Expression of novel amylases in Saccharomyces cerevisiae for the efficient conversion of raw starch to bioethanol

Cripwell, Rosemary Anne (2017-03)

Thesis (PhD)--Stellenbosch University, 2017.

Thesis

ENGLISH ABSTRACT: Starchy biomass is an ideal, abundant substrate for bioethanol production. The cost effective conversion of starch requires a fermenting yeast that is able to produce starch hydrolysing enzymes and ferment glucose to ethanol in one step called consolidated bioprocessing (CBP). Despite the advantages, CBP yeasts have not yet been employed for the industrial processing of raw starch during bioethanol production. Molecular biology has enabled the optimised expression of synthetically produced genes in Saccharomyces cerevisiae. The Aspergillus tubingensis raw starch hydrolysing α-amylase (amyA) and glucoamylase (glaA) encoding genes were codon optimised using different strategies and expressed in S. cerevisiae Y294. However, compared to the native coding sequences for the amyA and glaA genes, adapted synonymous codon usage resulted in a decrease in extracellular enzyme activity of 72% (30 nkat.ml-1) and 69% (4 nkat.ml-1), respectively. Additional fungal amylase encoding genes (native and codon optimised) were expressed in S. cerevisiae Y294 and then screened for starch hydrolysis. Subsequently, S. cerevisiae Y294 laboratory strains were constructed to co-express the best α-amylase and glucoamylase gene variants and evaluated for raw starch fermentation. During raw starch fermentations, the S. cerevisiae Y294[TemG_Opt-TemA_Nat] strain displayed the highest carbon conversion (based on the percentage starch converted on a mol carbon basis) of 85%, compared to 54% displayed by the S. cerevisiae Y294[AmyA-GlaA] benchmark strain. Therefore, the native α-amylase (temA_Nat) and codon optimised glucoamylase (temG_Opt) genes, both originating from Talaromyces emersonii, presented the best amylase combination and were selected for further evaluation. Amylolytic S. cerevisiae Ethanol Red™ and M2n industrial strains were constructed using the amdS marker (encoding for acetamidase). Strains co-expressing the temA_Nat and temG_Opt genes were selected for growth on acetamide as the sole nitrogen source. Amylolytic S. cerevisiae strains (Ethanol Red T12 and M2n T1) were compared in a CBP process (20% raw corn starch) at 30°C and 37°C. The maximum ethanol concentration produced at 30°C by the S. cerevisiae Ethanol Red T12 and M2n T1 strains was 86.5 g.l-1 and 99.4 g.l-1, respectively. Fermentations were supplemented with different dosages of STARGEN 002™, an exogenous GSHE (granular starch hydrolysing enzyme) cocktail, to compare the amylolytic yeast strains to an industrial simultaneous saccharification and fermentation (SSF) process. Fermentation results for the S. cerevisiae Ethanol Red T12 strain with 10% of the recommended STARGEN™ dosage compared well with the SSF using S. cerevisiae Ethanol Red™ containing the full recommended STARGEN™ dosage, both having carbon conversions of 50% after 48 hours and 93% after 192 hours. This study also highlights the application of novel industrial amylolytic yeasts in combination with STARGEN™ for decreased fermentations times. At 37°C, the amylolytic S. cerevisiae Ethanol Red T12 strain performed better than the S. cerevisiae M2n T1 strain, demonstrating its potential as a drop-in CBP yeast for existing bioethanol plants that use cold hydrolysis processes. The study also provided a novel enzyme combination (TemA_Nat and TemG_Opt) that efficiently hydrolyses raw corn starch. Finally, new light was shed on the importance of synonymous codon usage and the expression of native genes versus their codon optimised variants.

AFRIKAANSE OPSOMMING: Styselagtige biomassa is 'n ideale, volop substraat vir bio-etanol produksie. Die kosteeffektiewe omskakeling van stysel vereis 'n fermenterende gis wat styselafbrekende ensieme produseer en glukose na etanol in een stap omskakel, bekend as gekonsolideerde bioprosessering (GBP). Ten spyte van die voordele, word GBP-giste nog nie vir die industriële verwerking van rou stysel na bio-etanol gebruik nie. Molekulêre biologie het die optimale uitdrukking van sinteties-vervaardigde gene in Saccharomyces cerevisiae moontlik gemaak. Die kodonvolgorde van Aspergillus tubingensis gene wat vir die rou stysel hidroliserende α-amilase (amyA) en glukoamilase (glaA) kodeer, is met verskillende strategieë geoptimiseer en in S. cerevisiae uitgedruk. In vergelyking met die inheemse volgorde van die amyA en glaA gene, het aangepaste sinonieme kodongebruik egter onderskeidelik tot 'n afname van 72% (30 nkat.ml-1) en 69% (4 nkat.ml-1) in ekstrasellulêre ensiemaktiwiteit gelei. Addisionele fungi amilase-koderende gene (inheems en kodon-geoptimiseerd) is in S. cerevisiae Y294 uitgedruk en dan vir rou stysel hidrolise getoets. Die S. cerevisiae Y294 laboratoriumstamme wat die beste α-amilase en glukoamilase geenvariante gesamentlik uitdruk, is vervolgens geskep en vir rou stysel fermentasie geëvalueer. Tydens rou stysel fermentasies, het die S. cerevisiae Y294[TemG_Opt-TemA_Nat] gistam die hoogste rou stysel omskakeling getoon met 'n koolstof omskakeling van 85%, in vergelyking met 54% deur die S. cerevisiae Y294[AmyA-GlaA] verwysingstam. Die inheemse α-amilase (temA_Nat) en kodon-geoptimiseerde glukoamilase (temG_Opt) gene, beide van Talaromyces emersonii afkomstig, het die beste amilase kombinasie gelewer en is derhalwe vir verdere evaluering gekies. Amilolitiese S. cerevisiae Ethanol Red™ en M2n industriële stamme is ontwikkel met behulp van die amds merker (kodeer vir asetamidase). Stamme wat die temA_Nat en temG_Opt gene gesamentlik uitdruk, is op asetamied as enigste stikstofbron geselekeer. Amilolitiese S. cerevisiae stamme (Ethanol Red T12 en M2n T1) is in 'n GBP-proses (20% rou mieliestysel) by 30°C en 37°C vergelyk. Die maksimum etanolkonsentrasie deur die S. cerevisiae Ethanol Red T12 en M2n T1 stamme gelewer by 30°C, was onderskeidelik 86.5 g.l-1 en 99.4 g.l-1 . Fermentasies is met verskillende ladings van STARGEN 002™, 'n eksogene styselkorrel hidrolitiese ensiem-mengsel, aangevul ten einde die amilolitiese gisrasse in ‘n industriële gelyktydige versuikering en fermentasie (GVF) proses te vergelyk. Fermentasie resultate vir die S. cerevisiae Ethanol Red T12 stam met 10% van die aanbevole STARGEN™-lading het goed vergelyk met die S. cerevisiae Ethanol Red™ GVF met die volle aanbevole STARGEN™-lading. All twee het koolstof omskakelings van 50% na 48 uur en 93% na 192 ure. Hierdie studie beklemtoon ook die toepassing van unieke industriële amilolitiese giste in kombinasie met STARGEN™ vir verbeterde versuikering en fermentasie van rou mieliestysel. Die stysel-afbrekende S. cerevisiae Ethanol Red T12 gisras het by 37°C beter as die S. cerevisiae M2n T1 ras gedoen, wat sy potensiaal uitlig as 'n GBP-gis vir toevoeging tot bestaande bio-etanol fabrieke wat koue hidrolise-prosesse gebruik. Die studie het ook 'n unieke ensiemkombinasie (TemA_Nat en TemG_Opt) gelewer wat rou mieliestysel doeltreffend hidroliseer. Laastens is nuwe lig gewerp op die belang van sinonieme kodongebruik en die uitdrukking van inheemse gene teenoor kodon-geoptimiseerde variante.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/101363
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