Cloning and expression of fungal alpha-amylase genes in Saccharomyces cerevisiae with integrated glucoamylase gene for raw starch conversion into bioethanol

Increasing population numbers and the rapid growth of technology and industry have resulted in an increase in energy demand.Biomass-based fuels (biofuels) have received considerable interest as an alternative transport fuel as biomass is abundant, cheap and renewable.Starch is a good feedstock for bioethanol production with a mature technology established in the USA.However, the current starch-to-ethanol conversion process requires a high energy input and high amylolytic enzyme loadings for liquefaction, resulting in economic challenges.The yeast Saccharomyces cerevisiae is traditionally the preferred host for bioethanol production due to its high ethanol productivity, tolerance and high fermentation capacity, but is unable to utilise or ferment starch.Genetic engineering allows the construction of amylolytic S. cerevisiae strains that can convert starch to glucose and ferment the latter to ethanol.The application of raw starch hydrolysing enzymes could reduce the process time and cost of ethanol production, thus improving its economic feasibility.In this study, a literature and database search was conducted to obtain DNA sequences of genes encoding raw starch hydrolysing amylases.The Aureobasidium pullulans ApuA, Aspergillus terreus AteA, Cryptococcus sp.S-2 CryA and Saccharomycopsis fibuligera SfiA α-amylase encoding genes were synthesised and expressed on an episomal multicopy vector in a S. cerevisiae laboratory strain using the Trichoderma reesei xyn2 secretion signal.The S. cerevisiae Y294[AteA] and Y294[ApuA] strains displayed the highest levels of volumetric activity for the recombinant α-amylases (3.20 U.ml -1 and 2.57 U.ml -1 , respectively) when grown in SC -URA medium.The recombinant AteA and ApuA proteins were glycosylated and displayed pH optima between pH 4 and 5.Both enzymes were stable at 30°C and maintained up to 80% activity after 5 days.The ApuA and AteA genes were co-expressed with the Aspergillus tubingensis GlaA glucoamylase to generate the S. cerevisiae Y294[ApuA-GlaA] and Y294[AteA-GlaA] strains, respectively.When cultivated on 200 g.l -1 raw starch, the Y294[AteA-GlaA] strain produced 43.81 g.l -1 ethanol after 192 hours, which was significantly higher than the Y294[AmyA-GlaA] benchmark strain (41.02 g.l -1 ) and the Y294[ApuA-GlaA] strain (32.83 g.l -1 ).The Y294[AteA-GlaA] strain displayed a maximum yield of 57 g.l -1 ethanol in fermentations supplemented with STARGEN TM 002 (commercial enzyme cocktail), indicating the margin of improvement possible in improving process efficiency.Assessment of the Y294[AteA-GlaA] strain using various optimisation strategies concluded that additional Stellenbosch University https://scholar.sun.ac.zaOPSOMMING Toenemende bevolkingsgetalle en die vinnige groei in tegnologie en nywerheid het 'n toename in energiebehoeftes tot gevolg.Biomassa-gebaseerde brandstof (biobrandstof) geniet groot belangstelling omdat biomassa volop, goedkoop en hernubaar is.Stysel is 'n goeie roumateriaal vir bio-etanolproduksie met volwasse proses tegnologie wat in die VSA gevestig is.Die huidige stysel-tot-etanol omskakelingsproses verg egter 'n hoë energie-inset en hoë amilolitiese ensiemladings vir vervloeiing, wat tot ekonomiese uitdagings lei.Die gis Saccharomyces cerevisiae is tradisioneel die voorkeurgasheer vir bio-etanolproduksie weens sy hoë etanolproduktiwiteit, -verdraagsaamheid en hoë gistingskapasiteit, maar kan nie stysel benut of fermenteer nie.Genetiese manipulasie maak die konstruksie van amilolitiese S. cerevisiae stamme moontlik wat stysel na glukose kan omskakel en laasgenoemde tot etanol kan fermenteer.Die toepassing van rou stysel-hidroliserende ensieme kan die prosesseringtyd en koste van etanolproduksie verminder en gevolglik die ekonomiese lewensvatbaarheid daarvan verbeter.In hierdie studie is literatuur en databasisse deursoek vir DNS-volgordes van gene wat vir rou stysel hidroliserende amilases kodeer.Die geenvolgordes vir die Aureobasidium pullulans ApuA, Aspergillus terreus AteA, Cryptococcus sp.S-2 CryA en Saccharomycopsis fibuligera SfiA α-amilases is gesintetiseer en op 'n episomale multikopievektor in 'n S. cerevisiae laboratoriumstam uitgedruk deur dieTrichoderma reesei xyn2 sekresiesein te gebruik.Die S. cerevisiae Y294 [AteA] en S. cerevisiae Y294 [ApuA] rasse het die hoogste vlakke van volumetriese aktiwiteit vir die rekombinante α-amilases (3.20 U.ml -1 en 2.57 U.ml -1 , onderskeidelik) tydens groei op SC -URA medium getoon.Die rekombinante AteA en ApuA proteïene was versuiker en het pH optima tussen pH 4 en 5 getoon.Beide ensieme was by 30°C stabiel en het tot 80% aktiwiteit na 5 dae behou.Die ApuA en AteA gene is saam met die Aspergillus tubingensis GlaA glukoamilase in S. cerevisiae uitgedruk om onderskeidelik die S. cerevisiae Y294 [ApuA-GlaA] en S. cerevisiae Y294 [AteA-GlaA] rasse te skep.Die S. cerevisiae Y294 [AteA-GlaA] ras het 43.81 g.l -1 etanol vanaf 200 g.l -1 rou stysel na 192 ure geproduseer, wat beduidend meer as die S. cerevisiae Y294[AmyA-GlaA] verwysingsras (41.02 g.l -1 ) en die Y294[ApuA-GlaA] ras (32.83 g.l -1 ) was.Die S. cerevisiae Y294 [AteA-GlaA] ras het 'n maksimum opbrengs van 57 g.l -1 etanol getoon in fermentasies wat met STARGEN TM 002 ('n kommersiële ensiemmengsel) aangevul is, wat die ruimte vir verbetering van die proses-effektiwiteit aandui.Assessering van die