Heterologous production of redox-active fungal polyketides in Yarrowia lipolytica for sustainable energy storage
Year: 2026
Type: dissertation
Abstract: The increase in global energy demands requires a transition towards sustainable energy production and storage.Redox flow batteries (RFBs) with bioquinone electrolytes are a promising grid-scale energy storage solution.Bioquinones are synthesized by fungi, bacteria, and plants, conferring survival advantages, and notably their ability to participate in reversible redox reactions makes them attractive electrolyte candidates.In particular, fungal polyketide-derived quinones have shown the most promise based on molecular simulations of their standard reduction potentials.Biosynthesis of quinones using native filamentous fungi, however, is constrained by the regulatory intricacy of fungal biosynthetic gene clusters (BGCs), fungal morphology in submerged cultures, and limited genetic tractability.This dissertation aims to establish a heterologous production platform for fungal redox-active polyketides with potential application in sustainable energy storage.The heterologous organism of choice is the oleaginous yeast Yarrowia lipolytica, which, as a prolific lipid producer, has a rich pool of the polyketide building blocks acetyl-CoA and malonyl-CoA.Y. lipolytica has previously been used for the biosynthesis of plant-derived polyketides.To extend its application to fungal polyketides, a markerless CRISPR-Cas9 genome editing toolkit was adapted for reconstruction of select BGCs.6-Methyl salicylic acid (6-MSA), structurally one of the simplest polyketide products and a central intermediate in several quinone pathways, and the complex quinone bostrycoidin, were synthesized at titers of 403 mg/L and 35 mg/L, respectively, as a first demonstration of fungal polyketide expression in Y. lipolytica.For reconstruction of larger BGCs, polycistronic gene expression was explored as an alternative to the multiple-vector/multiple-promoter strategies.Seven 2A peptides were evaluated in Y. lipolytica, based on their efficiency to mediate translation of discrete proteins from a single open reading frame, using fluorescent reporter proteins in bicistronic constructs.Three of the 2A peptides were implemented for the first time in this organism.Subsequently, the PNPV2A1 peptide was used to produce m-cresol, a 6-MSA-derived compound, by expressing the responsible genes within a bicistronic cassette.The titers of 306 mg/L m-cresol produced by the bicistronic strain were only 25% lower than those of the monocistronic strain.In exchange, this polycistronic strategy enables integration of at least 2 genes per characterized genomic locus and reduces the number of Y. lipolytica transformation steps, thereby streamlining the BGC reconstruction process.To demonstrate the biotechnological potential in the context of sustainable energy iii Abstract storage, toluquinone and gentisyl quinone were selected for heterologous biosynthesis in Y. lipolytica and as a proof-of-concept their electrochemical properties were evaluated.The BGC of these 6-MSA-derived metabolites was identified in Aspergillus hancockii and reconstituted in Y. lipolytica.Since two of the BGC-encoded enzymes are CYP450 monooxygenases, efficient pathway reconstruction required co-expression of the native A. hancockii redox partner, highlighting the complexity of fungal pathway expression in heterologous hosts.Further, structural-and orthology-guided refinement was required to restore functionality to a misannotated CYP450 coding sequence.These pathway optimization strategies enabled production of 1.1 g/L toluquinol and 1.8 g/L gentisyl alcohol, which are the reduced forms of toluquinone and gentisyl quinone, respectively.Achieving these titers in shake flasks further demonstrates the metabolic compatibility of Y. lipolytica and the reconstructed biosynthetic pathway.Electrochemical characterization by cyclic voltammetry confirmed that the standard reduction potentials of heterologously produced polyketides are consistent with previous molecular simulations, supporting their suitability as candidate electrolytes.Toluquinol and gentisyl alcohol displayed quasi-reversible electron-transfer behavior, which might require additional mitigation strategies for battery implementation due to reduced energy and round-trip efficiency.However, with their positive half-wave potentials, toluquinol and gentisyl alcohol expand the relatively limited pool of bio-based positive electrolytes.Altogether, this dissertation establishes Y. lipolytica as a heterologous platform for fungal quinone biosynthesis and expands the synthetic biology toolbox required for their scalable production.The findings position microbially produced quinones as viable bio-based electrolyte candidates in RFB systems.Through the reconstitution of complex biosynthetic pathways and the production of redox-active compounds at gram-per-liter titers, the research presented in this thesis advances sustainable energy storage through microbiology and applied biotechnology.iv Dansk resuméStigningen i det globale energibehov kræver en omstilling til bæredygtig energiproduktion og lagring.Redox flow-batterier (RFB'er) med bioquinoner som elektrolytter er en lovende løsning til energilagring.Bioquinoner syntetiseres af svampe, bakterier og planter og bidrager til organismernes overlevelse.Især deres evne til at deltage i reversible redoxreaktioner gør dem til attraktive elektrolytkandidater. Særligt polyketidquinoner fra svampe har vist stort potentiale baseret på molekylære simuleringer af deres standardreduktionspotentialer.Biosyntese af quinoner i naturlige filamentøse svampe er imidlertid begrænset af den regulatoriske kompleksitet i deres biosyntetiske genklynger (BGC'er), deres morfologi i flydende kultivering samt begrænset genetisk manipulerbarhed.Denne afhandling har til formål at etablere en heterolog produktionsplatform for redoxaktive polyketider fra svampe med potentiel anvendelse inden for bæredygtig energilagring.Den valgte heterologe organisme er den olierige gær Yarrowia lipolytica, som er en effektiv lipidproducent, og har derfor en rig pulje af polyketid-byggestenene acetyl-CoA og malonyl-CoA.Y. lipolytica er tidligere blevet anvendt til biosyntese af polyketider fra planter.For at udvide organismens anvendelse til svampepolyketider blev et CRISPR-Cas9-genomredigeringsværktøjssæt tilpasset til rekonstruktion af udvalgte BGC'er.6-Methylsalicylsyre (6-MSA), strukturelt et af de simpleste polyketidprodukter og et centralt intermediært stof i flere quinon-biosynteseveje, samt det komplekse quinon bostrycoidin, blev produceret i udbytter på henholdsvis 403 mg/L og 35 mg/L som en første demonstration af produktion af polyketider fra svampe i Y. lipolytica.Til rekonstruktion af større BGC'er blev polycistronisk genekspression undersøgt som alternativ til strategier baseret på multiple vektorer og promotorer.Syv 2A-peptider blev undersøgt i Y. lipolytica med hensyn til deres effektivitet i at mediere translation af separate proteiner fra én enkelt åben læseramme ved anvendelse af fluorescerende reporterproteiner i bicistroniske konstruktioner.Tre af 2A-peptiderne blev evalueret i denne organisme for første gang.Efterfølgende blev PNPV2A1-peptidet brugt til produktion af m-cresol, en metabolit dannet ud fra 6-MSA, ved ekspression af de ansvarlige gener i en bicistronisk kassette.Udbyttet på 306 mg/L m-cresol produceret af den bicistroniske stamme var kun 25% lavere end for den monocistroniske stamme.Til gengæld muliggør denne polycistroniske strategi integration af mindst to gener per karakteriseret genomisk locus og reducerer antallet af transformationstrin i Y. lipolytica, hvilket dermed effektiviserer rekonstruktionen af BGC'er.For at demonstrere det bioteknologiske potentiale til bæredygtig energilagring blev v Resumé toluquinon og gentisylquinon udvalgt til heterolog produktion i Y. lipolytica, og deres elektrokemiske egenskaber blev evalueret.BGC'en for disse 6-MSA-baserede metabolitter blev identificeret i Aspergillus hancockii og rekonstitueret i Y. lipolytica.Da to af de BGC-kodede enzymer er CYP450-monooxygenaser, krævede de yderligere co-ekspression af den native redoxpartner fra A. hancockii.Dette fremhæver kompleksiteten ved ekspression af svampebiosynteseveje i heterologe værter.Derudover var strukturel og ortologibaseret forfinelse nødvendig for at reetablere funktionaliteten af en fejlannoteret CYP450-kodende sekvens.Disse optimeringsstrategier muliggjorde produktion af 1,1 g/L toluquinol og 1,8 g/L gentisylalkohol, som er de reducerede former af henholdsvis toluquinon og gentisylquinon.Opnåelsen af disse udbytter i rystekolber demonstrerer yderligere den metaboliske kompatibilitet mellem Y. lipolytica og den rekonstruerede biosyntesevej.Elektrokemisk karakterisering ved cyklisk voltammetri bekræftede, at standardreduktionspotentialerne for heterologt producerede quinoner er i overensstemmelse med tidligere molekylære simuleringer og understøtter deres egnethed som elektrolytkandidater. Toluquinol og gentisylalkohol udviste kvasi-reversibel elektronoverførsel, hvilket kan kræve yderligere evaluering ved anvendelse i batterisystemer på grund af reduceret energi-og cyklisk effektivitet.Med deres positive halvbølgepotentialer udvider toluquinol og gentisylalkohol den relativt begrænsede pulje af biobaserede positive elektrolytter.Samlet set etablerer denne afhandling Y. lipolytica som en heterolog platform for biosyntese af quinoner fra svampe og udvider de syntetisk-biologiske værktøjer, der er nødvendige for skalerbar produktion.Resultaterne positionerer mikrobielt producerede quinoner som levedygtige biobaserede elektrolytkandidater i RFB-systemer.Gennem rekonstruktion af komplekse biosynteseveje og produktion af redoxaktive stoffer i gram-per-liter-skala bidrager det præsenterede arbejde til udviklingen af bæredygtig energilagring baseret på mikrobiologi og anvendt bioteknologi.
Author: Mihaela Bejenari
Language: English
Subfield: Pharmacology
Field: Medicine
Domain: Health Sciences
Open Access status: gold
Funder: Novo Nordisk
OpenAlex ID: W7164843299