin-3-OH-O-methyltransferase candidates were enzymatically screened in vitro and introduced into the emodin-accumulating mutant in vivo to generate a physcion-producing strain showing the highest titre of 6.3 g/L in fed-batch fermentation. Thus, our study provides an alternative strategy for the highly efficient, economical production of physcion and a representative example for microbial synthetic biology.

Biosynthesis mechanism, genome mining and artificial construction of echinocandin O-sulfonation.

Micafungin, a semisynthetic derivative of the cyclic hexapeptide FR901379 produced by Coleophoma empetri fermentation, is the only O-sulfonated echinocandin-type antifungal drug. However, the detailed formation mechanism of O-sulfonate group, whether before or after the assembly of hexapeptide, remains elusive. Here, we confirmed that O-sulfonylation occurs after hexapeptide assembly as a kind of postmodification in the biosynthesis of FR901379. The released cyclic hexapeptide was hydroxylated by cytochrome P450 McfP and successively sulfonated by sulfotransferase McfS. And other three echinocandin sulfotransferases were identified through genome mining by using McfS as a sequence probe. Moreover, pneumocandin B0, the precursor of caspofungin, could be O-sulfonated by heterologously introducing the McfP-McfS into the pneumocandin B0-producing species Glarea lozoyensis. The water-solubility of sulfonated pneumocandin B0 is 4000 times higher than that of pneumocandin B0. The revealed O-sulfonation mechanism will provide new insights into the design and production of novel sulfonated echinocandins by metabolic engineering.

Identification of PKS-NRPS Hybrid Metabolites in Marine-Derived Penicillium oxalicum.

Filamentous fungi are abundant resources of bioactive natural products. Here, 151 marine-derived fungi were collected from the north Yellow Sea and identified by an internal transcribed spacer (ITS) sequence. The crude extracts of all strains were evaluated for their antimicrobial activities and analyzed by HPLC fingerprint. Based on these, strain Penicillium oxalicum MEFC104 was selected for further investigation. Two new polyketide-amino acid hybrid compounds with feature structures of tetramic acid, oxopyrrolidine A and B, were isolated. Their planner structures were assigned by HRESIMS and 1D/2D NMR experiments. The absolute configurations were determined by modified Mosher's method, J-based configuration analysis, and ECD calculations. Furthermore, the biosynthetic pathway was identified by bioinformatic analysis and gene-deletion experiments. This study established a link between oxopyrrolidines and the corresponding biosynthesis genes in P. oxalicum.

Characterization and Structural Analysis of Emodin-O-Methyltransferase from Aspergillus terreus.

All O-methylated derivatives of emodin, including physcion, questin, and 1-O-methylemodin, show potential antifungal activities. Notably, emodin and questin are two pivotal intermediates of geodin biosynthesis in Aspergillus terreus. Although most of the geodin biosynthetic steps have been investigated, the key O-methyltransferase (OMT) responsible for the O-methylation of emodin to generate questin has remained unidentified. Herein, through phylogenetic tree analysis and in vitro biochemical assays, the long-sought class II emodin-O-methyltransferase GedA has been functionally characterized. Additionally, the catalytic mechanism and key residues at the catalytic site of GedA were elucidated by enzyme-substrate-methyl donor analogue ternary complex crystal structure determination and site-directed mutagenesis. As we demonstrate, GedA adopts a typical general acid/base (E446/H373)-mediated transmethylation mechanism. In particular, residue D374 is also crucial for efficient catalysis through blocking the formation of intramolecular hydrogen bonds in emodin. This study will facilitate future engineering of GedA for the production of physcion or other site-specific O-methylated anthraquinone derivatives with potential applications as biopesticides.

Identification of a polyketide biosynthesis gene cluster by transcriptional regulator activation in Aspergillus terreus.

In filamentous fungi, the secondary metabolism is environmentally sensitive. Most of the biosynthetic gene clusters of secondary metabolites are silent under laboratory conditions. In this study, a highly conserved naphthopyrone PKS ATEG_06206 was identified in the genome sequence of A. terreus MEFC01. This gene is silent under laboratory conditions. To study the function of this PKS, we activated the silent biosynthetic pathway by replacing the promoter of cluster-specific transcriptional factor ATEG_06205 in A. terreus MEFC01. With this strategy, we confirmed that the products of this cryptic PKS are naphthoquinones. These naphthoquinones are soluble pigments, which could be secreted into the agar medium and culture broth. For this reason, the colour of mycelium and conidia of the activated mutant was significantly darker than that of the parental strain. The gene cluster and biosynthetic pathway were further elucidated through the reverse genetics approach and enzymatic assay in vitro.

Construction of an efficient Claviceps paspali cell factory for lysergic acid production.

Lysergic acid (LA) is the key precursor of ergot alkaloids, and its derivatives have been used extensively for the treatment of neurological disorders. However, the poor fermentation efficiency limited its industrial application. At the same time, the hardship of genetic manipulation has hindered the metabolic engineering of Claviceps strains to improve the LA titer further. In this study, an efficient genetic manipulation system based on the protoplast-mediated transformation was established in the industrial strain Claviceps paspali. On this basis, the gene lpsB located in the ergot alkaloids biosynthetic gene cluster was deleted to construct the LA-producing cell factory. Plackett-Burman and Box-Behnken designs were used in shaking flasks, achieving an optimal fermentation medium composition. The final titer of LA and iso-lysergic acid (ILA) reached 3.7 g·L-1, which was 4.6 times higher than that in the initial medium. Our work provides an efficient strategy for the biosynthesis of LA and ILA and lays the groundwork for its industrial production.

Bienzyme-Catalytic and Dioxygenation-Mediated Anthraquinone Ring Opening.

The C-10-C-4a bond cleavage of anthraquinone is believed to be a crucial step in fungal seco-anthraquinone biosynthesis and has long been proposed as a classic Baeyer-Villiger oxidation. Nonetheless, genetic, enzymatic, and chemical information on ring opening remains elusive. Here, a revised questin ring-opening mechanism was elucidated by in vivo gene disruption, in vitro enzymatic analysis, and 18O chasing experiments. It has been confirmed that the reductase GedF is responsible for the reduction of the keto group at C-10 in questin to a hydroxyl group with the aid of NADPH. The C-10-C-4a bond of the resultant questin hydroquinone is subsequently cleaved by the atypical cofactor-free dioxygenase GedK, giving rise to desmethylsulochrin. This proposed bienzyme-catalytic and dioxygenation-mediated anthraquinone ring-opening reaction shows universality.

Establishing an Efficient Genetic Manipulation System for Sulfated Echinocandin Producing Fungus Coleophoma empetri.

Micafungin is an important echinocandin antifungal agent for the treatment of invasive fungal infections. In industry, micafungin is derived from the natural product FR901379, which is a non-ribosomal cyclic hexapeptide produced by the filamentous fungus Coleophoma empetri. The difficulty of genetic manipulation in C. empetri restricts the clarification of FR901379 biosynthetic mechanism. In this work, we developed an efficient genetic manipulation system in the industrial FR901379-producing strain C. empetri MEFC009. Firstly, a convenient protoplast-mediated transformation (PMT) method was developed. Secondly, with this transformation method, the essential genetic elements were verified. Selectable markers hph, neo, and nat can be used for the transformation, and promotors Ppgk, PgpdA, and PgpdAt are functional in C. empetri MEFC009. Thirdly, the frequency of homologous recombination was improved from 4 to 100% by deleting the ku80 gene, resulting in an excellent chassis cell for gene-targeting. Additionally, the advantage of this genetic manipulation system was demonstrated in the identification of the polyketide synthase (PKS) responsible for the biosynthesis of dihydroxynapthalene (DHN)-melanin. This genetic manipulation system will be a useful platform for the research of FR901379 and further genome mining of secondary metabolites in C. empetri.

Aspergillus terreus as an industrial filamentous fungus for pharmaceutical biotechnology.

Aspergillus terreus is an important Aspergillus species, which has been applied in the industrial production of the bio-based chemical itaconic acid and the lipid-lowering drug lovastatin. The excellent fermentation capability has been demonstrated in these industrial applications. The genomic information revealed that the outstanding capacity of natural product synthesis by A. terreus remains to be further explored. With advances of the genome mining strategy, the products of several cryptic biosynthetic gene clusters have been discovered recently. In addition, a series of metabolic engineering studies have been performed in the industrial strains of lovastatin and itaconic acid to further improve the production processes. This review presents the current progress and the future outlook in the field of A. terreus biotechnology.

Discovery and Characterization of a PKS-NRPS Hybrid in Aspergillus terreus by Genome Mining.

Fungal polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) hybrids have been characterized to produce polyketide-amino acid compounds with striking structural features and biological activities. In this study, a PKS-NRPS hybrid enzyme was found in Aspergillus terreus by genome mining. By activating the cluster-specific transcriptional regulator, this cryptic PKS-NRPS gene cluster was successfully activated and ten products (1-10) were identified as pyranterreones. Using functional genetics, bioinformatics, and isotope-labeling feeding analysis, the biosynthetic pathway was revealed. This is the second PKS-NRPS hybrid identified in A. terreus.

Collaborative Biosynthesis of a Class of Bioactive Azaphilones by Two Separate Gene Clusters Containing Four PKS/NRPSs with Transcriptional Crosstalk in Fungi.

Azaphilones are a family of fungal polyketide metabolites with diverse chemical structures and biological activities with a highly oxygenated pyranoquinone bicyclic core. Here, a class of azaphilones possessing a 6/6/6/6 tetracyclic ring system was identified in Aspergillus terreus, and exhibited potential anticancer activities. The gene deletions and biochemical investigations demonstrated that these azaphilones were collaboratively synthesized by two separate clusters containing four core-enzymes, two nonreducing PKSs, one highly reducing PKS, and one NRPS-like. More interestingly, we found that the biosynthesis is coordinately regulated by a crosstalk mechanism between these two gene clusters based on three transcriptional factors. This is a meaningful mechanism of fungal secondary metabolism, which allows fungi to synthesize more complex compounds and gain new physiological functions. The results provide a new insight into fungal natural product biosynthesis.

Construction of an Efficient and Robust Aspergillus terreus Cell Factory for Monacolin J Production.

Monacolin J is a key precursor for the synthesis of the cholesterol-lowering drug simvastatin. Industrially, monacolin J is manufactured through the alkaline hydrolysis of the fungal polyketide lovastatin, which is relatively complex and environmentally unfriendly. A cell factory for monacolin J production was created by heterologously introducing lovastatin hydrolase into Aspergillus terreus in our previous study. However, residual lovastatin remained a problem for the downstream product purification. In this study, we used combined metabolic engineering strategies to create a more efficient and robust monacolin J-producing cell factory that completely lacks lovastatin residue. The complete deletion of the key gene lovF blocked the biosynthesis of lovastatin and led to a large accumulation of monacolin J without any lovastatin residue. Additionally, the overexpression of the specific transcription factor lovE under the P gpdAt promoter further increased the titer of monacolin J by 52.5% to 5.5 g L-1. Interestingly, the fermentation robustness was also significantly improved by the expression of lovE. This improvement not only avoids the process of alkaline hydrolysis but also simplifies the downstream separation process.

Enhanced Single-Step Bioproduction of the Simvastatin Precursor Monacolin J in an Industrial Strain of Aspergillus terreus by Employing the Evolved Lovastatin Hydrolase.

Biosynthesis of simvastatin, the active pharmaceutical ingredient of cholesterol-lowering drug Zocor, has drawn increasing global attention in recent years. Although single-step in vivo production of monacolin J, the intermediate biosynthetic precursor of simvastatin, has been realized by utilizing lovastatin hydrolase (PcEST) in our previous study, about 5% of residual lovastatin is still a problem for industrial production and quality control. In order to improve conversion efficiency and reduce lovastatin residues, modification of PcEST is carried out through directed evolution and a novel two-step high-throughput screening method. The mutant Q140L shows 18-fold improved whole-cell activity as compared to the wild-type, and one fold enhanced catalytic efficiency and 3 °C increased T5010 over the wild-type are observed by characterizing the purified protein. Finally, the engineered A. terreus strain overexpressing Q140L mutant exhibited the increased conversion efficiency and the reduced lovastatin residues by comparing with A. terreus strain overexpressing the wild-type PcEST, where almost 100% of the produced lovastatin is hydrolyzed to monacolin J. Therefore, this improved microbial cell factory can realize single-step bioproduction of monacolin J in a more efficient way, providing an attractive and eco-friendly substitute over the existing chemical synthetic routes of monacolin J and promoting complete bioproduction of simvastatin at industrial scale.

Single-step production of the simvastatin precursor monacolin J by engineering of an industrial strain of Aspergillus terreus.

Monacolin J is a key precursor for the synthesis of simvastatin (Zocor), an important drug for treating hypercholesterolemia. Industrially, monacolin J is manufactured through alkaline hydrolysis of lovastatin, a fungal polyketide produced by Aspergillus terreus. Multistep chemical processes for the conversion of lovastatin to simvastatin are laborious, cost expensive and environmentally unfriendly. A biocatalysis process for monacolin J conversion to simvastatin has been developed. However, direct bioproduction of monacolin J has not yet been achieved. Here, we identified a lovastatin hydrolase from Penicillium chrysogenum, which displays a 232-fold higher catalytic efficiency for the in vitro hydrolysis of lovastatin compared to a previously patented hydrolase, but no activity for simvastatin. Furthermore, we showed that an industrial A. terreus strain heterologously expressing this lovastatin hydrolase can produce monacolin J through single-step fermentation with high efficiency, approximately 95% of the biosynthesized lovastatin was hydrolyzed to monacolin J. Our results demonstrate a simple and green technical route for the production of monacolin J, which makes complete bioproduction of the cholesterol-lowering drug simvastatin feasible and promising.

Establishing an efficient gene-targeting system in an itaconic-acid producing Aspergillus terreus strain.

OBJECTIVES: To develop an efficient gene-targeting platform in an excellent itaconic acid producing strain Aspergillus terreus CICC40205. RESULTS: The frequency of homologous recombination was improved by deleting the ku80 gene. A nutritional transformation system based on the bidirectionally selectable marker, pyrG An , was established in the ku80-/pyrG-double mutant which is convenient for following marker rescue. The modified Cre/loxP recombination system was applied for the excision of the pyrG An marker by directly introducing Cre recombinase into the protoplasts. CONCLUSIONS: This gene-targeting system is an efficient platform for sequential and multiple genetic modifications in A. terreus and is conducive to study biosynthesis mechanisms and to improve the production ability of itaconic acid and other products.

Identification of an itaconic acid degrading pathway in itaconic acid producing Aspergillus terreus.

Itaconic acid, one of the most promising and flexible bio-based chemicals, is mainly produced by Aspergillus terreus. Previous studies to improve itaconic acid production in A. terreus through metabolic engineering were mainly focused on its biosynthesis pathway, while the itaconic acid-degrading pathway has largely been ignored. In this study, we used transcriptomic, proteomic, bioinformatic, and in vitro enzymatic analyses to identify three key enzymes, itaconyl-CoA transferase (IctA), itaconyl-CoA hydratase (IchA), and citramalyl-CoA lyase (CclA), that are involved in the catabolic pathway of itaconic acid in A. terreus. In the itaconic acid catabolic pathway in A. terreus, itaconic acid is first converted by IctA into itaconyl-CoA with succinyl-CoA as the CoA donor, and then itaconyl-CoA is hydrated into citramalyl-CoA by IchA. Finally, citramalyl-CoA is cleaved into acetyl-CoA and pyruvate by CclA. Moreover, IctA can also catalyze the reaction between citramalyl-CoA and succinate to generate succinyl-CoA and citramalate. These results, for the first time, identify the three key enzymes, IctA, IchA, and CclA, involved in the itaconic acid degrading pathway in itaconic acid producing A. terreus. The results will facilitate the improvement of itaconic acid production by metabolically engineering the catabolic pathway of itaconic acid in A. terreus.