Saccharomyces cerevisiae engineered for xylan utilisation

Thesis (PhD)--Stellenbosch University, 2016.ENGLISH ABSTRACT: Alternative fuels or sources of energy need to be generated to complement or replace fossil based fuels since the growing global energy demand will soon exceed the fossil fuel supply. Brazil and the USA are currently the world’s largest producers of bioethanol using sugar cane and corn starch as feedstock, respectively. However, financial feasibility of bioethanol technologies can only be attained when all of the fermentable carbon in plant biomass is converted to bioethanol. Cellulosic ethanol production has received much attention in the past decades due to the high cellulose composition of plant biomass. Yet xylan also represents a major component of lignocellulosic biomass that can be utilised for the cost-effective production of bioethanol. In this study, a recombinant xylan-utilising Saccharomyces cerevisiae strain was engineered by co-expression of the xylanase (xyn2) of Trichoderma reesei, the xylosidase (xlnD) of Aspergillus niger and the codon-optimised Bacteroides thetaiotaomicron xylose isomerase (xylA) genes. The addition of the Scheffersomyces stipitis xylulose kinase (xyl3) gene and the disruption of the native aldose reductase (GRE3) gene increased the carbon flux through the pentose phosphate pathway and minimised carbon loss due to the production of xylitol, respectively. Cultivation on xylose as sole carbohydrate source under oxygen-limitation resulted in the recombinant strain producing ethanol from xylose at a maximum theoretical yield of ~90%, while displaying a complete respiratory mode under aerobic conditions. An increase in biomass was observed that coincided with an increase in enzyme activity. Furthermore, strain adaptation on xylose resulted in a strain with an improved xylose conversion rate. A stable diploid S. cerevisiae strain overexpressing the B. thetaiotaomicron xylose isomerase encoding gene (xylA) and the S. stipitis xylulose kinase (xyl3) gene was constructed. The strain was used to compile metabolomics data at different time points when cultivated aerobically on xylose and glucose as respective sole carbohydrate sources. Cultivation on glucose resulted in a typical diauxic growth pattern on glucose and the production of ethanol due to the Crabtree effect. The UDP-D-glucose levels were approximately eight times higher with cultivation on xylose compared to glucose, indicating that the carbon is channeled towards biomass production. Glycerol was produced in response to ethanol and acetic acid toxicity and was substantially less with cultivation on xylose. Xylitol still accumulated despite the disruption of the GRE3 gene, which suggests the presence of additional non-specific aldose reductases. The concentration of phosphoenol pyruvate (PEP) was much lower with cultivation on xylose throughout the study, indicating that xylose does not induce the expression of pyruvate kinase (PYK1), which negatively affects the flux through the rest of glycolysis. The levels of fructose 1,6-bisphosphate (F1,6P) (an important modulator of the mitochondrial unspecific channel), was significantly lower with cultivation on xylose and contributed to the incomplete carbon catabolite response. Cultivation on xylose resulted in an increase in the pool size of the metabolites of the pentose phosphate pathway (PPP). The accumulation of sedoheptulose 7-phosphate suggests that the TAL1 enzyme is probably the rate-limiting enzyme activity of the PPP. This study is one of only a few that demonstrates xylose and xylan utilisation by a recombinant S. cerevisiae strain.AFRIKAANSE OPSOMMING: Alternatiewe brandstowwe of bronne van energie moet geskep word om fossielbrandstowwe te vervang, aangesien die groeiende globale energie-aanvraag binnekort die produksie van fossielbrandstowwe sal oorskry. Brasilië en die VSA is tans die wêreld se grootste bio-etanolprodusente en gebruik onderskeidelik suikerriet en mielie as voerstowwe. Die finansiële haalbaarheid van bio-etanoltegnologieë is slegs moontlik indien al die fermenteerbare koolstof in plantbiomassa na etanol omgeskakel word. Sellulolitiese etanolproduksie het in die afgelope dekades baie aandag ontvang as weens die hoë selluloseinhoud van plantbiomassa. Tog verteenwoordig xilaan ook 'n belangrike komponent van lignosellulose-agtige biomassa wat vir die koste-effektiewe produksie van bio-etanol gebruik kan word. In hierdie studie is 'n rekombinante xilaanbenuttende Saccharomyces cerevisiae-ras ontwerp deur gesamentlike uitdrukking van die Trichoderma reesei xilanase (xyn2), die Aspergillus niger xilosidase (xlnD) en die kodon-geoptimiseerde Bacteroides thetaiotaomicron xilose isomerase (xylA) gene. Die byvoeging van die Scheffersomyces stipitis xilulose kinase (xyl3)- geen en die ontwrigting van die natuurlike aldose reduktase (GRE3)-geen het onderskeidelik die koolstofvloei deur die pentosefosfaatweg verhoog en koolstofverlies weens die produksie van xylitol beperk. Die kweking op xilose as die enigste koolhidraatbron onder suurstofbeperkte toestande, het daartoe gelei dat die rekombinante ras etanol uit xilose teen 'n maksimum teoretiese opbrengs van ~90% vervaardig het, terwyl dit ‘n volledige respiratoriese metabolisme onder aërobiese toestande vertoon het. 'n Toename in biomassa is waargeneem wat met 'n toename in verder ensiemaktiwiteit ooreengestem het. Ras-aanpassing op xilose het verder tot 'n ras met 'n verbeterde xilosebenuttingstempo gelei. ‘n Stabiele diploïede S. cerevisiae ras is ontwerp waarin die B. thetaiotaomicron xilose isomerase (xylA)- en die S. stipitis xilulose kinase (xyl3)-gene oor-uitgedruk is. Die ras is vir die opstel van metaboloomdata op verskillende tydintervalle gebruik tydens die aërobiese kweking op onderskeidelik xilose en glukose as koolhidraatbronne. Kweking op glukose het tot 'n tipiese di-oukse groeipatroon en die produksie van etanol weens die Crabtree-effek gelei. Die UDF-D-glukosevlakke was ongeveer agt keer hoër met kweking op xilose in vergelyking met glucose, wat aandui dat die koolstof na biomassaproduksie gekanaliseer word. Gliserol was in reaksie op etanol- en asynsuurtoksisiteit geproduseer en aansienlik minder met kweking op xilose. Xilitol het steeds opgehoop ten spyte van die ontwrigting van die GRE3-geen, wat dui op die teenwoordigheid van nog nie-spesifieke aldose reduktase-gene. Die konsentrasie van fosfoenolpirovaat (FEP) was deurgaans baie laer met kweking op xylose, wat daarop dui dat xilose nie die produksie van pirovaat kinase (PYK1) induseer nie, wat die vloei deur die res van glikolise negatiewe beinvloed. Die vlakke van fruktose 1,6-bisfosfaat (F1,6P) ('n belangrike moduleerder van die mitochondriale nie-spesifieke kanaal), was aansienlik laer met kweking op xilose en het tot die onvolledige koolstofkatabolietreaksie bygedra. Kweking op xilose het 'n toename in die poelgroottes van metaboliete van die pentosefosfaatweg (PPP) meegebring. Die opeenhoping van sedoheptulose 7-fosfaat dui daarop dat die TAL1-ensiem waarskynlik die tempobeperkende ensiemaktiwiteit van die PPP is. Hierdie studie is een van net 'n paar wat xilose- en xilaanbenutting deur 'n rekombinante S. cerevisiae ras demonstreer.Doctora