Direct evidence that maltose transport activity Is affected by the lipid composition of brewer’s yeast

A brewer’s yeast strain was grown with maltose as sole carbon source under strictly anaerobic conditions with and without ergosterol and /or unsaturated fatty acid (Tween 80) supplements. Under all these conditions the MALx1 genes for maltose transporters were strongly expressed during growth. The fatty acid unsaturation indices of growing and stationary phase yeast were increased from about 20% to 56–69% by supplementation with Tween 80. Ergosterol contents were increased up to at least 4- fold by supplementation with ergosterol and Tween 80. Maltose transport activity measured at 20°C was not increased by supplementation with Tween 80 alone, but was increased 2-fold and 3-fold, respectively, in growing and stationary phase yeast by supplementation with ergosterol together with Tween 80. The stimulation of maltose transport by ergosterol was greater when the transport was measured at temperatures (10°C and 0°C) lower than 20°C. The results show that proper function of maltose transporters requires adequate amounts of ergosterol in the yeast. This effect may partly explain the low maltose (and maltotriose) uptake rates both in the second half of brewery fermentations, when the sterol content of yeast has fallen, and when fresh wort is pitched with sterol-deficient cropped yeast.Finnish brewing and malting industry (PBL) ; Leonardo da Vinci programme ; Fundação para a Ciência e a Tecnologia (FCT

Fermentation of high concentrations of lactose to ethanol by engineered flocculent saccharomyces cerevisiae

The development of microorganims that efficiently ferment lactose has a high biotechnological interest, particularly for cheese whey bioremediation processes with simultaneous bio-ethanol production. The lactose fermentation performance of a recombinant Saccharomyces cerevisiae flocculent strain was evaluated. The yeast consumed rapidly and completely lactose concentrations up to 150 g l-1 in either well- or micro-aerated batch fermentations. The maximum ethanol titre was 8% (v/v) and the highest ethanol productivity was 1.5–2 g l-1 h-1, in micro-aerated fermentations. The results presented here emphasise that this strain is an interesting alternative for the production of ethanol from lactose-based feedstocks.Fundação para a Ciência e a Tecnologia (FCT

Alcoholic fermentation of lactose by engineered flocculent Saccharomyces cerevisiae

The construction of Saccharomyces cerevisiae strains with the ability to ferment lactose has biotechnological interest, particularly for cheese whey fermentation to ethanol. Direct fermentation of whey to ethanol is generally not economically feasible because the low lactose content (ca. 5% w/v) results in low ethanol titre (2 – 3% v/v), making the distillation process too expensive. Concentration of whey lactose (e.g. by ultrafiltration) prior to fermentation is an option to obtain higher ethanol titres. Microbial strains are therefore needed that can efficiently convert high concentrations of lactose into ethanol. We describe here the engineering of a S. cerevisiae strain for efficient lactose fermentation, involving genetic and evolutionary engineering strategies. The evolved strain obtained fermented efficiently lactose concentrations up to 150 g L-1, including 3-fold concentrated cheese whey, producing ethanol titres up to 8% v/v. The strain is highly flocculent, a property that makes it particularly suitable for the development of high cell density fermentation processes

Fermentation of lactose to bio-ethanol by yeasts as part of integrated solutions for the valorisation of cheese whey

Cheese whey, the main dairy by-product, is increasingly recognized as a source of many bioactive valuable compounds. Nevertheless, the most abundant component in whey is lactose (ca. 5% w/v), which represents a significant environmental problem. Due to the large lactose surplus generated, its conversion to bio-ethanol has long been considered as a possible solution for whey bioremediation. In this review, fermentation of lactose to ethanol is discussed, focusing on wild lactose-fermenting yeasts, particularly Kluyveromyces marxianus, and recombinant Saccharomyces cerevisiae strains. The early efforts in the screening and characterization of the fermentation properties of wild lactose-consuming yeasts are reviewed. Furthermore, emphasis is given on the latter advances in engineering S. cerevisiae strains for efficient whey-to-ethanol bioprocesses. Examples of industrial implementation are briefly discussed, illustrating the viability of whey-to-ethanol systems. Current developments on strain engineering together with the growing market for biofuels will likely boost the industrial interest in such processes.Fundação para a Ciência e a Tecnologia (FCT) - Projecto ProBioethanol PTDC/BIO/66151/2006 ; bolsa SFRH/BD/13463/2003 and SFRH/BPD/44328/200

Lactose fermentation by recombinant Saccharomyces cerevisiae strains

The development of Saccharomyces cerevisiae strains with the ability to ferment lactose has a high biotechnological interest, particularly for cheese whey bioremediation processes with simultaneous bio-ethanol production. We have developed a flocculent S. cerevisiae strain that efficiently ferments lactose to ethanol, using a combination of genetic engineering and evolutionary engineering approaches. This strain fermented efficiently and nearly completely (residual lactose < 3 g·L -1) lactose concentrations up to 150 g·L-1, including 3-fold concentrated cheese whey, producing ethanol titres up to 8% (v/v). The ethanol productivity obtained with this strain (> 1.5 g·L -1·h-1) was higher than that reported for batch or fed-batch fermentations with other lactose-consuming recombinant S. cerevisiae strains. The strain is highly flocculent, a property that makes it interesting for the development of high cell density fermentation processes, which may attain much higher productivity

Ethanol production from high-glucose industrial substrates using ethanol-tolerant Saccharomyces cerevisiae strains

Ethanol is well known as a toxic metabolite for yeast cells. Thus, strains that can grow well under high ethanol stress condition are highly desirable. This work aims to select and characterize Saccharomyces cerevisiae strains with improved ethanol tolerance. Moreover, it aims to evaluate the feasibility of industrial residues as fermentation media and to optimize the composition of such media. The ethanol production and tolerance of the yeast strains have been evaluated, carrying out batch alcoholic fermentations with high-glucose YP medium. The most ethanol-tolerant strain was able to ferment 300 g/L glucose producing up to 17.4 % (v/v) of ethanol in trials carried out in anaerobic shake-flasks. Aiming to develop a fermentation medium based in industrial substrates, corn steep liquor (CSL) has been tested as medium supplement, in order to replace nutrients that are needed to allow both cellular growth and fermentation. Supplementation of 300 g/L glucose medium with CSL concentrations around 90 - 110 g/L has resulted in fermentation performance similar to that observed in YP medium with the same glucose concentration, thus confirming the feasibility of CSL as peptone and yeast extract substitute

Recombinant microbial systems for improved β-galactosidase production and biotechnological applications

β-Galactosidases (EC 3.2.1.23) constitute a large family of proteins that are known to catalyze both hydrolytic and transgalactosylation reactions. The hydrolytic activity has been applied in the food industry for decades for reducing the lactose content in milk, while the transgalactosylation activity has been used to synthesize galacto-oligosaccharides and galactose containing chemicals in recent years. The main focus of this review is on the expression and production of Aspergillus niger, Kluyveromyces lactis and bacterial β-galactosidases in different microbial hosts. Furthermore, emphasis is given on the reported applications of the recombinant enzymes. Current developments on novel β-galactosidases, derived from newly identified microbial sources or by protein engineering means, together with the use of efficient recombinant microbial production systems are converting this enzyme into a relevant synthetic tool. Thermostable β-galactosidases (cold-adapted or thermophilic) in addition to the growing market for functional foods will likely redouble its industrial interest.C. Oliveira and P. M. R. Guimaraes acknowledge support from Fundacao para a Ciencia e a Tecnologia (FCT), Portugal (grants SFRH/BDP/63831/2009 and SFRH/BDP/44328/2008, respectively)

Performance fermentativa de uma estirpe recombinante de Saccharomyces cerevisiae consumidora de lactose e floculante

In recent years, there has been a growing interest in bioreactors utilizing immobilized or flocculating cells in continuous process in order to improve the bioprocess productivity. One of possible promising implementations of continuous flocculation yeast system is bioremediation of cheese whey by means of alcoholic fermentation of lactose. The aim of this work was to carry out a kinetic analysis of alcoholic fermentation of lactose using strain NCYC869-A3/T1, a recombinant Saccharomyces cerevisiae flocculent strain expressing both the LAC4 (coding for b-galactosidase) and LAC12 (lactose permease) genes of Kluyveromyces lactis. Fermentations were performed in a 600 mL bubble column bioreactor, with different initial lactose concentrations. The lactose was completely consumed in all the fermentations. The maximum specific growth rate was found to increase with initial lactose concentration, reaching its maximum at 20 g L-1 initial lactose (doubling time of about 2 h). At higher initial lactose concentrations, specific growth rate decreased, indicating that the effect of substrate inhibition had become significant. The maximum ethanol concentration produced increased linearly when the initial lactose concentration was increased between 5 and 200 g L-1. However, the ethanol yields obtained were low (45 60% of the theoretical value), probably because of the high aeration rates used. In shake-flask fermentations, in conditions of micro aeration, the yeast was unable to completely consume 200 g L-1 initial lactose, producing a maximum of 57 g L-1 ethanol (which is about the same concentration produced in the bioreactor from complete consumption of 200 g L-1 lactose). Probably, the yeast has low ethanol tolerance and the ethanol produced inhibits further lactose fermentation. Ethanol productivity increased with increasing initial lactose concentration up to 150 g L-1 (1.23 g L-1 h-1). Further increase in initial lactose to 200 g L-1 led to a slight decrease in ethanol productivity.Recentemente, tem havido um interesse crescente em biorectores que utilizam células imobilizadas ou floculantes em processos contínuos, para melhorar a productividade dos bioprocessos. Uma das possíveis e promissoras aplicações de sistemas contínuos com células de levedura floculante é a bioremediação do soro do queijo, através da fermentação alcoólica da lactose. O objectivo deste trabalho foi fazer uma análise cinética da fermentação alcoólica da lactose utilizando a estirpe NCYC869-A3/T1, uma estirpe recombinante de S. cerevisiae floculante que expressa os genes LAC4 (codifica a b-galactosidase) e LAC12 (permease da lactose) de Kluyveromyces lactis. As fermentações foram feitas numa coluna de bolhas de 600 mL, com diferentes concentrações iniciais de lactose. A lactose foi consumida completamente em todas as fermentações. A taxa específica de crescimento máxima ampliou com o aumento da concentração inicial de lactose, atingindo o valor máximo para uma concentração inicial de lactose de 20 g L-1 (tempo de duplicação de aproximadamente 2 h). Com concentrações iniciais de lactose mais elevadas, a taxa específica de crescimento diminuiu, indicando que o efeito da inibição pelo substrato se tornou significativo. A concentração máxima de etanol produzido ampliou linearmente com o aumento da concentração incial de lactose entre 5 e 200 g L-1. Contudo, os rendimentos em etanol obtidos foram baixos (45 60 % do valor teórico), provavelmente devido às elevadas taxas de arejamento utilizadas. Em fermentações realizadas em matrazes agitados, em condições de micro-arejamento, a levedura foi incapaz de consumir completamente uma concentração inicial de lactose de 200 g L-1, produzindo um máximo de 57 g L-1 de etanol (aproximadamente a mesma concentração produzida no biorector a partir do consumo completo de 200 g L-1 de lactose). Provavelmente, a levedura tem uma baixa tolerância ao etanol, e o etanol produzido inibiu a fermentação da lactose que restava. A productividade em etanol ampliou com o aumento da concentração inicial de lactose até 150 g L-1 (1.23 g L-1 h-1). O aumento da concentração inicial de lactose para 200 g L-1 conduziu a um ligeiro decréscimo da productividade em etanol.Fundação para a Ciência e a Tecnologia (FCT)