Analysis of the rat JE gene promoter identifies an AP-1 binding site essential for basal expression but not for TPA induction.

We have cloned the immediate-early serum-reponsive JE gene from the rat in order to study the regulation of this gene. We show that sequences of the JE promoter region confer serum-inducibility to a reporter gene. Analysis of the promoter in transient assays reveals that: i) the -141/-88 region is required for the response to the phorbol ester TPA, ii) the -70/-38 region is essential for basal activity. This latter region harbors the sequence TGACTCC, which resembles the consensus site for AP-1 binding, TGACTCA. DNA-protein binding assays indicate that the JE AP-1 site and the consensus AP-1 site have an overlapping but not identical binding spectrum for AP-1 proteins. Our data suggest that the inability of some AP-1 sites to respond to TPA is caused by subtle differences in affinity for AP-1 proteins.     

Evolution of the human alpha-amylase multigene family through unequal, homologous, and inter- and intrachromosomal crossovers

Human amylase haplotypes differ from each other by different numbers of a long direct repeat unit of approximately 100 kb, encompassing two complete salivary amylase genes and one amylase pseudogene lacking the first three exons. The two salivary genes are part of a 75-kb-long inverted repeat. Two short sequences, hybridizing with a probe containing exons 1-3, were found in the central part of the inverted repeat. Sequencing showed that these fragments, designated r, contain exon 3 sequences. We present evidence that these r-fragments and the pseudogene most likely are remnants of the same ancestral pancreatic gene. We determined the orientation of the exon 3 sequences present in the r-fragment and show that an inversion can explain their origination. Hybridization studies, using random fragments from the intergenic region of the AMY gene cluster as probes, enabled us to detect more extended homologous regions in this cluster than were found previously on the basis of restriction maps only. Together, these results allow us to present a model for the evolution of the human amylase multigene family by a number of consecutive events involving inter- and intrachromosomal crossovers.     

Use of a bioelectrochemical cell for the synthesis of (bio)chemicals

A bioelectrochemical cell containing either d-glucose oxidase (β-d-glucose:oxygen 1-oxidoreductase, EC 1.1.3.4) or xanthine oxidase (xanthine:oxygen oxidoreductase, EC 1.2.3.2) plus dichlorophenol-indophenol as electron acceptor in one half-cell, and chloroperoxidase (chloride:hydrogen-peroxide oxidoreductase, EC 1.11.1.10) in the other half-cell is described. Due to a combination of chemical, biochemical and electrochemical reactions, electricity and specific (bio)chemicals can be produced in the cell simultaneously and in both compartments. Furthermore, the oxidases in a bioelectrochemical cell are not inactivated by H₂O₂ and as a result the operational lifetimes of the oxidases were increased about five-fold.     

No predictive value of GC phenotypes for HIV infection and progression to AIDS

Summary Linkage data on human factor H (HF) and 22 other human genetic markers are presented. Close linkage at 0<0.10 can be ruled out for a series of marker systems (Rh, PGM1, ACP1, Jk, Tf, Gc, MNSs, ME2, HLA, GLO1, ORM, Gt, PI, Hp, GPT). Strong evidence for linkage was obtained for peptidase A (PEPA) with lods >3.0 at =0.10 in males and at =0.20 for the sexes combined. From this result the HF locus can be provisionally assigned tochromosome 18.     

Genetics of urinary pepsinogen: A new hypothesis

Summary A new genetic model is proposed to explain the inheritance of the urinary pepsinogen (PG1) polymorphism. Each main fraction, 3, 4 and 5, in the multibanded electrophoretic pattern, is determined by its own specific gene, B, C and D respectively. The intensity ratio of the fractions is principally determined by the number of gene copies. Accordingly, the PG1 phenotypes are determined by gene combinations, haplotypes, some of which may be identical to alleles in previous one locus models. Some critical families, not interpretable using previous genetic models, are presented to support the hypothesis. Preliminary population data from the Netherlands are described. The molecular background of this polymorphism and its relevance for gastric (pre)malignancy is discussed.     

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.