Labelling of lipids by D-[1-14C]glucose, D-[6-14C]glucose and D-[3-3H]glucose in pancreatic islets from normal and GK rats

In pancreatic islets prepared from either normal or GK rats and incubated at either low (2.8 mM) or high (16.7 mM) D-glucose concentration, the labelling of both lipids and their glycerol moiety is higher in the presence of D-[1-¹⁴C]glucose than D-[6-¹⁴C]glucose. The rise in D-glucose concentration augments the labelling of lipids, the paired ¹⁴C/³H ratio found in islets exposed to both D-[1-¹⁴C]glucose or D-[6-¹⁴C]glucose and D-[3-³H]glucose being even slightly higher at 16.7 mM D-glucose than that found, under otherwise identical conditions, at 2.8 mM D-glucose. Such a paired ratio exceeds unity in islets exposed to D-[1-¹⁴C]glucose. The labelling of islet lipids by D-[6-¹⁴C]glucose is about 30 times lower than the generation of acidic metabolites from the same tracer. These findings indicate (i) that the labelling of islet lipids accounts for only a minor fraction of D-glucose catabolism in pancreatic islets, (ii) a greater escape to L-glycerol-3-phosphate of glycerone-3-phosphate generated from the C₁-C₂-C₃ moiety of D-glucose than D-glyceraldehyde-3-phosphate produced from the C₄-C₅-C₆ moiety of the hexose, (iii) that only a limited amount of [3-³H]glycerone 3-phosphate generated from D-[3-³H]glucose is detritiated at the triose phosphate isomerase level before being converted to L-glycerol-3-phosphate, and (iv) that a rise in D-glucose concentration results in an increased labelling of islet lipids, this phenomenon being somewhat more pronounced in the case of D-[1-¹⁴C]glucose or D-[6-¹⁴C]glucose rather than D-[3-³H]glucose.     

Enhanced Dark CO(2) Fixation by Preilluminated Chlorella pyrenoidosa and Anacystis nidulans

The products of short time photosynthesis and of enhanced dark ¹⁴CO₂ fixation (illumination in helium prior to addition of ¹⁴CO₂ in dark) by Chlorella pyrenoidosa and Anacystis nidulans were compared. Glycerate 3-phosphate, phosphoenolpyruvate, alanine, and aspartate accounted for the bulk of the ¹⁴C assimilated during enhanced dark fixation while hexose and pentose phosphates accounted for the largest fraction of isotope assimilated during photosynthesis. During the enhanced dark fixation period, glycerate 3-phosphate is carboxyl labeled and glucose 6-phosphate is predominantly labeled in carbon atom 4 with lesser amounts in the upper half of the C₆ chain and traces in carbon atoms 5 and 6. Tracer spread throughout all the carbon atoms of photosynthetically synthesized glycerate 3-phosphate and glucose 6-phosphate. During the enhanced dark fixation period, there was a slow formation of sugar phosphates which subsequently continued at 5 times the initial rate long after the cessation of ¹⁴CO₂ uptake. To explain the kinetics of changes in the labelling patterns and in the limited formation of the sugar phosphates during enhanced dark CO₂ fixation, the suggestion is made that most of the reductant mediating these effects did not have its origin in the preillumination phase.

It is concluded that a complete photosynthetic carbon reduction cycle operates to a limited extent, if at all, in the dark period subsequent to preillumination.

     

Cellobiose metabolism and cellobiohydrolase I biosynthesis by Trichoderma reesei

The relationship between β-linked disaccharide (cellobiose, sophorose) utilization and cellulase, particularly cellobiohydrolase I (CBH I) synthesis by Trichoderma reesei, was investigated. During growth on cellobiose and sophorose as carbon sources in batch as well as resting-cell culture, only sophorose induced cellulase formation. In the latter experiments, sophorose was utilized at a much lower rate than cellobiose, and the more cellulase produced, the lower its rate of utilization. Cellobiose and sophorose were utilized by the fungus mainly via hydrolysis by the cell wall- and cell membrane-bound β-glucosidase. Addition of sophorose to T. reesei growing on cellulose did not further stimulate cellulase synthesis, and addition of cellobiose was inhibitory. Cellobiose, however, promoted cellulase formation in both batch and resting cell cultures, when its hydrolysis by β-glucosidase was inhibited by nojirimycin. No cellulase formation was observed when the uptake of glucose (produced from cellobiose by β-glucosidase) was inhibited by 3-O-methylglucoside. Cellodextrins (C₂ to C₆) promoted formation of low levels of cellobiohydrolase I in indirect proportion to their rate of hydrolysis by β-glucosidase. Studies on the uptake of [³H]cellobiose, [³H]sophorose, and [¹⁴C]glucose in the presence of inhibitors of β-glucosidase (nojirimycin) and glucose transport (3-O-methylglucoside) show that glucose transport occurs at a much higher rate than disaccharide hydrolysis. Extracellular disaccharide hydrolysis accounts for at least 95% of their metabolism. The presence of an uptake system for cellobiose was established by demonstrating the presence of intracellular labeled [³H]cellobiose in T. reesei after its extracellular supply. The data are consistent with induction of cellulase and particularly CBH I formation in T. reesei by β-linked disaccharides under conditions where their uptake is favored at the expense of extracellular hydrolysis.     

Physiological properties of Cellulomonas fermentans,a mesophilic cellulolytic bacterium

Summary The fermentation of cellobiose, glucose and cellulose MN 300 by Cellulomonas fermentans was studied. The molar growth yields (i.e. grams of cells per mole of hexose equivalent) were similar on cellobiose and cellulose at low sugar consumption levels (47.8 and 46.5 respectively), but was lower on glucose (38.0). The occurrence of cellobiose phosphorylase activity, detected in cellobiose- and cellulose-grown cells, might explain this result. The specific growth rates measured in cultures on cellobiose, glucose and cellulose were 0.055 h^-1, 0.040 h^-1 and 0.013 h^-1 respectively. Growth inhibition was observed, and a drop in Y_H occurred after relatively low but different quantities of hexose were consumed (2.2 mM, 5 mM and 8 mM hexose equivalent with cellulose, glucose and cellobiose respectively), which coincided with a change in the fermentative metabolism from a typical mixed acid metabolism (1 ethanol, 1 acetate and 2 formate synthesized by consumed hexose) to a more ethanolic fermentation. When growth ceased in cellulose cultures, consumption of cellulose continued, as did production of ethanol.Molar growth yields of C. fermentans were similar in anaerobic and aerobic cellobiose cultures (47.8 g/mol and 42.2 g/mol respectively). Specific growth rates were also quite similar under both culture conditions (0.055±0.013 h^-1 and 0.070±0.007 h^-1 respectively). Aerobic metabolism was studied using ¹⁴C glucose. During the exponential growth phase, acetate, succinate and nonidentified compound(s) accumulated in the supernatant, but no ¹⁴CO₂ was produced. During the stationary phase, acetate was oxidized and ¹⁴CO₂ produced, but without any further biomass synthesis. It seems that a blocking of metabolite oxidation may have occurred in C. fermentans except in the case of acetate, but acetate oxidation was apparently not coupled with production of energy utilizable in biosynthesis.     

Ribose biosynthesis in methanogenic bacteria

NMR spectroscopy was used to determine the labeling patterns of the ribose moieties of ribonucleosides purified from Methanospirillum hungatei, Methanococcus voltae, Methanobrevibacter smithii, Methanosphaera stadtmanae, Methanosarcina barkeri and Methanobacterium bryantii labeled with ¹³C-precursors. In most methanogens tested ribose was labeled in a manner consistent with the operation of the oxidative branch of the pentose phosphate pathway. In contrast, transaldolase and transketolase reactions typical of a partial nonoxidative pentose phosphate pathway are hypothesized to explain the different labeling patterns and enrichments of carbon atoms observed in the ribose moiety of Methanococcus voltae. The source of erythrose 4-phosphate needed for the transaldolase reaction proposed in Methanococcus voltae, and for biosynthesis of aromatic amino acids in methanogenic bacteria in general, was assessed. Phenylalanine carbon atom C-7 was labeled by [1-¹³C]pyruvate in Methanospirillum hungatei, Methanococcus voltae, and Methanococcus jannaschii, the only methanogens which incorporated sufficient label from pyruvate for testing. Reductive carboxylation of a triose precursor (derived from pyruvate) to synthesize erythrose 4-phosphate is consistent with the labeling patterns observed in phenylalanine and ribose.Abbreviation TCA Tricarboxylic acid Issued as NRCC Publication No. 37382     

Sugar Transport in Immature Internodal Tissue of Sugarcane: II. Mechanism of Sucrose Transport

The mechanism by which sucrose is transported into the inner spaces of immature internodal parenchyma tissue of sugarcane (Saccharum officinarum L. var. H 49-5) was studied in short term experiments (15 to 300 seconds). Transport of sucrose, glucose, and fructose was each characterized by a V_max of 1.3 μmoles/gram fresh weight·2 hours, and each of these three sugars mutually and competitively inhibited transport of the other two. When ¹⁴C-glucose was supplied exogenously, ¹⁴C-glucose 6-phosphate and ¹⁴C-glucose were the first labeled compounds to appear in the tissue; no ¹⁴C-sucrose was detected until after 60-second incubation. After 15-second incubation in ¹⁴C-sucrose, all intracellular radioactivity was in glucose, fructose, glucose 6-phosphate, and fructose 6-phosphate; trace amounts of ¹⁴C-sucrose were found after 30 seconds and after 5 minutes, 71% of the intracellular radioactivity was in sucrose. Although it was possible that sucrose was transported intact into the inner space and then immediately hydrolyzed, it was shown that the rate of hydrolysis under these conditions was too low to account for the rate of hexose accumulation. Pretreatment of the tissue with rabbit anti-invertase antiserum eliminated sucrose transport, but had no effect on glucose transport. Since the antibodies did not penetrate the plasmalemma, it was concluded that sucrose was hydrolyzed by an invertase in the free space prior to transport. The glucose and fructose moieties, or their phosphorylated derivatives, were then transported into the inner space and sucrose was resynthesized. No evidence for the involvement of sucrose phosphate in transport was found in these experiments.     

Biotransformation of d-xylose to d-xylonate coupled to medium-chain-length polyhydroxyalkanoate production in cellobiose-grown Pseudomonas putida EM42

Co-production of two or more desirable compounds from low-cost substrates by a single microbial catalyst could greatly improve the economic competitiveness of many biotechnological processes. However, reports demonstrating the adoption of such co-production strategy are still scarce. In this study, the ability of genome-edited strain Pseudomonas putida EM42 to simultaneously valorize d -xylose and d -cellobiose – two important lignocellulosic carbohydrates – by converting them into the platform chemical d -xylonate and medium-chain-length polyhydroxyalkanoates, respectively, was investigated. Biotransformation experiments performed with P. putida resting cells showed that promiscuous periplasmic glucose oxidation route can efficiently generate extracellular xylonate with a high yield. Xylose oxidation was subsequently coupled to the growth of P. putida with cytoplasmic β-glucosidase BglC from Thermobifida fusca on d -cellobiose. This disaccharide turned out to be a better co-substrate for xylose-to-xylonate biotransformation than monomeric glucose. This was because unlike glucose, cellobiose did not block oxidation of the pentose by periplasmic glucose dehydrogenase Gcd, but, similarly to glucose, it was a suitable substrate for polyhydroxyalkanoate formation in P. putida. Co-production of extracellular xylose-born xylonate and intracellular cellobiose-born medium-chain-length polyhydroxyalkanoates was established in proof-of-concept experiments with P. putida grown on the disaccharide. This study highlights the potential of P. putida EM42 as a microbial platform for the production of xylonate, identifies cellobiose as a new substrate for mcl-PHA production, and proposes a fresh strategy for the simultaneous valorization of xylose and cellobiose.     

Penicillium verruculosum WA 30 a new source of cellulase

Summary Of fungi 110 strains were screened for extracellular cellulase production in shake flask experiments. Twelve strains produced the enzyme in significant quantity. Since the enzyme activity was assayed by different methods (liberation of reducing sugar from cotton, filter paper, carboxymethylcellulose and cellobiose), the estimation of the productivity of the strains differed according to the substrate used. The best cotton degrading activity per fermentation volume as well as per mg of secreted soluble protein was achieved by Penicillium verruculosum WA 30, a wild-type strain, for which the cellulase productivity has not yet been published. The cotton degrading (so-called C₁) activity was successfully enhanced nearly threefold in medium experiments. Analyses of saccharification digests showed that glucose was the predominant product, with negligible amounts of cellobiose. The pH and temperature optima for WA 30 cellulase complex were pH 4.2 and 60°C.     

Specific maltose labeling in the reducing glucose moiety.

Radioactive maltose with label in the reducing glucose moiety was prepared using a glucosyltransferase enzyme to catalyze exchange of [6-³H]glucose into unlabeled maltose. The enzyme was isolated from spinach by ammonium sulfate precipitation followed by DEAE column chromatography. A 77% yield of [6-³H]maltose was obtained after a reaction of 100 nmol of maltose with 0.0147 nmol of [6-³H]glucose was catalyzed by the most active column peak. The product was exclusively labeled in the reducing glucose moiety as indicated by the label occurring only in sorbitol following sodium borohydride reduction and sulfuric acid hydrolysis. Between 88.3 and 96.0% of the tritium in the synthesized preparation was present as [6-³H]maltose by Dowex 1-X4 chromatography. This column separates [6-³H]maltose-[U-¹⁴C]maltose mixtures and [6-³H]glucose-[U-¹⁴C]glucose mixtures apparently as a result of an isotope effect.     

The fermentation of hexose and pentose sugars by Candida shehatae and Pichia stipitis

Summary The fermentation by Candida shehatae and Pichia stipitis of xylitol and the various sugars which are liberated upon hydrolysis of lignocellulosic biomass was investigated. Both yeasts produced ethanol from d-glucose, d-mannose, d-galactose and d-xylose. Only P. stipitis fermented d-cellobiose, producing 6.5 g·l^-1 ethanol from 20 g·l^-1 cellobiose within 48 h. No ethanol was produced from l-arabinose, l-rhamnose or xylitol. Diauxie was evident during the fermentation of a sugar mixture. Following the depletion of glucose, P. stipitis fermented galactose, mannose, xylose and cellobiose simultaneously with no noticeable preceding lag period. A similar fermentation pattern was observed with C. shehatae, except that it failed to utilize cellobiose even though it grew on cellobiose when supplied as the sole sugar. P. stipitis produced considerably more ethanol from the sugar mixture than C. shehatae, primarily due to its ability to ferment cellobiose. In general P. stipitis exhibited a higher volumetric rate and yield of ethanol production. This yeast fermented glucose 30–50% more rapidly than xylose, whereas the rates of ethanol production from these two sugars by C. shehatae were similar. P. stipitis had no absolute vitamin requirement for xylose fermentation, but biotin and thiamine enhanced the rate and yield of ethanol production significantly.Nomenclature _max Maximum specific growth rate, h^-1 - Q _p Maximum volumetric rate of ethanol production, calculated from the slope of the ethanol vs. time curve, g·(l·h)^-1 - q _p Maximum specific rate of ethanol production, g·(g cells·h) - Y _p/s Ethanol yield coefficient, g ethanol·(g substrate utilized)^-1 - Y _x/s Cell yield coefficient, g biomass·(g substrate utilized)^-1 - E Efficiency of substrate utilization, g substrate consumed·(g initial substrate)^-1·100     

Phosphatidylglycerol synthesis in spinach chloroplasts: characterization of the newly synthesized molecule

Intact chloroplasts from spinach (Spinacia oleracea L., hybrid 424) readily incorporate [¹⁴C]glycerol-3-phosphate and [¹⁴C]acetate into diacylglycerol, monoacylglycerol, diacylglycrol, free fatty acids (only when acetate is the precursor), phosphatidic acid, phosphatidylcholine, and most notably phosphatidylglycerol. The fraction of phosphatidylglycerol synthesized is greatly increased by the presence of manganese chloride in the reaction mixture. Glycerol-3-phosphate-labeled phosphatidylglycerol is equally labeled in the two glycerol moieties of the molecule. Acetate-labeled phosphatidylglycerol is equally labeled in both acyl groups. Position one contains primarily oleate, linoleate and small amounts of palmitate. Position two contains primarily palmitate. No radioactive trans-Δ³-hexadecenoate was detected. The labeling patterns indicate that the radioactive phosphatidylglycerol is the product of de novo chloroplast lipid biosynthesis and furthermore, phosphatidylglycerol may be a substrate for fatty acid desaturation.     

Glycerolipid synthesis by homogenate and oil bodies from developing mustard (Sinapis alba L.) seed

[1-¹⁴C]Oleic acid and [14-¹⁴C]erucic acid were converted to their acyl-CoA derivatives and incorporated into acyl lipids by a homogenate from developing mustard (Sinapis alba L.) seed and oil bodies, as well as supernatant isolated by centrifugation at 20000 g. In both homogenate and oil bodies, the oleoyl moieties from exogenous [1-¹⁴C]oleoyl-CoA were most extensively incorporated into phosphatidic acids, but very little into phosphatidylcholines. The pattern of labelling of acyl lipids by oleoyl versus erucoyl moieties from either of the corresponding fatty acids, added individually or as a mixed substrate, indicates that oleoyl-CoA directly acylates sn-glycerol-3-phosphate to yield lysophosphatidic acids and phosphatidic acids that are subsequently converted to mono- and diacylglycerols. In contrast, erucoyl-CoA predominantly acylates preformed mono-and diacylglycerols containing oleoyl moieties to yield triacylglycerols containing erucoyl moieties.     

The metabolism of d-galactosamine and N-acetyl-d-galactosamine in rat liver

d-[1-¹⁴C]Galactosamine appears to be utilized mainly by the pathway of galactose metabolism in rat liver, as evidenced by the products isolated from the acid-soluble fraction of perfused rat liver. These products were eluted in the following order from a Dowex 1 (formate form) column and were characterized as galactosamine 1-phosphate, sialic acid, UDP-glucosamine, UDP-galactosamine, N-acetylgalactosamine 1-phosphate, N-acetylglucosamine 6-phosphate, UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine and an unidentified galactosamine-containing compound. In addition, [1-¹⁴C]glucosamine was found in the glycogen, an incorporation previously shown to result from the substitution of UDP-glucosamine for UDP-glucose in the glycogen synthetase reaction. Analysis of the [1-¹⁴C]glucosamine-containing disaccharides released from glycogen by β-amylase provided additional evidence that they consist of a mixture of glucose and glucosamine in a 1:1 ratio, but with glucose predominating on the reducing end. UDP-N-acetylgalactosamine was shown to result from the reaction of UTP with N-acetylgalactosamine 1-phosphate in the presence of a rat liver extract.     

Characterization of Lipid A,A Component of Lipopolysaccharides from Selenomonas ruminantium

Lipid components of a glycolipid, formerly designated as spot A, from the cells of Selenomonas ruminantium were investigated. The basic structure of this material had been previously shown to be β-glucosaminyl-l,6-glucosamine. The major component of O- and N-acyl side chains was β-OH C_13:0 acid when the cells were grown with added valerate. Approximately 85 % of the total amide linked fatty acids was this compound. A considerable amount of C_13:2 acid was also present as a component of O-acyl fatty acids. When the cells were grown in a glucose medium containing caproate, the major fatty acid component of the spot A compound was β-OH myristic and β-OH C_13:0: acids. ¹⁴C-Valerate or ¹⁴C-caproate, supplemented to the glucose medium, was incorporated into O- and N-acyl linked fatty acid moieties of the spot A compound. It was also shown that the spot A compound was the lipid A component of lipopolysaccharides of this organism.     

The inhibition of β-glucosidase activity in Trichoderma reesei C30 cellulase by derivatives and isomers of glucose

The inhibition of β-glucosidase in Trichoderma reesei C30 cellulase by D -glucose, its isomers, and derivatives was studied using cellobiose and ρ-nitrophenyl-β-glucoside (PNPG) as substrates for determining enzyme activity. The enzymatic hydrolysis of both substrates was inhibited competitively by glucose with approximate K_i values of 0.5mM and 8.7mM for cellobiose and PNPG as substrate, respectively. This inhibition by glucose was maximal at pH 4.8, and no inhibition was observed at pH 6.5 and above. The α anomer of glucose inhibited β-glucosidase to a greater extent than did the β form. Compared with D -glucose, L -glucose, D -glucose-6-phosphate, and D -glucose-1-phosphate inhibited the enzyme to a much lesser extent, unlike D -glucose-L -cysteine which was almost as inhibitory as glucose itself when cellobiose was used as substrate. Fructose (2?100mM) was found to be a poor inhibitor of the enzyme. It is suggested that high rates of cellobiose hydrolysis catalyzed by β-glucosidase may be prolonged by converting the reaction product glucose to fructose using a suitable preparation of glucose isomerase.     

Biosynthesis of radiolabeled cellodextrins by the Clostridium thermocellum cellobiose and cellodextrin phosphorylases for measurement of intracellular sugars

The Clostridium thermocellum cellobiose and cellodextrin phosphorylases (glucosyl transferases) in the cell extract were used to synthesize radiolabeled cellodextrins with a degree of polymerization (DP=2–6) from nonradioactive glucose-1-phosphate and radioactive glucose. Chain lengths of synthesized cellodextrin were controlled by the absence or presence of dithiothreitol and by reaction conditions. All cellodextrins have the sole radioactive glucose unit located at the reducing ends. Mixed cellodextrins (G2–G6) were separated efficiently by size-exclusion chromatography or less efficiently by thin-layer chromatography. A new rapid sampling device was developed using disposable syringes containing an ultracold methanol-quenching buffer. It was simple, less costly, and especially convenient for anaerobic fermentation. After an impulse feed of radiolabeled cellobiose, the intracellular sugar levels were measured after a series of operations—sampling, extracting, concentrating, separating, and reading. Results showed that the largest amount of radioactivity was cellobiose with lesser amounts of glucose, cellotriose, and cellotetraose, and an average DP of intracellular cellodextrins was ca. 2.     

Synthesis of proteins and nucleic acids by the pea aphid Acyrthosiphon pisum (aphididae) in the absence of a full complement of dietary amino acids

Protein, nucleic acids, and nucleotide syntheses were studied in pea aphids, Acyrthosiphon pisum (Harris), by feeding them labeled ¹⁴C-amino acids and [5-³H]-orotic acid in sucrose. It was demonstrated that in the absence of dietary essential amino acids, aphids were capable of synthesizing nucleic acids, nucleotides, and proteins when provided with a single dietary amino acid in sucrose. It is suggested that other required amino acids were possibly supplied by the symbionts present in the pea aphid and/or were obtained from the amino acid pool in the hemolymph or glucose, one of the end products of sucrose digestion. Of the various amino acids tested, synthesis of measurable amounts of protein or other compounds occurred when alanine, aspartic acid, glutamic acid, glycine, proline, or serine were provided, but no synthesis occurred with cysteine.     

Biochemistry of Suberization: Incorporation of [1-C]Oleic Acid and [1-C]Acetate into the Aliphatic Components of Suberin in Potato Tuber Disks (Solanum tuberosum)

Biosynthesis of the aliphatic components of suberin was studied in suberizing potato (Solanum tuberosum) slices with [1-¹⁴C]oleic acid and [1-¹⁴C]acetate as precursors. In 4-day aged tissue, [1-¹⁴C]oleic acid was incorporated into an insoluble residue, which, upon hydrogenolysis (LiA1H₄), released the label into chloroform-soluble products. Radio thin layer and gas chromatographic analyses of these products showed that ¹⁴C was contained exclusively in octadecenol and octadecene-1, 18-diol. OsO₄ treatment and periodate cleavage of the resulting tetraol showed that the labeled diol was octadec-9-ene-1, 18-diol, the product expected from the two major components of suberin, namely 18-hydroxyoleic acid and the corresponding dicarboxylic acid. Aged potato slices also incorporated [1-¹⁴C]acetate into an insoluble material. Hydrogenolysis followed by radio chromatographic analyses of the products showed that ¹⁴C was contained in alkanols and alkane-α,ω-diols. In the former fraction, a substantial proportion of the label was contained in aliphatic chains longer than C₂₀, which are known to be common constituents of suberin. In the labeled diol fraction, the major component was octadec-9-ene-1,18-diol, with smaller quantities of saturated C₁₆, C₁₈, C₂₀, C₂₂, and C₂₄-α,ω-diols. Soluble lipids derived from [1-¹⁴C]acetate in the aged tissue also contained labeled very long acids from C₂₀ to C₂₈, as well as C₂₂ and C₂₄ alcohols, but no labeled ω-hydroxy acids or dicarboxylic acids were detected. Label was also found in n-alkanes isolated from the soluble lipids, and the distribution of label among them was consistent with the composition of n-alkanes found in the wound periderm of this tissue; C₂₁ and C₂₃ were the major components with lesser amounts of C₁₉ and C₂₅. The amount of ¹⁴C incorporated into these bifunctional monomers in 0-, 2-, 4-, 6-, and 8-day aged tissue were 0, 1.5, 2.5, 0.8, and 0.3% of the applied [1-¹⁴C]oleic acid, respectively. Incorporation of [1-¹⁴C]acetate into the insoluble residue was low up to the 3rd day of aging, rapid during the next 4 days of aging, and subsequently the rate decreased. These changes in the rates of incorporation of exogenous oleic acid and acetate reflected the development of diffusion resistance of the tissue surface to water vapor. As the tissue aged, increasing amounts of the [1-¹⁴C]acetate were incorporated into longer aliphatic chains of the residue and the soluble lipids, but no changes in the distribution of radioactivity among the α-ω-diols were obvious. The above results demonstrated that aging potato slices constitute a convenient system with which to study the biochemistry of suberization.     

Metabolic engineering of E. coli for improving mevalonate production to promote NADPH regeneration and enhance acetyl-CoA supply

Microbial production of mevalonate from renewable feedstock is a promising and sustainable approach for the production of value-added chemicals. We describe the metabolic engineering of Escherichia coli to enhance mevalonate production from glucose and cellobiose. First, the mevalonate-producing pathway was introduced into E. coli and the expression of the gene atoB, which encodes the gene for acetoacetyl-CoA synthetase, was increased. Then, the deletion of the pgi gene, which encodes phosphoglucose isomerase, increased the NADPH/NADP⁺ ratio in the cells but did not improve mevalonate production. Alternatively, to reduce flux toward the tricarboxylic acid cycle, gltA, which encodes citrate synthetase, was disrupted. The resultant strain, MGΔgltA-MV, increased levels of intracellular acetyl-CoA up to sevenfold higher than the wild-type strain. This strain produced 8.0 g/L of mevalonate from 20 g/L of glucose. We also engineered the sugar supply by displaying β-glucosidase (BGL) on the cell surface. When cellobiose was used as carbon source, the strain lacking gnd displaying BGL efficiently consumed cellobiose and produced mevalonate at 5.7 g/L. The yield of mevalonate was 0.25 g/g glucose (1 g of cellobiose corresponds to 1.1 g of glucose). These results demonstrate the feasibility of producing mevalonate from cellobiose or cellooligosaccharides using an engineered E. coli strain.