Heterologous expression of cellulase genes in natural Saccharomyces cerevisiae strains

Davison, Steffi Angela (2016-03)

Thesis (MSc)--Stellenbosch University, 2016.

Thesis

ENGLISH ABSTRACT: The yeast Saccharomyces cerevisiae is regularly chosen for the heterologous production of industrial and medically relevant proteins, due to its rapid growth rate, high cell density fermentation capabilities, microbial safety and eukaryotic post-translational processing. Identifying strains with superior secretion and production of recombinant proteins, whether for pharmaceutical, agricultural or industrial processes, has the benefit of lowering production costs. This holds true for second generation (2G) cellulosic bioethanol production, where high titers of key cellulolytic enzymes are needed to break down complex lignocellulosic substrates. While several secretion-enhancing strategies have been attempted in heterologous production hosts, these strategies were limited by bottlenecks in the secretory pathway. Although protein characteristics and host restrictions are likely to contribute to these bottlenecks, these limitations are poorly understood. Exploiting naturally occurring yeast variants has shown great potential to identifying strains with varying fermentation profiles and tolerance to industrial stresses. The same variation is expected in the secreted and total heterologous cellulolytic activity levels between natural S. cerevisiae strains. Many natural yeast strains may not be suitable for direct industrial fermentation, however industrially relevant traits could be transferred to industrial strains, thereby creating a novel yeast strains with extra beneficial features. In this study, the potential of natural S. cerevisiae strains with regards to superior cellulolytic activity levels, robustness and other ideal characteristics for 2G cellulosic bioethanol production were evaluated. Preliminary screening of thirty natural strains for the production of Saccharomycopsis fibuligera Cel3A (S.f.Cel3A) activity demonstrated variation in secreted cellulase activity levels, allowing us to select seven strains with promising phenotypes. After cellulase genes were expressed on episomal and delta integrative plasmids in S .cerevisiae strains, the secreted activity yields of episomally produced Trichoderma reesei Cel5A (T.r.Cel5A) and Talaromyces emersonii Cel7A (T.e.Cel7A) were 3.5- and 3.7-fold higher in natural strain YI13 compared to reference strain S288c. However, no single strain had highest secreted activity for all three enzymes, suggesting cell specific activity levels is dependent on the genetic background of the host and properties of the protein. Nevertheless, YI13 was identified to be highly tolerant to secretion and cell wall stresses (predicted to result in higher cell specific activities). After evaluating other industrially relevant characteristics including growth vigour, fermentation vigour and tolerance to industrial stressors, natural strains were identified to have promising features for 2G cellulosic ethanol production. Variation in the fermentative (YP-glucose and Avicel cellulose) profiles of S. cerevisiae strains are observed, with the natural strains producing similar titers of ethanol (9.0 g/L) compared with the benchmark MH1000 strain in YP-glucose fermentation conditions. Multi-tolerance traits to industrial stresses were demonstrated by the YI13 strain including high ethanol tolerance (10% w/v), high temperature tolerance (37oC and 40oC), and tolerance to a cocktail of inhibitory compounds found in lignocellulosic hydrolysates, suggesting that this strain has a balance between an effective secretion pathway and robustness to withstand environmental conditions. These strains are a significant step toward creating an efficient cellulase secreting yeast for 2G bioethanol production.

AFRIKAANSE OPSOMMING: Die gis Saccharomyces cerevisiae word dikwels vir die heteroloë produksie van industriële en medies-toepaslike proteïene gebruik, weens sy vinnige groeitempo, hoë seldigtheid, fermentatiewe vermoëns, mikrobiese veiligheid en eukariotiese na-transleringsprosessering. Die identifisering van stamme met buitengewoon goeie sekresie en produksie van rekombinante proteïene, hetsy vir farmaseutiese, landbou of industriële prosesse, kan baie voordelig wees vir die vermindering van produksiekoste. Dit geld ook vir die produksie van tweede generasie (2G) sellulolitiese bio-etanol, waar 'n groot hoeveelheid sellulolitiese ensieme benodig word om komplekse sellulosesubstrate af te breek. Alhoewel daar reeds verskeie strategieë gebruik is om die sekresievermoë van heteroloë produseerders te verbeter, was hierdie strategieë beperk deur bottelnekke wat in die sekresie pad van die gasheerselle voorgekom het. Die beperkings van hierdie gasheerselle en die proteïeneienskappe wat bydra tot die knelpunte, word swak verstaan. Studies van gisvariante wat natuurlik voorkom het getoon dat daar groot potensiaal in die identifisering van stamme met wisselende fermentasie profiele en verdraagsaamheid vir industriële drukke lê. Dieselfde variasie word in die uitgeskeide en totale heteroloë sellulolitiese aktiwiteitsvlakke tussen natuurlike S. cerevisiae stamme verwag. Baie natuurlike gisstamme is geskik vir direkte industriële fermentasie, maar in die industrie kan betrokke eienskappe ook aan industriële stamme oorgedra word, om sodoende ‘n gisras te skep met ekstra voordelige funksies. In hierdie studie is die potensiaal van natuurlike S. cerevisiae stamme met betrekking tot beter sellulolitiese aktiwiteitsvlakke, robuustheid en ander ideale eienskappe van die 2G sellulosiese bio-etanol produksie geëvalueer. Voorlopige ondersoeke van dertig natuurlike stamme se produksie van Saccharomycopsis fibuligera Cel3A aktiwiteit het gedemonstreer dat daar ‘n variasie is in die afskeiding van sellulase-aktiwiteitsvlakke, wat ons toegelaat het om sewe van die stamme met belowende fenotipes te identifiseer. Nadat drie sellulasegene op episomale en delta geïntegreerde plasmiede in S. cerevisiae stamme uitgedruk was, was die uitgeskeide opbrengste van episomaal-geproduseerde Trichoderma reesei Cel5A en Talaromyces emersonii Cel7A aktiwiteite van die natuurlike ras YI13 onderskeidelik 3.5 - en 3.7 - keer hoër in aktiwiteit as die verwysingsras S288c. Wanneer die hoogste uitskeidingsaktiwiteit van die drie gene in verskillende stamme egter vergelyk word, het nie een van die stamme uitgestaan vir die hoogste sekresieproduksie van al die ensieme nie. Dit dui daarop dat selspesifieke aktiwiteits vlakke afhanklik van die genetiese agtergrond van die gasheersel en die eienskappe van die proteïne was. Nietemin, is YI13 as hoogs verdraagsaam vir sekresie en selwandspanning geidentifiseer. Na die evaluering van ander industrieel-relevante eienskappe, insluitend groeikrag, fermentasiekrag, en verdraagsaamheid van industriële stressors is daar getoon dat verskeie natuurlike stamme belowende eienskappe het vir die produksie van 2G sellulolitiese etanol. Variasies is in die fermentatiewe (YP-glukose en Avicel sellulose) profiele van S. cerevisiae stamme waargeneem, waar die natuurlike stamme soortgelyke hoeveelhede etanol (9.0 g/L) vervaardiging het, wat ooreenstem met die fermentasiekondisies van die verwysingsras MH1000 in YP-glukose. Multi-verdraagsaamheid eienskappe in industriële stamme is vir YI13 geïdentifiseer, waaronder hoë etanol verdraagsaamheid (10% w/v), temperatuur (37oC en 40oC), en die verdraagsaamheid van 'n mengsel van inhiberende verbindings gevind in sellulose hidrosilate ingesluit was. Dit dui daarop dat hierdie stam 'n balans het tussen 'n effektiewe afskeidingspad en duursaamheid om omgewingstoestande te weerstaan. Hierdie stamme verteenwoordig ‘n belangrike stap in die skep van 'n doeltreffende gis wat sellulases afskei vir produksie van 2G bio-etanol.

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