Heterologous Gene Expression as an Approach for Fungal Secondary Metabolite Discovery

Secondary metabolites (SM) are low molecular weight organic compounds which produce from some of the primary metabolic biosynthetic pathways and interconnected with primary metabolite to gain the necessary amount of energy, carbon, and nitrogen. Secondary metabolites are not essential for growth and produced after growth has terminated. However, they are used for survival function, antagonism, competition, communication, etc. and not a waste product. Fungi are a good source of SM and have been an extensive source of manufacturing pharmaceutical drugs, herbicides, insecticides, growth hormones, antifungal, antibiotics, mycotoxins, enzymes, and dyes.

There are four main classes of fungal SM.

Polyketides

Polyketides are the most abundant SM in fungi. Polyketides in fungi are synthesized by type I polyketide synthase (PKS) enzyme from acetyl coenzyme A and malonyl coenzyme A units. The condensation of primary metabolites; acetyl-CoA and malonyl-CoA to form β-ketoacetyl polymers are done 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 main components of fungal type I polyketide synthase. These enzymes are repeatedly adding a two-carbon unit when the peptide synthesis is occurring. Aflatoxin, lovastatin, fusaric acid, fusarubin are some of the well-known fungal polyketides.

Non-ribosomal peptides

Non-ribosomal peptides consist with proteinogenic amino acids and non-proteinogenic amino acids. These are synthesized by multimodular non-ribosomal peptide synthase. Non-ribosomal peptide synthase enzyme mainly consists of adenylation domain (A), and peptidyl carrier protein domain. Penicillin G, gilotoxin are some examples for fungal non-ribosomal peptides.

Terpenes

Terpenes are consist with 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 alkaloids

Indole alkaloids are form from shikimic acid pathway and mevalonate pathway. Normally, indole alkaloid precursors are aromatic amino acid tryptophan and dimethylallyl pyrophosphate. Ergopeptides, Fumitremorgen C are some fungal indole alkaloids. There are several approaches to discover fungal SM. These approaches can be classified as top-down and bottom-up approaches. Top-down approaches are started at the organism level such as collecting the biological samples, carry out the standard fermentation, extraction, and then isolation of SM for structural elucidation. Where in, bottom-up approaches are begun with a genetic level such as genome mining and genetic engineering. Molecular biological studies have shown that the genes which are responsible for biosynthesizing SM in fungi are clustered.

Most of the time the top-down approaches are encountered with known SM and difficult to find novel SMs. This is sometimes true even for genome mining approaches. This is mostly due to the gene clusters which producing SM are remain silent and not expressed under normal laboratory conditions. These gene clusters are called orphan or cryptic gene clusters. Normally, microorganisms do not express their genes all the time. Because it needs more resources and energy. They have a mechanism to regulate the gene expression.

The genetic engineering approaches such as homologous expression and heterologous expression can be used to overcome this issue. Homologous expression is the overexpression of a gene in the native organism. In contrast, heterologous expression is the expression of a gene or a portion of a gene of interest in a host organism which does not naturally have this gene. The advantages of the heterologous expression over homologous expression are the heterologous hosts normally have faster growth rate than native organism, wide precursor supply, amenable to genetic modification. 5, 6The main steps of heterologous expressions are, identify and isolate the SM of interest, identify the gene cluster which is responsible for producing the SM and the DNA fragment of interest. Then, transfer the fragment or significant piece of it to a vector with detectable fungal marker. Select a suitable host and carry out the genetic manipulation. After that the heterologous host need to be maintained until isolation of SM. There are some important facts that required when selecting a host. The nature of the biosynthetic gene cluster, the genetic and physiological characteristics of the native and host need to be studied. The approximately analogous functionality of the native and host organism also a good requirement in order to have successful heterologous expression. Common heterologous host for fungi are saccharomyces cerevisiae, aspergillus oryzae, and aspergillus nidulans. All of these organisms have well developed genetic tool box and minimal amount of endogenous SM production.

Here I present two successful examples of heterologous expression studies in fungi. Parker and coworkers have studied the heterologous biosynthesis of Nodulisporic acid F (NAF). Nodulisporic acids (NA) are a group of indole diterpene which is known to have insecticidal activity against blood feeding arthropods and these compounds are less harmful on mammals. Nodulisporic acids are produced by Hypoxylon pulicicidum and normally called Nodulisporium sp. However, the production of NAs from Hypoxylon pulicicidum is low and the total synthesis approaches are known to have multistep and complex. Therefore, Parker and group used genetic engineering approach to synthesis these important class of bioactive compounds. They have used Penicillium paxilli as the heterologous host which produces paxilline, an indole diterpene. The first three steps of biosynthesis pathway of paxilline in Penicillium paxilli is similar to the indole diterpene biosynthesis and has homologous genes. They identified the gene cluster which is responsible for producing NA in Hypoxylon pulicicidum and this gene cluster consists with thirteen genes. Hypoxylon pulicicidum has lack of geranylgeranyl pyrophosphate synthase encoding gene which needs in the first step in the biosynthesis of NA. The host organism has this enzyme which increased the production yield of NAF in the genetically modified host organism. After carrying out a series of studies with genetically modified plasmid in the host organism, they were able to find that the biosynthesis of NAF requires only five genes.

The next study that I present here has been done by Chooi and group. They have investigated the heterologous production of a benzoyl-primed tricarboxylic acid polyketide intermediate from the zaragozic Acid A (ZAA) biosynthetic pathway. Zaragozic Acid A is a polyketide which has a cholesterol lowering activity. The total synthesis of this polyketide has been done. However, the biosynthesis of the bicyclic core and benzoyl containing polyketide arm are still needing to be understood. In this study, they have used genetically engineered aspergillus nidulans heterologous host to study the biosynthesis pathway of ZAA especially the benzyl containing polyketide arm and was able to identify the intermediate 2 which is then act as a precursor to form ZAA. Curvularia lunata was the native organism that was used to identify the gene cluster responsible for producing ZAA. They were able to find that the biosynthesis of ZA intermediate 2 can be done using only three enzymes via heterologous expression. Benzoyl-CoA pool was the precursor and benzoyl CoA are naturally found in aspergillus nidulans.

In conclusion, heterologous expression is found as a robust approach for fungal secondary metabolite discovery. It provides the characterization of biosynthetic gene clusters from unculturable or poorly culturable microbes. However, there are some challenges in this technique that need to be addressed. The heterologous host always not produce the target molecule with good yield. Sometimes it is lower even than the native host. If the target molecule shows antifungal activity, the precautions such as expression of resistance gene need to be taken before modifying the genes. Therefore, the better understanding about the molecule of interest, native organism, and heterologous host is critical. Availability of necessary precursors, stability of biosynthetic enzyme in the host organism also need to be understood before the gene modification.