Caulobacter

Our project aims to engineer Caulobacter crescentus as a living platform for enzymatic surface display to support sustainable mine tailing remediation. By leveraging its crystalline S-layer system and resilience in harsh environments, we developed tools for displaying carbonic anhydrase (CA) and other functional proteins to enable calcium carbonate precipitation and biocementation in polluted sites.

Introduction

Remediation of Mine Tailings

The composition of waste material (tailings) produced from mines depends on the type of ore being extracted, but generally consists of a mix of residual minerals and chemical byproducts that leach into the soil, and consequently the surrounding environment[1]. Although the application of biocementation is a viable and sustainable remediation approach, few microbes can survive in these locations and existing studies have limited information on the practical implementation of this strategy. Some studies suggest the use of immobilized carbonic anhydrase (CA) to cement tailings, particularly through enzymatic display, which has emerged as a promising waste remediation platform[2]. While there are many bacterial candidates that can support whole cell enzymatic display, our project targets Caulobacter crescentus due to its ability to tolerant harsh living conditions and its robust surface display system[3].

C. crescentus is a Gram-negative, non-pathogenic, freshwater bacterium that has been extensively studied as a model organism for its dimorphic life cycle[4]. It divides into genetically identical but morphologically distinct progeny cells: a stalked cell that grows a holdfast structure to adhere to surfaces, and a swarmer cell with flagellum and pili structures for motility[4]. These features, along with its resilient stress adaptions, enable it to survive in low nutrient conditions.

While E. coli strains can be used for surface display of CAs in this context, Caulobacters have several characteristics that make it more desirable for use in meduCA. One factor is that C. crescentus is an obligate aerobe, which allows for more cost-effective cultivation in bioreactors. In comparison, E. coli switches from aerobic respiration to fermentation when oxygen levels become too low, which leads to rapid consumption of feedstock, acidification of the culture medium and ends the culture run in a bioreactor[5].

The Model Organism for Surface Display

Caulobacters produce a crystalline S-layer composed of a monomer, RsaA, that assembles into hexagonally packed structures that span the cell membrane[6]. This S-layer is anchored to the cell via long side chains connected to the lipopolysaccharides (LPS) on the outer membrane[6]. Uncommon to other S-layer harbouring organisms, the surface layer protein (Slp) of C. crescentus is secreted by the Type 1 mechanism[6]. This makes it an ideal host for adaptation of the S-layer for heterologous protein display, as the Type 1 pathway is more flexible in terms of the physical and chemical properties of the proteins secreted[7]. This system has been proven to display foreign peptides up to 200 amino acids in length and has potential to tolerate even larger peptides which was necessary for displaying CAs, as they can range from 260 to 459 amino acid residues [8]. Additionally, C. crescentus doesn’t secrete other proteins (in fact RsaA takes up to about 10 - 12% of all protein synthesis) so it’s easier to purify its the Slp compared to other systems.

RsaA contains a cell anchoring sequence in its N terminus, RTX (repeats in toxin) motifs, and a secretion signal in its C terminus. The middle domain is rich in glycine-aspartate amino acids which bind calcium ions and helps the S-layer form the crystalline lattice[5]. Heterologous proteins can be inserted at any site in this protein, but previous studies have identified specific locations that maximize display efficiency.

Previous work

Another appealing aspect of working with Caulobacters was the available support provided by the Hallam Lab, where the UBC-Vancouver team is based. Beth Davenport, a PhD student in the lab that specializes in Caulobacter bioremediation applications, provided valuable guidance throughout our cloning process. She previously displayed metal-binding proteins for bioremediation and established the groundwork for our display system.

Additionally, our team has displayed proteins on Caulobacter in two previous projects, although we weren’t able to recover a lot of previous work to build off of from 2014 and 2016. While other teams have explored Caulobacter for applications such as developing functional biofilms and producing glue from its polysaccharide proteins, there has been limited research on culturing this organism. Our team aimed to address this gap and provide tools for displaying proteins in Caulobacter.

Construct Design

To engineer C. crescentus to display fusion proteins, our design is inspired by the research done by the Smit Lab at UBC, who extensively characterized the Caulobacter S-layer system. The wild type Caulobacter strain CB2 is a laboratory strain that is commonly used to study the bacterial cell cycle, biofilm formation and other microbial mechanisms. We used the strain CB2A JS4038, a variant of CB2 that does not produce a surface layer because genomic copy of RsaA is non-functional[9], and has a disrupted SapA protease gene to avoid unintended cleavage of the fusion protein. In addition, it contains repBAC operon, enabling stable plasmid replication in . By introducing a plasmid with a functional copy of RsaA, we can recover and control the expression of fusion proteins in this chassis.

We used two plasmids, provided by the Hallam Lab, that were used to display and secrete fusion proteins respectively.

The first vector, henceforth referred to as pCB2A-Display, clones inserts in the middle of the protein. This vector has a dual ori (ColE1 and RSF1010), which enables plasmid replication in both E. coli and C. crescentus strains.

The display backbone contains a multiple cloning site at amino acid position 723 of the RsaA surface-layer protein. This position was selected based on findings from [10] whereby insertion of peptides at position 723 enabled protein surface display without disrupting the RsaA protein. We further modified this MCS by appending the flanking SapI recognition sites with additional nucleotides to stabilize enzyme binding during assembly. Moreover, we included a myc tag for protein recognition in downstream surface display and CA activity validation assays. While the original backbones contain an inducible promoter, we modified this to restore the constitutive WT rsaA promoter. Altogether, these features result in a recombinant backbone into which different CA candidates are compatible for insertion. Ultimately, this produces CA-RsaA fusion proteins which are anchored to the outer membrane of CB2A, allowing direct enzyme-substrate interactions at the extracellular interface.

The secretion construct contains the modified multiple cloning site between the inducible lac promoter and the C-terminus end of RsaA at amino acid position 336. As such, this allows the CA to be inserted directly fused to the C-terminus secretion signal domain. Additionally, the backbone contains the collagenase site (PLGP) which cleaves the protein of interest from the RsaA fragment after secretion. This results in the secretion of soluble CA.

Both display and secretion backbones confer chloramphenicol resistance by introducing the Chloramphenicol Acetyltransferase gene. This served as a selectable marker to enable the selective culturing of successfully transformed strains.

Cycle 1: Surface Display Vector Assembly

We created a new part based on this design, which can be found on the Registry at BBa_258NR5MC.

Cloning in E. coli

To create the display and secretion recombinant backbones, we performed gibson assembly to combine the necessary fragments, including the parent backbones, WT rsaA promoter, and modified MSC.

The assembled recombinant backbones were transformed and cloned in chemically competent DH5α E. coli. Transformants were grown on plates containing chloramphenicol on which only successfully transformed E. coli containing the chloramphenicol resistance gene form colonies. Subsequently, transformants containing the recombinant backbones were extracted via miniprep.

To insert the CA candidates into the recombinant backbones, we performed golden gate assembly using SapI. Transformants were grown on plates containing chloramphenicol and colonies were validated via PCR amplification of the MCS and restriction digest with BsaI.

Cycle 2: RsaA-CA Fusion Protein Design

Electroporation

We modified an existing electroporation protocol based on previous literature and experience from Hallam lab members.

Optimal parameters for electroporation of CB2A

Cycle 3: Electrotransformation of CB2A

Several constructs yielded colonies after CB2A electrotransformation, but we’ve made furthest progress with RsaA-BtCAII (codon optimized for E. coli). More information about this part can be found on the Registry, at BBa_25EMPHCH!