Towards Bioproduction of Yeast-Based Odd-Chain Fatty Acids
Doktorsavhandling, 2026
Odd-chain fatty acids (OCFAs) are high-value lipids with emerging medical and nutritional applications. However, their natural abundance is low, and conventional extraction or chemical synthesis methods are inefficient or unsustainable. Microbial fermentation using oleaginous yeasts offers a promising alternative, particularly when employing short-chain fatty acids digestates rich in propionic acid, the main precursor for OCFA synthesis.
In this thesis work yeast-based OCFA production has been explored by improving quantitative analytical methods, identifying suitable yeast cell factories, characterizing their performance, and initiating strain engineering efforts. First, a fatty acid methyl ester protocol was optimized for robust OCFA analysis. Second, eight strains capable of growth on 15 g/L propionic acid and production of OCFA were identified in a screening of nineteen oleaginous yeasts. Third, four yeast species were selected for further study: Cutaneotrichosporon oleaginosus for its high OCFA titers, Rhodotorula toruloides for its high OCFA yield, Yarrowia lipolytica for its high tolerance to propionic acid, and Blastobotrys adeninivorans for its ability to produce OCFAs, grow well in propionic acid and as a relatively underexplored yeast. Cultivation on mixtures of acetic and propionic acids at different carbon-to-nitrogen ratios revealed distinct and interesting performance characteristics for OCFA production in each yeast species. Fourth, a plasmid-based CRISPR-Cas9 metabolic engineering platform was established for B. adeninivorans through the identification of native autonomous replication systems, enabling the generation of mutants with enhanced OCFA content.
Overall, this work advances methodologies for fatty acid analysis, identifies promising oleaginous yeasts for OCFA production and characterize them, and establishes a new genetic engineering tool for strain development. These results provide a foundation for the development of efficient yeast cell factories and economically viable bioprocesses for sustainable OCFA production.
In this thesis work yeast-based OCFA production has been explored by improving quantitative analytical methods, identifying suitable yeast cell factories, characterizing their performance, and initiating strain engineering efforts. First, a fatty acid methyl ester protocol was optimized for robust OCFA analysis. Second, eight strains capable of growth on 15 g/L propionic acid and production of OCFA were identified in a screening of nineteen oleaginous yeasts. Third, four yeast species were selected for further study: Cutaneotrichosporon oleaginosus for its high OCFA titers, Rhodotorula toruloides for its high OCFA yield, Yarrowia lipolytica for its high tolerance to propionic acid, and Blastobotrys adeninivorans for its ability to produce OCFAs, grow well in propionic acid and as a relatively underexplored yeast. Cultivation on mixtures of acetic and propionic acids at different carbon-to-nitrogen ratios revealed distinct and interesting performance characteristics for OCFA production in each yeast species. Fourth, a plasmid-based CRISPR-Cas9 metabolic engineering platform was established for B. adeninivorans through the identification of native autonomous replication systems, enabling the generation of mutants with enhanced OCFA content.
Overall, this work advances methodologies for fatty acid analysis, identifies promising oleaginous yeasts for OCFA production and characterize them, and establishes a new genetic engineering tool for strain development. These results provide a foundation for the development of efficient yeast cell factories and economically viable bioprocesses for sustainable OCFA production.
Författare
Veronica Bonzanini
Chalmers, Life sciences, Industriell bioteknik
Bonzanini Veronica*, Palomo Sergio Morales*, Causevic Ariana, Haddad Momeni Majid, Olsson Lisbeth and Cecilia Geijer. Cutaneotrichosporon oleaginosus outperforms other oleaginous yeasts in producing odd-chain fatty acids from acetic and propionic acid.
Alalam Hanna*, Palomo Sergio Morales*, Bonzanini Veronica, Blanco Roldán Manuel Francisco, Causevic Ariana, Olsson Lisbeth, Haddad Momeni Majid and Cecilia Geijer. A plasmid-based CRISPR–Cas9 platform for metabolic engineering in the oleaginous yeast Blastobotrys adeninivorans.
Odd-Chain Fatty Acids (OCFAs) are a valuable type of lipids recently regarded as pro-health compounds which are gaining attention for their emerging medical and nutritional applications. Unfortunately, these fatty acids represent a minor fraction of the total fatty acids present in nature. OCFA extraction from plant or animal sources is inefficient and production through chemical synthesis is not environmentally sound. Instead, microbial fermentation offers a promising sustainable alternative for OCFA production, with oleaginous yeasts standing out as attractive platforms given their capability to accumulate high amounts of intracellular lipids. Short-chain fatty acids, that can be produced from microbial degradation of waste biomasses, can be used in ideal feeding substrates for OCFA production. These provide propionic acid, the main precursor for OCFAs, and represent sustainable and low-cost feedstock. Nevertheless, to attain an economically feasible OCFA bioproduction process, it is essential to develop a robust methodology to analyze yeast-derived OCFAs, identify yeast strains capable of producing OCFAs, characterize the identified yeast and improve the abilities of the characterized yeasts to achieve high amount of OCFAs.
In this thesis work, all of these key steps have been taken and explored. An existing protocol for fatty acid analysis has been carefully improved. A screening performed on nineteen different oleaginous yeast species identified eight yeasts capable of growing on propionic acid. Among the strains evaluated, four yeasts were selected for further characterization based on their distinct traits: Cutaneotrichosporon oleaginosus, Rhodotorula toruloides, Yarrowia lipolytica and Blastobotrys adeninivorans. Further characterization of these yeasts in a mixture of short chain fatty acids allowed us to explore their different growth abilities and OCFA production characteristics. Furthermore, a new tool to modify B. adeninivorans was developed and served to generate mutants with improved OCFA content.
Altogether, this research lays a foundation for further odd-chain fatty acid analysis and strain characterization while providing essential tools for the development of viable yeast cell factories for OCFA production.
In this thesis work, all of these key steps have been taken and explored. An existing protocol for fatty acid analysis has been carefully improved. A screening performed on nineteen different oleaginous yeast species identified eight yeasts capable of growing on propionic acid. Among the strains evaluated, four yeasts were selected for further characterization based on their distinct traits: Cutaneotrichosporon oleaginosus, Rhodotorula toruloides, Yarrowia lipolytica and Blastobotrys adeninivorans. Further characterization of these yeasts in a mixture of short chain fatty acids allowed us to explore their different growth abilities and OCFA production characteristics. Furthermore, a new tool to modify B. adeninivorans was developed and served to generate mutants with improved OCFA content.
Altogether, this research lays a foundation for further odd-chain fatty acid analysis and strain characterization while providing essential tools for the development of viable yeast cell factories for OCFA production.
Infrastruktur
Chalmers infrastruktur för masspektrometri
Ämneskategorier (SSIF 2025)
Mikrobiologi
DOI
10.63959/chalmers.dt/5912
ISBN
978-91-8103-455-4
Doktorsavhandlingar vid Chalmers tekniska högskola. Ny serie: 5912
Utgivare
Chalmers
VIVA
Opponent: Petri-Jaan Lahtvee