Can we now engineer Claviceps to produce ergot alkaloids without the toxicity of IDTs?

This work provides a hitherto missing functional proof for the involvement of [select genes] in IDT production in C. paspali, and opens the way for the use of molecular genetics for the optimization of this important fungus for the production of useful bioactive metabolites on an industrial scale.

In addition to being an important producer of ergot alkaloids and easy to handle in industrial and biotechnology contexts, Claviceps paspali produces toxins called indole-diterpenes (IDTs). These are harmful for agricultural crops, but may also have pharmaceutical use.

Although it is easy to handle in the lab, current transformation protocols for C. paspali are inefficient and inconsistent. In 2017, researchers in Hungary attempted to engineer a strain of C. paspali using Agrobacterium tumefaciens, an organism commonly used in synthetic plant biology for its versatility and specificity in inserting genes into various hosts. They aimed to optimize this transformation protocol for C. paspali, and managed to succeed in deleting the entire cluster of genes required for the synthesis of IDTs.

First, a deletion construct was built using an A. tumefaciens vector and the target DNA from C. paspali, with various other genes inserted as markers for later selection of transformed mutants. This construct was transformed into A. tumefaciens. The successfully transformed bacteria were mixed with C. paspali mycelia, and the two species co-incubated.

The fungi that had incorporated the deletion construct were selected for by various additions of antibiotics and selective media. Their non-expression of the IDT-associated genes was confirmed by LC-MS, and the efficiency of the whole approach was further optimized via tests for stability and optimal co-incubation, leading to substantial yields of transformed mutants. In addition to this, they also analysed the mutants’ ergot alkaloid profiles by HPLC, and found that deleting those IDT-associated genes had no effect on alkaloid production by the organism.

There are several significant implications for these discoveries.

We now have an optimized protocol for Agrobacterium-mediated transformation of C. paspali. This can increase efficiency and reduce costs associated with current transformation protocols involving C. paspali, which are largely dependent on techniques that disrupt the cell membrane.

We can now adapt this for other Claviceps species, for example to improve the yield of certain metabolites.

We have a contextualized understanding of secondary metabolites produced by Claviceps. The elucidation of the IDT biosynthesis pathway, and its independence from ergot alkaloid biosynthesis, can aid our understanding of the classification, evolution and ecology of these compounds.

We can further optimize production of useful metabolites by C. paspali. This can prove to be useful for large-scale industrial production of novel metabolites.

Reference:

Kozák, L., Szilágyi, Z., Vágó, B., Kakuk, A., Tóth, L., Molnár, I., & Pócsi, I. (2018). Inactivation of the indole-diterpene biosynthetic gene cluster of Claviceps paspali by Agrobacterium -mediated gene replacement. Applied Microbiology and Biotechnology, 102(7), 3255–3266. https://doi.org/10.1007/s00253-018-8807-x