Heterologous Gene Expression as an Approach for the Discovery of Fungal Secondary Metabolites interconnected with the primary metabolite to gain the necessary amount of energy, carbon and nitrogen. Secondary metabolites are not essential for growth and are produced after growth is complete. However, they are used for functions of survival, antagonism, competition, communication, etc. and do not constitute waste. Mushrooms are a good source of SM and have been an important source for manufacturing pharmaceutical drugs, herbicides, insecticides, growth hormones, antifungals, antibiotics, mycotoxins, enzymes and dyes. Say no to plagiarism. Get a tailor-made essay on “Why Violent Video Games Should Not Be Banned”? Get an original essay There are four main classes of fungal SM. Polyketides Polyketides are the most abundant SMs in fungi. Fungal polyketides are synthesized by the type I polyketide synthase (PKS) enzyme from acetylcoenzyme A and malonyl coenzyme A units. Condensation of primary metabolites; Acetyl-CoA and malonyl-CoA to form β-ketoacetyl polymers are made by PKS and they are linked to the enzyme by thioester bonds. Ketoacyl synthase (KS), acyl transferase (AT), and acyl carrier domain (ACP) are the major components of fungal polyketide synthase type I. These enzymes repeatedly add a two-carbon unit when the Peptide synthesis takes place. Aflatoxin, lovastatin, fusaric acid, and fusarubin are among the well-known fungal polyketides. Non-ribosomal peptides Non-ribosomal peptides consist of proteinogenic amino acids and non-proteinogenic amino acids. These are synthesized by non-ribosomal multimodular peptide synthase. The nonribosomal peptide synthase enzyme mainly consists of an adenylation (A) domain and a peptidyl carrier protein domain. Penicillin G and gilotoxin are some examples of non-ribosomal fungal peptides. Terpenes Terpenes are made up of several isoprene units and biosynthesized by terpene cyclase to produce different terpenes from different diphosphates. Some examples of fungal terpenes are aristolochene and gibberellin GA3. Indole AlkaloidsIndole alkaloids arise from the shikimic acid pathway and the mevalonate pathway. Normally, the precursors of indole alkaloids are the aromatic amino acid tryptophan and dimethylallyl pyrophosphate. Ergopeptides, Fumitremorgen C are fungal indole alkaloids. There are several approaches to discovering fungal SM. These approaches can be classified into top-down and bottom-up approaches. Top-down approaches are initiated at the organism level, such as collecting biological samples, performing standard fermentation, extracting, and then isolating the SM for structural elucidation. Bottom-up approaches are then launched at a genetic level such as genome mining and genetic engineering. Molecular biology studies have shown that the genes responsible for SM biosynthesis in fungi are clustered together. Most of the time, top-down approaches are encountered with known SMs and it is difficult to find new SMs. This is sometimes true even for genome mining approaches. This is mainly because the groups of genes that produce SM remainsilent and are not expressed under normal laboratory conditions. These groups of genes are called orphan or cryptic gene groups. Normally, microorganisms do not express their genes all the time. Because it needs more resources and energy. They have a mechanism to regulate gene expression. Genetic engineering approaches such as homologous expression and heterologous expression can be used to overcome this problem. Homologous expression is the overexpression of a gene in the native organism. In contrast, heterologous expression is the expression of a gene or part of a gene of interest in a host organism that does not naturally possess this gene. The advantages of heterologous expression over homologous expression are that heterologous hosts normally have a faster growth rate than the native organism, a large supply of precursors, and are amenable to genetic modification. 5, 6The main steps in heterologous expressions are the identification and isolation of the SM of interest, the identification of the gene cluster responsible for producing the SM, and the DNA fragment of interest. Then transfer the fragment or a large piece of it to a vector with a detectable fungal marker. Select a suitable host and carry out the genetic manipulation. After that, the heterologous host should be maintained until SM isolation. Some important facts are required when selecting a host. The nature of the biosynthetic gene cluster, genetic and physiological characteristics of the native species and the host need to be studied. The approximately analogous functionality of the native organism and the host organism is also a good condition for successful heterologous expression. Common heterologous hosts of fungi are Saccharomyces cerevisiae, Aspergillus oryzae and Aspergillus nidulans. All of these organisms have a well-developed genetic toolbox and a minimal amount of endogenous SM production. Here I present two successful examples of heterologous expression studies in fungi. Parker and colleagues studied the heterologous biosynthesis of nodulisporic acid F (NAF). Nodulisporic acids (NA) are a group of indole diterpenes known to have insecticidal activity against blood-feeding arthropods and these compounds are less harmful to mammals. Nodulisporic acids are produced by Hypoxylon pulicicidum and normally called Nodulisporium sp. However, production of NA from Hypoxylon pulicicidum is low and total synthesis approaches are known to be complex and multistep. Therefore, Parker and his group used a genetic engineering approach to synthesize this important class of bioactive compounds. They used Penicillium paxilli as a heterologous host that produces paxillin, an indole diterpene. The first three steps of the paxillin biosynthesis pathway in Penicillium paxilli are similar to the biosynthesis of the indole diterpene and have homologous genes. They identified the gene cluster responsible for NA production in Hypoxylon pulicicidum and this gene cluster includes thirteen genes. Hypoxylon pulicicidum lacks the gene encoding geranylgeranyl pyrophosphate synthase which it requires in the first step of NA biosynthesis. The host organism possesses this enzyme which increases the yield of NAF production in the genetically modified host organism. After carrying out a series of studies with genetically modified plasmids in the host organism, they were able to see that NAF biosynthesis requires only five genes. The next study I present here was carried out by Chooi and his group. They studied the heterologous production of a.

Essay / Heterologous Gene Expression as an Approach for the Discovery of Fungal Secondary Metabolites interconnected with the primary metabolite to gain the necessary amount of energy, carbon and nitrogen. Secondary metabolites are not essential for growth and are produced after growth is complete. However, they are used for functions of survival, antagonism, competition, communication, etc. and do not constitute waste. Mushrooms are a good source of SM and have been an important source for manufacturing pharmaceutical drugs, herbicides, insecticides, growth hormones, antifungals, antibiotics, mycotoxins, enzymes and dyes. Say no to plagiarism. Get a tailor-made essay on “Why Violent Video Games Should Not Be Banned”? Get an original essay There are four main classes of fungal SM. Polyketides Polyketides are the most abundant SMs in fungi. Fungal polyketides are synthesized by the type I polyketide synthase (PKS) enzyme from acetylcoenzyme A and malonyl coenzyme A units. Condensation of primary metabolites; Acetyl-CoA and malonyl-CoA to form β-ketoacetyl polymers are made by PKS and they are linked to the enzyme by thioester bonds. Ketoacyl synthase (KS), acyl transferase (AT), and acyl carrier domain (ACP) are the major components of fungal polyketide synthase type I. These enzymes repeatedly add a two-carbon unit when the Peptide synthesis takes place. Aflatoxin, lovastatin, fusaric acid, and fusarubin are among the well-known fungal polyketides. Non-ribosomal peptides Non-ribosomal peptides consist of proteinogenic amino acids and non-proteinogenic amino acids. These are synthesized by non-ribosomal multimodular peptide synthase. The nonribosomal peptide synthase enzyme mainly consists of an adenylation (A) domain and a peptidyl carrier protein domain. Penicillin G and gilotoxin are some examples of non-ribosomal fungal peptides. Terpenes Terpenes are made up of several isoprene units and biosynthesized by terpene cyclase to produce different terpenes from different diphosphates. Some examples of fungal terpenes are aristolochene and gibberellin GA3. Indole AlkaloidsIndole alkaloids arise from the shikimic acid pathway and the mevalonate pathway. Normally, the precursors of indole alkaloids are the aromatic amino acid tryptophan and dimethylallyl pyrophosphate. Ergopeptides, Fumitremorgen C are fungal indole alkaloids. There are several approaches to discovering fungal SM. These approaches can be classified into top-down and bottom-up approaches. Top-down approaches are initiated at the organism level, such as collecting biological samples, performing standard fermentation, extracting, and then isolating the SM for structural elucidation. Bottom-up approaches are then launched at a genetic level such as genome mining and genetic engineering. Molecular biology studies have shown that the genes responsible for SM biosynthesis in fungi are clustered together. Most of the time, top-down approaches are encountered with known SMs and it is difficult to find new SMs. This is sometimes true even for genome mining approaches. This is mainly because the groups of genes that produce SM remainsilent and are not expressed under normal laboratory conditions. These groups of genes are called orphan or cryptic gene groups. Normally, microorganisms do not express their genes all the time. Because it needs more resources and energy. They have a mechanism to regulate gene expression. Genetic engineering approaches such as homologous expression and heterologous expression can be used to overcome this problem. Homologous expression is the overexpression of a gene in the native organism. In contrast, heterologous expression is the expression of a gene or part of a gene of interest in a host organism that does not naturally possess this gene. The advantages of heterologous expression over homologous expression are that heterologous hosts normally have a faster growth rate than the native organism, a large supply of precursors, and are amenable to genetic modification. 5, 6The main steps in heterologous expressions are the identification and isolation of the SM of interest, the identification of the gene cluster responsible for producing the SM, and the DNA fragment of interest. Then transfer the fragment or a large piece of it to a vector with a detectable fungal marker. Select a suitable host and carry out the genetic manipulation. After that, the heterologous host should be maintained until SM isolation. Some important facts are required when selecting a host. The nature of the biosynthetic gene cluster, genetic and physiological characteristics of the native species and the host need to be studied. The approximately analogous functionality of the native organism and the host organism is also a good condition for successful heterologous expression. Common heterologous hosts of fungi are Saccharomyces cerevisiae, Aspergillus oryzae and Aspergillus nidulans. All of these organisms have a well-developed genetic toolbox and a minimal amount of endogenous SM production. Here I present two successful examples of heterologous expression studies in fungi. Parker and colleagues studied the heterologous biosynthesis of nodulisporic acid F (NAF). Nodulisporic acids (NA) are a group of indole diterpenes known to have insecticidal activity against blood-feeding arthropods and these compounds are less harmful to mammals. Nodulisporic acids are produced by Hypoxylon pulicicidum and normally called Nodulisporium sp. However, production of NA from Hypoxylon pulicicidum is low and total synthesis approaches are known to be complex and multistep. Therefore, Parker and his group used a genetic engineering approach to synthesize this important class of bioactive compounds. They used Penicillium paxilli as a heterologous host that produces paxillin, an indole diterpene. The first three steps of the paxillin biosynthesis pathway in Penicillium paxilli are similar to the biosynthesis of the indole diterpene and have homologous genes. They identified the gene cluster responsible for NA production in Hypoxylon pulicicidum and this gene cluster includes thirteen genes. Hypoxylon pulicicidum lacks the gene encoding geranylgeranyl pyrophosphate synthase which it requires in the first step of NA biosynthesis. The host organism possesses this enzyme which increases the yield of NAF production in the genetically modified host organism. After carrying out a series of studies with genetically modified plasmids in the host organism, they were able to see that NAF biosynthesis requires only five genes. The next study I present here was carried out by Chooi and his group. They studied the heterologous production of a.

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