RESUMEN
The effect of time and pressure on the selective extraction of sugar beet pectin using steam pre-treatment on unprocessed Sugar Beet Pulp was evaluated using a design of experiments approach. This process gave the highest solubilisation of pectin oligomers at a relatively low pressure and longer time (5Bar, 24min), whilst leaving the majority of the cellulose fraction intact. This method of steam pre-treatment fits into the concept of a sugar beet biorefinery as it valorises an existing waste stream without requiring any further physical processing such as milling or dilution with water. The residual cellulose fraction was enriched in cellulose and could be effectively fermented into ethanol by yeast after enzymatic digestion, producing 0.48g ethanol per gram of glucose.
Asunto(s)
Beta vulgaris/química , Fraccionamiento Químico/métodos , Fermentación , Temperatura , Arabinosa/análisis , Reactores Biológicos , Carbohidratos/análisis , Cromatografía Líquida de Alta Presión , Etanol/metabolismo , Hidrólisis , Modelos Teóricos , Proteínas/aislamiento & purificación , Reproducibilidad de los Resultados , Saccharomyces cerevisiae/metabolismo , SolubilidadRESUMEN
The environmental value of sustainably producing bioproducts from biomass is now widely appreciated, with a primary target being the economic production of fuels such as bioethanol from lignocellulose. The application of thermophilic prokaryotes is a rapidly developing niche in this field, driven by their known catabolic versatility with lignocellulose-derived carbohydrates. Fundamental to the success of this work has been the development of reliable genetic and molecular systems. These technical tools are now available to assist in the development of other (hyper)thermophilic strains with diverse phenotypes such as hemicellulolytic and cellulolytic properties, branched chain alcohol production and other 'valuable bioproduct' synthetic capabilities. Here we present an insight into the historical limitations, recent developments and current status of a number of genetic systems for thermophiles. We also highlight the value of reliable genetic methods for increasing our knowledge of thermophile physiology. We argue that the development of robust genetic systems is paramount in the evolution of future thermophilic based bioprocesses and make suggestions for future approaches and genetic targets that will facilitate this process.
Asunto(s)
Biotecnología/métodos , Celulosa/metabolismo , Enzimas/genética , Enzimas/metabolismo , Ingeniería Metabólica/métodos , Células Procariotas/enzimología , Células Procariotas/metabolismo , Biocombustibles , Hidrólisis , TemperaturaRESUMEN
We describe the metabolic engineering of two strains of Geobacillus thermoglucosidasius to divert their fermentative carbon flux from a mixed acid pathway, to one in which ethanol becomes the major product. This involved elimination of the lactate dehydrogenase and pyruvate formate lyase pathways by disruption of the ldh and pflB genes, respectively, together with upregulation of expression of pyruvate dehydrogenase. Unlike the situation in Escherichia coli, pyruvate dehydrogenase is active under anaerobic conditions in thermophilic bacilli, but expressed sub-optimally for a role as the primary fermentation pathway. Mutants were initially characterised in batch culture using glucose as carbon substrate and strains with all three modifications shown to form ethanol efficiently and rapidly at temperatures in excess of 60 degrees C in yields in excess of 90% of theoretical. The strain containing the 3 modifications, TM242, was also shown to efficiently ferment cellobiose and a mixed hexose and pentose feed.
Asunto(s)
Acetiltransferasas/metabolismo , Etanol/metabolismo , Eliminación de Gen , Mejoramiento Genético/métodos , Geobacillus/fisiología , L-Lactato Deshidrogenasa/metabolismo , Acetiltransferasas/genética , L-Lactato Deshidrogenasa/genética , Ingeniería de Proteínas/métodosRESUMEN
We have previously reported the disappearance of a specific strain degrading chlorobenzene from a functionally stable bioreactor. In the present work, we investigated this species succession and isolated a new dominant strain, identified as Pandoraea pnomenusa sp. strain MCB032. A specific 16S rRNA-targeted oligonucleotide probe was designed and validated to identify strain MCB032 using fluorescence in situ hybridisation (FISH). The results confirmed the presence of strain MCB032 in samples collected over time, and showed that it was primarily located within the biofilm. Denaturing gradient gel electrophoresis (DGGE) provided evidence that the species succession occurred early in the operating period. The application of these biomolecular tools highlighted the remarkable stability of this new strain during the 15 months of reactor operation. The succession was attributed to the competitive kinetic behaviour of strain MCB032, which exhibited faster growth (micro(max) = 0.34 h(-1)) and higher substrate affinity (K(s) = 0.35 mg L(-1)) than strain JS150. Finally, this study contributed to the characterisation of the recently established Pandoraea genus, an emerging group in the biodegradation field.
Asunto(s)
Reactores Biológicos/microbiología , Clorobencenos/metabolismo , Modelos Biológicos , Proteobacteria/citología , Proteobacteria/fisiología , Biodegradación Ambiental , Diferenciación Celular , Simulación por Computador , Proteobacteria/aislamiento & purificación , Especificidad de la EspecieRESUMEN
3-Hydroxybenzoate 6-hydroxylase from Klebsiella pneumoniae M5a1 is an enzyme that utilizes 3-hydroxybenzoate (3-HBA) as substrate yielding gentisate. Site-directed mutagenesis was carried out to define which residues may be involved in catalytic reaction. Substitution of arginine to glutamate at position 169 of the enzyme resulted in the complete loss of catalytic activity. This indicated Arg169 may play an important role in 3-HBA 6-hydroxylase catalysis.
Asunto(s)
Arginina/metabolismo , Klebsiella pneumoniae/enzimología , Oxigenasas de Función Mixta/metabolismo , Secuencia de Aminoácidos , Arginina/química , Gentisatos/metabolismo , Hidroxibenzoatos/metabolismo , Oxigenasas de Función Mixta/química , Oxigenasas de Función Mixta/genética , Datos de Secuencia Molecular , Mutación , Alineación de SecuenciaRESUMEN
Thermoanaerobacter ethanolicus JW200 Fe(7) was grown in continuous culture, using xylose as the primary carbon source, with progressively lower concentrations of supplementary yeast extract. This enabled the comparison of metabolic flux to fermentation end-products under carbon-limited and carbon-sufficient (yeast extract-limited) conditions and the determination of process data under fully mass-balanced conditions. Under carbon-limitation, the specific ethanol-formation rate was described by q (p)=40.34 micro +3.74, the specific rate of substrate utilisation for maintenance was 0.31+/-0.02 g x g(-1) x h(-1) and the maximum cell yield on xylose, corrected for maintenance requirements, was 0.15+/-0.04 g x g(-1). Based on the product profiles, these corresponded to a maintenance coefficient of m(ATP)=4.1+/-0.5 mmol x g(-1) x h(-1) and a maximum cell yield of = 14.7+/-0.8 x g x mol(-1). Limitation by a component in yeast extract resulted in incomplete xylose utilisation, increased catabolic flux rates (primarily resulting in increased lactate production, due to limitations in the flux through the phosphoroclastic reaction), a reduction in cell yield = 10.0+/-1.0 g x mol(-1) and an increase in maintenance energy requirements of m(ATP)=7.95+/-0.7 mmol x g(-1). The latter was also reflected in a shift from ethanol to acetate production at lower growth rates. An analysis of ethanol and acetate tolerance indicated that any high-intensity process employing this strain would require a bioreactor design which incorporated continuous ethanol stripping.
Asunto(s)
Bacillaceae/metabolismo , Reactores Biológicos , Medios de Cultivo/farmacología , Microbiología Industrial/instrumentación , Xilosa/metabolismo , Acetatos/metabolismo , Bacillaceae/efectos de los fármacos , Bacillaceae/crecimiento & desarrollo , Técnicas Bacteriológicas , Biomasa , Carbono/metabolismo , Etanol/metabolismo , Etanol/farmacología , Fermentación , Lactatos/metabolismo , Oxidación-Reducción , Extractos Vegetales/farmacología , Propilenglicol/metabolismo , LevadurasRESUMEN
Benzene dioxygenase (BDO; EC 1.14.12.3) from Pseudomonas putida ML2 dihydroxylates benzene to produce cis-1,2-dihydroxy-cyclohexa-3,5-diene. As well as oxidising benzene and toluene, cell-free extracts of Escherichia coli JM109 expressing recombinant BDO oxidised cyclohexene, 1-methylcyclohexene and 3-methylcyclohexene. In an attempt to construct a novel metabolic pathway for the degradation of cyclohexene (via an initial BDO-mediated dihydroxylation of cyclohexene), cis-1,2-cyclohexanediol-degrading bacteria were isolated by enrichment culture. The bedC1C2BA genes encoding BDO (under the control of the tac promoter) were sub-cloned into pLAFR5, successfully conjugated into seven of the Gram-negative cis-1,2-cyclo-hexanediol-degrading isolates and stably maintained and expressed in three of them. However, despite their ability to grow on cis-1,2-cyclohexanediol as sole carbon source, express an active BDO and oxidise cyclohexene, none of the three strains was able to grow on cyclohexene as sole carbon source. Analysis revealed that BDO oxidised cyclohexene to a mixture of two products, a monohydroxylated (2-cyclohexen-1-ol) product and a dihydroxylated (cis-1,2-cyclohexanediol) product; and failure to grow on cyclohexene was attributed to the toxicity of metabolic intermediates accumulating from the 2-cyclohexen-1-ol metabolism.
Asunto(s)
Ciclohexanos/metabolismo , Ciclohexanoles/metabolismo , Oxigenasas de Función Mixta/metabolismo , Pseudomonas putida/enzimología , Pseudomonas/enzimología , Biodegradación Ambiental , Clonación Molecular , Medios de Cultivo , Ciclohexenos , Escherichia coli/genética , Genes Bacterianos , Oxigenasas de Función Mixta/genética , Oxidación-Reducción , Plásmidos , Pseudomonas/genética , Pseudomonas/aislamiento & purificación , Pseudomonas aeruginosa/enzimología , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/aislamiento & purificación , Pseudomonas putida/genética , Pseudomonas putida/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
The genes encoding the six polypeptide components of the alkene monooxygenase from Xanthobacter strain Py2 (Xamo) have been located on a 4.9-kb fragment of chromosomal DNA previously cloned in cosmid pNY2. Sequencing and analysis of the predicted amino acid sequences indicate that the components of Xamo are homologous to those of the aromatic monooxygenases, toluene 2-, 3-, and 4-monooxygenase and benzene monooxygenase, and that the gene order is identical. The genes and predicted polypeptides are aamA, encoding the 497-residue oxygenase alpha-subunit (XamoA); aamB, encoding the 88-residue oxygenase gamma-subunit (XamoB); aamC, encoding the 122-residue ferredoxin (XamoC); aamD, encoding the 101-residue coupling or effector protein (XamoD); aamE, encoding the 341-residue oxygenase beta-subunit (XamoE); and aamF, encoding the 327-residue reductase (XamoF). A sequence with >60% concurrence with the consensus sequence of sigma54 (RpoN)-dependent promoters was identified upstream of the aamA gene. Detailed comparison of XamoA with the oxygenase alpha-subunits from aromatic monooxygenases, phenol hydroxylases, methane monooxygenase, and the alkene monooxygenase from Rhodococcus rhodochrous B276 showed that, despite the overall similarity to the aromatic monooxygenases, XamoA has some distinctive characteristics of the oxygenases which oxidize aliphatic, and particularly alkene, substrates. On the basis of the similarity between Xamo and the aromatic monooxygenases, Xanthobacter strain Py2 was tested and shown to oxidize benzene, toluene, and phenol, while the alkene monooxygenase-negative mutants NZ1 and NZ2 did not. Benzene was oxidized to phenol, which accumulated transiently before being further oxidized. Toluene was oxidized to a mixture of o-, m-, and p-cresols (39.8, 18, and 41.7%, respectively) and a small amount (0.5%) of benzyl alcohol, none of which were further oxidized. In growth studies Xanthobacter strain Py2 was found to grow on phenol and catechol but not on benzene or toluene; growth on phenol required a functional alkene monooxygenase. However, there is no evidence of genes encoding steps in the metabolism of catechol in the vicinity of the aam gene cluster. This suggests that the inducer specificity of the alkene monooxygenase may have evolved to benefit from the naturally broad substrate specificity of this class of monooxygenase and the ability of the host strain to grow on catechol.
Asunto(s)
Benceno/metabolismo , Bacterias Gramnegativas/enzimología , Oxigenasas/metabolismo , Fenol/metabolismo , Tolueno/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Biodegradación Ambiental , Genes Bacterianos , Bacterias Gramnegativas/genética , Datos de Secuencia Molecular , Oxigenasas/química , Oxigenasas/genética , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
A 3.5 kb EcoRI-BamHI fragment of Bacillus subtilis chromosomal DNA carrying the ribR gene, involved in regulation of the B. subtilis riboflavin operon, was cloned in the B. subtilis-Escherichia coli shuttle vector pCB20. DNA sequence analysis of this fragment revealed several ORFs, one of which encodes a polypeptide of 230 amino acids with up to 45% sequence identity with FAD synthetases from a number of micro-organisms, such as Corynebacterium ammoniagenes, E. coli and Pseudomonas fluorescens, and also to the ribC gene product of B. subtilis. The ribR gene was amplified by PCR, cloned and expressed in E. coli. Measurement of flavokinase activity in cell extracts demonstrated that ribR encodes a monofunctional flavokinase which converts riboflavin into FMN but not to FAD, and is specific for the reduced form of riboflavin.
Asunto(s)
Bacillus subtilis/genética , Regulación Bacteriana de la Expresión Génica , Operón , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Riboflavina/metabolismo , Secuencia de Aminoácidos , Bacillus subtilis/enzimología , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Clonación Molecular , Escherichia coli/genética , Mononucleótido de Flavina/metabolismo , Flavina-Adenina Dinucleótido/metabolismo , Genes Bacterianos/genética , Datos de Secuencia Molecular , Mutación , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Sistemas de Lectura Abierta/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Regiones Promotoras Genéticas/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Mapeo Restrictivo , Riboflavina/análogos & derivados , Riboflavina/química , Riboflavina/genética , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
Few strains of thermophilic Bacillus spp are readily transformable with plasmid DNA. Given the considerable phylogenetic and phenotypic diversity amongst thermophilic bacilli, we have examined whether transformability is a trait associated with a particular phylogenetic group, by sequencing the 16S ribosomal RNA genes from transformable strains NUB3621, K1041, and NRRL1174. Although all of these strains were described in the literature as B. stearothermophilus, only NRRL1174 is closely related to the type strain of this species. Based on its 16S rDNA sequence and physiological data K1041 appeared to belong to the species B. thermodenitrificans, while NUB3621 showed a slightly closer relationship to B. thermoglucosidasius than to B. stearothermophilus. Therefore we conclude that the trait of transformability, though possibly strain-specific, is not limited to a single species of thermophilic Bacillus.
Asunto(s)
Geobacillus stearothermophilus/genética , Filogenia , Transformación Bacteriana , ADN Bacteriano/genética , ADN Ribosómico/genética , Genes de ARNr , Geobacillus stearothermophilus/crecimiento & desarrollo , Geobacillus stearothermophilus/fisiología , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa/métodos , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADNRESUMEN
The genes encoding the six polypeptide components of the alkene monooxygenase from Xanthobacter Py2 have been sequenced. The predicted amino acid sequence of the first ORF shows homology with the iron binding subunits of binuclear non-haem iron containing monooxygenases including benzene monooxygenase, toluene 4-monooxygenase (> 60% sequence similarity) and methane monooxygenase (> 40% sequence similarity) and that the necessary sequence motifs associated with iron co-ordination are also present. Secondary structure prediction based on the amino acid sequence showed that the predominantly alpha-helical structure that surrounds the binuclear iron binding site was conserved allowing the sequence to be modelled on the co-ordinates of the methane monooxygenase alpha-subunit. Significant differences in the residues forming the hydrophobic cavity which forms the substrate binding site are discussed with reference to the differences in reaction specificity and stereospecificity of binuclear non-haem iron monooxygenases.
Asunto(s)
Bacterias Aerobias Gramnegativas/enzimología , Modelos Moleculares , Oxigenasas/química , Secuencia de Aminoácidos , Sitios de Unión , Secuencia Conservada/genética , Genes Bacterianos/genética , Bacterias Aerobias Gramnegativas/genética , Hierro/química , Datos de Secuencia Molecular , Oxigenasas/genética , Estructura Secundaria de Proteína , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
The epoxide hydrolase (EH) from Corynebacterium sp. C12, which grows on cyclohexene oxide as sole carbon source, has been purified to homogeneity in two steps, involving anion exchange followed by hydrophobic-interaction chromatography. The purified enzyme is multimeric (probably tetrameric) with a subunit size of 32,140 Da. The gene encoding Corynebacterium EH was located on a 3.5-kb BamHI fragment of C12 chromosomal DNA using a DNA probe generated by PCR using degenerate primers based on the N-terminal and an internal amino acid sequence. Sequencing and database comparison of the predicted amino acid sequence of Corynebacterium EH shows that it is similar to mammalian and plant soluble EH, and the recently published sequence of epichlorohydrin EH from Agrobacterium radiobacter AD1 [Rink, R., Fennema, M., Smids, M., Dehmel, U. & Janssen, D. B. (1997) J. Biol. Chem. 272, 14650- 14657), particularly around the catalytic site. All of these proteins belong to the alpha/beta-hydrolase-fold family of enzymes. Similarity to the mammalian microsomal EH is weaker.
Asunto(s)
Corynebacterium/enzimología , Epóxido Hidrolasas/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Composición de Base , Secuencia de Bases , Sitios de Unión , Clonación Molecular , Codón/genética , Corynebacterium/genética , Corynebacterium/crecimiento & desarrollo , Ciclohexanos/metabolismo , Ciclohexenos , Cartilla de ADN/genética , ADN Bacteriano/química , ADN Bacteriano/genética , Epóxido Hidrolasas/química , Epóxido Hidrolasas/genética , Genes Bacterianos , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Homología de Secuencia de AminoácidoRESUMEN
1,2-dibromoethane (DBE) is a common environmental contaminant; it is potentially carcinogenic and has been detected in soil and groundwater supplies. Most of the biodegradation studies to date have been performed under anaerobic conditions or in the context of soil remediation, where the pollutant concentration was in the parts per billion range. In this work a mixed bacterial culture capable of complete aerobic mineralization of concentrations of DBE up to 1 g liter(-1) under well-controlled laboratory conditions was enriched. In order to verify biodegradation, formation of biodegradation products as well as the disappearance of DBE from the biological medium were measured. Complete mineralization was verified by measuring stoichiometric release of the biodegradation products. This mixed culture was found to be capable of degrading other halogenated compounds, including bromoethanol, the degradation of which has not been reported previously.
Asunto(s)
Contaminantes Ambientales/metabolismo , Dibromuro de Etileno/metabolismo , Aerobiosis , Biodegradación AmbientalRESUMEN
Epoxypropane isomerase from Xanthobacter Py2 has been resolved into at least two components (A and B) by ion-exchange chromatography. Both components were required for the degradation of epoxypropane and were purified further. Component A was apparently homohexameric with a subunit M(r) of about 44,000, and possessed NAD(+)-dependent dihydrolipoamide dehydrogenase activity and lipoamide reductase activity. It was sensitive to inhibition by o-phenanthroline and the thiol-specific reagents N-ethylmaleimide(NEM)and p-chloromercuribenzoate. Component B was homodimeric with a subunit M(r), of 62,170 and contained 2 mol.mol-1 FAD. It had an NADPH-dependent lipoamide reductase activity which was sensitive to NEM and p-chloromercuribenzoate. The N-terminal amino acid sequences and monomer sizes of components A and B correspond to those of ORF1 and ORF3 respectively (ORF = open reading frame) of a recently published sequence of a clone which complements mutants unable to degrade epoxypropane. NADPH was found to replace the need for a low-M(r), fraction in epoxypropane degradation assays containing components A and B and NAD+. The predicted amino acid sequence of component A (ORF1) has been analysed and shown to contain a potential ADP binding site near the N-terminus and putative cofactor binding domain near the C-terminus, with sequence similarity to the biotinyl and lipoyl binding domains of biotin-dependent carboxylases and 2-oxoacid dehydrogenases respectively. A reaction mechanism for epoxypropane degradation, incorporating recent evidence for combined isomerization and carboxylation to acetoacetate, is discussed.
Asunto(s)
Compuestos Epoxi/metabolismo , Bacterias Aerobias Gramnegativas/enzimología , Oxidorreductasas Intramoleculares , Isomerasas/aislamiento & purificación , Complejos Multienzimáticos/aislamiento & purificación , Oxidorreductasas/aislamiento & purificación , Secuencia de Aminoácidos , Isomerasas/genética , Isomerasas/metabolismo , Datos de Secuencia Molecular , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/metabolismo , Sistemas de Lectura Abierta , Oxidación-Reducción , Oxidorreductasas/genética , Oxidorreductasas/metabolismoRESUMEN
Continuous fermentation with cell recycle proved very effective in increasing the ethanol volumetric productivity of the thermophilic facultative anaerobe, Bacillus stearothermophilus strain LLD-15, on sucrose at 70 degrees C. When complete cell recycle was used, cell viability decreased after a few residence times and sucrose consumption was reduced. Operation using a constant bleed rate resulted in greater stability and higher ethanol volumetric productivities. A mathematical model based on maintenance energy requirements provided an adequate description of the system.
RESUMEN
Whole cells of the propene utilizing Mycobacterium sp. M156 (NCIMB 40156) oxidised styrene, 2-,3- and 4-fluorostyrene, 3- and 4-chlorostyrene and 3- and 4-methylstyrenes to their respective epoxides. Rates of oxidation were comparable to that of styrene. alpha-Methylstyrene was also epoxidised at a lower rate, while trans-beta-methylstyrene and 1,2-dihydronaphthalene were poor substrates. In those cases that were investigated, epoxidation occurred with a high degree of stereospecificity.
Asunto(s)
Compuestos Epoxi/síntesis química , Mycobacterium/metabolismo , Estirenos/síntesis química , Biotransformación , Cromatografía de Gases , Compuestos Epoxi/química , Compuestos Epoxi/metabolismo , Cromatografía de Gases y Espectrometría de Masas , Cinética , Rotación Óptica , Estereoisomerismo , Estireno , Estirenos/química , Estirenos/metabolismo , Especificidad por SustratoRESUMEN
A synthetic medium was developed by the pulse and medium-shift technique for the continuous cultivation of Bacillus stearothermophilus strain LLD-15 (NCIMB 12428) under anaerobic conditions. This mutant strain lacks L-lactate dehydrogenase activity, and is a promising candidate for the production of ethanol from pentoses and hexoses, using a high-temperature two-stage process. The final medium contained four amino acids and five vitamins, and growth characteristics in this medium compared well with those in complex medium containing yeast extract and tryptone. At 70 degrees C, the medium was capable of supporting good anaerobic and aerobic growth at 10 g input sucrose l-1. High ethanol production indicated that pyruvate metabolism probably occurred via the combined activity of the pyruvate-formate-lyase pathway and pyruvate dehydrogenase.
Asunto(s)
Medios de Cultivo , Geobacillus stearothermophilus/crecimiento & desarrollo , L-Lactato Deshidrogenasa/metabolismo , Aminoácidos/metabolismo , Anaerobiosis , Técnicas Bacteriológicas , Extractos Celulares , Lactatos/biosíntesis , Ácido Láctico , Mutación , Peptonas/metabolismo , Vitaminas/metabolismoRESUMEN
Choline, a component of the wall teichoic acid of Streptococcus pneumoniae, was converted to cytidine diphosphocholine via choline phosphate by enzymes which were identified in cell-free extracts of the pneumococcus. The first enzyme, choline kinase, was investigated in some detail. It appeared to have a pH optimum of 7.3 to 7.4 and was stimulated by Mg2+. Kinetic studies gave an apparent Michaelis constant (Km) for ATP of I mM, and for choline of 0.19 mM, with Vmax values of 3 nmol min-1 (mg protein)-1 and 0.5 nmol min-1 (mg protein)-1 respectively. The second enzyme, CDPcholine pyrophosphorylase was specific for CTP and had a requirement for Mg2+ with an optimum at 7 mM.