FunOrder: A robust and semi-automated method for the identification of essential biosynthetic genes through computational molecular co-evolution.

Secondary metabolites (SMs) are a vast group of compounds with different structures and properties that have been utilized as drugs, food additives, dyes, and as monomers for novel plastics. In many cases, the biosynthesis of SMs is catalysed by enzymes whose corresponding genes are co-localized in the genome in biosynthetic gene clusters (BGCs). Notably, BGCs may contain so-called gap genes, that are not involved in the biosynthesis of the SM. Current genome mining tools can identify BGCs, but they have problems with distinguishing essential genes from gap genes. This can and must be done by expensive, laborious, and time-consuming comparative genomic approaches or transcriptome analyses. In this study, we developed a method that allows semi-automated identification of essential genes in a BGC based on co-evolution analysis. To this end, the protein sequences of a BGC are blasted against a suitable proteome database. For each protein, a phylogenetic tree is created. The trees are compared by treeKO to detect co-evolution. The results of this comparison are visualized in different output formats, which are compared visually. Our results suggest that co-evolution is commonly occurring within BGCs, albeit not all, and that especially those genes that encode for enzymes of the biosynthetic pathway are co-evolutionary linked and can be identified with FunOrder. In light of the growing number of genomic data available, this will contribute to the studies of BGCs in native hosts and facilitate heterologous expression in other organisms with the aim of the discovery of novel SMs.

An overview on the biosynthesis and metabolic regulation of monacolin K/lovastatin.

Lovastatin/monacolin K (MK) is used as a lipid lowering drug, due to its effective hypercholesterolemic properties, comparable to synthetic statins. Lovastatin's biosynthetic pathway and gene cluster composition have been studied in depth in Aspergillus terreus. Evidence shows that the MK biosynthetic pathway and gene cluster in Monascus sp. are similar to those of lovastatin in A. terreus. Currently, research efforts have been focusing on the metabolic regulation of MK/lovastatin synthesis, and the evidence shows that a combination of extracellular and intracellular factors is essential for proper MK/lovastatin metabolism. Here, we comprehensively review the research progress on MK/lovastatin biosynthetic pathways, its synthetic precursors and inducing substances and metabolic regulation, with a view to providing reference for future research on fungal metabolism regulation and metabolic engineering for MK/lovastatin production.

Exploring the Brazilian diversity of Aspergillus sp. strains for lovastatin and itaconic acid production.

Filamentous fungi are well known for producing secondary metabolites applied in various industrial segments. Among these, lovastatin and itaconic acid, produced by Aspergillus terreus, have applications in the pharmaceutical and chemical industries. Lovastatin is primarily used for the control of hypercholesterolemia, while itaconic acid is a building block for the production of synthetic fibers, coating adhesives, among others. In this study, for the first time, 35 strains of Aspergillus sp. from four Brazilian culture collections were evaluated for lovastatin and itaconic acid production and compared to a reference strain, ATCC 20542. From an initial screening, the strains ATCC 20542, URM 224, URM1876, URM 5061, URM 5254, URM 5256, URM 5650, and URM 5961 were selected for genomic comparison. Among tested strains, the locus corresponding to the lovastatin genomic cluster was assembled, showing that all genes essential for lovastatin biosynthesis were present in producing URM 5961 and URM 5650 strains, with 100% and 98.5% similarity to ATCC 20542, respectively. However, in the no producing URM 1876, URM 224, URM 5254, URM 5061, and URM 5256 strains, this cluster was either fragmented or missing. Among the 35 strains evaluated for itaconic acid production in this study, only three strains had titers above 0.5 g/L, 16 strains had production below 0.5 g/L, and the remaining 18 strains had no production, with the highest production of itaconic acid observed in the URM 5254 strain with 2.2 g/L. The essential genes for itaconic acid production, mttA, cadA msfA were also mapped, where all three genes linked to itaconic acid production were found in a single contig in the assembly of each strain. In contrast to lovastatin loci, there is no correlation between the level of itaconic acid production and genetic polymorphisms in the genes associated with its 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.

Overexpression of Monacolin K Biosynthesis Genes in the Monascus purpureus Azaphilone Polyketide Pathway.

Monascus purpureus is an important food and drug microbial resource through the production of a variety of secondary metabolites, including monacolin K, a well-recognized cholesterol-lowering agent. However, the high production costs and naturally low contents of monacolin K have restricted its large-scale production. Thus, in this study we sought to improve the production of monacolin K in M. purpureus through overexpression of four genes ( mokC, mokD, mokE, and mokI). Four overexpression strains were successfully constructed by protoplast electric shock conversion, which resulted in a 234.3%, 220.8%, 89.5%, and 10% increase in the yield of monacolin K, respectively. The overexpression strains showed clear changes to the mycelium surface with obvious folds and the spores with depressions, whereas the pBC5 mycelium had a fuller structure with a flatter surface. Further investigation of these strains can provide the theoretical basis and technical support for the development of functional Monascus varieties.

Recent advances in reconstructing microbial secondary metabolites biosynthesis in Aspergillus spp.

High throughput genome sequencing has revealed a multitude of potential secondary metabolites biosynthetic pathways that remain cryptic. Pathway reconstruction coupled with genetic engineering via heterologous expression enables discovery of novel compounds, elucidation of biosynthetic pathways, and optimization of product yields. Apart from Escherichia coli and yeast, fungi, especially Aspergillus spp., are well known and efficient heterologous hosts. This review summarizes recent advances in heterologous expression of microbial secondary metabolite biosynthesis in Aspergillus spp. We also discuss the technological challenges and successes in regard to heterologous host selection and DNA assembly behind the reconstruction of microbial secondary metabolite biosynthesis.

Engineered monoculture and co-culture of methylotrophic yeast for de novo production of monacolin J and lovastatin from methanol.

As a promising one-carbon renewable substrate for industrial biotechnology, methanol has attracted much attention. However, engineering of microorganisms for industrial production of pharmaceuticals using a methanol substrate is still in infancy. In this study, the methylotrophic yeast Pichia pastoris was used to produce anti-hypercholesterolemia pharmaceuticals, lovastatin and its precursor monacolin J, from methanol. The biosynthetic pathways for monacolin J and lovastatin were first assembled and optimized in single strains using single copies of the relevant biosynthetic genes, and yields of 60.0mg/L monacolin J and 14.4mg/L lovastatin were obtained using methanol following pH controlled monoculture. To overcome limitations imposed by accumulation of intermediates and metabolic stress in monoculture, approaches using pathway splitting and co-culture were developed. Two pathway splitting strategies for monacolin J, and four for lovastatin were tested at different metabolic nodes. Biosynthesis of monacolin J and lovastatin was improved by 55% and 71%, respectively, when the upstream and downstream modules were separately accommodated in two different fluorescent strains, split at the metabolic node of dihydromonacolin L. However, pathway distribution at monacolin J blocked lovastatin biosynthesis in all designs, mainly due to its limited ability of crossing cellular membranes. Bioreactor fermentations were tested for the optimal co-culture strategies, and yields of 593.9mg/L monacolin J and 250.8mg/L lovastatin were achieved. This study provides an alternative method for production of monacolin J and lovastatin and reveals the potential of a methylotrophic yeast to produce complicated pharmaceuticals from methanol.

Function-impairing polymorphisms of the hepatic uptake transporter SLCO1B1 modify the therapeutic efficacy of statins in a population-based cohort.

BACKGROUND: The efficacy of statins, which are used commonly in primary and secondary prevention of cardiovascular diseases, shows a wide range of interindividual variability. Genetic variants of OATP1B1, a hepatic uptake transporter, can modify access of statins to its therapeutic target, thereby potentially altering drug efficacy. We studied the impact of genetic variants of OATP1B1 on the lipid-lowering efficacy of statins in a population-based setting. MATERIALS AND METHODS: The basis of the analysis was the Study of Health in Pomerania, a cohort of 2732 men and women aged 20-81 years. Included in the statistical analysis to evaluate the impact of OATP1B1 on therapeutic efficacy of statins were 214 individuals diagnosed with dyslipidaemia during initial recruitment and receiving statins during the 5-year follow-up. RESULTS: Analysing the impact of the OATP1B1 genotype, we observed a trend for lower statin-induced total cholesterol reduction in carriers of the SLCO1B1 512C variant. Restricting the analysis to patients receiving simvastatin, pravastatin, lovastatin and fluvastatin indicated a statistically significant association of the OATP1B1 genotype on lipid parameters at the 5-year follow-up. No such effect was observed for atorvastatin. Calculation of achievement of treatment goals according to the NCEP-ATPIII guidelines showed a lower rate of successful treatment when harbouring the mutant allele for patients taking simvastatin (46.7 vs. 73.9%). A similar trend was observed for pravastatin (34.4 vs. 70.4%). CONCLUSION: Genetic variants of OATP1B1 leading to impaired hepatic uptake of statins translated into reduced drug efficacy in a population-based cohort.

Culture-based and sequence-based insights into biosynthesis of secondary metabolites by Aspergillus terreus ATCC 20542.

Aspergillus terreus ATCC 20542 was cultivated in various culture media in order to activate its genome-encoded biosynthetic pathways and explore the secondary metabolic repertoire. In addition to mevinolinic acid (lovastatin) and its precursor monacolin L, a number of other secondary metabolites were found in the analyzed cultures, namely terreic acid, citrinin, (+)-geodin, terrein, and dehydrocurvularin. In contrast to previously described gene clusters responsible for production of lovastatin, monacolin L, (+)-geodin and dehydrocurvularin, the gene clusters of A. terreus associated with the formation of terreic acid, citrinin and terrein still await identification. Putative gene clusters potentially related to citrinin and terreic acid biosynthesis were suggested in the publicly available genome of A. terreus NIH 2624. The functions of putative genes in the previously identified cluster of (+)-geodin biosynthesis were predicted by confronting the annotation results with the proposed biosynthetic pathway and published biochemical studies on the underlying enzymes. Since there were no available data regarding genetic aspects of terrein biosynthesis, the candidate gene cluster potentially responsible for the production of terrein was not suggested.

Double oxidation of the cyclic nonaketide dihydromonacolin L to monacolin J by a single cytochrome P450 monooxygenase, LovA.

Lovastatin, a cyclic nonaketide from Aspergillus terreus, is a hypercholesterolemic agent and a precursor to simvastatin, a semi-synthetic cholesterol-lowering drug. The biosynthesis of the lovastatin backbone (dihydromonacolin L) and the final 2-methylbutyryl decoration have been fully characterized. However, it remains unclear how two central reactions are catalyzed, namely, introduction of the 4a,5-double bond and hydroxylation at C-8. A cytochrome P450 gene, lovA, clustered with polyketide synthase lovB, has been a prime candidate for these reactions, but inability to obtain LovA recombinant enzyme has impeded detailed biochemical analyses. The synthetic codon optimization and/or N-terminal peptide replacement of lovA allowed the lovA expression in yeast (Saccharomyces cerevisiae). Both in vivo feeding and in vitro enzyme assays showed that LovA catalyzed the conversion of dihydromonacolin L acid to monacolin L acid and monacolin J acid, two proposed pathway intermediates in the biosynthesis of lovastatin. LovA was demonstrated to catalyze the regio- and stereo-specific hydroxylation of monacolin L acid to yield monacolin J acid. These results demonstrate that LovA is the single enzyme that performs both of the two elusive oxidative reactions in the lovastatin biosynthesis.

Cloning and bioinformatic analysis of lovastatin biosynthesis regulatory gene lovE.

BACKGROUND: Lovastatin is an effective drug for treatment of hyperlipidemia. This study aimed to clone lovastatin biosynthesis regulatory gene lovE and analyze the structure and function of its encoding protein. METHODS: According to the lovastatin synthase gene sequence from genebank, primers were designed to amplify and clone the lovastatin biosynthesis regulatory gene lovE from Aspergillus terrus genomic DNA. Bioinformatic analysis of lovE and its encoding animo acid sequence was performed through internet resources and software like DNAMAN. RESULTS: Target fragment lovE, almost 1500 bp in length, was amplified from Aspergillus terrus genomic DNA and the secondary and three-dimensional structures of LovE protein were predicted. CONCLUSION: In the lovastatin biosynthesis process lovE is a regulatory gene and LovE protein is a GAL4-like transcriptional factor.

Screening and selection of lovastatin hyper-producing mutants of Aspergillus terreus using cyclic mutagenesis.

134 fungal cultures isolated from different soil samples were screened for lovastatin production. Of these, 38 isolates produced different levels of lovastatin. An Aspergillus terreus strain GD13, producing 190 mg/l of lovastatin was selected and subjected to a rational mutation-selection programme based on the resistance to lovastatin and fatty acid synthase (FAS) inhibitors, viz., iodoacetamide and N-ethylmaleimide. After three cycles of mutagenesis, a hyper-producing mutant (EM19) exhibiting 7.5-fold (1424 mg/l) higher levels of lovastatin when compared to wild type parent strain was obtained.

MlcR, a zinc cluster activator protein, is able to bind to a single (A/T)CGG site of cognate asymmetric motifs in the ML-236B (compactin) biosynthetic gene cluster.

All of the binding sequences for MlcR, a transcriptional activator of ML-236B (compactin) biosynthetic genes in Penicillium citrinum, were identified by an in vitro gel-shift assay. All the identified sequences contain an asymmetric direct repeat comprised of conserved tetrad bases (A/T)CGG with a spacer sequence of high similarity; in particular, G at position 2 and T at position 3 in the spacer are well conserved. The first (A/T)CGG repeat was essential for MlcR-binding and MlcR could bind to this monomeric site, probably as a monomer. This binding feature might enable MlcR to tolerate the variation of the spacer length and compositions in vitro. From these data, we propose that the consensus binding motif for MlcR is an asymmetric direct repeat, 5'-(A/T)CGG-NGTN(3-6)-TCGG-3'.

Identification of the specific sequence recognized by Penicillium citrinum MlcR, a GAL4-type transcriptional activator of ML-236B (compactin) biosynthetic genes.

MlcR is a pathway-specific transcriptional activator of the ML-236B biosynthetic genes in Penicillium citrinum. The MlcR-binding sequences were identified by an in vitro gel-shift assay and an in vivo reporter assay for the region between mlcA and mlcC as a model. The gel-shift assay showed that recombinant MlcR bound to the DNA sequence 5'-ACGGCGTTATTCGG-3' and most of the bases in this motif were required for the interaction between MlcR and DNA. In the reporter assay using beta-glucuronidase (GUS), substitution of the bases in this binding sequence resulted in the drastic reduction of GUS activities. These data clearly indicate that this MlcR-binding sequence is essential for the transcriptional activation of mlcA and mlcC in P. citrinum. Similar motifs were found in other loci of the ML-236B biosynthetic gene cluster and the consensus-binding motif for MlcR was predicted to be a direct repeat, 5'-WCGG-N(6)-TCGG-3'.

Cloning and characterization of monacolin K biosynthetic gene cluster from Monascus pilosus.

Monacolin K is a secondary metabolite synthesized by polyketide synthases (PKS) from Monascus, and it has the same structure as lovastatin, which is mainly produced by Aspergillus terreus. In the present study, a bacterial artificial chromosome (BAC) clone, mps01, was screened from the BAC library constructed from Monascus pilosus BCRC38072 genomic DNA. The putative monacolin K biosynthetic gene cluster was found within a 42 kb region in the mps01 clone. The deduced amino acid sequences encoded by the nine genes designated as mokA- mokI, which share over 54% similarity with the lovastatin biosynthetic gene cluster in A. terreus, were assumed to be involved in monacolin K biosynthesis. A gene disruption construct designed to replace the central part of mokA, a polyketide synthase gene, in wild-type M. pilosus BCRC38072 with a hygromycin B resistance gene through homologous recombination, resulted in a mokA-disrupted strain. The disruptant did not produce monacolin K, indicating that mokA encoded the PKS responsible for monacolin K biosynthesis in M. pilosus BCRC38072.

LaeA, a regulator of secondary metabolism in Aspergillus spp.

Secondary metabolites, or biochemical indicators of fungal development, are of intense interest to humankind due to their pharmaceutical and/or toxic properties. We present here a novel Aspergillus nuclear protein, LaeA, as a global regulator of secondary metabolism in this genus. Deletion of laeA (DeltalaeA) blocks the expression of metabolic gene clusters, including the sterigmatocystin (carcinogen), penicillin (antibiotic), and lovastatin (antihypercholesterolemic agent) gene clusters. Conversely, overexpression of laeA triggers increased penicillin and lovastatin gene transcription and subsequent product formation. laeA expression is negatively regulated by AflR, a sterigmatocystin Zn2Cys6 transcription factor, in a unique feedback loop, as well as by two signal transduction elements, protein kinase A and RasA. Although these last two proteins also negatively regulate sporulation, DeltalaeA strains show little difference in spore production compared to the wild type, indicating that the primary role of LaeA is to regulate metabolic gene clusters.

[Study on space flight-mutants of Monascus purpureus using Random Amplified Polymorphic DNA].

OBJECTIVE: To study the mechanism of microbial mutation in aerospace. METHOD: Random Amplified Polymorphic DNA (RAPD) was used to analyze the polymorphism of high Lovastatin-producing mutants. RESULT: Variations occurred in genomes of these mutants. CONCLUSION: The high Lovastatin producibility of the mutants is due to the variation of genomes, therefore the microbial mutation in the aerospace is achieved via the variation of hereditary genes in the microbes.

Integrating transcriptional and metabolite profiles to direct the engineering of lovastatin-producing fungal strains.

We describe a method to decipher the complex inter-relationships between metabolite production trends and gene expression events, and show how information gleaned from such studies can be applied to yield improved production strains. Genomic fragment microarrays were constructed for the Aspergillus terreus genome, and transcriptional profiles were generated from strains engineered to produce varying amounts of the medically significant natural product lovastatin. Metabolite detection methods were employed to quantify the polyketide-derived secondary metabolites lovastatin and (+)-geodin in broths from fermentations of the same strains. Association analysis of the resulting transcriptional and metabolic data sets provides mechanistic insight into the genetic and physiological control of lovastatin and (+)-geodin biosynthesis, and identifies novel components involved in the production of (+)-geodin, as well as other secondary metabolites. Furthermore, this analysis identifies specific tools, including promoters for reporter-based selection systems, that we employed to improve lovastatin production by A. terreus.

[Effect of space flight on yield of Monascus purpureus].

OBJECTIVE: To select high Lovastatin-producing microbial breed by space flight. METHOD: Monascus purpureus species was carried into space by the recoverable spaceship, "Shenzhou 3". After flight, the strain was rejuvenized, segregated and selected. The content of Lovastatin produced in the solid fermentation was examined. RESULT: Mutants with high productivity of Lovastatin were obtained. A series of tests showed that the acquired character of the mutants was stable. CONCLUSION: Space flight is an effective method for the selection of fine strains.

Functional analysis of mlcR, a regulatory gene for ML-236B (compactin) biosynthesis in Penicillium citrinum.

The mlcR gene encodes a putative 50.2-kDa protein with a Zn(II)(2)Cys(6) DNA-binding domain, which may be involved in the regulation of ML-236B biosynthesis in Penicillium citrinum. The induction of ML-236B production appears to correlate with the expression of mlcR, and the ML-236B biosynthetic genes mlcA- mlcH, and occurs mostly during the stationary phase. The present study was designed to examine the effects of alterations in mlcR expression on ML-236B biosynthesis. We first set out to increase the mlcR copy number in the chromosome of P. citrinum. Transformants with additional copies of native mlcR showed increased transcription of mlcR and produced larger amounts of ML-236B than the parent strain. Altered mlcR expression was also achieved by introducing a construct, designated pgkA(P)::mlcR, that contained the mlcR coding region fused to the (constitutively active) promoter and terminator sequences of the Aspergillus nidulans 3-phospho-glycerate kinase (pgkA) gene. Transformants carrying the pgkA(P)::mlcR construct expressed mlcR constitutively, and produced ML-236B during the exponential growth phase, suggesting that the pgkA(P)::mlcR construct does affect the regulation of ML-236B biosynthesis. Comparative expression analysis by RT-PCR showed that altering the expression profile of mlcR influenced the expression of some of the ML-236B biosynthetic genes. The evidence suggests that mlcR may indeed be involved in the transcriptional activation of some of the pathway-specific genes required for ML-236B biosynthesis.