Chitinase display system

The importance of chitinases
Chitin is a polysaccharide molecule naturally found in a variety of biological structures, mostly exoskeletons of insects and crustaceans, similar to cellulose in structure and likewise able to form strong crystalline fibers. Most notably however, chitin is the most important building block of the fungal cell wall, making it an extremely interesting biomarker for the detection of fungal infections [1]. Being able to break down the long chitin polymers into their individual monomeric units, N-acetyl glucosamine molecules, hence represents the first step towards the development of a molecular fungal sensing system.  Chitinases are a particular class of glycosyl hydrolases capable of cleaving the glycosidic bonds of chitin polymers [2]: these enzymes are found in a wide range of different organisms, spanning a vast array of biological and ecological functions. In bacteria, for example, they contribute to the conversion of chitin into a usable carbon source, in insects to the moulting of old chitin exoskeletons, in yeasts to morphogenesis and cell division, while their inducible overexpression in plants allows for a natural defence against different biotic stresses [3]. In all domains of life, however, chitinases activity plays a fundamental role in enhancing resistance against fungal pathogens infections, making it the ideal candidate for the development of natural fungicides [4].


The spore structure
B. subtilis spores are covered by multiple set of membranes not normally found in vegetative cells, which result in their superior resistance to heat and similar unsuitable growth conditions. The dehydrated core, hosting a copy of the chromosome, is surrounded by an inner and outer membrane, between which layers of peptidoglycan molecules form the cortex. This complex is in turn surrounded by a basement layer, itself encapsulated in a two-layered protective spore coat and, in some case, an outermost thin crust.The spore coat is one of the most interesting feature of the spore, consisting of more than 70 different proteins and glycoproteins pivotal to the spore’s structural integrity [5].

Figure 1: B. subtilis spore structure

The system
Some of these endogenous structural proteins have successfully been used as “molecular anchors” for the display of a wide range foreign molecules on the surface of recombinant B. subtilis spores[6]. Importantly to the scope of this project, the work of Rostami et al., [7] showcased the possibility of building a chitinase spore surface display system in B. subtilis,by designing a novel fusion protein featuring ChiS, an exochitinase derived from B. pumilus, and CotG, an endogenous structural protein of the outer sporecoat. The ChiS chitinase, in addition to its intrinsic antifungal properties, has also shown the potential, until now only believed to belong to some plant chitinases, to act as a lysozyme, exhibiting even stronger cell lysis activity, an interesting trait for potential biocontrol applications [8] [9]. Anchor protein CotG, is one of the most abundant structural proteins of the outer spore coat, leading to higher display density, but has only a minor role in the morphogenesis of the spore coat compared to other structural proteins, meaning that modifications to its structure are less likely to cause problems to the correct assembly of the coat [10]. We decided to build upon this work, introducing an entirely novel element within the fusion protein design: a linker, a feature that has been shown to significantly improve the enzymatic bioactivity of similar proteins previously displayed on the surface of B. subtilis spores.  As an example, a linker-featuring an esterase from Clostridium thermocellum displayed on B. subtilis spores achieved 1.2-fold higheractivity compared to a non-linker fusion protein, as a greater separation between the anchor protein and enzyme of interest was achieved, preventing steric hindrance within the enzyme [11]. In addition, we set out to explore the activity of the construct both in silico and vitro featuring a different anchor protein, CotZ,[12] found instead on the outermost layer of the spore, the thin crust encapsulating the outer coat membrane.

Modelling and cloning
In the process of finding the optimal linker sequence to include in ourdesign, structural modeling highlighted the better performance of flexible linkers over rigid ones, while sequence-based approaches allowed to identify the linkers with the lowest interaction with neighboring residues. Investigation of disulphide bonds formation between the chitinase and the anchor protein allowed to compare in silico the predicted performance of CotG and CotZ, completed by docking simulations to assess affinity.  Golden Gate was chosen as the cloning methodology for the assembly of the construct, taking advantage of modular parts belonging to an existing toolkit for B. subtilis developed by Dr. Joaquin Caro Astorga. To validate experimentally the modelling work regarding interactions between the anchor proteins and the chitinase, it was decided to build two fusion proteins featuring CotG and CotZ respectively, to be able to further assess in vitro their performance.
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Self Digesting Plasmid

The system was largely inspired by the work of Quijano et al. [13], who developed a self-digesting plasmid using a sporulation-activated promoter to trigger expression of Cas9 to induce plasmid digestion. Their system consists in a single operon, feauturing PsspA, a promoter that gets activated only in stage T3 of sporulation (when forespore and mother cell are completely separated by their respective membranes), a Cas9 CDS followed by either one or two gRNAs, targeting the origin of replication of the plasmid, D15, an exonuclease from the T5 phage effectively responsible for plasmid digestion, and a terminator. On the same plasmid an expression cassette is also mounted: in their work, Quijano et al. demonstrated the efficacy of their system expressing anchor protein CotG fused with GFP, showing that the resulting spores retained surface fluorescence without containing any foreign DNA.

Our system uses very similar components to the one developed by Quijano et al., but with a few differences, result of both design consideration, parts availability and toolkit requirements. For starters, to improve the system’s sensitivity, it was decided to take out the gRNA from the operon and control its expression by a constitutive promoter independent of the sporulation cascade, ensuring large presence of gRNA at the time of Cas9 activation, and only one gRNA was included in the final design. Moreover, sporulation-induced promoter PsspB was used instead of PsspA, as readily available in the Ellis’ lab collection and in any case activated in the same sporulation phase as the original one.

Orthogonal Receptor Engineering

Knockout strain creation
Dormant spores can turn into metabolically functional, vegetative bacteria cells via a process called germination, following a series of well-defined steps [14]. Briefly, the process starts with the expulsion of monovalent cations from the spore, followed by Ca²⁺ and DPA, increasing the pH from 6.5 yto roughly 7.7, a fundamental prerequisite for the development of a vegetative germ cell. This allows water to permeate the spore, triggering hydrolysis of the spore coat and restarting the cell metabolic activity. While the germ cell developmental steps have been thoroughly studied and characterized to a good extent, the exact triggering mechanism that initiates germination is far less established. Spores are known to respond by initiating the germination cascade to a class of nutrients called germinants, including L-alanine, asparagine and simple sugars such as glucose or fructose [15].  

The putative set of proteins responsible for the recognition of these germinants and the subsequent initiation of the germination cascade are known as germinant receptors, sitting on the inner membrane of the spore. In B. subtilis, three receptors are believed to control the majority of germination: GerA, GerB and GerK. The former is capable of inducing complete germination of the bacterial spore in response to L-alanine alone, while the other two require stimulation from a variety of different germinants and their signaling often doesn’t result in full germination. Due to its orthogonality towards a single germinant, in addition to its superior germination-inducing capacities, GerA was chosen in this project as the target for the development of achitin-sensing receptors.  

Prior to that, however, given the essential requirement of our sensing system to be orthogonal to chitin and chitin only, a strain feauturing knockouts of all the other receptors had to be developed, to avoid random germination of the spore in the presence of other germinants readily available in nature. The knockout strain was acquired with the generous contribution of Dr. Graham Christie atthe University of Cambridge, who agreed to share with the team a strain named Ger3 feauturing gene knockouts of GerA, GerB and GerK.[16]  

Germinant receptor engineering
Initially, we considered conducting directed evolution using error-prone PCR to alter receptor specificity to chitin monomer N-acetyl glucosamine (NAG). However, we only had the capacity to produce and test a mutant library on the order of 10⁵ combinations whereas the number of possible combinations is on the order of 20¹⁰⁰⁰ (for a receptor of around 1000 residues), which would have left us with a negligible chance of success.  

Preliminary homology modelling and existing literature suggested that GerA acts as a water and ion channel, triggering germination via spore rehydration, and indicated the receptor’s subunit GerAB as the putative binding site for L-alanine [17] [18]. In order to increase the complexity of our mutant library, we therefore decided to target our mutagenesis process only to this specific region of the receptor. More specifically, given the very limited knowledge of the structure and dynamics of germinant receptor proteins, we setout to identify the key binding residues involved in the receptor’s functionand specificity.  

After building the receptor using AlphaFold and validating its structure via extensive modelling of protein-protein interactions and structural alignment, conserved and highly variable regions were investigated using Multiple Sequence Alignment (MSA) with other subunits form the germinant receptors’ family. This step was crucial in the identification of the binding residues, as strongly conserved regions are thought to be responsible of the structural integrity of the receptor, while the variable ones connected to specificity.  

Out of this pool of identified regions, potential binding residues were selected via a combination of methods, focusing around the flexible docking ofNAG to the modelled receptor, allowing to narrow down the pool to a final selection of regions with difference prediction scores. We planned to order our combinatorial mutant library from TWIST Biosciences, with a maximum of 10⁸ variations. The service allows for the user to define any amino acid bias or exclusion at each residue position:  given NAG’s greater affinity to polar and hydrophobic residues [19], we limited the potential mutations to amino acids such as asparagine, tyrosine, and tryptophan. Other residues such as the ones lining the channel were limited to smaller amino acids to allows pace for the larger germinant, as suggested by Dr Luke Yates.

Directed evolution pipeline 
In order to assess the successful engineering of the receptor and its orthogonality to N-acetyl glucosamine only, a testing pipeline featuring a positive and negative selection specificity assay was developed.  Initially, the library ordered from TWIST would have been transformed into B. subtilis, the resulting colonies subsequently sporulated and spores harvested and purified.

The spores would then be transferred into different minimal media formulation, specifically designed not to contain any knowngerminant factors but to support growth of vegetative cells, and then treated with a 70℃ heatshock for 20mins. This step constitutes the negative selection assay of the system, as it ensures that all the spores that randomly germinated without the presence of any germinant get killed. This selection step is of paramount importance, as it allows to get rid of the genetic combinations that lead to structural defects in the editing of the receptor, causing it to act as a continuously open channel and hence always triggering spore rehydration and germination.

The media would then be supplemented with 10mM N-acetyl-glucosamine: the germinated spores, now vegetative cells, would be separated from the ungerminated spores following the protocol devised by Sacks and Alderton [20], which uses polyethylene glycol 4000 (PEG) and a strong potassium phosphate buffer to harness the difference in hydrophobicity between the two. This constitutes the positive selection step of the pipeline, as only spores effectively responsive to N-acetyl glucosamine would be able to germinate, confirming the successful engineering of GerA. The isolated cells would then be plated, liquid cultured,miniprepped and finally sequenced, in order to understand the precise nature of the mutations that lead to the desired change in binding specificity.