The isolation and characterisation of isoprenoid/carotenoid mutants in Xanthophyllomyces dendrorhous (Phaffia) as renewable sources of high value pigments.

Xanthophyllomyces dendrorhous, formally (Phaffia rhodozyma) is a basidiomycetous yeast that has the ability to produce astaxanthin. It is one of the only microbes know that is capable of producing these high-value industrial carotenoids. The global market for carotenoids is expected to be worth over $1.4 billion by 2018. The market for phytoene is also growing rapidly with supply outstripping demand. This is due to its UV protection and anti-wrinkle properties that have led to the molecule being used in cosmetics. Phaffia is a GRAS organism and recently we have shown amenability to the production of mutants using methylnitronitrosoguanidine (MNNG), a chemical mutagen. To date we have generated high astaxanthin, b-carotene, phytoene, no carotenoids (geranylgeranyl pyrophosphate, GGPP accumulator) and a range of sterol mutants. These are unique in their own right.
These mutants will be subjected to subsequent rounds of mutagenesis and the b-carotene mutant (orange in colour) used to obtain a lycopene containing mutant (red in colour). It is aimed to reach levels that will deliver yields at 5 to 10mg/gDW.

The aim of this project is to exploit Xanthophyllomyces dendrorhous as a renewable source and production platform for high value carotenoids. Main objectives include:
Subsequent mutagenesis rounds (using methylnitronitrosoguanidine (MNNG) and EMS) of existing isoprenoid/carotenoid mutants to create super producers of sterol precursors, and high value carotenoids such as phytoene, lycopene, -carotene, zeaxanthin and astaxanthin.

Characterisation of those mutants generated using transcriptomic and metabolomics approaches in association with biochemical and molecular validation.
Optimisation of growth conditions and carotenoid formation using different media and epigenetic modulators.

Like most isoprenoids of industrial and nutritional value, chemical synthesis is the production method of choice. The synthesis of these molecules in most cases is difficult, expensive and has a detrimental environmental impact, both from the utilisation of petrochemical refining precursors and use of hazardous organic solvents. In the proposed studentship the red yeast Phaffia will be subjected to mutagenesis to create and develop existing strains capable of producing high levels of key carotenoids and isoprenoids. These strains can form the basis for industrial exploitation and act as a cross-cutting resources for the production of value isoprenoids using a Synthetic Biology approach.

Experimental approach.

Task 1. Generation of carotenoid mutants in Xanthophyllomyces. Previous rounds of mutagenesis using the chemical mutagen methylnitronitrosoguanidine (MNNG) have generated stable mutants perturbed in most steps within the carotenoid pathway. A selection of these mutants will enter the next cycle of mutagenesis and addition rounds using the alternative mutagen Ethyl methane Sulphonate (EMS). The procedures for the generation and screening are all established in the laboratory. The use of a colour screen ensures rapidity and robustness.
Task 2. Biochemical and molecular characterisation of the mutants generated. Detailed analysis of the carotenoids present in the mutants and wild type will be performed by HPLC-PDA/MS. Transcript levels of the pathway components will be determined by QRT-PCR. From the perturbation candidate genes will be designated and sequencing of the putative mutant genes performed. In selected mutants metabolomics and transcriptomics will be carried out to assess global changes across metabolism in the strains.
Task 3. Validation of the mutated carotenoids genes. Those candidate mutant gene sequences will be validated for activity through functional complementation in E.coli and Phaffia. The former will use E.coli containing complementation vectors producing the relevant precursors and in the case of Phaffia mutants will be transformed with the wild type genes. In addition in vitro mutagenesis will be carried out to replicate the mutation found.
Task 4. Optimisation of carotenoid production. Using a select group of mutants growth characteristics associated with different carotenoids will be determined. The ability of these strains to grow on a range of cheap Agroindustry waste materials will be explored and the enhancement of carotenoid formation using epigenetic modifiers.

This PhD studentship is funded for 3 years. Applicants are expected to hold, or to be awarded a first class or a good upper second class BSc Degree, Masters or an equivalent qualification in a relevant field by October 2016. The course will commence in October 2016. Funding includes an annual tax-free stipend at the standard Research Council rate (£16,057 for 2015-2016, to be confirmed for 2016-2017 but typically increases annually in line with inflation) and covers tuition fees at the UK/EU rate.

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