Utilisation of wheat bran as a substrate for bioethanol production using recombinant cellulases and amylolytic yeast

Wheat bran, generated from the milling of wheat, represents a promising feedstock for the production of bioethanol. This substrate consists of three main components: starch, hemicellulose and cellulose. The optimal conditions for wheat bran hydrolysis have been determined using a recombinant cellulase cocktail (RCC), which contains two cellobiohydrolases, an endoglucanase and a beta-glucosidase. The 10% (w/v, expressed in terms of dry matter) substrate loading yielded the most glucose, while the 2% loading gave the best hydrolysis efficiency (degree of saccharification) using unmilled wheat bran. The ethanol production of two industrial amylolytic Saccharomyces cerevisiae strains, MEL2[TLG1-SFA1] and M2n [TLG1-SFA1], were compared in a simultaneous saccharification and fermentation (SSF) for 10% wheat bran loading with or without the supplementation of optimised RCC. The recombinant yeasts. cerevisiae MEL2[TLG1-SFA1] and M2n[TLG1-SFA1] completely hydrolysed wheat bran's starch producing similar amounts of ethanol (5.3 +/- 0.14 g/L and 5.0 +/- 0.09 g/L, respectively). Supplementing SSF with RCC resulted in additional ethanol production of about 2.0 g/L. Scanning electron microscopy confirmed the effectiveness of both RCC and engineered amylolytic strains in terms of cellulose and starch depolymerisatio

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

Thesis (PhD)--Stellenbosch University, 2017.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.Doctora

Construction of industrial Saccharomyces cerevisiae strains for the efficient consolidated bioprocessing of raw starch

CITATION: Cripwell, R. A., et al. 2019. Construction of industrial Saccharomyces cerevisiae strains for the efficient consolidated bioprocessing of raw starch. Biotechnology for Biofuels, 12:201, doi:10.1186/s13068-019-1541-5.Background: Consolidated bioprocessing (CBP) combines enzyme production, saccharification and fermentation into a one-step process. This strategy represents a promising alternative for economic ethanol production from starchy biomass with the use of amylolytic industrial yeast strains. Results: Recombinant Saccharomyces cerevisiae Y294 laboratory strains simultaneously expressing an α-amylase and glucoamylase gene were screened to identify the best enzyme combination for raw starch hydrolysis. The codon optimised Talaromyces emersonii glucoamylase encoding gene (temG_Opt) and the native T. emersonii α-amylase encoding gene (temA) were selected for expression in two industrial S. cerevisiae yeast strains, namely Ethanol Red™ (hereafter referred to as the ER) and M2n. Two δ-integration gene cassettes were constructed to allow for the simultaneous multiple integrations of the temG_Opt and temA genes into the yeasts’ genomes. During the fermentation of 200 g l−1 raw corn starch, the amylolytic industrial strains were able to ferment raw corn starch to ethanol in a single step with high ethanol yields. After 192 h at 30 °C, the S. cerevisiae ER T12 and M2n T1 strains (containing integrated temA and temG_Opt gene cassettes) produced 89.35 and 98.13 g l−1 ethanol, respectively, corresponding to estimated carbon conversions of 87 and 94%, respectively. The addition of a commercial granular starch enzyme cocktail in combination with the amylolytic yeast allowed for a 90% reduction in exogenous enzyme dosage, compared to the conventional simultaneous saccharification and fermentation (SSF) control experiment with the parental industrial host strains. Conclusions: A novel amylolytic enzyme combination has been produced by two industrial S. cerevisiae strains. These recombinant strains represent potential drop-in CBP yeast substitutes for the existing conventional and raw starch fermentation processes.https://biotechnologyforbiofuels.biomedcentral.com/articles/10.1186/s13068-019-1541-5Publisher's versio

Developing novel yeast strains for the consolidated bioprocessing of starchy substrates into bioethanol

Bioethanol belongs to the large family of renewable fuels that can be used as alternatives to fossil oil, with hundreds of billions of liters produced in the US and Brazil from maize and sugarcane. However, criticism due to the rise of food prices and food shortage is impeding the global implementation of bioethanol production from crops. Second-generation bioethanol could be made from non-food feedstocks rich in starch and lignocellulose, consisting of food waste or processing residues. The conventional first-generation bioethanol production from starch is a well-established technology, based on a multiple-step process that is hardly sustainable: high energy demand is firstly required to pretreat the material at high temperatures, then expensive commercial enzymes are needed to breakdown the granules in order to obtain free glucose for the final fermentation step. In this scenario, large cost reductions can be achieved through process integration (consolidated bioprocessing, CBP) by using a new amylolytic and fermenting microbe able to directly convert starchy biomass into fuel in a single bioreactor. To date, no natural CBP microorganism has been described. Saccharomyces cerevisiae is the most common fermenting yeast, traditionally used in the food industry. The high fermenting activity, the Generally Regarded As Safe (GRAS) status and industrial robustness are desirable characteristics for large scale bioethanol production. Unfortunately, S. cerevisiae lacks hydrolytic enzymes and cannot use starch as a carbon source. A new S. cerevisiae strain genetically engineered with fungal amylases can significantly contribute to improve the feasibility of granular starch hydrolysis. This study aimed at searching for novel yeast strains with high fermenting abilities on starchy by-products to be used as host strains for the development of new and efficient CBP yeast. A cluster of twenty-one novel S. cerevisiae isolates was screened for their fermenting potential and the industrial Ethanol RedTM was used as the benchmark. The fermenting performances were assessed on starchy substrates, broken rice, and raw corn starch, at high loading (20% w/v) in simultaneous saccharification and fermentation (SSF) set up at 30\ub0 C. The starch hydrolysis was carried out with the commercial amylases cocktail STARGENTM 002. The novel S. cerevisiae L20 strain outperformed the industrial benchmark and was selected as the host strain for the development of novel starch-to-ethanol CBP strains through the engineering of two efficient starch-hydrolyzing genes from Aspergillus tubigensis: the alpha-amylase AmyA and the glucoamylase GlaA. To this aim, both \uf064-integration and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 technologies are being currently applied. The transformants strains obtained so far have been assessed for enzymatic activity, resulting in excellent performances, and will be further employed for fermentation of starchy substrates

Additional glucoamylase genes increase ethanol productivity on rice and potato waste streams by a recombinant amylolytic yeast

: The implementation of consolidated bioprocessing for converting starch to ethanol relies on a robust yeast that produces enough amylases for rapid starch hydrolysis. Furthermore, using low-cost substrates will assist with competitive ethanol prices and support a bioeconomy, especially in developing countries. This paper addresses both challenges with the expression of additional glucoamylase gene copies in an efficient amylolytic strain (Saccharomyces cerevisiae ER T12) derived from the industrial yeast, Ethanol Red™. Recombinant ER T12 was used as a host to increase ethanol productivity during raw starch fermentation; the ER T12.7 variant, selected from various transformants, displayed enhanced raw starch conversion and a 36% higher ethanol concentration than the parental strain after 120 h. Unripe rice, rice bran, potato waste and potato peels were evaluated as alternative starchy substrates to test ER T12.7's fermenting ability. ER T12.7 produced high ethanol yields at significantly improved ethanol productivity, key criteria for its industrial application

Engineering industrial yeast strains for Consolidated Bioprocessing of starchy substrates and by-products to ethanol.

Introduction. Commercial bioethanol is currently obtained from starchy substrates, with a relatively mature technology for corn in the USA. However, starch-to-ethanol processes are still expensive and the development of a Consolidated Bioprocessing (CBP) through amylolytic yeast could considerably reduce commercial costs (van Zyl et al. 2012). This research project aimed to construct efficient amylolytic CBP Saccharomyces cerevisiae strains for the industrial ethanol production from starchy feedstock. Materials and methods. In the last eight years, ten fungal glucoamylase and alpha-amylase genes, native and codon-optimized, were screened in different combinations for their high activity into the laboratory strain S. cerevisiae Y294. The most proficient sequences were \u3b4-integrated into industrial yeast strains (van Zyl et al. 2011; Favaro et al., 2012; Favaro et al. 2015). Results. This report gives an overview of the research outcomes we obtained towards the CBP of starchy materials into ethanol. So far, the most effective raw starch-hydrolyzing combination was found to be the codon-optimized glucoamylase of Thermomyces lanuginosus glucoamylase (TLG1) and \u3b1-amylase of Saccharomycopsis fibuligera (SFA1) and their gene were \u3b4-integrated into the industrial S. cerevisiae strains M2n and MEL2. The resulting recombinant yeast displayed high activities on raw starch (up to 4461 nkat/g dry cell weight) and produced in a bioreactor about 64 g/L ethanol from 200 g/L raw corn starch, corresponding to 55% of the theoretical yield (g of ethanol/g of glucose equivalent). Their starch conversion efficiencies were even higher on sorghum and triticale (62 and 73% of the theoretical yield, respectively). Moreover, both recombinant strains were efficiently used also for the CBP of starchy by-products, such as wheat bran and rice husk, where starch content is about 10-30% of the biomass. Supplementing the CBP with recombinant cellulases was beneficial to hydrolyze also the cellulose content of the agricultural residues, thus increasing the overall ethanol yield. Discussion. This is the first report of CBP from natural starchy substrates and by-products using industrial yeast strains co-secreting glucoamylase and \u3b1-amylase. The high ethanol yields achieved at bioreactor scale pave the way for their large scale CBP applications