The relationship between the in situ reduction level of the cytochrome c pool of Azorhizobium caulinodans growing in a chemostat with NH4+ or N2 as the N source and the total activity of cytochrome c oxidases

Abstract The in situ method for determination of reduction levels of cytochromes b and c pools during steady-state growth (Pronk et al., Anal. Biochem. 214, 149–155, 1993) was applied to chemostat cultures of the wild-type, a cytochrome aa₃ single mutant and a cytochrome aa₃/d double mutant of Azorhizobium caulinodans . For growth with NH₄⁺ as the N source, the results indicate that (i) the aa₃ mutant strains growing at a dissolved O₂ tension of 0.5% possess an active alternative cytochrome c oxidase, which is hardly present during fully aerobic growth, and assuming that (i) also pertains to the wild-type, (ii) the wild-type uses cytochrome aa₃ under fully aerobic conditions. For growth with N₂ as the N source, it was found that the aa₃ mutant strains growing at dissolved O₂ tensions ranging from 0.5 to 3.0% also contain an active alternative cytochrome c oxidase.     

Catalase Overexpression Reduces Lactic Acid-Induced Oxidative Stress in Saccharomyces cerevisiae

Industrial production of lactic acid with the current pyruvate decarboxylase-negative Saccharomyces cerevisiae strains requires aeration to allow for respiratory generation of ATP to facilitate growth and, even under nongrowing conditions, cellular maintenance. In the current study, we observed an inhibition of aerobic growth in the presence of lactic acid. Unexpectedly, the cyb2Δ reference strain, used to avoid aerobic consumption of lactic acid, had a specific growth rate of 0.25 h⁻¹ in anaerobic batch cultures containing lactic acid but only 0.16 h⁻¹ in identical aerobic cultures. Measurements of aerobic cultures of S. cerevisiae showed that the addition of lactic acid to the growth medium resulted in elevated levels of reactive oxygen species (ROS). To reduce the accumulation of lactic acid-induced ROS, cytosolic catalase (CTT1) was overexpressed by replacing the native promoter with the strong constitutive TPI1 promoter. Increased activity of catalase was confirmed and later correlated with decreased levels of ROS and increased specific growth rates in the presence of high lactic acid concentrations. The increased fitness of this genetically modified strain demonstrates the successful attenuation of additional stress that is derived from aerobic metabolism and may provide the basis for enhanced (micro)aerobic production of organic acids in S. cerevisiae.Lactic acid is an organic acid with a wide range of applications. In the food industry, lactic acid has traditionally been used as an antimicrobial as well as a flavor enhancer. Besides having applications in textile, cosmetic, and pharmaceutical industries (5), lactic acid has been applied for the manufacture of lactic acid polymers (11, 40). These polymers have properties that are similar to those of petroleum-derived plastics. Skyrocketing oil prices caused by dwindling fossil fuel reserves coupled with pressures to tackle environmental issues are creating increased demand for bioderived, and often biodegradable, polymers, such as poly-lactic acid.Current industrial lactic acid fermentations are based on different species of lactic acid bacteria. These bacteria have complex nutrient requirements due to their limited ability to synthesize B vitamins and amino acids (8) and are intolerant to acidic conditions with a pH between 5.5 and 6.5 required for growth (40). Acidification of the growth medium during lactic acid fermentation is typically counteracted by the addition of neutralizing agents (e.g., CaCO₃), resulting in the formation of large quantities of insoluble salts, such as gypsum, during downstream processing.Saccharomyces cerevisiae has received attention as a possible alternative biocatalyst. This organism is relatively tolerant to low pH and has simple nutrient requirements. The production of lactic acid with metabolically engineered S. cerevisiae was achieved by introducing a NAD⁺-dependent lactate dehydrogenase, leading to the simultaneous formation of both ethanol and lactate (1a, 12, 31, 32, 36). Further improvements were made by constructing a pyruvate decarboxylase-negative (Pdc⁻) S. cerevisiae strain (1a, 31, 44) that converted glucose to lactic acid as the sole fermentation product.Although the redox balance and ATP generation in lactic acid fermentation are analogous to those in alcoholic fermentation, engineered homolactic S. cerevisiae strains could not sustain anaerobic growth (44). In addition, the lactate formation rate under anaerobic conditions in the presence of excess glucose was significantly lower than the specific ethanol production rate of the wild-type strain. Moreover, exposure of the anaerobic cell suspension to oxygen immediately led to a 2.5-fold increase in the lactate formation rate. The stimulatory effect of oxygen on lactic acid fermentation may reflect an energetic constraint in lactate fermentation, probably as a consequence of energy-dependent product export (42, 44). In agreement with this hypothesis, intracellular ATP concentrations and the related energy charge decrease rapidly during anaerobic homolactic fermentation by S. cerevisiae (1). Consequently, industrial production of lactic acid with S. cerevisiae may require (micro)aerobic conditions to allow for the generation of sufficient ATP to enable cell growth and, even under nongrowing conditions, maintenance.The formation of reactive oxygen species (ROS) during cellular respiration is an unavoidable side effect of aerobic life relying on oxygen as the final electron acceptor. At least 2% of oxygen consumed in mitochondrial respiration undergoes only one electron reduction, mainly by the semiquinone form of coenzyme Q, generating superoxide radicals (O₂⁻) (26). In addition, the prooxidant effects of organic acids have been demonstrated using sod mutants (30). An in vitro study by Ali et al. (3) also linked ROS formation to weak organic acids and showed enhanced hydroxy radical (OH) generation in the presence of lactic acid.Among different ROS, the hydroxy radical that originates from H₂O₂ in the metal-mediated Fenton/Haber-Weiss reactions is especially reactive. It indiscriminately oxidizes intracellular proteins, nucleic acids, and lipids in the cell membranes (4, 38). Lactate interacts with the ferric ion (Fe³⁺) to form a stable complex of Fe³⁺-lactate at a molar ratio of 1:2. This complex then reacts with H₂O₂ to enhance the OH generation via the Fenton reaction (2, 3). Although a similar in vivo mechanism has not yet been proven, previous research indicates that lactic acid and other weak organic acids enhance oxidative stress of aerobic yeast cultures.Like other eukaryotic organisms, S. cerevisiae possesses enzymatic defense mechanisms, including several crucial antioxidant enzymes, such as catalase and superoxide dismutase (SOD). SOD removes O₂⁻ by converting it to H₂O₂, which, in turn, can be disproportionated to water by catalase or glutathione peroxidase. Cytosolic catalase, Ctt1p, is thought to play a general role, as CTT1 expression is regulated by various stresses, including oxidative stress, osmotic stress, and starvation (15, 23, 33). More recently, catalase has also been implicated in response to acetic acid tolerance and acetic acid-induced programmed cell death (17, 47).The goals of the present study were to assess the in vivo relevance of lactate-mediated oxidative stress in S. cerevisiae and to investigate whether its effects could be ameliorated by enhanced expression of catalase.     

Novel Evolutionary Engineering Approach for Accelerated Utilization of Glucose,Xylose, and Arabinose Mixtures by Engineered Saccharomyces cerevisiae Strains

Lignocellulosic feedstocks are thought to have great economic and environmental significance for future biotechnological production processes. For cost-effective and efficient industrial processes, complete and fast conversion of all sugars derived from these feedstocks is required. Hence, simultaneous or fast sequential fermentation of sugars would greatly contribute to the efficiency of production processes. One of the main challenges emerging from the use of lignocellulosics for the production of ethanol by the yeast Saccharomyces cerevisiae is efficient fermentation of d-xylose and l-arabinose, as these sugars cannot be used by natural S. cerevisiae strains. In this study, we describe the first engineered S. cerevisiae strain (strain IMS0003) capable of fermenting mixtures of glucose, xylose, and arabinose with a high ethanol yield (0.43 g g⁻¹ of total sugar) without formation of the side products xylitol and arabinitol. The kinetics of anaerobic fermentation of glucose-xylose-arabinose mixtures were greatly improved by using a novel evolutionary engineering strategy. This strategy included a regimen consisting of repeated batch cultivation with repeated cycles of consecutive growth in three media with different compositions (glucose, xylose, and arabinose; xylose and arabinose; and only arabinose) and allowed rapid selection of an evolved strain (IMS0010) exhibiting improved specific rates of consumption of xylose and arabinose. This evolution strategy resulted in a 40% reduction in the time required to completely ferment a mixture containing 30 g liter⁻¹ glucose, 15 g liter⁻¹ xylose, and 15 g liter⁻¹ arabinose.In recent years, the need for biotechnological manufacturing based on lignocellulosic feedstocks has become evident (6, 10). In contrast to the readily fermentable, mainly starch- or sucrose-containing feedstocks used in current biotechnological production processes, lignocellulosic biomass requires intensive pretreatment and hydrolysis, which yield complex mixtures of sugars (3, 7, 14, 27). For cost-effective and efficient industrial processes, complete and fast conversion of all sugars present in lignocellulosic hydrolysates is a prerequisite. The major hurdles encountered in implementing these production processes are the conversion of substrates that cannot be utilized by the organism of choice and, even more importantly, the subsequent improvement of sugar conversion rates and product yields.The use of evolutionary engineering has proven to be very valuable for obtaining phenotypes of (industrial) microorganisms with improved properties, such as an expanded substrate range, increased stress tolerance, and efficient substrate utilization (16, 17). Also, for the yeast Saccharomyces cerevisiae, the preferred organism for large-scale ethanol production for the past few decades, evolutionary engineering has been extensively used to select for industrially relevant phenotypes. For ethanol production from lignocellulose by S. cerevisiae, one of the main challenges is efficient conversion of the pentoses d-xylose and l-arabinose to ethanol. To deal with this challenge, S. cerevisiae strains have been metabolically engineered since the early 1990s for the conversion of xylose into ethanol by the introduction of heterologous xylose utilization pathways (for recent reviews, see references 9 and 20). Arabinose utilization, however, has been addressed only quite recently. The most successful approach for obtaining arabinose consumption in S. cerevisiae has been the introduction of a bacterial arabinose utilization pathway (5, 26). In addition to metabolic engineering, extensive evolutionary engineering (by prolonged cultivation of recombinant S. cerevisiae strains in either anaerobic chemostat or repeated anaerobic batch cultures) was required to obtain S. cerevisiae strains that ferment either xylose (13, 19) or arabinose (5, 26) fast or to improve fermentation performance with mixtures containing glucose and xylose (12). In contrast, (evolutionary) engineering has still not resulted in fast and efficient fermentation of both xylose and arabinose to ethanol by a single recombinant S. cerevisiae strain. At best, simultaneous utilization of xylose and arabinose yielded large amounts of the undesirable side products xylitol and arabinitol (11). Hence, a major remaining challenge is the conversion of both xylose and arabinose with high ethanol production rates and yields.In a previous study, an S. cerevisiae strain was metabolically engineered to obtain both xylose and arabinose utilization. For this, the Piromyces XylA, S. cerevisiae XKS1, and Lactobacillus plantarum araA, araB, and araD genes, as well as the endogenous genes of the pentose phosphate pathway (RPE1, RKI1, TKL1, and TAL1), were overexpressed. Selection by sequential batch cultivation under conditions with arabinose as the sole carbon source resulted in strain IMS0002, which is capable of fermenting arabinose to ethanol under anaerobic conditions (26). Unfortunately, the ability to ferment xylose to ethanol was largely lost during long-term selection for improved l-arabinose fermentation. During anaerobic batch cultivation of strain IMS0002 in a glucose-xylose-arabinose mixture, xylose was not consumed completely and was converted to almost equimolar amounts of xylitol. This loss of xylose metabolism illustrates the limitations of selection in media supplemented with a single carbon and energy source.The goal of the present study was to evaluate and optimize selection strategies for evolutionary optimization of the utilization of substrate mixtures. Fermentation of glucose, xylose, and arabinose mixtures by engineered S. cerevisiae strains was used as the model.     

Steady-state and transient-state analysis of growth and metabolite production in a Saccharomyces cerevisiae strain with reduced pyruvate-decarboxylase activity

Pyruvate decarboxylase is a key enzyme in the production of low-molecular-weight byproducts (ethanol, acetate) in biomass-directed applications of Saccharomyces cerevisiae. To investigate whether decreased expression levels of pyruvate decarboxylase can reduce byproduct formation, the PDC2 gene, which encodes a positive regulator of pyruvate-decarboxylase synthesis, was inactivated in the prototrophic strain S. cerevisiae CEN. PK113-7D. This caused a 3-4-fold reduction of pyruvate-decarboxylase activity in glucose-limited, aerobic chemostat cultures grown at a dilution rate of 0.10 h(-1). Upon exposure of such cultures to a 50 mM glucose pulse, ethanol and acetate were the major byproducts formed by the wild type. In the pdc2Delta strain, formation of ethanol and acetate was reduced by 60-70%. In contrast to the wild type, the pdc2Delta strain produced substantial amounts of pyruvate after a glucose pulse. Nevertheless, its overall byproduct formation was ca. 50% lower. The specific rate of glucose consumption after a glucose pulse to pdc2Delta cultures was about 40% lower than in wild-type cultures. This suggests that, at reduced pyruvate-decarboxylase activities, glycolytic flux is controlled by NADH reoxidation. In aerobic, glucose-limited chemostat cultures, the wild type exhibited a mixed respiro-fermentative metabolism at dilution rates above 0.30 h(-1). Below this dilution rate, sugar metabolism was respiratory. At dilution rates up to 0.20 h(-1), growth of the pdc2Delta strain was respiratory and biomass yields were similar to those of wild-type cultures. Above this dilution rate, washout occurred. The low micro(max) of the pdc2Delta strain in glucose-limited chemostat cultures indicates that occurrence of respiro-fermentative metabolism in wild-type cultures is not solely caused by competition of respiration and fermentation for pyruvate. Furthermore, it implies that inactivation of PDC2 is not a viable option for reducing byproduct formation in industrial fermentations.     

Fed-batch cultivation of the docosahexaenoic-acid-producing marine alga Crypthecodinium cohnii on ethanol

The heterotrophic marine microalga Crypthecodinium cohnii produces docosahexaenoic acid (DHA), a polyunsaturated fatty acid with food and pharmaceutical applications. So far, DHA production has been studied with glucose and acetic acid as carbon sources. This study investigates the potential of ethanol as an alternative carbon source for DHA production by C. cohnii. In shake-flask cultures, the alga was able to grow on ethanol. The specific growth rate was optimal with 5 g l(-1) ethanol and growth did not occur at 0 g l(-1) and above 15 g l(-1). By contrast, in fed-batch cultivations with a controlled feed of pure ethanol, cumulative ethanol addition could be much higher than 15 g l(-1), thus enabling a high final cell density and DHA production. In a representative fed-batch cultivation of C. cohnii with pure ethanol as feed, 83 g dry biomass l(-1), 35 g total lipid l(-1) and 11.7 g DHA l(-1) were produced in 220 h. The overall volumetric productivity of DHA was 53 mg l(-1 )h(-1), which is the highest value reported so far for this alga.     

Homofermentative lactate production cannot sustain anaerobic growth of engineered Saccharomyces cerevisiae: possible consequence of energy-dependent lactate export

Due to a growing market for the biodegradable and renewable polymer polylactic acid, the world demand for lactic acid is rapidly increasing. The tolerance of yeasts to low pH can benefit the process economy of lactic acid production by minimizing the need for neutralizing agents. Saccharomyces cerevisiae (CEN.PK background) was engineered to a homofermentative lactate-producing yeast via deletion of the three genes encoding pyruvate decarboxylase and the introduction of a heterologous lactate dehydrogenase (EC 1.1.1.27). Like all pyruvate decarboxylase-negative S. cerevisiae strains, the engineered strain required small amounts of acetate for the synthesis of cytosolic acetyl-coenzyme A. Exposure of aerobic glucose-limited chemostat cultures to excess glucose resulted in the immediate appearance of lactate as the major fermentation product. Ethanol formation was absent. However, the engineered strain could not grow anaerobically, and lactate production was strongly stimulated by oxygen. In addition, under all conditions examined, lactate production by the engineered strain was slower than alcoholic fermentation by the wild type. Despite the equivalence of alcoholic fermentation and lactate fermentation with respect to redox balance and ATP generation, studies on oxygen-limited chemostat cultures showed that lactate production does not contribute to the ATP economy of the engineered yeast. This absence of net ATP production is probably due to a metabolic energy requirement (directly or indirectly in the form of ATP) for lactate export.     

Metabolic engineering of glycerol production in Saccharomyces cerevisiae

Inactivation of TPI1, the Saccharomyces cerevisiae structural gene encoding triose phosphate isomerase, completely eliminates growth on glucose as the sole carbon source. In tpi1-null mutants, intracellular accumulation of dihydroxyacetone phosphate might be prevented if the cytosolic NADH generated in glycolysis by glyceraldehyde-3-phosphate dehydrogenase were quantitatively used to reduce dihydroxyacetone phosphate to glycerol. We hypothesize that the growth defect of tpi1-null mutants is caused by mitochondrial reoxidation of cytosolic NADH, thus rendering it unavailable for dihydroxyacetone-phosphate reduction. To test this hypothesis, a tpi1delta nde1delta nde2delta gut2delta quadruple mutant was constructed. NDE1 and NDE2 encode isoenzymes of mitochondrial external NADH dehydrogenase; GUT2 encodes a key enzyme of the glycerol-3-phosphate shuttle. It has recently been demonstrated that these two systems are primarily responsible for mitochondrial oxidation of cytosolic NADH in S. cerevisiae. Consistent with the hypothesis, the quadruple mutant grew on glucose as the sole carbon source. The growth on glucose, which was accompanied by glycerol production, was inhibited at high-glucose concentrations. This inhibition was attributed to glucose repression of respiratory enzymes as, in the quadruple mutant, respiratory pyruvate dissimilation is essential for ATP synthesis and growth. Serial transfer of the quadruple mutant on high-glucose media yielded a spontaneous mutant with much higher specific growth rates in high-glucose media (up to 0.10 h(-1) at 100 g of glucose. liter(-1)). In aerated batch cultures grown on 400 g of glucose. liter(-1), this engineered S. cerevisiae strain produced over 200 g of glycerol. liter(-1), corresponding to a molar yield of glycerol on glucose close to unity.     

Pyruvate Decarboxylase Catalyzes Decarboxylation of Branched-Chain 2-Oxo Acids but Is Not Essential for Fusel Alcohol Production by Saccharomyces cerevisiae

The fusel alcohols 3-methyl-1-butanol, 2-methyl-1-butanol, and 2-methyl-propanol are important flavor compounds in yeast-derived food products and beverages. The formation of these compounds from branched-chain amino acids is generally assumed to occur via the Ehrlich pathway, which involves the concerted action of a branched-chain transaminase, a decarboxylase, and an alcohol dehydrogenase. Partially purified preparations of pyruvate decarboxylase (EC 4.1.1.1) have been reported to catalyze the decarboxylation of the branched-chain 2-oxo acids formed upon transamination of leucine, isoleucine, and valine. Indeed, in a coupled enzymatic assay with horse liver alcohol dehydrogenase, cell extracts of a wild-type Saccharomyces cerevisiae strain exhibited significant decarboxylation rates with these branched-chain 2-oxo acids. Decarboxylation of branched-chain 2-oxo acids was not detectable in cell extracts of an isogenic strain in which all three PDC genes had been disrupted. Experiments with cell extracts from S. cerevisiae mutants expressing a single PDC gene demonstrated that both PDC1- and PDC5-encoded isoenzymes can decarboxylate branched-chain 2-oxo acids. To investigate whether pyruvate decarboxylase is essential for fusel alcohol production by whole cells, wild-type S. cerevisiae and an isogenic pyruvate decarboxylase-negative strain were grown on ethanol with a mixture of leucine, isoleucine, and valine as the nitrogen source. Surprisingly, the three corresponding fusel alcohols were produced in both strains. This result proves that decarboxylation of branched-chain 2-oxo acids via pyruvate decarboxylase is not an essential step in fusel alcohol production.Saccharomyces cerevisiae has been used for centuries in the production of bread and alcoholic beverages. Along with ethanol and carbon dioxide, fermenting cultures of this yeast produce a variety of low-molecular-weight flavor compounds (including alcohols, diacetyl, esters, organic acids, organic sulfides, and carbonyl compounds). The compounds 3-methyl-1-butanol, 2-methyl-1-butanol, and 2-methyl-1-propanol, commonly known as fusel alcohols, and their esters make an important contribution to the flavor of alcoholic beverages and bread (1, 14).A metabolic pathway for production of fusel alcohols by yeast was first proposed by Ehrlich (6). The Ehrlich pathway starts with the enzyme-catalyzed decarboxylation of branched-chain 2-oxo acids to the corresponding aldehydes. Subsequently, the aldehyde is reduced to the corresponding fusel alcohol by an alcohol dehydrogenase (11, 16, 24). The branched-chain 2-oxo acid substrates for the Ehrlich pathway can be produced by the deamination of l-leucine, l-isoleucine, or l-valine. Growth of S. cerevisiae with any of these three amino acids as the nitrogen source results in the accumulation of the corresponding fusel alcohol (2, 3, 21). Alternatively, branched-chain 2-oxo acids may be synthesized de novo from carbohydrates as intermediates of branched-chain amino acid synthesis (13).The conversion of branched-chain oxo acids into their respective aldehydes and alcohols via the Ehrlich pathway resembles the fermentative metabolism of pyruvate, which yields ethanol and carbon dioxide. In both cases, the decarboxylation of a 2-oxo acid is followed by the reduction of the resulting aldehyde. Partially purified preparations of yeast pyruvate decarboxylase have been shown to catalyze the decarboxylation of various 2-oxo acids, including the putative intermediates of the Ehrlich pathway (8, 12, 16, 21). However, it has not been conclusively proven that pyruvate decarboxylase is essential for or even involved in fusel alcohol production by S. cerevisiae.Dickinson and Dawes (4) have reported that, at least under some conditions, oxidative decarboxylation by a mitochondrial branched-chain oxo acid dehydrogenase complex (17) is involved in the catabolism of branched-chain 2-oxo acids. Mutants that did not express the lipoamide dehydrogenase subunit of this enzyme complex accumulated branched-chain oxo acids in batch cultures grown on media containing leucine, isoleucine, or valine (4), thus casting some doubt on the exclusive role of pyruvate decarboxylase in the decarboxylation of branched-chain oxo acids.The aim of this study was to reinvestigate the role of pyruvate decarboxylase in the production of fusel alcohols by S. cerevisiae. The S. cerevisiae genome harbors three structural genes (PDC1, PDC5, and PDC6) that can each encode an active pyruvate decarboxylase (9). In wild-type yeast strains, PDC6 expression is either very low or absent (7, 9). However, revertants of pdc1-pdc5 double mutants, in which a recombination event has caused a fusion of the PDC1 promoter and the PDC6 open reading frame, express a functional enzyme (10). Therefore, studies on the physiological effects of pyruvate decarboxylase deficiency are most easily interpreted when they are performed with strains in which all three PDC genes are disrupted.In the present study, the decarboxylation of branched-chain 2-oxo acids was studied in cell extracts of wild-type S. cerevisiae and in extracts of an isogenic pyruvate decarboxylase-negative mutant. Furthermore, conversion of branched-chain amino acids to the corresponding fusel alcohols by intact cells was analyzed in ethanol-grown cultures of a wild-type S. cerevisiae strain and in those of the Pdc⁻ mutant.