For ethanol production from lignocellulose, the fermentation of xylose is an economic necessity. Saccharomyces cerevisiae has been metabolically engineered with a xylose-utilizing pathway. However, the high ethanol yield and productivity seen with glucose have not yet been achieved. To quantitatively analyze metabolic fluxes in recombinant S. cerevisiae during metabolism of xylose-glucose mixtures, we constructed a stable xylose-utilizing recombinant strain, TMB 3001. The XYL1 and XYL2 genes from Pichia stipitis, encoding xylose reductase (XR) and xylitol dehydrogenase (XDH), respectively, and the endogenous XKS1 gene, encoding xylulokinase (XK), under control of the PGK1 promoter were integrated into the chromosomal HIS3 locus of S. cerevisiae CEN.PK 113-7A. The strain expressed XR, XDH, and XK activities of 0.4 to 0.5, 2.7 to 3.4, and 1.5 to 1.7 U/mg, respectively, and was stable for more than 40 generations in continuous fermentations. To obtain an economically feasible industrial process for ethanol production from lignocellulose, it is necessary to ferment all sugars present with high yields and productivities (53). The commonly used Saccharomyces cerevisiae has many advantages as an ethanol producer, such as fast sugar consumption, high ethanol yield from hexoses, and high resistance to inhibitory compounds that are present in the hydrolysates. However, a major drawback is that S. cerevisiae cannot utilize the pentose sugar xylose, only its isomer xylulose. In xylose-utilizing yeasts, the conversion from xylose to xylulose is a two-step process catalyzed by xylose reductase (XR) and xylitol dehydrogenase (XDH) (10), whereas bacteria perform the conversion in one step with xylose isomerase (XI) (23).Xylose fermentation by recombinant S. cerevisiae carrying heterologous XYL1 and XYL2 genes from Pichia stipitis, which encode XR and XDH, respectively, has resulted mainly in xylitol formation (24,44,48). Similarly, if xylA from Thermus thermophilus, which encodes XI, is introduced into S. cerevisiae, then only limited xylose fermentation is observed (47). Limited xylose fermentation by recombinant S. cerevisiae has been ascribed to poor xylose uptake (9, 24, 25), a cofactor imbalance generated by the discrepancy in cofactor usage by XR and XDH (8,24,49), limitations in the pentose phosphate pathway (12,24,38,48), and insufficient induction or activation of ethanologenic enzymes (5,17,20,29). When homologous XKS1, which encodes xylulokinase (XK), was overexpressed in a Saccharomyces sp. strain carrying XYL1 and XYL2, the ethanol yield and the xylose uptake rate increased under oxygenlimited conditions, but xylitol was still a major by-product (22).Although the shortcomings of xylose fermentation by recombinant S. cerevisiae have been investigated in several studies, data from anaerobic fermentations do not exist and quantitative data are sparse. Chemostat cultivations in which growth rate and concentrations of substrates and products are constant enable quantitative determinations of metabolic fluxes. Analysis o...
Fermentation of the pentose sugar xylose to ethanol in lignocellulosic biomass would make bioethanol production economically more competitive. Saccharomyces cerevisiae, an efficient ethanol producer, can utilize xylose only when expressing the heterologous genes XYL1 (xylose reductase) and XYL2 (xylitol dehydrogenase). Xylose reductase and xylitol dehydrogenase convert xylose to its isomer xylulose. The gene XKS1 encodes the xylulose-phosphorylating enzyme xylulokinase. In this study, we determined the effect of XKS1 overexpression on two different S. cerevisiae host strains, H158 and CEN.PK, also expressing XYL1 and XYL2. H158 has been previously used as a host strain for the construction of recombinant xylose-utilizing S. cerevisiae strains. CEN.PK is a new strain specifically developed to serve as a host strain for the development of metabolic engineering strategies. Fermentation was carried out in defined and complex media containing a hexose and pentose sugar mixture or a birch wood lignocellulosic hydrolysate. XKS1 overexpression increased the ethanol yield by a factor of 2 and reduced the xylitol yield by 70 to 100% and the final acetate concentrations by 50 to 100%. However, XKS1 overexpression reduced the total xylose consumption by half for CEN.PK and to as little as one-fifth for H158. Yeast extract and peptone partly restored sugar consumption in hydrolysate medium. CEN.PK consumed more xylose but produced more xylitol than H158 and thus gave lower ethanol yields on consumed xylose. The results demonstrate that strain background and modulation of XKS1 expression are important for generating an efficient xylose-fermenting recombinant strain of S. cerevisiae.
“Microthrix parvicella” strain RN1 was isolated from an activated sludge treatment plant in Italy using micromanipulation techniques. The strain grows as thin unbranched filaments which are Gram‐positive with Neisser‐positive granules. The isolate was characterized by analysis of the 16S rDNA which was amplified directly from cell biomass by the polymerase chain reaction and sequenced. “Microthrix parvicella” strain RN1 presents a very high similarity (100%) with another “M. parvicella” strain recently isolated in Australia, suggesting that this micro‐organism, a novel, deep branching member of the actinomycetes subphylum, is the same causing the common events of bulking and foaming phenomena in activated sludge treatment plants throughout the world.
Five isolates of a filamentous bacterial morphotype with the distinctive diagnostic microscopic features of Eikelboom Type 1863 were obtained from activated sludge sewage treatment plants in Victoria, Australia. On the basis of phenotypic evidence and 16S rDNA sequence data, these isolates proved to be polyphyletic. Two (Ben 06 and Ben 06C) are from the Chryseobacterium subgroup which is in the Cytophaga group, subdivision I of the Flexibacter–Cytophaga–Bacteroides phylum. Two (Ben 56 and Ben 59) belong to the genus Acinetobacter, and one (Ben 58) is a Moraxella sp., closest to Mor. osloensis. The significance of these findings to the reliance on microscopic features for identification of these filamentous bacteria in activated sludge is discussed.
The name Tetracoccus cechii is proposed for two strains of the tetrad arranged cocci, previously known as ‘G’ bacteria, which were isolated from laboratory scale activated sludge plants in the Czech Republic and in Italy. They were morphologically, phenotypically and phylogenetically characterized and found to comprise a novel lineage in the alpha‐3 group of the proteobacterial phylum in the domain Bacteria. The strains are Gram‐negative and produce intracellular inclusions of poly‐β‐hydroxybutyrate. Although commonly seen in activated sludge mixed liquor as cocci 1–2 μm in diameter, arranged in tetrads, in pure culture they can also grow in amorphous aggregations and the cells are generally more variable in their size and shape with coccobacilli as well as cocci being present. They are not able to grow phototrophically, nor can they reduce nitrate beyond nitrite nor grow anaerobically. The closest phylogenetic neighbours of T. cechii are Rhodobacter sphaeroides and R. capsulatus which are 93% similar by 16S rDNA comparison. Tetracoccus cechii is oxidase‐ and catalase‐positive, non‐motile and has an optimal growth temperature between 25° and 35°C. The 16S rRNA of T. cechii has a 21 nucleotide deletion in the V9 region (Escherichia coli positions 1258–1278) and this feature is a unique molecular synapomorphy in the alpha‐3 group.
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