Roskilde University Yeast Cell Factory-Platform for the Screening and the Industrial Production of Flavonoids and other Phenolic Compounds PhD thesis by Beata Joanna Lehka Lehka, Beata Joanna Publication date: 2017 Document Version Publisher's PDF, also known as Version of record Citation for published version (APA): Lehka, B. J. (2017). Yeast Cell Factory-Platform for the Screening and the Industrial Production of Flavonoids and other Phenolic Compounds: PhD thesis by Beata Joanna Lehka. Roskilde Universitet. 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Oct. 2021 Yeast Cell Factory-Platform for the Screening and the Industrial Production of Flavonoids and other Phenolic Compounds PhD THESIS BY BEATA JOANNA LEHKA MAY 2017 Supervisors: Ernesto Simon and Assoc. Prof. Håvard Jenssen Submission date: 31/05/2017 The thesis has been submitted to the Department of Science and Environment, Roskilde University, Denmark i PREFACE AND ACKNOWLEDGEMENTS This thesis presents work done during my PhD study. The work was carried out at a biotech company – EVOLVA – from April 2014 to May 2017 and at Roskilde University – Department of Science and Environment – from June to December 2016. I would like to sincerely thank my supervisors for their inspiration, encouragement and support. Ernesto Simon, thank you for your guidance throughout the whole process, for helping with experimental work and for your enormous patience. Håvard Jenssen, since my master studies you encouraged me to become a PhD student, thank you for your support and believing in me. I would also like to thank Niels Bjerg Jensen, who showed me how to be efficient in the laboratory and supported me during my last months at Evolva. I would like to thank my colleagues from Evolva and RUC for creating an inspiring and fun working environment as well as for the constructive discussions. A special thanks goes to Katherina Garcia Vanegas, Michael Eichenberger and Michael Naesby for collaborating with me. It was a great pleasure working with you! I also would like to thank Bo Svensmark and Steffen Larsen for their help in the mathematical problem solving described in the current thesis. Finally, I want to thank my husband for driving me to the laboratory during weekends and for supporting me during tough times and my parents for motivating me throughout my life. i ABSTRACT Flavonoids are secondary plant metabolites derived from the phenylpropanoid pathway. These bioactive compounds are of great commercial interest due to their varied properties, such as anti- oxidative, anti-tumor and/or antibacterial. However, industrial production of flavonoids based on purification from plants or on their organic synthesis can be problematic. On the other hand, the so called bio-industrial production of a variety of compounds in microorganisms is gaining great popularity. The objective of this study is to produce naringenin and some of its derivatives in Saccharomyces cerevisiae as a model for industrial production of flavonoids. By combining a balanced heterologous expression of (phenylpropanoid) naringenin biosynthetic pathway genes and the optimisation of yeast metabolism we developed a strain producing 430 mg/L of naringenin from glucose. In this set up naringenin was produced from phenylalanine by action of a phenylalanine ammonia lyase (PAL) in a strain overproducing phenylalanine and tyrosine. As tyrosine can also be a precursor for naringenin production via tyrosine ammonia lyase (TAL), we evaluated in vivo the activity of several TAL enzymes in S. cerevisiae. Most of the ammonia lyases have affinity to both phenylalanine and tyrosine, with a strong preference for phenylalanine. Some TALs have been described before but these either have very low overall activity on tyrosine or are also capable of using phenylalanine as substrate. Here we identified a novel TAL, an enzyme from Aeromonas salmonicida with relatively high activity towards tyrosine and no activity towards phenylalanine. Production of flavonoids and stilbenoids in S. cerevisiae is challenging, partially due to carbon loss towards phloretic acid by the action of an unknown endogenous reductase. Through the screening of 26 putative double bond reductase knockout strains we identified Tsc13 as the enzyme responsible for the reduction of p-coumaroyl-CoA to phloretic acid. Since Tsc13 is an essential enzyme, a combination of homology modelling and site-saturation mutagenesis was used to identify residues that would impair its activity towards p-coumaroyl-CoA without compromising cell growth. Several mutations leading to lower phloretic acid production were found and discussed. As an alternative approach we complemented the deletion of the TSC13 gene with homologues from various plants. Genes from Arabidopsis thaliana, Gossypium hirsutum and Malus domestica were able to sustain cell progression while not resulting in p- coumaroyl-CoA reduction to phloretic acid. These findings should be of great value for the industrial production of p-coumaric acid-derived molecules in S. cerevisiae. As naringenin is a precursor for other commercially relevant flavonoids we developed a platform for the production of a library of flavonoid derivatives and screened them for antibacterial properties. Seven different yeast strains producing flavonoids (naringenin, kaempferol, dihydrokaempferol, apigenin and afzelechin), a stilbenoid (resveratrol) and a dihydrochalcone (phloretin) were used as a base for the introduction of a library of decorating enzymes known to be active towards some of these compounds. Cariogenic bacteria were selected as a model for the screening of the combinatorial library for antibacterial potential. Although no compounds with significant antibacterial properties were identified, the method for the construction of flavonoid libraries that is presented here could serve as a base for future screenings. ii DANSK RESUME Flavonoider er sekundære plantemetabolitter derivatiseret fra enten phenylalanin eller tyrosin. Disse bioaktive stoffer har en stor kommerciel interesse grundet deres forskellige egenskaber som fx anti-oxidative, anti-tumor og/eller antibakterielle egenskaber. Industriel produktion af flavonoider, baseret på oprensning fra planter eller organisk synthese, kan dog være problematisk. På den anden side er den såkaldte bioindustrielle produktion af en vifte stoffer vha. mikroorganismer ved at bliver mere og mere populært. Målet med dette studie er at producere naringenin samt nogle af dens derivater i Saccharomyces cerevisiae som model for industriel produktion af flavonoider. Ved at kombinere et balanceret heterologt udtryk af de gener, der er involveret i de biosyntetiske reaktioner ved fremstilling af (phenylpropanoid) naringenin samt en optimering af gærmetabolismen har vi udviklet en stamme, der producerer 430 mg/L naringenin fra glukose. Under disse omstændigheder blev naringenin produceret fra phenylalanin ved hjælp af phenylalanin ammonia lyase (PAL) i en stamme, der overproducerer phenylalanin og tyrosin. Da tyrosin også kan være en forløber for naringenin-produktion via tyrosin ammonia lyase (TAL), har vi undersøgt in vivo aktiviteten af flere TAL enzymer i S. cerevisiae. De fleste ammonia lyaser har affinitet for både phenylalanin og tyrosin, med præference for phenylalanin. Nogle enzymer er tidligere beskrevet som værende TAL enzymer, men de har enten en meget lav aktivitet på tyrosin, eller også er de i stand til at bruge phenylalanin som substrat. Vi har her identificeret et nyt TAL, et enzym fra Aeromonas salmonicida med relativ høj aktivitet mod tyrosin og ingen aktivitet mod phenylalanin. Produktion af flavonoider og stilbenoider i S. cerevisiae er udfordrende, delvist pga. kulstoftab til phloretic syre som resultat af aktiviteten af en ukendt endogen reduktase. Gennem undersøgelser af 26 formodede c-c dobbeltbindingsreduktase knockout stammer, har vi identificeret Tsc13 som det enzym, der er ansvarlig for reduktionen af p-coumaroyl-CoA til phloretic syre. Da Tsc13 er et essentielt enzym blev en kombination af homologi-modellering og site-mættet mutagenese brugt til at identificere aminosyrerester som kan hæmme aktiviteten på p-coumaroyl-CoA uden at kompromittere cellevæksten. Vi har fundet og diskuteret flere mutationer som resulterede i lavere phloretic syre produktion. Som et alternativ har vi komplementeret deletionen af TSC13 genet med homologer fra forskellige planter. Gener fra Arabidopsis thaliana, Gossypium hirsutum og Malus domestica var i stand til at opretholde cellevæksten uden at resultere i p-coumaroyl-CoA reduktion til phloretic syre. Disse resultater kan være af stor værdi for industriel produktion af p-coumaric-syre-fremstillede molekyler i S. cerevisiae. Eftersom naringenin er en forløber for andre kommercielt