Engineering Phaeodactylum for vaccine production
Lead Research Organisation:
University College London
Department Name: Biochemical Engineering
Abstract
The use of photosynthetic chassis strains has the potential to offer sustainability advantages for industrial biotechnology. This project seeks to explore how synthetic biology approaches in microalgae can be employed to optimise expression of recombinant proteins, using vaccine production as our model system.
A protocol1 for delivery of episomes to the marine diatom Phaeodactylum tricornutum has recently been devised, considerably expanding the toolbox for synthetic biology in microalgae. However, for microalgae to be considered as viable expression systems, product titre must be increased.
Several recombinant vaccines currently use bacterial and fungal systems for expression. Hepatitis B, based on the formation of VLPs based either on the surface antigen or the hepatitis core protein are currently expressed in Pichia pastoris and Saccharomyces cerevisiae. While both these strains are able to produce high biomass, there are limitations in engineering the strain to obtain correct folding of modified HepB proteins, total expression yield, induction mechanism that are not dependent on substrates toxic to the cell, such as with methanol induction in yeast, or inefficient induction of the glucose/galactose switch in S. cerevisiae. Furthermore, release of the vaccine often requires cell disruption to release the intracellular proteins. This operation can lead to loss of the product as upto 50% of the product associates with the insoluble cell debris and is lost from the purification process.
Using HepB as a starting point, strategies for the expression of HepBSAg (Hepatitis B surface antigen) and its potential secretion will be investigated in Phaeodactylum tricormutum.
An optimisation strategy for vaccine production in this host strain will be explored by combining synthetic biology approaches for construction of gene expression system together with manipulation of growth conditions. For instance, while the promoter and terminators of nitrate reductase has been used in other studies 2, considerable scope remains for investigation into the regulation of recombinant gene expression.
1. Karas, B. J. et al. Designer diatom episomes delivered by bacterial conjugation. Nat. Commun. 6, 6925 (2015).
2. Vanier, G. et al. Biochemical Characterization of Human Anti-Hepatitis B Monoclonal Antibody Produced in the Microalgae Phaeodactylum tricornutum. PLoS One 10, e0139282 (2015).
A protocol1 for delivery of episomes to the marine diatom Phaeodactylum tricornutum has recently been devised, considerably expanding the toolbox for synthetic biology in microalgae. However, for microalgae to be considered as viable expression systems, product titre must be increased.
Several recombinant vaccines currently use bacterial and fungal systems for expression. Hepatitis B, based on the formation of VLPs based either on the surface antigen or the hepatitis core protein are currently expressed in Pichia pastoris and Saccharomyces cerevisiae. While both these strains are able to produce high biomass, there are limitations in engineering the strain to obtain correct folding of modified HepB proteins, total expression yield, induction mechanism that are not dependent on substrates toxic to the cell, such as with methanol induction in yeast, or inefficient induction of the glucose/galactose switch in S. cerevisiae. Furthermore, release of the vaccine often requires cell disruption to release the intracellular proteins. This operation can lead to loss of the product as upto 50% of the product associates with the insoluble cell debris and is lost from the purification process.
Using HepB as a starting point, strategies for the expression of HepBSAg (Hepatitis B surface antigen) and its potential secretion will be investigated in Phaeodactylum tricormutum.
An optimisation strategy for vaccine production in this host strain will be explored by combining synthetic biology approaches for construction of gene expression system together with manipulation of growth conditions. For instance, while the promoter and terminators of nitrate reductase has been used in other studies 2, considerable scope remains for investigation into the regulation of recombinant gene expression.
1. Karas, B. J. et al. Designer diatom episomes delivered by bacterial conjugation. Nat. Commun. 6, 6925 (2015).
2. Vanier, G. et al. Biochemical Characterization of Human Anti-Hepatitis B Monoclonal Antibody Produced in the Microalgae Phaeodactylum tricornutum. PLoS One 10, e0139282 (2015).
Organisations
People |
ORCID iD |
| Brenda Parker (Primary Supervisor) | |
| Greta Csalane Besenyei (Student) |
Studentship Projects
| Project Reference | Relationship | Related To | Start | End | Student Name |
|---|---|---|---|---|---|
| EP/N509577/1 | 01/10/2016 | 24/03/2022 | |||
| 1807434 | Studentship | EP/N509577/1 | 01/10/2016 | 06/09/2020 | Greta Csalane Besenyei |
| Description | For the purposes of placing the current and future proposed work within context, this work builds on a previously developed bioprocess model for the recovery of recombinant proteins from microalgae cultures. The predictive technoeconomic model highlighted yield and production volume as major factors influencing economic feasibility of microalgal platforms relative to more established eukaryotic host organisms. The current study aimed to apply emerging molecular biology tools to express specific recombinant proteins in Phaeodactylum tricornutum such that the bioprocess model can be tested using actual exemplars. The challenges primarily targeted within the scope of this project include the (1) stable expression of recombinant proteins at sufficiently high concentrations, (2) replace antibiotic selection markers with auxotrophic ones to avoid the potential risk of horizontal gene transfer for whole cell products, and (3) reducing high downstream processing costs with secretion of the product into the culture medium. To investigate the opportunity presented through the expression of recombinant proteins in Phaeodactylum, a model system was created through a specific design and assembly of constructs incorporating the sequence for the salmonid alphavirus disease (SAV) structural protein. Alternate versions of the SAV construct were produced via the Golden Gate cloning method utilizing an array of standard modular parts to facilitate comparison of expression levels and production costs in the presence/absence of either a constitutive or inducible promoter and a signal peptide. These were designed to explore the utility of controllable protein expression levels using constitutive or inducible promoters and secreted and non-secreted product formation. For SAV constructs with a constitutive fcpA promoter, we have defined optimum range for seeding concentration, inoculum culture age and harvesting time point for highest level of expression. We have monitored the expression level in constructs harbouring an alkaline phosphatase inducible promoter over time post induction compared to constitutive expression levels. In addition, we have investigated the amount of SAV protein secreted into the culture medium in constructs that included the signal peptide. Introducing auxotrophic selection markers facilitates the timely isolation of clones expressing the target protein without the risk of horizontal gene transfer. One example is the previously isolated uracil auxotrophic mutant of P. tricornutum. Deficient cells of this strain lack the biosynthesis pathway to produce uridine-5'-monophosphate (UMP) - a precursor of uracil - in the absence of which the cells gradually starve to death. This function is then restored by simultaneous insertion of the gene of interest and the ptUMPS gene to correct for the disrupted pathway, allowing successfully transformed cells to survive without supplementation of uracil. We investigated transformation efficiency and selection time for auxotrophic selection compared to the standard antibiotic selection process. In our model system, using Golden Gate cloning, we designed a single plasmid integrating two genes, one encoding a fusion protein of the salmonid alphavirus (SAV) and the auxotrophic marker gene exclusively. Using a standard biolistic method, we transformed uracil auxotrophic P. tricornutum cells and obtained SAV expressing clones in uracil-free selective medium with a transformation efficiency comparable to that observed in an antibiotic selection process. In addition, a faster selection process in liquid medium has been established, shortening the starvation phase to 6 weeks, which is half the time that is required on solid medium. To our knowledge, this is the first time when no antibiotic selection marker was involved in the selection for P. tricornutum cells expressing a recombinant protein. In order to evaluate techno-economics of an auxotrophic microalgae expression platform, we created a predictive model as a decisional support tool based on scale up cultivation in single use, disposable bag systems to calculate operational costs of production and downstream processing. |
| Exploitation Route | Characterisation of the isolated clones has started but requires further investigation which could include the following: 1) optimization of culture conditions using automated, robotic platforms, followed by 2) the scale-up of cultivation in single-use, disposable bag systems and 3) development of predictive decisional tool for the evaluation of primary recovery stages both using ultra scale-down experiments and at pilot-scale 4) exploration of alternate methods to improve commercial relevance (e.g. continuous cultivation mode in combination with inducible promoters ) 5) further product development and formulation of an edible vaccine to facilitate clinical trials. The mentioned research can potentially be carried out as part of another PhD project or post-doctoral research. |
| Sectors | Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology |
| Description | The increasing demand for therapeutic proteins over the last decade has driven the industry to explore new, alternative expression platforms. Microalgae represent a promising platform for biological manufacturing as synthetic biology expression systems. Eukaryotic microalgae combine the advantages of pro and eukaryotic platforms as well as plants; being photosynthetic microorganisms, they can utilize a source of light and inexpensive culture medium for their cultivation. Most microalgae have rapid doubling times and grow in suspension as single cells. Perhaps more importantly, industrial microalgal chassis strains perform complex post-translational modifications of polypeptides similar to that observed in higher eukaryotic organisms. Phaeodactylum tricornutum, the heterokont diatom has proven itself as a versatile platform for the expression of recombinant proteins. More recently, a unique pathway for protein trafficking has been identified, whereby partially glycosylated proteins are targeted into the chloroplast via an ER-chloroplast membrane fusion. This offers the opportunity to express and accumulate recombinant proteins that have been proven to be challenging in the past using other platforms. In addition to the accumulation of recombinant proteins in the chloroplast or intracellularly, Phaeodactylum also offers the option to target high value recombinant proteins into the culture medium. Diatom milking, which means the secretion or continuous extraction of an intracellular product, could potentially enable the elimination of the cell disruption step. Furthermore, continuous harvesting such as perfusion filtration can facilitate recycling of the cells allowing a more cost-efficient cultivation and processing. In relation to vaccine production, the current use of Phaeodactylum within aquaculture feed suggests that this platform holds promise for application within oral vaccine strategies. The simple cultivation requirements for this organism, combined with, for instance, inexpensive single-use disposable bags could enable a production system to be set up at the point of care, eliminating costs associated with maintaining a cold chain for vaccine delivery. As part of meeting future industry needs, demonstration and process development for the use of an auxotrophic selection marker is essential for the oral application of whole cell microalgal products. It is well known that in creating genetically modified (GM) seeds and plants, antibiotic resistance genes are commonly used as marker genes for the selection of transformed plant cells. In parallel, concern has been addressed about whether horizontal genes transfer (HGT) of these genes from the plant material to environmental microorganisms can take place, thereby - in the next step - compromising the therapeutic value of antibiotics in human and veterinary medicine. In a report funded by the European Union (EU), it was concluded that the acquisition of new genes, such as antibiotic resistance genes from plant to environmental bacteria, might be possible. Therefore, it was recommended that only genes coding for antibiotics not used in human and veterinary medicine should be allowed when making GM seeds and plants. Development of the current selection system based on complementation of the auxotrophic gene eliminates hurdles that are currently in the way of using microalgae as whole cell edible vaccines. |
| First Year Of Impact | 2020 |
| Sector | Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology |
| Impact Types | Economic |