Use of the KlADH4 Promoter for Ethanol-Dependent Production of Recombinant Human Serum Albumin in Kluyveromyces lactis

KlADH4 is a gene of Kluyveromyces lactis encoding a mitochondrial alcohol dehydrogenase activity which is specifically induced by ethanol. The promoter of this gene was used for the expression of heterologous proteins in K. lactis, a very promising organism which can be used as an alternative host to Saccharomyces cerevisiae due to its good secretory properties. In this paper we report the ethanol-driven expression in K. lactis of the bacterial β-glucuronidase and of the human serum albumin (HSA) genes under the control of the KlADH4 promoter. In particular, we studied the extracellular production of recombinant HSA (rHSA) with integrative and replicative vectors and obtained a significant increase in the amount of the protein with multicopy vectors, showing that no limitation of KlADH4 trans-acting factors occurred in the cells. By deletion analysis of the promoter, we identified an element (UAS_E) which is sufficient for the induction of KlADH4 by ethanol and, when inserted in the respective promoters, allows ethanol-dependent activation of other yeast genes, such as PGK and LAC4. We also analyzed the effect of medium composition on cell growth and protein secretion. A clear improvement in the production of the recombinant protein was achieved by shifting from batch cultures (0.3 g/liter) to fed-batch cultures (1 g/liter) with ethanol as the preferred carbon source.     

Effect of Specific Growth Rate on Fermentative Capacity of Baker’s Yeast

The specific growth rate is a key control parameter in the industrial production of baker’s yeast. Nevertheless, quantitative data describing its effect on fermentative capacity are not available from the literature. In this study, the effect of the specific growth rate on the physiology and fermentative capacity of an industrial Saccharomyces cerevisiae strain in aerobic, glucose-limited chemostat cultures was investigated. At specific growth rates (dilution rates, D) below 0.28 h⁻¹, glucose metabolism was fully respiratory. Above this dilution rate, respirofermentative metabolism set in, with ethanol production rates of up to 14 mmol of ethanol · g of biomass⁻¹ · h⁻¹ at D = 0.40 h⁻¹. A substantial fermentative capacity (assayed offline as ethanol production rate under anaerobic conditions) was found in cultures in which no ethanol was detectable (D < 0.28 h⁻¹). This fermentative capacity increased with increasing dilution rates, from 10.0 mmol of ethanol · g of dry yeast biomass⁻¹ · h⁻¹ at D = 0.025 h⁻¹ to 20.5 mmol of ethanol · g of dry yeast biomass⁻¹ · h⁻¹ at D = 0.28 h⁻¹. At even higher dilution rates, the fermentative capacity showed only a small further increase, up to 22.0 mmol of ethanol · g of dry yeast biomass⁻¹ · h⁻¹ at D = 0.40 h⁻¹. The activities of all glycolytic enzymes, pyruvate decarboxylase, and alcohol dehydrogenase were determined in cell extracts. Only the in vitro activities of pyruvate decarboxylase and phosphofructokinase showed a clear positive correlation with fermentative capacity. These enzymes are interesting targets for overexpression in attempts to improve the fermentative capacity of aerobic cultures grown at low specific growth rates.     

Improved bioethanol production in an engineered Kluyveromyces lactis strain shifted from respiratory to fermentative metabolism by deletion of NDI1

In this paper, we report the metabolic engineering of the respiratory yeast Kluyveromyces lactis by construction and characterization of a null mutant (Δklndi1) in the single gene encoding a mitochondrial alternative internal dehydrogenase. Isolated mitochondria of the Δklndi1 mutant show unaffected rate of oxidation of exogenous NADH, but no oxidation of matrix NADH; this confirms that KlNdi1p is the only internal NADH dehydrogenase in K. lactis mitochondria. Permeabilized cells of the Δklndi1 mutant do not show oxidation of matrix NADH, which suggests that shuttle systems to transfer the NADH from mitochondrial matrix to cytosol, for being oxidized by external dehydrogenases, are not functional. The Δklndi1 mutation decreases the chronological life span in absence of nutrients. The expression of KlNDI1 is increased by glutathione reductase depletion. The Δklndi1 mutation shifts the K. lactis metabolism from respiratory to fermentative: the Δklndi1 strain shows reduced respiration rate and increased ethanol production from glucose, while it does not grow in non-fermentable carbon sources such as lactate. The biotechnological benefit of the Δklndi1 mutant for bioethanol production from waste cheese whey lactose was proved.     

Effects of Cultural Conditions on Mononucleotide Phosphorylating Activity of Yeast Cells

It was found that the AMP phosphorylating activity of Candida sp. N–25–2 (a hydrocarbon assimilating yeast) was affected extremely by the liquid volume of cultural medium and the concentration of inorganic salts in medium. The yeast cells having no fermentative activity showed a strong activity of AMP phosphorylation when they were cultured under relative anaerobic conditions. It was observed that the glucose consumption of yeast cells was promoted by the addition of Mg²⁺ ion and AMP into the reaction system, and that the AMP phosphorylation was promoted in the presence of F-1,6-DP or phosphaenolpyruvate.

The cells of Candida sp. N–25–2 grown on glucose medium had a remarkable fermentative activity, while the cells grown on acetate or ethanol medium had a weak activity. On the other hand, it was found that the cells grown at strong aeration on glucose medium were able to produce remarkably the phosphorylated substances from mononucleotides, when F-1,6-DP was added as a phosphate donor. Similar phenomenon was observed in case of the cells grown on the carbon sources such as acetate, ethanol and hydrocarbon.     

Utilization of nitrate abolishes the “Custers effect” in Dekkera bruxellensis and determines a different pattern of fermentation products

Nitrate is one of the most abundant nitrogen sources in nature. Several yeast species have been shown to be able to assimilate nitrate and nitrite, but the metabolic pathway has been studied in very few of them. Dekkera bruxellensis can use nitrate as sole nitrogen source and this metabolic characteristic can render D. bruxellensis able to overcome S. cerevisiae populations in industrial bioethanol fermentations. In order to better characterize how nitrate utilization affects carbon metabolism and the yields of the fermentation products, we investigated this trait in defined media under well-controlled aerobic and anaerobic conditions. Our experiments showed that in D. bruxellensis, utilization of nitrate determines a different pattern of fermentation products. Acetic acid, instead of ethanol, became in fact the main product of glucose metabolism under aerobic conditions. We have also demonstrated that under anaerobic conditions, nitrate assimilation abolishes the “Custers effect”, in this way improving its fermentative metabolism. This can offer a new strategy, besides aeration, to sustain growth and ethanol production for the employment of this yeast in industrial processes.     

STUDIES IN THE PHYSIOLOGY OF FUSARIA. THE RESPIRATORY AND FERMENTATIVE MECHANISMS

1.Fusarium tricothecoides was selected for a study of the respiratory and fermentative activities of Fusaria. "Resting cell" suspensions were investigated by the Barcroft manometric technique. 2. The results of the investigation indicate clearly that the mechanism of endogenous metabolism (respiration) is distinct from the exogenous mechanism (fermentation). Anaerobically no significant CO₂ production is apparent without added substrate. In the presence of glucose the anaerobic CO₂ evolution is practically equal to the added CO₂ evolved aerobically in the presence of added glucose. Low concentrations of iodoacetate or fluoride selectively poison the exogenous mechanism but do not affect the endogenous mechanism. Alcohol is not produced in the course of endogenous metabolism, but is produced in the presence of added glucose. 3. A study of the metabolism of the organism throughout its entire growth phase from 1 to 7 days has been made. 4. The ability of suspensions of Fusarium sp. H., obtained by growth on a variety of common substrates, to attack a large number of carbon sources with the production of exogenous CO₂ was determined. It is found that organisms grown on glucose will attack only glucose, mannose, and fructose, but none of the common intermediary metabolites except pyruvic acid. Organisms grown on galactose attack galactose, as well as the other hexoses, indicating an adaptive mechanism. 5. An identical mechanism for the dissimilation of glucose, mannose, and galactose is indicated since no additive effects with these substrates were observed. Growths on non-hexose carbon sources attack glucose slightly under the experimental conditions with the evolution of CO₂, but do not attack any other substrate. This would indicate a residual glucose-dissimilating mechanism in all growths investigated. 6. Striking similarities between the general metabolism of resting suspensions of Fusarium sp. H. and resting suspensions of yeast cells are apparent.     

Effect of carbon sources on the growth and ethanol production of native yeast Pichia kudriavzevii ITV-S42 isolated from sweet sorghum juice

The importance of non-Saccharomyces yeast species in fermentation processes is widely acknowledged. Within this group, Pichia kudriavzevii ITV-S42 yeast strain shows particularly desirable characteristics for ethanol production. Despite this fact, a thorough study of the metabolic and kinetic characteristics of this strain is currently unavailable. The aim of this work is to study the nutritional requirements of Pichia kudriavzevii ITV-S42 strain and the effect of different carbon sources on the growth and ethanol production. Results showed that glucose and fructose were both assimilated and fermented, achieving biomass and ethanol yields of 0.37 and 0.32 gg⁻¹, respectively. Glycerol was assimilated but not fermented; achieving a biomass yield of 0.88 gg⁻¹. Xylose and sucrose were not metabolized by the yeast strain. Finally, the use of a culture medium enriched with salts and yeast extract favored glucose consumption both for growth and ethanol production, improving ethanol tolerance reported for this genre (35 g L⁻¹) to 90 g L⁻¹ maximum ethanol concentration (over 100%). Furthermore Pichia kudriavzevii ITV-S42 maintained its fermentative capacity up to 200 g L⁻¹ initial glucose, demonstrating that this yeast is osmotolerant.

     

Production of ethanol from thin stillage by metabolically engineered Escherichia coli

Thin stillage is a by-product generated in large amounts during the production of ethanol that is rich in carbon sources like glycerol, glucose and maltose. Unfortunately, the fermentation of thin stillage results in a mixture of organic acids and ethanol and minimum utilization of glycerol, the latter a compound that can represent up to 80% of the available substrates in this stream. We report here the efficient production of ethanol from thin stillage by a metabolically engineered strain of Escherichia coli. Simultaneous utilization of glycerol and sugars was achieved by overexpressing either the fermentative or the respiratory glycerol-utilization pathway. However, amplification of the fermentative pathway (encoded by gldA and dhaKLM) led to more efficient consumption of glycerol and promoted the synthesis of reduced products, including ethanol. A previously constructed strain, EH05, containing mutations that prevented the accumulation of competing by-products (i.e. lactate, acetate, and succinate) and overexpressing the fermentative pathway for glycerol utilization [i.e. strain EH05 (pZSKLMgldA)], efficiently converted thin stillage supplemented with only mineral salts to ethanol at yields close to 85% of the theoretical maximum. Ethanol accounted for about 90% (w/w) of the product mixture. These results, along with the comparable performance of strain EH05 (pZSKLMgldA) in 0.5 and 5 l fermenters, indicate a great potential for the adoption of this process by the biofuels industry.     

The wine yeasts of the Cape

Summary The claims made byCusters in connection with the disproportionate yields of ethanol and carbon dioxide from the fermentative dissimilation of glucose in yeast water byBrett. claussenii were investigated forBrett. intermedius. All results obtained showed thatBrett. intermedius produced the normal equimolar amounts of ethanol and carbon dioxide, irrespective of whether the studies were conducted in yeast water or in dilute yeast water supplemented with a suitable nitrogen source. Re-investigation of the fermentative dissimilation of glucose in yeast water byBrett. claussenii gave the same results as withBrett. intermedius. Part I and II: Antonie van Leeuwenhoek24, 239, 1958;25, 145, 1959.     

ACR1, a gene encoding a protein related to mitochondrial carriers,is essential for acetyl-CoA synthetase activity in Saccharomyces cerevisiae

The utilization of ethanol via acetate by the yeast Saccharomyces cerevisiae requires the presence of the enzyme acetyl-coenzyme A synthetase (acetyl-CoA synthetase), which catalyzes the activation of acetate to acetyl-coenzyme A (acetyl-CoA). We have isolated a mutant, termed acr1, defective for this activity by screening for mutants unable to utilize ethanol as a sole carbon source. Genetic and biochemical characterization show that, in this mutant, the structural gene for acetyl-CoA synthetase is not affected. Cloning and sequencing demonstrated that the ACR1 gene encodes a protein of 321 amino acids with a molecular mass of 35 370 Da. Computer analysis suggested that the ACR1 gene product (ACR1) is an integral membrane protein related to the family of mitochondrial carriers. The expression of the gene is induced by growing yeast cells in media containing ethanol or acetate as sole carbon sources and is repressed by glucose. ACR1 is essential for the utilization of ethanol and acetate since a mutant carrying a disruption in this gene is unable to grow on these compounds.     

Cloning of a second gene encoding 6-phosphofructo-2-kinase in yeast, and characterization of mutant strains without fructose-2,6-bisphosphate

We have identified a new gene, PFK27, that encodes a second inducible 6-phosphofructo-2-kinase in the yeast Saccharomyces cerevisiae. Sequencing shows an open reading frame of 397 amino acids and 45.3 kDa. Amino acid sequence comparisons with other bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase isoenzymes of various organisms revealed similarities only to the kinase domains. Expression of PFK27 was induced severalfold by glucose and sucrose, but not by galactose or maltose, suggesting that sugar transport might be involved in triggering the induction signal. We have constructed a mutant strain devoid of any fructose-2,6-bisphosphate. The mutant strain grew well on several kinds and concentrations of carbon sources. The levels of hexose phosphates in the cells were increased, but flux rates for glucose utilization and ethanol production were similar to the wild-type strain. However, after the transfer of the mutant cells from respiratory to fermentative growth conditions, growth, glucose consumption and ethanol production were delayed in a transition phase. Our results show that fructose-2,6-bisphosphate is an important effector in vivo of the 6-phosphofructo-1-kinase/fructose-1,6-bisphospha-tase enzyme pair, and is involved in the initiation of glycolysis during the transition to a fermentative mode of metabolism. Nevertheless, it can be effectively replaced by other effectors and regulatory mechanisms during growth on glucose.     

Fermentative metabolism impedes p53-dependent apoptosis in a Crabtree-positive but not in Crabtree-negative yeast

Tumour cells distinguish from normal cells by fermenting glucose to lactate in presence of sufficient oxygen and functional mitochondria (Warburg effect). Crabtree effect was invoked to explain the biochemical basis of Warburg effect by suggesting that excess glucose suppresses mitochondrial respiration. It is known that the Warburg effect and Crabtree effect are displayed by Saccharomyces cerevisiae, during growth on abundant glucose. Beyond this similarity, it was also demonstrated that expression of human pro-apoptotic proteins in S. cerevisiae such as Bax and p53 caused apoptosis. Here, we demonstrate that p53 expression in S. cerevisiae (Crabtree-positive yeast) causes increase in ROS levels and apoptosis when cells are growing on non-fermentable carbon sources but not on fermentable carbon sources, a feature similar to tumour cells. In contrast, in Kluyveromyces lactis (Crabtree-negative yeast) p53 causes increase in ROS levels and apoptosis regardless of the carbon source. Interestingly, the increased ROS levels and apoptosis are correlated to increased oxygen uptake in both S. cerevisiae and K. lactis. Based on these results, we suggest that at least in yeast, fermentation per se does not prevent the escape from apoptosis. Rather, the Crabtree effect plays a crucial role in determining whether the cells should undergo apoptosis or not.     

Homo- and heterofermentative lactobacilli differently affect sugarcane-based fuel ethanol fermentation

Bacterial contamination during industrial yeast fermentation has serious economic consequences for fuel ethanol producers. In addition to deviating carbon away from ethanol formation, bacterial cells and their metabolites often have a detrimental effect on yeast fermentative performance. The bacterial contaminants are commonly lactic acid bacteria (LAB), comprising both homo- and heterofermentative strains. We have studied the effects of these two different types of bacteria upon yeast fermentative performance, particularly in connection with sugarcane-based fuel ethanol fermentation process. Homofermentative Lactobacillus plantarum was found to be more detrimental to an industrial yeast strain (Saccharomyces cerevisiae CAT-1), when compared with heterofermentative Lactobacillus fermentum, in terms of reduced yeast viability and ethanol formation, presumably due to the higher titres of lactic acid in the growth medium. These effects were only noticed when bacteria and yeast were inoculated in equal cell numbers. However, when simulating industrial fuel ethanol conditions, as conducted in Brazil where high yeast cell densities and short fermentation time prevail, the heterofermentative strain was more deleterious than the homofermentative type, causing lower ethanol yield and out competing yeast cells during cell recycle. Yeast overproduction of glycerol was noticed only in the presence of the heterofermentative bacterium. Since the heterofermentative bacterium was shown to be more deleterious to yeast cells than the homofermentative strain, we believe our findings could stimulate the search for more strain-specific antimicrobial agents to treat bacterial contaminations during industrial ethanol fermentation.     

Increased aerobic metabolism is essential for the beneficial effects of caloric restriction on yeast life span

Calorie restriction is a dietary regimen capable of extending life span in a variety of multicellular organisms. A yeast model of calorie restriction has been developed in which limiting the concentration of glucose in the growth media of Saccharomyces cerevisiae leads to enhanced replicative and chronological longevity. Since S. cerevisiae are Crabtree-positive cells that present repression of aerobic catabolism when grown in high glucose concentrations, we investigated if this phenomenon participates in life span regulation in yeast. S. cerevisiae only exhibited an increase in chronological life span when incubated in limited concentrations of glucose. Limitation of galactose, raffinose or glycerol plus ethanol as substrates did not enhance life span. Furthermore, in Kluyveromyces lactis, a Crabtree-negative yeast, glucose limitation did not promote an enhancement of respiratory capacity nor a decrease in reactive oxygen species formation, as is characteristic of conditions of caloric restriction in S. cerevisiae. In addition, K. lactis did not present an increase in longevity when incubated in lower glucose concentrations. Altogether, our results indicate that release from repression of aerobic catabolism is essential for the beneficial effects of glucose limitation in the yeast calorie restriction model. Potential parallels between these changes in yeast and hormonal regulation of respiratory rates in animals are discussed. G. A. Oliveira and E. B. Tahara contributed equally to this work.     

RAG3 gene and transcriptional regulation of the pyruvate decarboxylase gene in Kluyveromyces lactis

The RAG3 gene has been cloned from a Kluyveromyces lactis genomic library by complementation of the rag3 mutation, which shows impaired fermentative growth on glucose in the presence of respiratory inhibitors. From the nucleotide sequence of the cloned DNA, which contained an open reading frame of 765 codons, the predicted protein is 49.5% identical to the Pdc2 protein of Saccharomyces cerevisiae, a regulator of pyruvate decarboxylase in this yeast. Measurement of the pyruvate decarboxylase activity in the original rag3–1 mutant and in the null mutant confirmed that the RAG3 gene is involved in pyruvate decarboxylase synthesis in K. lactis. The effect is exerted at the mRNA level of the pyruvate decarboxylase structural gene KIPDCA. Despite analogies between the RAG3 gene of K. lactis and the PDC2 gene of S. cerevisiae, these genes were unable to reciprocally complement.     

Glycolysis Controls Plasma Membrane Glucose Sensors To Promote Glucose Signaling in Yeasts

Sensing of extracellular glucose is necessary for cells to adapt to glucose variation in their environment. In the respiratory yeast Kluyveromyces lactis, extracellular glucose controls the expression of major glucose permease gene RAG1 through a cascade similar to the Saccharomyces cerevisiae Snf3/Rgt2/Rgt1 glucose signaling pathway. This regulation depends also on intracellular glucose metabolism since we previously showed that glucose induction of the RAG1 gene is abolished in glycolytic mutants. Here we show that glycolysis regulates RAG1 expression through the K. lactis Rgt1 (KlRgt1) glucose signaling pathway by targeting the localization and probably the stability of Rag4, the single Snf3/Rgt2-type glucose sensor of K. lactis. Additionally, the control exerted by glycolysis on glucose signaling seems to be conserved in S. cerevisiae. This retrocontrol might prevent yeasts from unnecessary glucose transport and intracellular glucose accumulation.