Lysine-91 of the tetraheme <Emphasis Type="Italic">c</Emphasis>-type cytochrome CymA is essential for quinone interaction and arsenate respiration in <Emphasis Type="Italic">Shewanella</Emphasis> sp. strain ANA-3

The tetraheme c-type cytochrome, CymA, is essential for arsenate respiratory reduction in Shewanella sp. ANA-3, a model arsenate reducer. CymA is predicted to mediate electron transfer from quinols to the arsenate respiratory reductase (ArrAB). Here, we present biochemical and physiological evidence that CymA interacts with menaquinol (MQH₂) substrates. Fluorescence quench titration with the MQH₂ analog, 2-n-heptyl-4-hydroxyquinoline-N-oxide (HOQNO), was used to demonstrate quinol binding of E. coli cytoplasmic membranes enriched with various forms of CymA. Wild-type CymA bound HOQNO with a K _d of 0.1–1 μM. It was also shown that the redox active MQH₂ analog, 2,3-dimethoxy-1,4-naphthoquinone (DMNH₂), could reduce CymA in cytoplasmic membrane preparations. Based on a CymA homology model made from the NrfH tetraheme cytochrome structure, it was predicted that Lys91 would be involved in CymA-quinol interactions. CymA with a K91Q substitution showed little interaction with HOQNO. In addition, DMNH₂-dependent reduction of CymA-K91Q was diminished by 45% compared to wild-type CymA. A ΔcymA ANA-3 strain containing a plasmid copy of cymA-K91Q failed to grow with arsenate as an electron acceptor. These results suggest that Lys91 is physiologically important for arsenate respiration and support the hypothesis that CymA interacts with menaquinol resulting in the reduction of the cytochrome.     

Aerobic Respiration of Kappa Particles from Paramecium aurelia,Stock 51*

SYNOPSIS. Kappa particles from killer cultures of stock 51 Paramecium aurelia were purified and their respiration measured polarographically. The slight bacterial contaminations in the kappa preparations were not significant. Freshly collected kappa in dilute buffer at room temperature had an endogenous Q_O2 of 17.0 ± 1.6 μl/mg dry weight/hr (mean ± standard error). The Q_O2 decayed 50% in 5 hr. Among the sugars tested only glucose and sucrose increased the respiratory rate of kappa. The di- and tri-carboxylates of the tricarboxylic acid cycle stimulated the respiration of kappa. KCN, CO and 2-heptyl-4-hydroxyquinoline N-oxide (HOQNO) inhibited respiration. These findings ensure an organismic status for kappa and justify the belief that it is bacterial in origin.     

Plastoquinone as a common link between photosynthesis and respiration in a blue-green alga

The role of plastoquinone in a thermophilic blue-green alga, Shynechococcus sp., was studied by measuring reduction kinetics of cytochrome 553 which was oxidized with red flash preferentially exciting photosystem I. Sensitivity of the cytochrome reduction to DBMIB indicates that cytochrome 553 accepts electrons from reduced plastoquinone. Plastoquinone is in turn reduced in cells without electrons from photosystem II, since DCMU, which inhibited methyl viologen photoreduction more strongly than DBMIB, failed to affect the cytochrome reduction. Participation of cyclic electron transport around photosystem I in cytochrome reduction in the presence of DCMU was excluded, because methyl viologen and antimycin A had no effect on the cytochrome kinetics. On the other hand, electron donation from endogenous substrates to plastoquinone was suggested from decreases in rate of the cytochrome reduction by dark starvation of cells and also from restoration of fast reduction kinetics by the addition of exogenous substrates to or by reillumination of starved cells.KCN, which completely suppressed respiratory O₂-uptake, induced a marked acceleration of the cytochrome reduction in starved cells. The poison was less or not effective in stimulating the cytochrome reduction in more extensively starved or reilluminated cells.Results indicate that plastoquinone is functioning not only in the photosynthetic but also in the respiratory electron transport chain, thereby forming a common link between the two energy conservation systems of the blue-green alga.

---

     

ISOLATION,GROWTH, AND RESPIRATION OF A THERMOPHILIC BLUE-GREEN ALGA

Holton , Raymond W. (U. Texas, Austin.) Isolation, growth, and respiration of a thermophilic blue-green alga. Amer. Jour. Bot. 49(1): 1–6. Illus. 1962.—The isolation of a bacteria-free culture of Hapalosiphon laminosus (= Mastigocladus laminosus) from an algal mass obtained from Liard Hot Springs, British Columbia, is described. Seven different bacterial media at 2 incubation temperatures were used to test for bacteria in suspensions of partially broken up algae. The algal growth rate was obtained by calculating the slope of the straight line obtained in a plot of the cube root of the dry wt against time. In cultures aerated with air the growth was optimal between 35 and 50 C, with an upper limit at 55 and a lower limit at 25. Aeration with 1% CO₂ in air significantly increased growth rates and the optimum was at 45–50 C. Unaerated flasks grew more slowly than aerated ones. The endogenous Qo₂ (in μl/hr–mg dry wt) at 45 was 8.0 for growing cells and 2.8 for starved cells kept in darkness 18 hr. The Qo₂ was maximal between pH 7.0 and 8.5. The concentration of phosphate buffer affected the Qo₂ and rates in 0.02 M were slightly greater than those in distilled water and much greater than those in 0.4 M. The Qo₂ of growing cells was slightly stimulated by 2,4-dinitrophenol (DNP), whereas glucose or glucose plus DNP did not affect it. In contrast, the endogenous Qo₂ of starved cells was nearly doubled in glucose and tripled in DNP plus glucose.     

Studies on the Metabolism of Aspergilli

Studies were made on the endogenous respiration of Aspergillus sojae K.S. Observing the changes of Kjeldahl-nitrogen in each fraction of the mycelial components, the author concluded that pool amino acids, bound amino acids, protein, nucleic acids and nucleotides covered whole of the nitrogenous reserves available for endogenous respiration in the mycelia. A study was carried out on the effect of preincubation with glucose or amino acids on endogenous respiration. Stimulation of either oxygen uptake, protein breakdown or ammonia formation was observed during respiration of the mycelia incubated with a suitable concentration of azide, 2,4-dinitrophenol, potossium fluoride, monoiodoacetic acid or ethylenediaminetetraacetic acid. Ammonia formation accompanied with endogenous respiration seemed to proceed inversely by the influence of energy yielding reaction.     

Comparative Physiology of Respiration in Aquatic Fungi

A comparative study of terminal respiration was undertaken in five genera of aquatic fungi in the Leptomitales. The cytochrome system in this group of fungi contained cytochrome a-a₃ (605 nm), cytochrome c (551 nm), cytochrome b (557 nm), and cytochomo b (564 nm). A representative of each of three aerobic genera, Leptomitus, Apodachlya, and Sapromyces, had a total cytochrome content of about 2×10^?10 mol/mg dry weight. An endogenous respiration rate of 21 μl O₂ uptake/ (h × mg dry weight) at 21.7°C was found in Leptomitus and Apodmhlya and 14 in Sapromyces. The strain belonging to the fermenlative genus Mindeniella had approximately one-third of the total cytochrome content and one-third of the endogenous respiration rate observed in Leptomitus and Apodachlya. Mindeniella and Sapromyces contained less total cytochrome when grown under reduced oxygen tension than when grown in air. Only about one-half of the b-type cytochrome was redueible by endogenous substrates. Both cytochrome a₃ and an unidentified pigment bound CO. The endogenous respiration of Leptomitus, Apodachlyo, and Sapromyces was strongly Inhibited by sodium cyanide, sodium azide, antimycin A, and sodium fluoroacetate.     

Changes in proline level in response to osmotic stress and exogenous amino acids in Raphanus sativus L. seedlings

The changes in endogenous proline levels of Raphanus sativus L. seedlings was monitored in presence of exogenous amino acids in normal and osmotically stressed seedlings. In unstressed seedlings, proline uptake was detected only at higher (1 mM) concentration of applied L-proline. however, proline uptake was promoted at all (1 μM to 1000 μM) concentrations of applied L-proline under osmotic stress conditions. Amongst other exogenous amino acids, L-leucine, L-glutamic acid, L-alanine, and L-histidine enhanced endogenous levels of proline, while exogenous hydorxyproline and γ-amino butyric acid reduced it.     

Oxidative phosphorylation in Micrococcus denitrificans under autotrophic growth conditions

Summary Cell-free preparations from autotrophically grown M. denitrificans yielded P/O ratios of 0.6–1.6 with H₂, 0.4–1.2 with NADH, 0.7–1.0 with succinate, and 0.3–0.5 with ascorbate as the oxidizable substrates.The phosphorylation in all cases was inhibited effectively by DNP, DBP, PCP, CCCP, and dicumarol at concentrations which did not cause any significant inhibition of oxygen uptake.The respiratory chain inhibitors such as antimycin A, HOQNO, cyanide, and azide were the potent inhibitors of the phosphate esterification coupled to the oxidation of H₂, NADH, and succinate; the ascorbate-linked phosphorylation was inhibited by cyanide or azide only.While the NADH oxidation and associated phosphorylation was markedly sensitive to rotenone and other flavoprotein inhibitors, the oxidation of H₂ was relatively insensitive although there was a partial inhibition of the coupled phosphorylation. The experimental results indicated that bulk of the electron transfer from H₂ bypassed the NADH-dehydrogenase or the rotenone sensitive site.Non-standard Abbreviations BAL British Anti-Lewisite (2,3-Dimercaptopropanol) - CCCP Carbonyl-cyanide-m-chlorophenylhydrazone - DBP 2,6-Dibromophenol - DNP 2,4-Dinitrophenol - GSH reduced glutathione - HOQNO 2-n-Heptyl-4-hydroxyquinoline-N-oxide - PCMB p-hydroxymercuribenzoate - PCP Pentachlorphenol - TTFA Thenoyltrifluoracetone - TMPD N,N,N,N-tetramethyl-p-phenylenediamine - BSA Bovine serum albumin - EDTA Ethylenediamine tetraacetic acid Post doctorate fellow of the Deutsche ForschungsgemeinschaftNational Science Foundation Research Associate.     

Growth of Certain Aquatic Omycetes on Amino Acids I. Saprolegnia, Achlya, Leptolegnia, and Dictyuchus

Four fungi in the order Saprolegniales —Saprolegnia sp., Achlya ambisexualis, Leptolegnia eccentrica and Dictyuchus sterile– were tested for growth in synthetic media containing one of the following carbon sources: glucose, maltose, sucrose, alanine, proline, glutamate, leucine, arginine and phenylalanine. All of these compounds were effective substrates for one or more of the four fungi. The ability of Saprolegnia sp. to utilize other substrates was studied. Saprolegnia sp. can metabolize soluble starch, fructose, ornithine, aspartate, serine, and lysine but did not grow on galactose and eight additional amino acids.     

The oxidation and reduction of pyridine nucleotides by Rhodopseudomonas spheroides and Chlorobium thiosulfatophilum

Summary The light-induced formation of NADH by whole cells of Rhodopseudomonas spheroides has been followed fluorimetrically and found to lag slightly behind cytochrome c oxidation. The uncoupler, FCCP¹, abolished NADH formation which was also inhibited by HOQNO¹. Electron flow from NADH to oxygen or cytochrome c was inhibited in chromatophores of R. spheroides by HOQNO, antimycin A and rotenone. From the known properties of the inhibitors used it is deduced that NADH formation in the light is dependent upon reversed electron flow. No light-induced formation of NAD(P)H by whole cells or chromatophores of Chlorobium thiosulfatophilum was detected either fluorimetrically or by extraction followed by enzymic assay although cytochrome c oxidation was extensive in whole cells. Extracts of C. thiosulfatophilum catalysed the rapid reduction of endogenous or mammalian cytochrome c; unlike R. spheroides this activity was found almost entirely in the soluble fraction and was insensitive to HOQNO, antimycin A and rotenone. No cytochrome b was detected in C. thiosulfatophilum by difference spectroscopy of pyridine haemochromes of acetone powders. The K _m for NADH of NADH-cytochrome c reductase in both organisms was about 3 mol; the reductase was inhibited by NAD. The rates of NADPH-cytochrome c reductase in R. spheroides particles were too low for K _m determination; for C. thiosulfatophilum particles the K _m for NADPH was about 300 mol. The addition of NADH to soluble extracts of either organism caused the reduction of endogenous flavin that was reoxidised by ferricyanide. The NADH-cytochrome c reductase of C. thiosulfatophilum was not separated from ferredoxin on a DEAE column. It is concluded that in C. thiosulfatophilum the formation of NADH in an energy-linked reaction is unlikely; the possibility of a cyclic electron flow involving chlorophyll, ferredoxin, flavoprotein and cytochrome c is discussed.     

Effect of aerobic and anaerobic conditions on the in vivo nitrate reductase assay in spinach leaves

¹⁵N-labelled nitrate was used to show that nitrate reduction by leaf discs in darkness was suppressed by oxygen, whereas nitrite present within the cell could be reduced under aerobic dark conditions. In other experiments, unlabelled nitrite, allowed to accumulate in the tissue during the dark anaerobic reduction of nitrate was shown by chemical analysis to be metabolised during a subsequent dark aerobic period. Leaves of intact plants resembled incubated leaf discs in accumulating nitrite under anaerobic conditions. Nitrate, n-propanol and several respiratory inhibitors or uncouplers partly reversed the inhibitory effect of oxygen on nitrate reduction in leaf discs in the dark. Of these nitrate and propanol acted synergistically. Reversal was usually associated with inhibition of respiration but some concentrations of 2,4-dinitrophenol (DNP) and ioxynil reversed inhibition without affecting respiratory rates. Respiratory inhibitors and uncouplers stimulated nitrate reduction in the anaerobic in vivo assay i.e. in conditions where the respiratory process is non-functional. Freezing and thawing leaf discs diminished but did not eliminate the sensitivity of nitrate reduction to oxygen inhibition.Abbreviations DNP 2,4-dinitrophenol - HOQNO 8-hydroxyquinoline-N-oxide - DCPIP 2,6-dichlorophenolindophenol - CCCP Carbonyl cyanide m-chlorophenylhydrazone - TES N-tris(hydroxymethyl)methyl-2-amino ethanesulphonic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid     

Studies on the Physiology of Host-parasite Relationship in Orobanche. I. Respiratory Metabolism of Host and Parasite

The respiratory rate of the roots of mustard (Brassica cam-pestris L.) and tomato (Lycopersicum esculentum Mill.) serving as hosts for the total root parasites Orobanche aegyptiaca Pers. and O.cernua Loefll. was measured using Warburg manometric technique. At the same time determinations were made of the respiration of the apical, basal and root regions of the parasites. The effects of sodium fluoride, malonic acid, sodium azide and DNP (2,4-dinitrophenol) on the rate of respiration of the host roots as well as of the parasites were studied. The Orobanche infection results in a marked increase in the respiratory rate near the host-parasite contact region. The damaging effect of infection seems to be due mainly to a continuous flow of water, minerals and metabolites from host to parasite. The haustorial invasion creates an obstruction in the translocation of metabolites. The respiration rate is lower in Orobanche than in the host, which might be related to its slower growth rate, inefficient oxidative processes and an escaping of certain energy-requiring interconversion processes. Roots of O. aegyptiaca are more well-developed and have higher rate of respiration. They can absorb more water and minerals from the soil. This fact might be connected with the specificity of the two species. NaF and malonic acid inhibit the respiration to a similar extent in healthy and infected roots. This indicates that the pathway of respiration does not change materially after infection. The EMP and Krebs cycle seem to operate at a lower intensity in Orobanche, which is proved by the lower inhibition of the respiration as compared to in the host. Azide causes a stronger reduction of the respiration in infected than in healthy roots. It would imply that the infection stimulates the activity of metal containing oxidases. The weaker inhibition of the respiration in Orobanche tissues indicates a mediation of other enzymes in the oxidation processes than in the host. The respiration is less stimulated by DNP in infected than in healthy roots. Contrary to the general effect of DNP, this substance decreases the O₂ uptake in the parasite tissues. This fact may be explained by the occurrence of exceptionally high amounts of endogenous phenolic compounds and an insufficient production of ATP in the parasite.     

Deletion of cytochrome c oxidase genes from the cyanobacterium Synechocystis sp. PCC6803: Evidence for alternative respiratory pathways

An oligonucleotide directed against a highly conserved region of aa₃-type cytochrome c oxidases was used to clone the cox genes from the cyanobacterium Synechocystis sp. PCC6803. Several overlapping clones were obtained that contained the coxB, coxA, and coxC genes, transcribed in the same direction in that order, coding for subunits II, I, and III, respectively. The deduced protein sequences of the three subunits showed high sequence similarity with the corresponding subunits of all known aa₃-type cytochrome c oxidases. A 1.94-kb HindII fragment containing most of coxA and about half of coxC was deleted and replaced by a cassette coding for kanamycin resistance. Mutant cells that were homozygous for the deleted cox locus were obtained. They were viable under photoautotrophic and photoheterotrophic conditions, but contained no cytochrome c oxidase activity. Nevertheless, these mutant cells showed almost normal respiration, defined as cyanide-inhibitable O₂ uptake by whole cells in the dark. It is concluded, therefore, that aa₃-type cytochrome c oxidase is not the only terminal respiratory oxidase in Synechocystis sp. PCC6803.Abbreviations CM cytoplasmic membrane - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - HQNO 2-heptyl-4-hydroxyquinoline N-oxide - ICM intracytoplasmic membranes - SU subunit - TES (N-tris(hydroxymethyl)methyl)-2-aminoethane sulfonic acid     

Respiration in Organic Acid-Forming Molds

Cytochrome c, coenzyme Q and lactic dehydrogenase (l-lactate: NAD oxidoreductase, EC 1, 1, 1, 27) in Rhizopus oryzae were studied in order to investigate the connection between the mechanism of lactate formation and terminal respiration.

Cytochrome c was extracted easily and in good yield by the addition of cetyltrimethyl ammonium bromide to mycelial suspensions. It was purified by calcium phosphate gel and Amberlite IRC-50 resin chromatography.

Coenzyme Q was extracted with ethanol, purified by chromatography on silicic acid, and, following crystallization from a mixture of ethanol and methanol, was identified as coenzyme Q₀.

Lactic dehydrogenase was partially purified and some of its properties were investigated.

Rhizopus oryzae at an early growth stage in shake culture produced almost no lactate. At this stage, the mycelia were rich in cytochrome c and FAD. On the contrary, those of later growth stages fermented a larger amount of the glucose to lactate and the contents of cytochrome c and FAD were lower than in the young mycelia.

Surface cultures produced lactate at a rate very nearly equivalent to the rate of glucose consumption. Addition of zinc to the medium resulted in decreased lactate production, but no increase was observed in the mycelial content of either cytochrome c or FAD in this case. On the other hand, increased quantities of FMN were found in mycelia from shake or surface cultures when zinc was added.     

Inhibitory effects of myxothiazol and 2-n-heptyl-4-hydroxyquinoline-N-oxide on the auxiliary electron transport pathways of Rhodobacter capsulatus

The effects of various electron transport inhibitors upon the rates of reduction NO ₃ ^- , dimethyl sulphoxide (DMSO) and N₂O in anaerobic suspensions of Rhodobacter capsulatus have been studied. A new method for the determination of the rates of reduction of these auxiliary oxidants in intact cells is presented, based on the proportionality observed between the concentration of oxidant and the duration of the electrochromic carotenoid bandshift. For NO ₃ ^- and N₂O good agreement was found between rates of reduction determined using electrodes and those determined by the electrochromic method.Myxothiazol and antimycin A had no effect on the rates of reduction of NO ₃ ^- and DMSO suggesting that the cytochrome b/c ₁complex is not involved in electron transport to these oxidants. 2-n-heptyl-4-hydroxyquinoline-N-oxide (HOQNO) inhibited at two sites, one within the cytochrome b/c ₁complex and the other on the nitrate reducing pathay, but had no effect on electron transport to N₂O or DMSO. In both intact cells and cell free extracts, HOQNO had no effect on the nitrate dependent re-oxidation of reduced methylviologen (MVH₂), a direct electron donor to nitrate reductase.Our data are consistent with a branch point for the auxiliary electron transport pathways at the level of the ubiquinone pool.Non-standard abbreviations HOQNO 2-n-heptyl-4-hydroxyquinoline-N-oxide - TMAO trimethylamine-N-oxide - DMSO dimethyl-sulphoxide - membrane potential - MVH₂ reduced methyl viologen     

Oxidative metabolism of dermatophytes

A method for preparing young, actively respiring dermatophyte mycelia was obtained through the use of concentrated spore inocula and short growth periods in static culture. These hyphal elements were uniform in appearance, and vacuoles were absent. Concentrated mycelial suspensions were obtained which could be transferred easily and accurately. Glucose stimulated oxygen uptake in young mycelia which had been grown in a medium with low carbohydrate content. The level of endogenous respiration was affected by exogenous glucose only when this substrate stimulated oxygen uptake by less than 14%. Low nicotinamide adenine dinucleotide phosphate (NADP) dehydrogenase activity was noted in microconidia which have a low endogenous Q_o2 value, whereas the activity of this enzyme was greater in macroconidia and mycelia which possess higher endogenous Q_o2 values. Microsporum gypseum oxidizes 50% of exogenous glucose and assimilates the remainder. A large percentage of this substrate was assimilated into nitrogenous substances.     

Kinetics and Specificity of Amino Acid Uptake by the Duckweed Spirodela polyrhiza (L.) Schleiden

Borstlap, A. G, Meenks, J. L. D., van Eck, W. F. and Bicker,J. T. E. 1986. Kinetics and specificity of amino acid uptakeby the duckweed Spirodela polyrhiza (L.) Schleiden.—J.exp. Bot. 37: 1020–1035. Uptake of ¹⁴C-labelled amino acids by intact, axenically grownplants of Spirodela polyrhiza (L.) Schleiden was investigated.Experiments in which uptake was measured from the decrease inthe amino acid concentration in the medium, indicated that saturableuptake conforms to the sum of two Michaelis-Menten terms, possiblycorresponding with a high-affinity and a low-affinity system.Further experiments with L-leucine, L-glutamic acid, and L-lysine,in which uptake was measured by assaying the amount of ¹⁴ inthe plants, showed the presence of a non-saturable componentin addition to the dual saturable uptake. Uptake of L-glutamic acid precipitously declined between pH4?0 and 6? and that of L-leucine between pH 4?0 and 8? whereasL-lysine uptake was optimal at pH 6?0. No evidence was foundthat the apparent high-affinity and low-affinity systems respondeddifferently to changes in external pH or to the addition ofCCCP. The non-saturable uptake component was not affected bychanges in external pH or by adding CCCP, and might have beendue to free space uptake. Mutual inhibition of uptake was found between acidic and neutralamino acids (L-leucine, L-methionine, L-glutamic acid) and betweenbasic amino acids (L-lysine, L-ornithine). The basic amino acidshad no effect on the uptake of L-leucine, L-methionine and L-glutamicacid, although the uptake of basic amino acids was inhibitedby glutaminc acid and several neutral amino acids. It is suggested that the duckweed has a high-affinity transportsystem for neutral and acidic amino acids, and a distinct high-affinitysystem for basic amino acids. It is argued that the first systemtransports zwitterionic amino acids (z-system), and that thesecond system transports cationic amino acids(y⁺-system). Thespecificity of the low-affinity system is less certain, butthere is some evidence that it is similar to that of their high-affinitycounterparts. Key words: Kinetics, membrane transport, pH-dependency, transport systems, uptake isotherms     

Detection of ‘long-haired’ Saprolegnia (S. parasitica) isolates using monoclonal antibodies

The ability of five monoclonal antibodies (Mabs) raised against a pathogenic Saprolegnia parasitica isolate from brown trout to detect and differentiate between isolates with bundles of long hairs (S. parasitica) and other Saprolegnia species was determined by means of an indirect immunofluorescence assay. Four of the Mabs used recognized some of the long-haired S. parasitica isolates but also cross-reacted with other Saprolegnia species without bundles of hairs and with Achlya sp. The other Mab (named 18A6) was able to differentiate between the asexual and most of the sexual isolates in the group of long-haired S. parasitica isolates, but did not recognize Achlya sp. or the Saprolegnia species without bundles of hairs, with the exception of S. hypogyna. These results indicate that isolates with bundles of long hairs are closely related with other members of genus Saprolegnia and share several antigens. However, Mab 18A6 seems to recognize an epitope that is expressed mainly in the asexual isolates in the long-haired S. parasitica isolates.     

丙酮酸和乙酰-CoA协同促进L-亮氨酸的合成

【目的】通过理性改造柠檬酸合酶(citrate synthase,CS)、丙酮酸脱氢酶系E1p (pyruvate dehydrogenase complex,PDHC,编码基因aceE)和ATP-柠檬酸裂解酶(ATP-Citrate lyase,ACL),有效供应胞内丙酮酸和乙酰-CoA,以提高L-亮氨酸产量。【方法】以谷氨酸棒杆菌(Corynebacterium glutamicum)为底盘细胞,分析不同CS和PDHC酶活水平对L-亮氨酸合成的影响。随后,考查协同改造CS和PDHC或引入绿硫菌(Chlorobium tepidum)中ACL对L-亮氨酸合成的影响。【结果】低强度的CS酶活(即重组菌XL-3 P_(dapA-R2)gltA)有利于L-亮氨酸的合成,L-亮氨酸产量达到17.5±0.6 g/L。而改变PDHC酶活水平不利于L-亮氨酸的合成。此外,以启动子P_(dapA-R2)控制CS表达,而以启动子P_(gapA)控制PDHC表达时(即重组菌XL-4),可实现胞内丙酮酸和乙酰-CoA的有效供给,L-亮氨酸产量达到20.2±1.7 g/L,且显著降低副产物产量。若在重组菌XL-4中引入C.tepidum,ACL会显著抑制菌体生长而不利于L-亮氨酸合成,而引入到出发菌XL-3中因胞内丙酮酸和乙酰-CoA得到有效供给,目标重组菌XL-5L-亮氨酸产量达到18.5±1.2 g/L,比出发菌株XL-3增加了14.2%。【结论】重组菌XL-4中因协同控制CS和PDHC酶活,从而实现胞内丙酮酸和乙酰-CoA有效供给,促进L-亮氨酸的合成。该研究结果对后续利用代谢工程技术强化微生物合成L-亮氨酸等支链氨基酸具有重要的参考价值。