Increasing Lovastatin Production by Re-routing the Precursors Flow of Aspergillus terreus via Metabolic Engineering

Hasan, Hanan; Rahim, Muhamad Hafiz Abd; Campbell, Leona T.; Carter, Dee; Abbas, Ali; Montoya, Alejandro

doi:10.1007/s12033-021-00393-w

Cited by 12 publications

(6 citation statements)

References 44 publications

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“…Taken together, we constructed an effective MJ heterologous biosynthetic system in A. niger, which could provide a possible strategy for the production of other valuable compounds. In future work, to further improve the production level of MJ, precursor (such as acetyl-CoA and malonyl-CoA) supply could be an important strategy for improving the production of MJ derivative statins such as lovastatin [37,38] and simvastatin [39].…”

Section: Discussionmentioning

confidence: 99%

Heterologous Synthesis of Monacolin J by Reconstructing Its Biosynthetic Gene Cluster in Aspergillus niger

Zheng

et al. 2022

JoF

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Monacolin J (MJ), a key precursor of Lovastatin, could synthesize important statin drug simvastatin by hydrolyzing lovastatin and adding different side chains. In this study, to reduce the cumbersome hydrolysis of lovastatin to produce MJ in the native strain Aspergillus terreus, the MJ biosynthetic pathway genes (lovB, lovC, lovG, and lovA) were heterologously integrated into the genome of Aspergillus. niger CBS513.88 with strong promoters and suitable integration sites, via yeast 2μ homologous recombination to construct expression cassettes of long-length genes and CRISPR/Cas9 homology-directed recombination (CRISPR-HDR) to integrate MJ genes in the genome of A. niger. RT-PCR results proved that pathway synthesis-related genes could be heterologously expressed in A. niger. Finally, we constructed an engineered strain that could produce monacolin J, detected by LC-HR-ESIMS (MJ, 339.22 [M-H]+). The yield of MJ reached 92.90 mg/L after 7-day cultivation. By optimizing the cultivation conditions and adding precursor, the final titer of MJ was 142.61 mg/L on the fourth day of fed-batch cultivation, which was increased by 53.5% compared to the original growth conditions. Due to the wide application of A. niger in industrial fermentation for food and medicine, the following work will be dedicated to optimizing the metabolic network to improve the MJ production in the engineered strain.

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Section: Discussionmentioning

confidence: 99%

Heterologous Synthesis of Monacolin J by Reconstructing Its Biosynthetic Gene Cluster in Aspergillus niger

Zheng

et al. 2022

JoF

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“…Among them, the mutant LovE-b19, whose lovE gene under the control of P4 promoter, produced the highest level of lovastatin at the titer of 1512 mg/L (Fig. 7A), representing the highest yield recorded, which was significantly higher than that of the highest production level of lovastatin reported (1342 mg/L) [24][25][26][27][28][29][30][31] (Table 2). The transcription of lovE in these mutants was then analyzed.…”

Section: Overexpressing Of Love To Increase Lovastatin Using Two Stro...mentioning

confidence: 92%

Screening and genetic engineering of marine-derived Aspergillus terreus for high-efficient production of lovastatin

Na,

Zheng,

Jia

et al. 2024

Microb Cell Fact

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Background Lovastatin has widespread applications thanks to its multiple pharmacological effects. Fermentation by filamentous fungi represents the major way of lovastatin production. However, the current lovastatin productivity by fungal fermentation is limited and needs to be improved. Results In this study, the lovastatin-producing strains of Aspergillus terreus from marine environment were screened, and their lovastatin productions were further improved by genetic engineering. Five strains of A. terreus were isolated from various marine environments. Their secondary metabolites were profiled by metabolomics analysis using Ultra Performance Liquid Chromatography–Mass spectrometry (UPLC–MS) with Global Natural Products Social Molecular Networking (GNPS), revealing that the production of secondary metabolites was variable among different strains. Remarkably, the strain of A. terreus MJ106 could principally biosynthesize the target drug lovastatin, which was confirmed by High Performance Liquid Chromatography (HPLC) and gene expression analysis. By one-factor experiment, lactose was found to be the best carbon source for A. terreus MJ106 to produce lovastatin. To improve the lovastatin titer in A. terreus MJ106, genetic engineering was applied to this strain. Firstly, a series of strong promoters was identified by transcriptomic and green fluorescent protein reporter analysis. Then, three selected strong promoters were used to overexpress the transcription factor gene lovE encoding the major transactivator for lov gene cluster expression. The results revealed that compared to A. terreus MJ106, all lovE over-expression mutants exhibited significantly more production of lovastatin and higher gene expression. One of them, LovE-b19, showed the highest lovastatin productivity at a titer of 1512 mg/L, which represents the highest production level reported in A. terreus. Conclusion Our data suggested that combination of strain screen and genetic engineering represents a powerful tool for improving the productivity of fungal secondary metabolites, which could be adopted for large-scale production of lovastatin in marine-derived A. terreus.

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“…Though A. terreus causes severe agricultural damage and acts as a human pathogen, it is also a well-known industrial workhorse. For example, it is a well-known producer of itaconic acid 16 , 17 and lovastatin 18 , 19 , the former of which is heavily used in the chemical industries, and the latter of which is an important cholesterol-lowering drug. A. terreus also produce many carbohydrate-active enzymes (CAZymes) including cellulases, xylanases, lipases, and phytases that are employed in plant biomass degradation 20 – 24 .…”

Section: Introductionmentioning

confidence: 99%

Whole genome sequence characterization of Aspergillus terreus ATCC 20541 and genome comparison of the fungi A. terreus

Mortensen

Chang

et al. 2023

Sci Rep

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Aspergillus terreus is well-known for lovastatin and itaconic acid production with biomedical and commercial importance. The mechanisms of metabolite formation have been extensively studied to improve their yield through genetic engineering. However, the combined repertoire of carbohydrate-active enzymes (CAZymes), cytochrome P450s (CYP) enzymes, and secondary metabolites (SMs) in the different A. terreus strains has not been well studied yet, especially with respect to the presence of biosynthetic gene clusters (BGCs). Here we present a 30 Mb whole genome sequence of A. terreus ATCC 20541 in which we predicted 10,410 protein-coding genes. We compared the CAZymes, CYPs enzyme, and SMs across eleven A. terreus strains, and the results indicate that all strains have rich pectin degradation enzyme and CYP52 families. The lovastatin BGC of lovI was linked with lovF in A. terreus ATCC 20541, and the phenomenon was not found in the other strains. A. terreus ATCC 20541 lacked a non-ribosomal peptide synthetase (AnaPS) participating in acetylaszonalenin production, which was a conserved protein in the ten other strains. Our results present a comprehensive analysis of CAZymes, CYPs enzyme, and SM diversities in A. terreus strains and will facilitate further research in the function of BGCs associated with valuable SMs.

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Increasing Lovastatin Production by Re-routing the Precursors Flow of Aspergillus terreus via Metabolic Engineering

Cited by 12 publications

References 44 publications

Heterologous Synthesis of Monacolin J by Reconstructing Its Biosynthetic Gene Cluster in Aspergillus niger

Heterologous Synthesis of Monacolin J by Reconstructing Its Biosynthetic Gene Cluster in Aspergillus niger

Screening and genetic engineering of marine-derived Aspergillus terreus for high-efficient production of lovastatin

Whole genome sequence characterization of Aspergillus terreus ATCC 20541 and genome comparison of the fungi A. terreus

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