Engineering of Rhodosporidium toruloides for the production of value-added chemicals

Abstract

There are currently increasing concerns about escalating crude oil prices, depleted petroleum resources, and environmental damages caused by plastics. In light of this, bio-based and biodegradable materials, such as polyhydroxyalkanoates (PHAs), have gained global attention from industry and academia as sustainable alternatives to petroleum-based materials. Poly-hydroxybutyrate (PHB) is the simplest PHA and has a high potential to replace petrochemical polymers due to its similar thermoplastic and mechanical properties. Several different microorganisms can naturally produce PHB as a form of carbon storage. Metabolic engineering where the PHB biosynthetic pathway is recombinantly expressed in alternative microbial hosts might however be crucial to reach industrially relevant levels of PHB production, for instance by using a host that can utilize complex renewable feedstocks as substrates, and to avoid the fact that natural producers can depolymerize PHB and use it as a carbon source.

The non-conventional, oleaginous yeast Rhodosporidium toruloides BOT-A2 was previously engineered with the PHB pathway from Cupriavidus necator, but it was demonstrated to produce lower PHB levels in terms of titer and yield than natural producers. In this study, a PHB production optimization strategy was attempted by subjecting BOT-A2 to nitrogen starvation cultivation conditions, a condition known in oleaginous yeasts to lead to high flux through acetyl-CoA, the precursor metabolite of PHB and lipids. Three promoters potentially induced by nitrogen depletion identified in a recent transcriptomic study of BOT-A2 were each coupled in front of a green fluorescent protein (GFP) and introduced into the R. toruloides BOT-A2 genome to be evaluated in a nitrogen-limited medium via flow cytometry. The most suitable nitrogen-induced promoter was chosen for the expression of the phaA gene, which encodes the first enzyme of the PHB pathway and possibly the rate-limiting step. The new PHB pathway was successfully randomly integrated into the genome of R. toruloides through electroporation. Six clones were screened for PHB production through a shake flask experiment and only one was demonstrated to synthesize PHB above the limit of detection. The best clone reached a PHB titer of 25mg PHB/L and PHB yield of 1,21 mg PHB/g glucose after cultivation in the nitrogen-limited medium for 72h. However, the previous best strain reached similar levels (22mg PHB/L and 1,07 mg PHB/g glucose) in this medium, showing that the nitrogen-induced promoter strategy did not result in any major improvements in PHB productivity.

Popular Abstract

Plastic is the most used material in our daily life, especially for packaging applications. Because of their non-biodegradability, they are considered a non-eco-friendly material. In addition, plastic is made from petroleum which is a non-renewable resource. Bioplastics are a valid alternative to plastic thanks to their similar characteristics. Moreover, they are degradable in nature and can be produced from renewable resources.

PHB is a biopolymer that can be used to produce bioplastic and it is made by different organisms in nature. These microorganisms have in their DNA the genes required for PHB production. A yeast called Rhodosporidum toruloides was previously used to produce PHB because it is thought to be a good producer. Previously, the three genes required for PHB synthesis were assembled into a large circular fragment of DNA called plasmid. The plasmid was inserted in this microorganism since it is not a natural producer through a process called electroporation, which led it to integrate the genes into its chromosomal. The yeast was then grown with high concentrations of glucose and nitrogen in the medium. These cultivation conditions made it produce PHB but not in an efficient way though.

In this project, a new plasmid carrying PHB genes with a different promoter in front of the first gene involved in PHB production was constructed. A promoter is a region of DNA upstream of a gene that tells the RNA polymerase where to initiate the transcription process. The promoter used in this study is a promoter that is on and works only when nitrogen is limited in the medium. Then, the plasmid was used to insert the PHB genes into R. toruloides chromosomal through electroporation. The engineered yeast cells were grown in a medium with a low concentration of nitrogen for 72 hours. At the start and end points of the cultivation samples to see if PHB was produced and how glucose was consumed were taken.

In this study, hence, a different promoter induced by nitrogen starvation for the expression of the first gene involved in PHB synthesis and a different growing condition were used to see if a higher amount of PHB could be produced by R. toruloides. It was demonstrated that R. toruloides can produce PHB when growing with a low concentration of nitrogen in the medium, but without getting any improvement in the amount of PHB obtained.

• author: Badiali, Sofia
• supervisor: Daniel Brink
• organization: Applied Microbiology
• alternative title: Modifiering av Rhodosporidium toruloides för produktion av förädlade kemikalier
• course: KMBM05 20221
• year: 2022
• type: H2 - Master's Degree (Two Years)
• subject: Technology and Engineering; Biology and Life Sciences
• keywords: Applied microbiology, value-added chemicals, rhodosporidium toruloides, nitrogen-induced promoter strategy
• language: English
• id: 9103661
• date added to LUP: 2022-12-06 09:39:54
• date last changed: 2022-12-06 09:39:54