Amino acid production from rice straw and wheat bran hydrolysates by recombinant pentose-utilizing <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Corynebacterium glutamicum wild type lacks the ability to utilize the pentose fractions of lignocellulosic hydrolysates, but it is known that recombinants expressing the araBAD operon and/or the xylA gene from Escherichia coli are able to grow with the pentoses xylose and arabinose as sole carbon sources. Recombinant pentose-utilizing strains derived from C. glutamicum wild type or from the l-lysine-producing C. glutamicum strain DM1729 utilized arabinose and/or xylose when these were added as pure chemicals to glucose-based minimal medium or when they were present in acid hydrolysates of rice straw or wheat bran. The recombinants grew to higher biomass concentrations and produced more l-glutamate and l-lysine, respectively, than the empty vector control strains, which utilized the glucose fraction. Typically, arabinose and xylose were co-utilized by the recombinant strains along with glucose either when acid rice straw and wheat bran hydrolysates were used or when blends of pure arabinose, xylose, and glucose were used. With acid hydrolysates growth, amino acid production and sugar consumption were delayed and slower as compared to media with blends of pure arabinose, xylose, and glucose. The ethambutol-triggered production of up to 93 ± 4 mM l-glutamate by the wild type-derived pentose-utilizing recombinant and the production of up to 42 ± 2 mM l-lysine by the recombinant pentose-utilizing lysine producer on media containing acid rice straw or wheat bran hydrolysate as carbon and energy source revealed that acid hydrolysates of agricultural waste materials may provide an alternative feedstock for large-scale amino acid production.     

Ethanol production during D-xylose,L-arabinose,and D-ribose fermentation by Bacteroides polypragmatus

Summary The specific growth rate () during cultivation of Bacteroides polypragmatus in 2.51 batch cultures in 4–5% (w/v) l-arabinose medium was 0.23 h^-1 while that in either d-xylose or d-ribose medium was lower (=0.19 h^-1). Whereas growth on arabinose or xylose occurred after about 6–8 h lag period, growth on ribose commenced after a 30 h lag phase. The maximum substrate utilization rate for arabinose, ribose and xylose in media with an initial substrate concentration of 4–5% (w/v) was 0.77, 0.76, and 0.60 g/l/h respectively. In medium containing a mixture of glucose, arabinose, and xylose, the utilization of all three substrates occurred concurrently. The maximum amount of ethanol produced after 72 h growth in 4–5% (w/v) of arabinose, xylose, and ribose was 9.4, 6.5, and 5.3 g/l, respectively. The matabolic end products (mol/mol substrate) of growth in 4.4% (w/v) xylose medium were 0.73 ethanol, 0.49 acetate, 1.39 CO₂, 1.05 H₂, and 0.09 butyrate.National Research Council of Canada No. 23406     

The metabolic significance of octulose phosphates in the photosynthetic carbon reduction cycle in spinach

¹⁴C-Labelled octulose phosphates were formed during photosynthetic ¹⁴CO₂ fixation and were measured in spinach leaves and chloroplasts. Because mono- and bisphosphates of d-glycero- d-ido-octulose are the active 8-carbon ketosugar intermediates of the L-type pentose pathway, it was proposed that they may also be reactants in a modified Calvin–Benson–Bassham pathway reaction scheme. This investigation therefore initially focussed only on the ido-epimer of the octulose phosphates even though ¹⁴C-labelled d-glycero- d-altro-octulose mono- and bisphosphates were also identified in chloroplasts and leaves. ¹⁴CO₂ predominantly labelled positions 5 and 6 of d-glycero- d-ido-octulose 1,8-P₂ consistent with labelling predictions of the modified scheme. The kinetics of ¹⁴CO₂ incorporation into ido-octulose was similar to its incorporation into some traditional intermediates of the path of carbon, while subsequent exposure to ¹²CO₂ rapidly displaced the ¹⁴C isotope label from octulose with the same kinetics of label loss as some of the confirmed Calvin pathway intermediates. This is consistent with octulose phosphates having the role of cyclic intermediates rather than synthesized storage products. (Storage products don’t rapidly exchange isotopically labelled carbons with unlabelled CO₂.) A spinach chloroplast extract, designated stromal enzyme preparation (SEP), catalysed and was used to measure rates of CO₂ assimilation with Calvin cycle intermediates and octulose and arabinose phosphates. Only pentose (but not arabinose) phosphates and sedoheptulose 7-phosphate supported CO₂ fixation at rates in excess of 120 μmol h⁻¹ mg⁻¹ Chl. Rates for octulose, sedoheptulose and fructose bisphosphates, octulose, hexose and triose monophosphates were all notably less than the above rate and arabinose 5-phosphate was inactive. Altro-octulose phosphates were more active than phosphate esters of the ido-epimer. The modified scheme proposed a specific phosphotransferase and SEP unequivocally catalysed reversible phosphate transfer between sedoheptulose bisphosphate and d-glycero- d-ido-octulose 8-phosphate. It was also initially hypothesized that arabinose 5-phosphate, an L-Type pentose pathway reactant, may have a role in a modified Calvin pathway. Arabinose 5-phosphate is present in spinach chloroplasts and leaves. Radiochromatography showed that ¹⁴C-arabinose 5-phosphate with SEP, but only in the presence of an excess of unlabelled ribose 5-phosphate, lightly labelled ribulose 5-phosphate and more heavily labelled hexose and sedoheptulose mono- and bisphosphates. However, failure to demonstrate any CO₂ fixation by arabinose 5-phosphate as sole substrate suggested that the above labelling may have no metabolic significance. Despite this arabinose and ribose 5-phosphates are shown to exhibit active roles as enzyme co-factors in transaldolase and aldolase exchange reactions that catalyse the epimeric interconversions of the phosphate esters of ido- and altro-octulose. Arabinose 5-phosphate is presented as playing this role in a New Reaction Scheme for the path of carbon, where it is concluded that slow reacting ido-octulose 1,8 bisphosphate has no role. The more reactive altro-octulose phosphates, which are independent of the need for phosphotransferase processing, are presented as intermediates in the new scheme. Moreover, using the estimates of phosphotransferase activity with altro-octulose monophosphate as substrate allowed calculation of the contributions of the new scheme, that ranged from 11% based on the intact chloroplast carboxylation rate to 80% using the carboxylation rate required for the support of octulose phosphate synthesis and its role in the phosphotransferase reaction.     

Bioconversion of lignocellulose-derived sugars to ethanol by engineered Saccharomyces cerevisiae

Lignocellulosic biomass from agricultural and agro-industrial residues represents one of the most important renewable resources that can be utilized for the biological production of ethanol. The yeast Saccharomyces cerevisiae is widely used for the commercial production of bioethanol from sucrose or starch-derived glucose. While glucose and other hexose sugars like galactose and mannose can be fermented to ethanol by S. cerevisiae, the major pentose sugars D-xylose and L-arabinose remain unutilized. Nevertheless, D-xylulose, the keto isomer of xylose, can be fermented slowly by the yeast and thus, the incorporation of functional routes for the conversion of xylose and arabinose to xylulose or xylulose-5-phosphate in Saccharomyces cerevisiae can help to improve the ethanol productivity and make the fermentation process more cost-effective. Other crucial bottlenecks in pentose fermentation include low activity of the pentose phosphate pathway enzymes and competitive inhibition of xylose and arabinose transport into the cell cytoplasm by glucose and other hexose sugars. Along with a brief introduction of the pretreatment of lignocellulose and detoxification of the hydrolysate, this review provides an updated overview of (a) the key steps involved in the uptake and metabolism of the hexose sugars: glucose, galactose, and mannose, together with the pentose sugars: xylose and arabinose, (b) various factors that play a major role in the efficient fermentation of pentose sugars along with hexose sugars, and (c) the approaches used to overcome the metabolic constraints in the production of bioethanol from lignocellulose-derived sugars by developing recombinant S. cerevisiae strains.     

Fermentation of corn fiber hydrolysate to lactic acid by the moderate thermophile <Emphasis Type="Italic">Bacillus coagulans</Emphasis>

A strain of Bacillus coagulans that converted mixed sugars of glucose, xylose, and arabinose to l-lactic acid with 85% yield at 50°C was isolated from composted dairy manure. The strain was tolerant to aldehyde growth inhibitors at 2.5 g furfural/l, 2.5 g 5-hydroxymethylfurfural/l, 2.5 g vanillin/l, and 1.2 g p-hydroxybenzaldehyde/l. In a simultaneous saccharification and fermentation process, the strain converted a dilute-acid hydrolysate of 100 g corn fiber/l to 39 g lactic acid/l in 72 h at 50°C. Because of its inhibitor tolerance and ability to fully utilize pentose sugars, this strain has potential to be developed as a biocatalyst for the conversion of agricultural residues into valuable chemicals.     

Endogeneous β-<Emphasis Type="SmallCaps">d</Emphasis>-xylosidase and α-<Emphasis Type="SmallCaps">l</Emphasis>-arabinofuranosidase activity in flax seed mucilage

Flax seed mucilage (FM) contains a mixture of highly doubly substituted arabinoxylan as well as rhamnogalacturonan I with unusual side group substitutions. Treatment of FM with a GH11 Bacillus subtilis XynA endo 1,4-β-xylanase (BsX) gave limited formation of reducing ends but when BsX and FM were incubated together on different wheat arabinoxylan substrates and birchwood xylan, significant amounts of xylose were released. Moreover, arabinose was released from both water-extractable and water-unextractable wheat arabinoxylan. Since no xylose or arabinose was released by BsX addition alone on these substrates, nor without FM or BsX addition, the results indicate the presence of endogenous β-d-xylosidase and α-l-arabinofuranosidase activities in FM. FM also exhibited activity on both p-nitrophenyl α-l-arabinofuranoside (pNPA) and p-nitrophenyl β-d-xylopyranoside (pNPX). Based on K _M values, the FM enzyme activities had a higher affinity for pNPX (K _M 2 mM) than for pNPA (K _M 20 mM).     

Metabolic evolution of non-transgenic <Emphasis Type="Italic">Escherichia coli</Emphasis> SZ420 for enhanced homoethanol fermentation from xylose

Efficient utilization of pentose sugars (xylose and arabinose) is an essential requirement for economically viable ethanol production from cellulosic biomass. The desirable pentose-fermenting ethanologenic biocatalysts are the native microorganisms or the engineered derivatives without recruited exogenous gene(s). We have used a metabolic evolution (adaptive selection) approach to improve a non-transgenic homoethanol Escherichia coli SZ420 (ldhA pflB ackA frdBC pdhR::pflBp6-aceEF-lpd) for xylose fermentation. An improved mutant, E. coli KC01, was evolved through a 3 month metabolic evolution process. This evolved mutant increased pyruvate dehydrogenase activity by 100%, cell growth rate (h⁻¹) by 23%, volumetric ethanol productivity by 65% and ethanol tolerance by 200%. These improvements enabled KC01 to complete 50 g xylose l⁻¹ fermentations with an ethanol titer of 23 g l⁻¹ and a yield of 90%. The improved cell growth and ethanol production of KC01 are likely attributed to its three fold increased ethanol tolerance.     

Recombinant Escherichia coli engineered for production of L-lactic acid from hexose and pentose sugars

Recombinant Escherichia coli have been constructed for the conversion of glucose as well as pentose sugars into L-lactic acid. The strains carry the lactate dehydrogenase gene from Streptococcus bovis on a low copy number plasmid for production of L-lactate. Three E. coli strains were transformed with the plasmid for producing L-lactic acid. Strains FBR9 and FBR11 were serially transferred 10 times in anaerobic cultures in sugar-limited medium containing glucose or xylose without selective antibiotic. An average of 96% of both FBR9 and FBR11 cells maintained pVALDH1 in anaerobic cultures. The fermentation performances of FBR9, FBR10, and FBR11 were compared in pH-controlled batch fermentations with medium containing 10% w/v glucose. Fermentation results were superior for FBR11, an E. coli B strain, compared to those observed for FBR9 or FBR10. FBR11 exhausted the glucose within 30 h, and the maximum lactic acid concentration (7.32% w/v) was 93% of the theoretical maximum. The other side-products detected were cell mass and succinic acid (0.5 g/l). Journal of Industrial Microbiology & Biotechnology (2001) 27, 259–264. Received 05 November 2000/ Accepted in revised form 03 July 2001     

Growth of engineered Pseudomonas putida KT2440 on glucose,xylose, and arabinose: Hemicellulose hydrolysates and their major sugars as sustainable carbon sources

Lignocellulosic biomass is the most abundant bioresource on earth containing polymers mainly consisting of d ‐glucose, d ‐xylose, l ‐arabinose, and further sugars. In order to establish this alternative feedstock apart from applications in food, we engineered Pseudomonas putida KT2440 as microbial biocatalyst for the utilization of xylose and arabinose in addition to glucose as sole carbon sources. The d ‐xylose‐metabolizing strain P. putida KT2440_xylAB and l ‐arabinose‐metabolizing strain P. putida KT2440_araBAD were constructed by introducing respective operons from Escherichia coli. Surprisingly, we found out that both recombinant strains were able to grow on xylose as well as arabinose with high cell densities and growth rates comparable to glucose. In addition, the growth characteristics on various mixtures of glucose, xylose, and arabinose were investigated, which demonstrated the efficient co‐utilization of hexose and pentose sugars. Finally, the possibility of using lignocellulose hydrolysate as substrate for the two recombinant strains was verified. The recombinant P. putida KT2440 strains presented here as flexible microbial biocatalysts to convert lignocellulosic sugars will undoubtedly contribute to the economic feasibility of the production of valuable compounds derived from renewable feedstock.     

Hierarchy in Pentose Sugar Metabolism in Clostridium acetobutylicum

Bacterial metabolism of polysaccharides from plant detritus into acids and solvents is an essential component of the terrestrial carbon cycle. Understanding the underlying metabolic pathways can also contribute to improved production of biofuels. Using a metabolomics approach involving liquid chromatography-mass spectrometry, we investigated the metabolism of mixtures of the cellulosic hexose sugar (glucose) and hemicellulosic pentose sugars (xylose and arabinose) in the anaerobic soil bacterium Clostridium acetobutylicum. Simultaneous feeding of stable isotope-labeled glucose and unlabeled xylose or arabinose revealed that, as expected, glucose was preferentially used as the carbon source. Assimilated pentose sugars accumulated in pentose phosphate pathway (PPP) intermediates with minimal flux into glycolysis. Simultaneous feeding of xylose and arabinose revealed an unexpected hierarchy among the pentose sugars, with arabinose utilized preferentially over xylose. The phosphoketolase pathway (PKP) provides an alternative route of pentose catabolism in C. acetobutylicum that directly converts xylulose-5-phosphate into acetyl-phosphate and glyceraldehyde-3-phosphate, bypassing most of the PPP. When feeding the mixture of pentose sugars, the labeling patterns of lower glycolytic intermediates indicated more flux through the PKP than through the PPP and upper glycolysis, and this was confirmed by quantitative flux modeling. Consistent with direct acetyl-phosphate production from the PKP, growth on the pentose mixture resulted in enhanced acetate excretion. Taken collectively, these findings reveal two hierarchies in clostridial pentose metabolism: xylose is subordinate to arabinose, and the PPP is used less than the PKP.     

Improved strains of recombinant Escherichia coli for ethanol production from sugar mixtures

Hemicellulose hydrolysates of agricultural residues often contain mixtures of hexose and pentose sugars. Ethanologenic Escherichia coli that have been previously investigated preferentially ferment hexose sugars. In some cases, xylose fermentation was slow or incomplete. The purpose of this study was to develop improved ethanologenic E. coli strains for the fermentation of pentoses in sugar mixtures. Using fosfomycin as a selective agent, glucose-negative mutants of E. coli KO11 (containing chromosomally integrated genes encoding the ethanol pathway from Zymomonas mobilis) were isolated that were unable to ferment sugars transported by the phosphoenolpyruvate-dependent phosphotransferase system. These strains (SL31 and SL142) retained the ability to ferment sugars with independent transport systems such as arabinose and xylose and were used to ferment pentose sugars to ethanol selectively in the presence of high concentrations of glucose. Additional fosfomycin-resistant mutants were isolated that were superior to strain KO11 for ethanol production from hexose and pentose sugars. These hyperproductive strains (SL28 and SL40) retained the ability to metabolize all sugars tested, completed fermentations more rapidly, and achieved higher ethanol yields than the parent. Both SL28 and SL40 produced 60 gl^–1 ethanol from 120 gl^–1 xylose in 60 h, 20% more ethanol than KO11 under identical conditions. Further studies illustrated the feasibility of sequential fermentation. A mixture of hexose and pentose sugars was fermented with near theoretical yield by SL40 in the first step followed by a second fermentation in which yeast and glucose were added. Such a two-step approach can combine the attributes of ethanologenic E. coli for pentoses with the high ethanol tolerance of conventional yeasts in a single vessel.     

The impact of supplemental carbon sources on <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> growth,chlorophyll content and anthocyanin accumulation

This research explores the impacts of a broad range of supplemental carbon sources on growth and development of Arabidopsis thaliana. Parameters measured include dark-germinated hypocotyl length, light-germinated root growth, rosette growth, chlorophyll concentration and anthocyanin content. Treatment sugars include sucrose, maltose, d-glucose, d-fructose, l-arabinose, l-fucose, d-galactose, d-mannose, l-rhamnose and d-xylose each supplied at 4, 20 or 100 mM. This comparison of the effect of different carbon sources on multiple parameters and under identical conditions showed that every carbon source had unique qualitative and quantitative effects on Arabidopsis growth and development. Root growth was particularly sensitive to supplemental carbon source. Growth on 100 mM sucrose, maltose, glucose or xylose stimulated root growth by ~100%. Growth on arabinose, fucose, galactose, mannose or rhamnose inhibited root growth by 50% or more. Several sugars that strongly inhibited root growth had either no effect (galactose and fucose) or a positive effect (arabinose) on hypocotyl elongation and rosette growth. Rhamnose was the only carbon source that inhibited hypocotyl elongation across all concentrations. Sucrose, maltose, glucose, fructose, arabinose or xylose stimulated rosette growth by ~50%. Chlorophyll content was strongly reduced by mannose while sucrose, glucose, galactose and rhamnose caused smaller reductions. Anthocyanin accumulation was strongly induced by both galactose and mannose. Only mannose impacted all parameters across all concentrations. Based on these data it can be concluded that the effect of each carbon source on Arabidopsis growth and development is specific in terms of both magnitude and the parameters impacted.     

Utilization of Xylose for Growth by the Eukaryotic Alga, Chlorella

A green alga, Chlorella, was found to be capable of utilizing xylose or other pentose sugars (xylitol, arabinose) for enhanced growth rates when grown in the light, but not when grown heterotrophically in the dark. With selection for growth in xylose-containing medium, it was possible to improve dramatically the ability of selected Chlorella strains to grow on xylose mixotrophically. Growth on arabinose or xylitol was not changed in the xylose-selected strains. Received: 5 November 1998 / Accepted: 25 January 1999     

Occurrence of O-methylated sugars in surface glycoconjugates in Chlamydomonas eugametos

Previously, we have shown that the monomeric-sugar composition of cell-surface-associated glycoconjugates of two strains of Chlamydomonas eugametos, of different mating type, differs strikingly (Gerwig et al. 1984, Carbohydr. Res. 127, 245–251). Besides the common occurrence of various pentoses and hexoses, the glycoconjugates of one strain contain 4-O-methyl xylose, a 2-O-methyl pentose (probably 2-O-methyl arabinose) and 3-O-methyl galactose, whereas those of the other strain contain 6-O-methyl mannose and 3-O-methyl glucose. In order to investigate whether these differences are relevant to the mating process of this organism, the sugar composition of the sexual progeny of these strains was analyzed. The ability to produce 4-O-methyl xylose, 2-O-methyl pentose and 3-O-methyl galactose on the one hand, and the ability to produce 6-O-methyl mannose and 3-O-methyl glucose on the other hand, appear to be genetically linked. However, the ability to produce either set of O-methyl sugars was inherited independently of mating type. O-Methylated sugars do not occur in the cell wall of C. eugametos, or in the cell-free medium, but only in surface-membrane-associated glycoconjugates, extractable with salt or detergent solutions.Abbreviation mt ^+/- mating-type plus or minus     

Pentose transport by the ruminal bacterium Butyrivibrio fibrisolvens

Abstract Butyrivibrio fibrisolvens is a fibrolytic ruminal bacterium that degrades hemicellulose and ferments the resulting pentose sugars. Washed cells of strain D1 accumulated radiolabelled xylose ( K _m= 1.5 μ M) and arabinose ( K _m= 0.2 μ M) when the organism was grown on xylose, arabinose, or glucose, but cultures grown on sucrose or cellobiose had little capacity to transport pentose. Glucose and xylose inhibited transport of each other non-competitively. Both sugars were utilized preferentially over arabinose, but since they did not inhibit transport of arabinose, it appeared that the preference was related to an internal metabolic step. Although the protonmotive force was completely abolished by ionophores, cells retained some ability to transport pentose. In contrast, the metabolic inhibitors iodoacetate, arsenate, and fluoride had little effect on protonmotive force but caused a large decrease in intracellular ATP and xylose and arabinose uptake. These results suggested that high-affinity, ATP-dependent mechanisms were responsible for pentose transport and hexose sugars affected the utilization of xylose and arabinose.     

Production of L-arabitol from L-arabinose by Candida entomaea and Pichia guilliermondii

 Lignocellulosic biomass, particularly corn fiber, represents a renewable resource that is available in sufficient quantities from the corn wet milling industry to serve as a low cost feedstock for production of fuel alcohol and valuable coproducts. Several enzymatic and chemical processes have potential for the conversion of cellulose and hemicellulose to fermentable sugars. The hydrolyzates are generally rich in pentoses (D-xylose and L-arabinose) and D-glucose. Yeasts produce a variety of polyalcohols from pentose and hexose sugars. Many of these sugar alcohols have food applications as low-calorie bulking agents. During the screening of 49 yeast strains capable of growing on L-arabinose, we observed that two strains were superior secretors of L-arabitol as a major extracellular product of L-arabinose. Candida entomaea NRRL Y-7785 and Pichia guilliermondii NRRL Y-2075 produced L-arabitol (0.70 g/g) from L-arabinose (50 g/l) at 34°C and pH 5.0 and 4.0, respectively. Both yeasts produced ethanol (0.32–0.33 g/g) from D-glucose (50 g/l) and only xylitol (0.43–0.51 g/g) from D-xylose (50 g/l). Both strains preferentially utilized D-glucose>D-xylose>L-arabinose from mixed substrate (D-glucose, D-xylose and L-arabinose, 1:1:1, 50 g/l, total) and produced ethanol (0.36–0.38 g/g D-glucose), xylitol (0.02–0.08 g/g D-xylose) and L-arabitol (0.70–0.81 g/g L-arabinose). The yeasts co-utilized D-xylose (6.2–6.5 g/l) and L-arabinose (4.9–5.0 g/l) from corn fiber acid hydrolyzate simultaneously and produced xylitol (0.10 g/g D-xylose) and L-arabitol (0.53–0.54 g/g L-arabinose). Received: 24 April 1995/Received revision: 9 August 1995/Accepted: 7 September 1995     

H<Subscript>2</Subscript> synthesis from pentoses and biomass in <Emphasis Type="Italic">Thermotoga</Emphasis> spp.

We have investigated H₂ production on glucose, xylose, arabinose, and glycerol in Thermotoga maritima and T. neapolitana. Both species metabolised all sugars with hydrogen yields of 2.7–3.8 mol mol⁻¹ sugar. Both pentoses were at least comparable to glucose with respect to their qualities as substrates for hydrogen production, while glycerol was not metabolised by either species. Glycerol was also not metabolised by T. elfii. We also demonstrated that T. neapolitana can use wet oxidised wheat straws, in which most sugars are stored in glycoside polymers, for growth and efficient hydrogen production, while glucose, xylose and arabinose are consumed in parallel.     

Deletion of methylglyoxal synthase gene (<Emphasis Type="Italic">mgsA</Emphasis>) increased sugar co-metabolism in ethanol-producing <Emphasis Type="Italic">Escherichia coli</Emphasis>

The use of lignocellulose as a source of sugars for bioproducts requires the development of biocatalysts that maximize product yields by fermenting mixtures of hexose and pentose sugars to completion. In this study, we implicate mgsA encoding methylglyoxal synthase (and methylglyoxal) in the modulation of sugar metabolism. Deletion of this gene (strain LY168) resulted in the co-metabolism of glucose and xylose, and accelerated the metabolism of a 5-sugar mixture (mannose, glucose, arabinose, xylose and galactose) to ethanol.     

Revealing oxidative pentose metabolism in new Pseudomonas putida isolates

The Pseudomonas putida group in the Gammaproteobacteria has been intensively studied for bioremediation and plant growth promotion. Members of this group have recently emerged as promising hosts to convert intermediates derived from plant biomass to biofuels and biochemicals. However, most strains of P. putida cannot metabolize pentose sugars derived from hemicellulose. Here, we describe three isolates that provide a broader view of the pentose sugar catabolism in the P. putida group. One of these isolates clusters with the well-characterized P. alloputida KT2440 (Strain BP6); the second isolate clustered with plant growth-promoting strain P. putida W619 (Strain M2), while the third isolate represents a new species in the group (Strain BP8). Each of these isolates possessed homologous genes for oxidative xylose catabolism (xylDXA) and a potential xylonate transporter. Strain M2 grew on arabinose and had genes for oxidative arabinose catabolism (araDXA). A CRISPR interference (CRISPRi) system was developed for strain M2 and identified conditionally essential genes for xylose growth. A glucose dehydrogenase was found to be responsible for initial oxidation of xylose and arabinose in strain M2. These isolates have illuminated inherent diversity in pentose catabolism in the P. putida group and may provide alternative hosts for biomass conversion.     

Incorporation of 14C-l-arabinose into polysaccharides of maize root-tips

Summary [1-¹⁴C]-l-arabinose was supplied to maize roots over a range of concentrations extending from 0.1 M to 0.04 mM. In each case, only xylose and arabinose units in the cell wall polysaccharides became labelled. However, although uptake increased with concentration, the conversion of l-arabinose to these cell wall units was not greatly influenced by raising the external sugar concentration, and there was no marked accumulation of UDP-pentose under any of the experimental conditions tested. Furthermore, specific activity of the arabinose isolated from the cell wall hydrolysates was always higher than that of the xylose. Because the labelling was so specific, patterns of pentose deposition could be followed by preparing autoradiographs of sections from roots fed with ¹⁴C-l-arabinose. In the pith and cortex, which are typically parenchymatous tissues, the maximum rate of incorporation was observed in cell walls at around 2 mm from the cap-stele junction. These cells had just reached their full width and were about to undergo a phase of rapid elongation. Results are in essential aggrement with those obtained earlier with d-glucuronate in similar experiments.