Identification of novel mutations in MLC1 responsible for megalencephalic leukoencephalopathy with subcortical cysts

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is an inherited neurologic disorder with macrocephaly before the age of one and slowly progressive deterioration of motor functions. Magnetic resonance imaging shows diffusely abnormal and swollen white matter of the cerebral hemispheres and the presence of subcortical cysts in the anterior-temporal region and often also in the frontoparietal region. Mutations in the MLC1 gene, encoding a putative membrane protein, have been recently identified as a cause for MLC. Here, we describe 14 new mutations in 18 patients. Two identified polymorphisms lead to alterations of amino acid residues. The role, suggested by others, of a mutation in the MLC1gene in catatonic schizophrenia and the possible function of the MLC1 protein as a cation channel are discussed.     

Hydrolytic activity in baker's yeast limits the yield of asymmetric 3-oxo ester reduction

Microbial reductions of ketones hold great potential for the production of enantiopure alcohols, as long as highly selective redox enzymes are not interfered with by competing activities. During reduction of ethyl 3-oxobutanoate by baker's yeast (Saccharomyces cerevisiae) to ethyl (S)-3-hydroxybutanoate, a high enantiomeric excess (> 99%) can be obtained. However, reported yields do not exceed 50-70%. In this article, three main causes are shown to be responsible for these low to moderate yields. These are evaporation of the substrate and product esters, absorption or adsorption of the two esters by the yeast cells and hydrolysis of the two esters by yeast enzymes. The hydrolysis products are further metabolized by the yeast. By reducing the evaporation and absorption losses, the reduction yield can easily be improved to about 85%. Improvement of the efficiency of the reduction and hence the reduction/hydrolysis ratio should lead to a further increase in yield.     

An interlaboratory comparison of physiological and genetic properties of four Saccharomyces cerevisiae strains

To select a Saccharomyces cerevisiae reference strain amenable to experimental techniques used in (molecular) genetic, physiological and biochemical engineering research, a variety of properties were studied in four diploid, prototrophic laboratory strains. The following parameters were investigated: 1) maximum specific growth rate in shake-flask cultures; 2) biomass yields on glucose during growth on defined media in batch cultures and steady-state chemostat cultures under controlled conditions with respect to pH and dissolved oxygen concentration; 3) the critical specific growth rate above which aerobic fermentation becomes apparent in glucose-limited accelerostat cultures; 4) sporulation and mating efficiency; and 5) transformation efficiency via the lithium-acetate, bicine, and electroporation methods. On the basis of physiological as well as genetic properties, strains from the CEN.PK family were selected as a platform for cell-factory research on the stoichiometry and kinetics of growth and product formation.     

Regulation of fermentative capacity and levels of glycolytic enzymes in chemostat cultures of Saccharomyces cerevisiae

Regulation of fermentative capacity was studied in chemostat cultures of two Saccharomyces cerevisiae strains: the laboratory strain CEN.PK113-7D and the industrial bakers’ yeast strain DS28911. The two strains were cultivated at a fixed dilution rate of 0.10 h⁻¹ under various nutrient limitation regimes: aerobic and anaerobic glucose limitation, aerobic and anaerobic nitrogen limitation on glucose, and aerobic ethanol limitation. Also the effect of specific growth rate on fermentative capacity was compared in glucose-limited, aerobic cultures grown at dilution rates between 0.05 h⁻¹ and 0.40 h⁻¹. Biomass yields and metabolite formation patterns were identical for the two strains under all cultivation conditions tested. However, the way in which environmental conditions affected fermentative capacity (assayed off-line as ethanol production rate under anaerobic conditions) differed for the two strains. A different regulation of fermentative capacity in the two strains was also evident from the levels of the glycolytic enzymes, as determined by in vitro enzyme assays. With the exception of phosphofructokinase and pyruvate decarboxylase in the industrial strain, no clear-cut correlation between the activities of glycolytic enzymes and the fermentative capacity was found. These results emphasise the need for controlled cultivation conditions in studies on metabolic regulation in S. cerevisiae and demonstrate that conclusions from physiological studies cannot necessarily be extrapolated from one S. cerevisiae strain to the other.     

Engineering of Saccharomyces cerevisiae for efficient anaerobic alcoholic fermentation of L-arabinose

For cost-effective and efficient ethanol production from lignocellulosic fractions of plant biomass, the conversion of not only major constituents, such as glucose and xylose, but also less predominant sugars, such as l-arabinose, is required. Wild-type strains of Saccharomyces cerevisiae, the organism used in industrial ethanol production, cannot ferment xylose and arabinose. Although metabolic and evolutionary engineering has enabled the efficient alcoholic fermentation of xylose under anaerobic conditions, the conversion of l-arabinose into ethanol by engineered S. cerevisiae strains has previously been demonstrated only under oxygen-limited conditions. This study reports the first case of fast and efficient anaerobic alcoholic fermentation of l-arabinose by an engineered S. cerevisiae strain. This fermentation was achieved by combining the expression of the structural genes for the l-arabinose utilization pathway of Lactobacillus plantarum, the overexpression of the S. cerevisiae genes encoding the enzymes of the nonoxidative pentose phosphate pathway, and extensive evolutionary engineering. The resulting S. cerevisiae strain exhibited high rates of arabinose consumption (0.70 g h(-1) g [dry weight](-1)) and ethanol production (0.29 g h(-1) g [dry weight](-1)) and a high ethanol yield (0.43 g g(-1)) during anaerobic growth on l-arabinose as the sole carbon source. In addition, efficient ethanol production from sugar mixtures containing glucose and arabinose, which is crucial for application in industrial ethanol production, was achieved.     

Formate as an Auxiliary Substrate for Glucose-Limited Cultivation of Penicillium chrysogenum: Impact on Penicillin G Production and Biomass Yield

Production of β-lactams by the filamentous fungus Penicillium chrysogenum requires a substantial input of ATP. During glucose-limited growth, this ATP is derived from glucose dissimilation, which reduces the product yield on glucose. The present study has investigated whether penicillin G yields on glucose can be enhanced by cofeeding of an auxiliary substrate that acts as an energy source but not as a carbon substrate. As a model system, a high-producing industrial strain of P. chrysogenum was grown in chemostat cultures on mixed substrates containing different molar ratios of formate and glucose. Up to a formate-to-glucose ratio of 4.5 mol·mol⁻¹, an increasing rate of formate oxidation via a cytosolic NAD⁺-dependent formate dehydrogenase increasingly replaced the dissimilatory flow of glucose. This resulted in increased biomass yields on glucose. Since at these formate-to-glucose ratios the specific penicillin G production rate remained constant, the volumetric productivity increased. Metabolic modeling studies indicated that formate transport in P. chrysogenum does not require an input of free energy. At formate-to-glucose ratios above 4.5 mol·mol⁻¹, the residual formate concentrations in the cultures increased, probably due to kinetic constraints in the formate-oxidizing system. The accumulation of formate coincided with a loss of the coupling between formate oxidation and the production of biomass and penicillin G. These results demonstrate that, in principle, mixed-substrate feeding can be used to increase the yield on a carbon source of assimilatory products such as β-lactams.     

Chromosomal Copy Number Variation in Saccharomyces pastorianus Is Evidence for Extensive Genome Dynamics in Industrial Lager Brewing Strains

Lager brewing strains of Saccharomyces pastorianus are natural interspecific hybrids originating from the spontaneous hybridization of Saccharomyces cerevisiae and Saccharomyces eubayanus. Over the past 500 years, S. pastorianus has been domesticated to become one of the most important industrial microorganisms. Production of lager-type beers requires a set of essential phenotypes, including the ability to ferment maltose and maltotriose at low temperature, the production of flavors and aromas, and the ability to flocculate. Understanding of the molecular basis of complex brewing-related phenotypic traits is a prerequisite for rational strain improvement. While genome sequences have been reported, the variability and dynamics of S. pastorianus genomes have not been investigated in detail. Here, using deep sequencing and chromosome copy number analysis, we showed that S. pastorianus strain CBS1483 exhibited extensive aneuploidy. This was confirmed by quantitative PCR and by flow cytometry. As a direct consequence of this aneuploidy, a massive number of sequence variants was identified, leading to at least 1,800 additional protein variants in S. pastorianus CBS1483. Analysis of eight additional S. pastorianus strains revealed that the previously defined group I strains showed comparable karyotypes, while group II strains showed large interstrain karyotypic variability. Comparison of three strains with nearly identical genome sequences revealed substantial chromosome copy number variation, which may contribute to strain-specific phenotypic traits. The observed variability of lager yeast genomes demonstrates that systematic linking of genotype to phenotype requires a three-dimensional genome analysis encompassing physical chromosomal structures, the copy number of individual chromosomes or chromosomal regions, and the allelic variation of copies of individual genes.     

Comparative proteomics of colon cancer stem cells and differentiated tumor cells identifies BIRC6 as a potential therapeutic target

Patients with liver metastases from colon carcinoma show highly variable responses to chemotherapy and tumor recurrence is frequently observed. Therapy-resistant cancer stem cells have been implicated in drug resistance and tumor recurrence. However, the factors determining therapy resistance and tumor recurrence are poorly understood. The aim of this study was to gain insight into these mechanisms by comparing the proteomes of patient-derived cancer stem cell cultures and their differentiated isogenic offspring. We established colonosphere cultures derived from resection specimens of liver metastases in patients with colon cancer. These colonospheres, enriched for colon cancer stem cells, were used to establish isogenic cultures of stably differentiated nontumorigenic progeny. Proteomics based on one-dimensional gel electrophoresis coupled to nano liquid chromatography tandem MS was used to identify proteome differences between three of these paired cultures. The resulting data were analyzed using Ingenuity Pathway Software. Out of a total data set of 3048 identified proteins, 32 proteins were at least twofold up-regulated in the colon cancer stem cells when compared with the differentiated cells. Pathway analysis showed that "cell death " regulation is strikingly different between the two cell types. Interestingly, one of the top-up-regulated proteins was BIRC6, which belongs to the class of Inhibitor of Apoptosis Proteins. Knockdown of BIRC6 sensitized colon cancer stem cells against the chemotherapeutic drugs oxaliplatin and cisplatin. This study reveals that differentiation of colon cancer stem cells is accompanied by altered regulation of cell death pathways. We identified BIRC6 as an important mediator of cancer stem cell resistance against cisplatin and oxaliplatin. Targeting BIRC6, or other Inhibitors of Apoptosis Proteins, may help eradicating colon cancer stem cells.