Fermentation of L-arabinose,D-xylose and D-glucose by ethanologenic recombinant Klebsiella oxytoca strain P2

Summary Recombinant Klebsiella oxytoca strain P2 carrying genes for pyruvate decarboxylase and alcohol dehydrogenase from Zymomonas mobilis was evaluated for its ability to ferment arabinose, xylose and glucose alone and in mixtures in pH-controlled batch fermentations. This organism produced 0.34–0.43 g ethanol/g sugar at pH 6.0 and 30°C on 8% sugar substrate and demonstrated a preference for glucose. Sugar utilization was glucose > arabinose > xylose and ethanol production was xylose > glucose > arabinose.     

Fed-batch fermentor synthesis of 3-dehydroshikimic acid using recombinant Escherichia coli.

3-Dehydroshikimic acid (DHS), in addition to being a potent antioxidant, is the key hydroaromatic intermediate in the biocatalytic conversion of glucose into aromatic bioproducts and a variety of industrial chemicals. Microbial synthesis of DHS, like other intermediates in the common pathway of aromatic amino acid biosynthesis, has previously been examined only under shake flask conditions. In this account, synthesis of DHS using recombinant Escherichia coli constructs is examined in a fed-batch fermentor where glucose availability, oxygenation levels, and solution pH are controlled. DHS yields and titers are also determined by the activity of 3-deoxy-D-arabino-heptulosonic acid 7-phosphate (DAHP) synthase. This enzyme's expression levels, sensitivity to feedback inhibition, and the availability of its substrates, phosphoenolpyruvate (PEP) and D-erythrose 4-phosphate (E4P), dictate its in vivo activity. By combining fed-batch fermentor control with amplified expression of a feedback-insensitive isozyme of DAHP synthase and amplified expression of transketolase, DHS titers of 69 g/L were synthesized in 30% yield (mol/mol) from D-glucose. Significant concentrations of 3-dehydroquinic acid (6.8 g/L) and gallic acid (6.6 g/L) were synthesized in addition to DHS. The pronounced impact of transketolase overexpression, which increases E4P availability, on DHS titers and yields indicates that PEP availability is not a limiting factor under the fed-batch fermentor conditions employed.     

Uptake of D-xylose and D-glucose by Spirochaeta aurantia.

Uptake of D-[14C]glucose and D-[14C]xylose by Spirochaeta aurantia was demonstrated to be osmotic shock sensitive and to require a high-energy phosphorylated compound rather than a proton motive force. These features are similar to those of binding protein-mediated transport systems in other gram-negative bacteria.     

Reactions of D-xylose and D-glucose in alkaline,aqueous solutions

Treatment of D-xylose and D-glucose with 0.63M sodium hydroxide at 96° in an atmosphere of nitrogen yielded, in addition to acidic, aliphatic degradation-products, the following cyclic enols and phenolic compounds: 2-hydroxy-3-methyl-2-cyclopenten-1-one (1), 2-hydroxy-3,4-dimethyl-2-cyclopenten-1-one (2), pyrocatechol (3), 3-methyl-1,2-benzenediol (4), 4-methyl-1,2-benzenediol (5), 3,4-di-methyl-1,2-benzenediol (6), 2-methyl-1,4-benzenediol (7), 2,5-dihydroxyacetophe-none (8), 3-hydroxy-5-methylacetophenone (9), 3,4-dihydroxyacetophenone (10), 3,4-hydroxybenzaldehyde (11), 2,3,4-trihydroxy-5-methylacetophenone (12), and 2,3-dihydroxy-6-methylacetophenone (13).     

Induction of NADPH-linked D-xylose reductase and NAD-linked xylitol dehydrogenase activities in Pachysolen tannophilus by D-xylose, L-arabinose, or D-galactose

Considerable interest in the D-xylose catabolic pathway of Pachysolen tannophilus has arisen from the discovery that this yeast is capable of fermenting D-xylose to ethanol. In this organism D-xylose appears to be catabolized through xylitol to D-xylulose. NADPH-linked D-xylose reductase is primarily responsible for the conversion of D-xylose to xylitol, while NAD-linked xylitol dehydrogenase is primarily responsible for the subsequent conversion of xylitol to D-xylulose. Both enzyme activities are readily detectable in cell-free extracts of P. tannophilus grown in medium containing D-xylose, L-arabinose, or D-galactose and appear to be inducible since extracts prepared from cells growth in media containing other carbon sources have only negligible activities, if any. Like D-xylose, L-arabinose and D-galactose were found to serve as substrates for NADPH-linked reactions in extracts of cells grown in medium containing D-xylose, L-arabinose, or D-galactose. These L-arabinose and D-galactose NADPH-linked activities also appear to be inducible, since only minor activity with L-arabinose and no activity with D-galactose is detected in extracts of cells grown in D-glucose medium. The NADPH-linked activities obtained with these three sugars may result from the actions of distinctly different enzymes or from a single aldose reductase acting on different substrates. High-performance liquid chromatography and gas-liquid chromatography of in vitro D-xylose, L-arabinose, and D-galactose NADPH-linked reactions confirmed xylitol, L-arabitol, and galactitol as the respective conversion products of these sugars. Unlike xylitol, however, neither L-arabitol nor galactitol would support comparable NAD-linked reaction(s) in cellfree extracts of induced P. tannophilus. Thus, the metabolic pathway of D-xylose diverges from those of L-arabinose or D-galactose following formation of the pentitol.     

6-Deoxy-D-glucose and D-xylose: Analogs for the study of D-glucose transport by mouse 3T3 cells

6-Deoxy-D-glucose and D-xylose, structural homomorphs of D-glucose that lack a 6-hydroxyl group or a 6-hydroxymethyl group, respectively, are transported efficiently by mouse 3T3 cells, with good affinity and high specificity for the D-glucose transport system. Since these analogs lack the 6-hydroxyl group, which is the site of phosphorylation of glucose by hexokinase, they are taken up and are recoverable from cells in an unchanged state. Thus, 6-deoxy-D-glucose and D-xylose offer advantages as transport substrates over 2-deoxy-D-glucose, which is phosphorylated by intercellular hexokinases, and 3-O-methyl-D-glucose, which shows a lower specificity for the D-glucose transport system.     

Regulation and butanol inhibition of D-xylose and D-glucose uptake in Clostridium acetobutylicum.

Clostridium acetobutylicum exhibited diauxie growth in the presence of mixtures of glucose and xylose. Both glucose- and xylose-grown cells had a glucose uptake activity. On the other hand, growth on xylose was associated with the induction of a xylose permease activity, which was repressed by glucose in xylose-induced cells. The rate of sugar uptake with increasing sugar concentrations showed saturation kinetics with an apparent Km of 1.25 X 10(-5) M for glucose and 5 X 10(-3) M for xylose. Concomitant with the production of solvents, the activities of the glucose and xylose transport systems decreased. Among the main products of fermentation, butanol was shown to be a potent inhibitor of the growth of the organism and of the rate of sugar uptake as well as of sugar incorporation into cell materials. These inhibitory effects of butanol were more pronounced in xylose-grown cells than in glucose-grown cells. Butanol completely inhibited growth at a concentration of 14 g/liter for cultures growing on glucose and 8 g/liter for cultures growing on xylose. Concentrations of 7 and 10.5 g/liter of butanol caused a 50% inhibition of the xylose and glucose incorporations into cell materials. These inhibitory levels of butanol were found in typical glucose or xylose fermentation.     

Comparison of the periplasmic receptors for L-arabinose, D-glucose/D-galactose, and D-ribose. Structural and Functional Similarity.

The primary sequence of the receptor for L-arabinose or Ara-binding protein (ABP) composed of 306 residues is very different from the D-glucose/D-galactose-binding protein (GGBP) which consists of 309 residues. Nevertheless, superimpositioning of the well-refined high resolution structures of ABP in complex with D-galactose and the GGBP in complex with D-glucose shows very similar structures; 220 of the residues (or about 70%) have a root mean square deviation of 2.0 A. From the superpositioning, nine pairs of continuous segments (consisting of 8-51 residues), mainly alpha-helices and beta-strands that form the core of the two lobes of the bilobate proteins were found to exhibit strong sequence homology. The equivalenced structures and aligned sequences show that many of the polar, as well as aromatic residues, in the sugar-binding sites located in the cleft between the two lobes are highly conserved. Surprisingly, however, the exact mode of binding of the D-galactose in ABP is totally different from that of the D-glucose in GGBP. Using the structurally aligned sequences of the ABP and GGBP as a template, we have matched the sequence of the ribose-binding protein (RBP) which consists of 271 residues with the ABP/GGBP pair. Although the nine aligned segments of all three proteins show little sequence identity, they have significant homology. Four additional segments of RBP were matched only with GGBP, leading to the alignment of about 90% of the RBP sequence with the GGBP sequence. Many of the conserved residues in the binding sites of ABP and GGBP matched with similar residues in RBP. Additional observations indicate that the GGBP/RBP pair is more closely related than the ABP/RBP or ABP/GGBP pair. All three binding proteins, which may have diverged from a common ancestor, serve as primary receptors for bacterial high affinity active transport systems. Moreover, GGBP and RBP, but not ABP, also act as receptors for chemotaxis. An exposed site located in one domain, which includes Gly74, for interacting with the trg transmembrane signal transducer that is involved in triggering chemotaxis has been located in the structure of GGBP (Vyas, N.K., Vyas, M.N., and Quiocho, F.A. (1988) Science 242, 1290-1295). Whereas the site is absent in the structure of ABP, it is strongly predicted to be present in RBP which shares the same trg transducer with GGBP. The knowledge-based alignment of RBP further revealed two possible additional peripheral chemotactic sites that show high structural and sequence similarity between GGBP and RBP only. At least one of these sites, together with the one proven to exist in the other domain, could be used by the signal transducer with which both binding proteins interact in a way which the substrate-loaded "closed cleft" structure could be discriminated from the unliganded "open cleft" form by the transducer.     

Pentose degradation in archaea: Halorhabdus species degrade D-xylose,L-arabinose and D-ribose via bacterial-type pathways

Extremophiles - The degradation of the pentoses d-xylose, l-arabinose and d-ribose in the domain of archaea, in Haloferax volcanii and in Haloarcula and Sulfolobus species, has been shown to...     

Transcriptional analysis of catabolite repression in Clostridium acetobutylicum growing on mixtures of D-glucose and D-xylose

Clostridium acetobutylicum is a strict anaerobic organism that is used for biotechnological butanol fermentation. It ferments various hexoses and pentoses to solvents but prefers glucose presumably using a catabolite repression mechanism. Accordingly during growth on a mixture of D-glucose and D-xylose a typical diauxic growth pattern was observed. We used DNA microarrays and real-time RT-PCR to study gene expression during growth on D-glucose, D-xylose mixtures on a defined minimal medium together with monitoring substrate consumption and product formation. We identified two putative operons involved in D-xylose degradation. The first operon (CAC1344-CAC1349) includes a transporter, a xylulose-kinase, a transaldolase, a transketolase, an aldose-1-epimerase and a putative xylose isomerase that has been annotated as an arabinose isomerase. This operon is induced by D-xylose but was catabolite repressed by D-glucose. A second operon (CAC2610-CAC2612) consists of a xylulose-kinase, a hypothetical protein and a gene that has been annotated as a L-fucose isomerase that might in fact code for a xylose isomerase. This operon was induced by D-xylose but was not subject to catabolite repression. In accordance with these results we identified a CRE site in the catabolite repressed operon but not in the operon that was not subject to catabolite repression.     

Convenient conversion of wheat hemicelluloses pentoses (D-xylose and L-arabinose) into a common intermediate

The transformation of D-xylose and L-arabinose, the two major components of wheat straw and bran, into a unique multifunctional, optically pure, five-carbon synthon has been achieved. The synthetic sequence requires three steps: suitable protection of the hydroxyl groups of the pentoses, introduction of an iodide at the C-5 position and zinc-mediated opening of the furanose ring leading to the formation of a common substituted pent-4-enal.     

Amplification of D-xylose and D-glucose isomerase activities in Escherichia coli by gene cloning.

A recombinant plasmid, designated pUC1002, was constructed by ligation of a HindIII restriction endonuclease fragment of Escherichia coli chromosomal DNA to vector plasmid pMB9. Strains carrying this plasmid were selected by transformation of an E. coli strain bearing the xyl-7 mutation to a xylose-positive (Xyl+) phenotype. Strains containing pUC1002 produced coordinately elevated levels of D-xylose isomerase and D-xylulose kinase. Under appropriate conditions, the isomerase also efficiently catalyzed the conversion of D-glucose to D-fructose.     

Simultaneous utilization of D-cellobiose, D-glucose, and D-xylose by recombinant Corynebacterium glutamicum under oxygen-deprived conditions

Corynebacterium glutamicum R was metabolically engineered to broaden its sugar utilization range to d-xylose and d-cellobiose contained in lignocellulose hydrolysates. The resultant recombinants expressed Escherichia coli xylA and xylB genes, encoding d-xylose isomerase and xylulokinase, respectively, for d-xylose utilization and expressed C. glutamicum R bglF ^317A and bglA genes, encoding phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) β-glucoside-specific enzyme IIBCA component and phospho-β-glucosidase, respectively, for d-cellobiose utilization. The genes were fused to the non-essential genomic regions distributed around the C. glutamicum R chromosome and were under the control of their respective constitutive promoter trc and tac that permitted their expression even in the presence of d-glucose. The enzyme activities of resulting recombinants increased with the increase in the number of respective integrated genes. Maximal sugar utilization was realized with strain X5C1 harboring five xylA–xylB clusters and one bglF ^317A –bglA cluster. In both d-cellobiose and d-xylose utilization, the sugar consumption rates by genomic DNA-integrated strain were faster than those by plasmid-bearing strain, respectively. In mineral medium containing 40 g l⁻¹ d-glucose, 20 g l⁻¹ d-xylose, and 10 g l⁻¹ d-cellobiose, strain X5C1 simultaneously and completely consumed these sugars within 12 h and produced predominantly lactic and succinic acids under growth-arrested conditions.     

Microbial synthesis of p-hydroxybenzoic acid from glucose.

A series of recombinant Escherichia coli strains have been constructed and evaluated for their ability to synthesize p-hydroxybenzoic acid from glucose under fed-batch fermentor conditions. The maximum concentration of p-hydroxybenzoic acid synthesized was 12 g/L and corresponded to a yield of 13% (mol/mol). Synthesis of p-hydroxybenzoic acid began with direction of increased carbon flow into the common pathway of aromatic amino acid biosynthesis. This was accomplished in all constructs with overexpression of a feedback-insensitive isozyme of 3-deoxy-D-arabino-heptulosonic acid 7-phosphate synthase. Expression levels of enzymes in the common pathway of aromatic amino acid biosynthesis were also increased in all constructs to deliver increased carbon flow from the beginning to the end of the common pathway. A previously unreported inhibition of 3-dehydroquinate synthase by L-tyrosine was discovered to be a significant impediment to the flow of carbon through the common pathway. Chorismic acid, the last metabolite of the common pathway, was converted into p-hydroxybenzoic acid by ubiC-encoded chorismate lyase. Constructs differed in the strategy used for overexpression of chorismate lyase and also differed as to whether mutations were present in the host E. coli to inactivate other chorismate-utilizing enzymes. Use of overexpressed chorismate lyase to increase the rate of chorismic acid aromatization was mitigated by attendant decreases in the specific activity of DAHP synthase and feedback inhibition caused by p-hydroxybenzoic acid. The toxicity of p-hydroxybenzoic acid towards E. coli metabolism and growth was also evaluated.