Heterologous expression of biosurfactant transporters in Debaryomyces hansenii

Ewald Hettema, University of Sheffield



“Due to their tractable nature, model microorganisms are attractive candidates for industrial processes. However, limitations in their inherent biological capability lead to challenges in production and commercialisation. Organisms such as D. hansenii perform industrially attractive biochemistry but require projects such as this to develop the tools to manipulate them. I look forward to continued collaboration with Dr Hettema and Dr Gilmour.”

Dr Jeremy Bartosiak-Jentys, R&D Manager, Biotechnology, Croda

The Challenge

Surfactants are ubiquitous performance ingredients that allow formulators to create the products we all use. Although surfactants are often made from petrochemical sources, there is increasing global demand to shift to production using sustainable, biological resources and technologies. Some microbes, including certain yeast, can produce and secrete biosurfactants, often to high levels. However, manipulating synthesis to produce specific structures has proved challenging.

In order to produce biosurfactants in an industrial setting, a robust and tractable production host is required. One potential organism is the halotolerant, non-pathogenic yeast Debaryomyces hansenii, which naturally accumulates high levels of lipids, that can provide the hydrophobic portion of the surfactant molecule.

The Research

University of Sheffield logoDr Ewald Hettema is a Reader at the University of Sheffield. The research in his laboratory focuses mainly on the mechanisms underlying peroxisome biogenesis. Dr Hettema and his colleague Dr Jim Gilmour (also from the University of Sheffield), have a long-standing collaboration to investigate the physiology and molecular biology of D. hansenii.

Croda is a British company that creates, makes and sells speciality chemicals that deliver real benefits to a range of diverse products across personal, health, crop and home care markets.

Dr Hettema and Dr Gilmour applied for a CBMNet Business Interaction Voucher with Croda. Their project aimed to engineer D. hansenii to express known fungal transporters that may play a role in improving the biosurfactant production capability of the organism.

The Result

Tools for protein expression and/or tagging are limited in D. hansenii. This project successfully generated a number of novel resources that will be useful for the future development of D. hansenii as an industrial organism. In the first instance, a stable shuttle plasmid, capable of replication in both E. coli and D. hansenii, was engineered. Essential plasmid elements have also been identified and cloned, including: (1) a strong, constitutive promoter; (2) an auxotrophic marker; (3) fluorescent tags; (4) immunolabelling tags.

Since D. hansenii translates the CTG codon into both leucine and serine, instead of just leucine, all of these genes were engineered accordingly. In addition, an ORF encoding a biosurfactant transporter was generated, again with appropriate codons for expression.

The Future

In the immediate future, important next steps will involve proving that it is possible to express a heterologous transporter in D. hansenii. The tools needed to do this are now in place and Dr Hettema plans to expand the resources at their disposal by, e.g., evaluating different promoters.

In the longer term, the tools generated in this project will be of general use to all researchers using CTG clade yeast as model organisms and should allow its development as an industrial biotechnology organism.

Croda are particularly interested in engineering an alternative robust production host for the biosynthesis of biosurfactants. To this end, Dr Hettema and Croda are actively continuing their collaboration and have successfully applied for Impact, Innovation & Knowledge Exchange funding from the University of Sheffield. They plan to further explore future research directions and how to fund their collaboration in the long-term.