Biotechnological production of L-ribose from L-arabinose.

L-Ribose is a rare and expensive sugar that can be used as a precursor for the production of L-nucleoside analogues, which are used as antiviral drugs. In this work, we describe a novel way of producing L-ribose from the readily available raw material L-arabinose. This was achieved by introducing L-ribose isomerase activity into L-ribulokinase-deficient Escherichia coli UP1110 and Lactobacillus plantarum BPT197 strains. The process for L-ribose production by resting cells was investigated. The initial L-ribose production rates at 39 degrees C and pH 8 were 0.46 +/- 0.01 g g(-1) h(-1) (1.84 +/- 0.03 g l(-1) h(-1)) and 0.27 +/- 0.01 g g(-1) h(-1) (1.91 +/- 0.1 g l(-1) h(-1)) for E. coli and for L. plantarum, respectively. Conversions were around 20% at their highest in the experiments. Also partially purified protein precipitates having both L-arabinose isomerase and L-ribose isomerase activity were successfully used for converting L-arabinose to L-ribose.

Metabolic engineering of Lactobacillus plantarum for production of L-ribulose.

L-Ribulose is a rare and expensive sugar that can be used as a precursor for the production of other rare sugars of high market value such as L-ribose. In this work we describe a production process for L-ribulose using L-arabinose, a common component of polymers of lignocellulosic materials, as the starting material. A ribulokinase-deficient mutant of the heterofermentative lactic acid bacterium Lactobacillus plantarum NCIMB8826 was constructed. Expression of araA, which encodes the critical enzyme L-arabinose isomerase, was repressed by high glucose concentrations in batch cultivations. A fed-batch cultivation strategy was therefore used to maximize L-arabinose isomerase production during growth. Resting cells of the ribulokinase-deficient mutant were used for the production of L-ribulose. The isomerization of L-arabinose to L-ribulose was very unfavorable for L-ribulose formation. However, high L-ribulose yields were obtained by complexing the produced L-ribulose with borate. The process for L-ribulose production in borate buffer by resting cells was optimized using central composite designs. The experiment design suggested that the process has an optimal operation point around an L-arabinose concentration of 100 g liter(-1), a borate concentration of 500 mM, and a temperature of 48 degrees C, where the statistical software predicted an initial L-ribulose production rate of 29.1 g liter(-1) h(-1), a best-achievable process productivity of 14.8 g liter(-1) h(-1), and a conversion of L-arabinose to L-ribulose of 0.70 mol mol(-1).

Cloning and expression of a xylitol-4-dehydrogenase gene from Pantoea ananatis.

The Pantoea ananatis ATCC 43072 mutant strain is capable of growing with xylitol as the sole carbon source. The xylitol-4-dehydrogenase (XDH) catalyzing the oxidation of xylitol to L-xylulose was isolated from the cell extract of this strain. The N-terminal amino acid sequence of the purified protein was determined, and an oligonucleotide deduced from this peptide sequence was used to isolate the xylitol-4-dehydrogenase gene (xdh) from a P. ananatis gene library. Nucleotide sequence analysis revealed an open reading frame of 795 bp, encoding the xylitol-4-dehydrogenase, followed by a 5' region of another open reading frame encoding an unknown protein. Results from a Northern analysis of total RNA isolated from P. ananatis ATCC 43072 suggested that xdh is transcribed as part of a polycistronic mRNA. Reverse transcription-PCR analysis of the transcript confirmed the operon structure and suggested that xdh was the first gene of the operon. Homology searches revealed that the predicted amino acid sequence of the P. ananatis XDH shared significant identity (38 to 51%) with members of the short-chain dehydrogenase/reductase family. The P. ananatis xdh gene was successfully overexpressed in Escherichia coli, XDH was purified to homogeneity, and some of its enzymatic properties were determined. The enzyme had a preference for NAD+ as the cosubstrate, and in contrast to previous reports, the enzyme also showed a side activity for the D-form of xylulose. Xylitol was converted to L-xylulose with a high yield (>80%) by the resting recombinant cells, and the L-xylulose was secreted into the medium. No evidence of D-xylulose being synthesized by the recombinant cells was found.

Actinopolyspora halophila has two separate pathways for betaine synthesis.

The extremely halophilic actinomycete Actinopolyspora halophila is a rare example of a heterotrophic eubacterium producing betaine from simple carbon sources. A. halophila synthesized remarkably high intracellular concentrations of betaine. The highest betaine concentration, determined at 24% (w/v) NaCl, was 33% of the cellular dry weight. Trehalose was synthesized as a compatible solute, accounting for up to 9.7% of the cellular dry weight. The betaine concentration was shown to increase with increasing NaCl concentration, whereas the trehalose concentration was highest at the lowest NaCl concentration used (15% w/v). A. halophila was capable of accumulating betaine from the medium, while at the same time betaine was also excreted back into the medium by the cells. Along with the de novo synthesis of betaine, A. halophila was able to take up choline from the medium and oxidize it to betaine. Some basic characteristics of the choline oxidation system are described. Choline was oxidized to betaine aldehyde in a reaction in which H2O2 generation and oxygen consumption were coupled. Betaine aldehyde was also oxidized, but with lesser efficiency. In addition, betaine aldehyde was oxidized further to betaine in a reaction in which NAD(P)+ was reduced.

Improved osmotolerance of recombinant Escherichia coli by de novo glycine betaine biosynthesis.

The genes from the extreme halophile Ecto-thiorhodospira halochloris encoding the biosynthesis of glycine betaine from glycine were cloned into Escherichia coli. The accumulation of glycine betaine and its effect on osmotolerance of the cells were studied. In mineral medium with NaCl concentrations from 0.15 to 0.5 M, the accumulation of both endogenously synthesized and exogenously provided glycine betaine stimulated the growth of E. coli. The intracellular levels of glycine betaine and the cellular yields were clearly higher for cells receiving glycine betaine exogenously than for cells synthesizing it. The lower level of glycine betaine accumulation in cells synthesizing it is most likely a consequence of the limited availability of precursors (e.g. S-adenosylmethionine) rather than the result of a low expression level of the genes. Glycine betaine also stimulated the growth of E. coli and decreased acetate formation in mineral medium with high sucrose concentrations (up to 200 g.l(-1)).

Characterization of glycine sarcosine N-methyltransferase and sarcosine dimethylglycine N-methyltransferase.

Glycine betaine is accumulated in cells living in high salt concentrations to balance the osmotic pressure. Glycine sarcosine N-methyltransferase (GSMT) and sarcosine dimethylglycine N-methyltransferase (SDMT) of Ectothiorhodospira halochloris catalyze the threefold methylation of glycine to betaine, with S-adenosylmethionine acting as the methyl group donor. These methyltransferases were expressed in Escherichia coli and purified, and some of their enzymatic properties were characterized. Both enzymes had high substrate specificities and pH optima near the physiological pH. No evidence of cofactors was found. The enzymes showed Michaelis-Menten kinetics for their substrates. The apparent K(m) and V(max) values were determined for all substrates when the other substrate was present in saturating concentrations. Both enzymes were strongly inhibited by the reaction product S-adenosylhomocysteine. Betaine inhibited the methylation reactions only at high concentrations.

Extreme halophiles synthesize betaine from glycine by methylation.

Glycine betaine is a compatible solute, which is able to restore and maintain osmotic balance of living cells. It is synthesized and accumulated in response to abiotic stress. Betaine acts also as a methyl group donor and has a number of important applications including its use as a feed additive. The known biosynthetic pathways of betaine are universal and very well characterized. A number of enzymes catalyzing the two-step oxidation of choline to betaine have been isolated. In this work we have studied a novel betaine biosynthetic pathway in two phylogenically distant extreme halophiles, Actinopolyspora halophila and Ectothiorhodospira halochloris. We have identified a three-step series of methylation reactions from glycine to betaine, which is catalyzed by two methyltransferases, glycine sarcosine methyltransferase and sarcosine dimethylglycine methyltransferase, with partially overlapping substrate specificity. The methyltransferases from the two organisms show high sequence homology. E. halochloris methyltransferase genes were successfully expressed in Escherichia coli, and betaine accumulation and improved salt tolerance were demonstrated.