ERASynBio2: An orthogonal, organism-independent expression platform based on extracytoplasmic function (ECF) sigma factors

Lead Research Organisation: John Innes Centre
Department Name: Molecular Microbiology

Abstract

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Technical Summary

Orthogonality is a key feature of classical engineering approaches but a major challenge to Synthetic Biology (SynBio) due to the high degree of context dependence and interconnectivity in biological systems. ECFexpress will develop a SynBio design framework based on Extracytoplasmic Function sigma factors (ECFs) to implement highly orthogonal regulatory switches and circuits. ECFs represent ideal building blocks for SynBio applications, because they are modular, inherently orthogonal, universal, and scalable. Initially, we will evaluate the organism-independent potential of ECFs by implementing orthogonal ECF-based regulation in four phylogenetically highly diverse bacteria, including two biotechnological workhorses. This will allow extracting and establishing universal design rules for choosing and implementing orthogonal ECF-based natural switches in any bacterium. Subsequently, we will design and engineer novel synthetic ECF switches with increased orthogonality. This goal will be achieved by applying design strategies based on combinatorial synthesis and structure-guided mutational approaches. Finally, we will use rational forward design to build and evaluate complex synthetic circuits based on ECF switches. This combined theoretical and experimental evaluation of novel ECF-based circuits will allow us to benchmark their orthogonality and to explore the ECF circuit design space. ECFexpress strictly adheres to engineering principles by (i) applying defined standards in assembly, measurements and data management, (ii) establishing orthogonal parts and switches for biological circuit design, and (iii) developing a bioinformatics platform for data management, mathematical modelling and forward design. Taken together, ECFexpress aims at a foundational advance by implementing true orthogonality to bacterial cells. It will be directly applicable to projects in basic research and in the knowledge-based bio-economy.

Planned Impact

Not required.
 
Description 1. Use of heterologous ECF sigma-antisigma pairs to control gene expression in Streptomyces.

We have successfully implemented a heterologous ECF sigma factor, PspX (CCQ18694), in Streptomyces venezuelae as proof-of-principle that non-native ECFs can be used in S. venezuelae for orthologous expression. PspX comes from the actinomycete species Planomonospora alba and is involved in regulating the biosynthesis of the antibiotic planosporicin.

We have built strains of S. venezuelae that contain a PspX target promoter fused to a gene encoding ß-glucuronidase (GUS) and at the same time constitutively express PspX. In this way, GUS activity can be used to assess promoter activity (Figure 1). In the absence of PspX, low levels of GUS were detected. In contrast, the constitutive expression of the PspX sigma factor resulted in considerably increased levels of GUS activity. When the cognate antisigma, PspW, was co-expressed with PspX expressed, the levels of GUS activity were similar to those detected in the absence of sigma factor, presumably as a result of sequestration of the sigma factor by the antisigma factor. These results confirm that S. venezuelae can be used as a host for non-native sigma factors, and that the activity of these heterologously expressed sigma factors can be regulated by a cognate antisigma partner.

Similar experiments were attempted using MibX (NCBI accession # ADK32553) and its cognate antisigma MibW from the actinomycete Microbispora corallina, but these gave more ambiguous answers.

2. Structure of the BldN/RsbN complex

We have successfully solved the crystal structure of a novel ECF sigma/antisigma complex: Streptomyces venezuelae BldN/RsbN (in collaboration with Maria Schumacher and Dick Brennan, Duke University, USA). This ECF sigma/antisigma pair functions to control a key stage in the morphological development of these filamentous bacteria.

Background. The first dramatic event of the Streptomyces lifecycle is the emergence of the aerial hyphae. These reproductive structures have to break surface tension in order to escape from the aqueous environment of the vegetative mycelium and grow into the air. To achieve this, streptomycetes have to cover the reproductive structures in an extremely hydrophobic sheath, comprised of two families of proteins called the chaplins and the rodlins.

The expression of the genes encoding the chaplins and rodlins is controlled by an ECF sigma factor known as sBldN. bldN expression is subject to transcriptional regulation but, in addition, sBldN activity is also controlled post-translationally. In this mechanism, a cognate anti-sigma factor, RsbN, recruits sBldN to the membrane, preventing it from binding to RNA polymerase. RsbN has three well-defined parts: a ~270-residue extracytoplasmic domain, a single transmembrane helix, and a ~100-residue cytoplasmic anti-sigma factor domain (ASD).

This means that a signal that inactivates RsbN is needed to release sBldN and turn on expression of the components of the hydrophobic sheath. The nature of that signal is still unknown, however, given the bulk of RsbN (residues 140-412) sits on the outside of the membrane, it seems likely the signal that inactivates RsbN to release sBldN is extracytoplasmic. An attractive possibility is that this signal is the assembly of the hydrophobic sheath itself, creating a positive feedback loop to reinforce sheath assembly once it has started.

The structure. Although the nature of the inactivating signal is still uncertain, what is now clear is that this signal leads to the proteolytic processing of RsbN. Using an N-terminal his-tag on RsbN, it has been possible to purify from S. venezuelae a soluble cytoplasmic complex of sBldN bound to the ~100-residue RsbN cytoplasmic ASD, and the crystal structure of this complex has recently been determined (unpublished data; in collaboration with Profs Maria Schumacher and Richard Brennan, Duke University, Durham, USA). The structure of the anti-sigma and the structure of the complex as a whole are novel. The structures of the individual sigma domains 2 and 4 closely resemble those of sigmas characterized previously.
Exploitation Route PspX could be used to construct an inducible expression system based on the fact that the anti-sigma factor, PspW, releases the sigma factors in the presence of planosporicin, the product of the biosynthetic pathway that PspW regulates. Therefore, the addition of planosporicin could be used to induce expression of genes that are under the control of the PspX target promoter.
Sectors Pharmaceuticals and Medical Biotechnology