BlueBio4Future Training Courses – Keynote speakers Lectures

Four recognized synthetic biology researchers are participating in the Short Course in Synthetic Biology, part of the BlueBio4Future ERA Chair project. They will share their most recent findings with the Short Course attendees and the CIIMAR research community.

We hereby present the opportunity for those who were unable to take the entire three-day course  to attend lectures by Stephen Wallace, Paul D’Agostino, Vincent Libis, and Julie Zedler:

(Re-)Organising Heterologous Enzymes in the Cyanobacterial Cell

Julie Zedler – Friedrich Schiller University Jena, Germany

Cyanobacteria have attracted attention for the development of sustainable industrial processes owed to their genetic tractability and fast phototrophic growth utilising sunlight as a “free” source of energy. Over the years, the engineering of cyanobacteria has advanced substantially and their metabolic capacity as cell factories has been demonstrated by a large array of different products reported in the literature. Producing a target compound often requires engineering of multi-enzyme, heterologous metabolic pathways. However, achieving efficient metabolic channeling and the spatial organisation of heterologous enzymes is challenging. This is partly due to the complex cyanobacterial ultrastructure and subcellular compartmentalisation. Prokaryotic protein translocation mechanisms are conserved but fundamental questions (e.g. composition and membrane localisation of translocation pores) remain unanswered in cyanobacteria.
Here, different strategies of targeting heterologous enzymes to native, subcellular localizations as well as secretion (out of the cell) will be presented. Furthermore, the application of Synthetic Biology concepts to unlock new-to-nature compartmentalisation and scaffolding strategies will be discussed. (Re-)organising enzymes in the cellular context can substantially improve the metabolic flux towards a product of interest and, thus, advance cyanobacterial biotechnology.

Direct Pathway Cloning (DiPaC) towards the efficient capture and expression of diverse cyanobacterial biosynthetic gene clusters

Paul D’Agostino – Dresden University of Technology, Germany

Microbial natural products are a vital source of inspiration for drug development. Therefore, the discovery of novel and bioactive natural products is essential to combat emerging new pathogens and to counteract the exacerbating crisis of microbial resistance. While cyanobacteria have proven to be an excellent source of structurally intriguing and bioactive natural products, several factors impede the efficient discovery and exploitation of these molecules including slow growth rates, challenging or ineffective genetic manipulation techniques and low molecule titres. To overcome these difficulties and speed-up discovery rates, we recently developed a synthetic biology cloning strategy termed Direct Pathway Cloning (DiPaC) for the capture and heterologous expression of small- to mid-sized cyanobacterial gene clusters. DiPaC utilises long-range PCR and in vitro DNA assembly to facilitate pathway assembly, cluster refactoring and direct transfer into any vector backbone in vitro.

In the first part of my talk, I will describe our application of DiPaC towards the experimental validation of the cyanobacterin biosynthetic gene cluster. In the second part of my talk, I will describe how we further exploited our knowledge gained of cyanobacterin biosynthesis for further discovery, including discovery of cryptic pathways and combinatorial (bio-)synthesis to generate novel structures.

Due to its simplicity, we envisage DiPaC to become an essential method for biosynthetic gene cluster capture and metabolic pathway construction with significant applications for the functional characterisation of novel natural product pathways well into the future.

Scalable microbial metabolite discovery through synthetic biology

Vincent Libis  – INSERM – Paris, France

Discovery of bioactive secondary metabolites of microbial origin have declined in the past decades, depriving clinical pipelines from a key source of novel lead molecules. Encouragingly, the natural repertoire of microbial secondary metabolites remains vastly underexplored, and recent developments in microbial genome mining technologies offer ways to accelerate the pace of discoveries. Sequencing and bioinformatics allow prioritization of biosynthetic genes predicted to encode new metabolites, and cloning and heterologous expression of such genes can speed up the discovery of therapeutically relevant molecules. Here, an approach allowing to massively parallelize these processes will be presented. The streamlined interrogation of a large number of biosynthetic genes contained in a strain collection led us to discover several previously uncharacterized natural products, including a novel antibiotic. I will discuss the importance of leveraging economies of scale with cloning strategies and their potential to become a major source of novel leads. On the side, I will present how citizens can be involved on specific bottlenecks and take part in the global effort to find novel molecules, in particular urgently needed novel antibiotics.

Designer Microorganisms for Sustainable Chemical Synthesis

Stephen Wallace – University of Edinburgh, United Kingdom

Reducing the carbon emissions of the modern chemical industry is one of the most pressing challenges facing modern research in biotechnology. Here, engineered microorganisms have emerged as an elegant solution, where de novo biosynthetic pathways can be designed to convert a range of renewable feedstocks into industrial small molecules via fermentation.

In my talk, I will discuss some of our most recent work in this area focussed on the use of PET plastic ‘waste’ as a new carbon feedstock for microbial biosynthesis. This approach simultaneously creates sustainable routes to industrial petrochemicals whilst also addressing the plastic waste crisis. I will also introduce some of our unpublished work in the field of biocompatible chemistry – the use of non-enzymatic catalysis and microbial metabolism simultaneously to create new-to-nature products. Here, I will outline how we have interfaced engineered metabolism and biocompatible Pd catalysis at the cell membrane to enable the bio-production of industrial products that cannot be accessed via synthetic biology alone.

Together, these studies will showcase how synthetic biology can be applied to enable the bioconversion of plastic waste into industrial chemicals that are currently manufactured from fossil fuels, and how synthetic biology can be merged with synthetic chemistry to enable the use of abiotic chemistry in living cells.