Ralfuranones contribute to mushroom‐type biofilm formation by Ralstonia solanacearum strain OE1‐1

After invasion into intercellular spaces of tomato plants, the soil‐borne, plant‐pathogenic Ralstonia solanacearum strain OE1‐1 forms mushroom‐shaped biofilms (mushroom‐type biofilms, mBFs) on tomato cells, leading to its virulence. The strain OE1‐1 produces aryl‐furanone secondary metabolites, ralfuranones (A, B, J, K and L), dependent on the quorum sensing (QS) system, with methyl 3‐hydroxymyristate (3‐OH MAME) synthesized by PhcB as a QS signal. Ralfuranones are associated with the feedback loop of the QS system. A ralfuranone productivity‐deficient mutant (ΔralA) exhibited significantly reduced growth in intercellular spaces compared with strain OE1‐1, losing its virulence. To analyse the function of ralfuranones in mBF formation by OE1‐1 cells, we observed cell aggregates of R. solanacearum strains statically incubated in tomato apoplast fluids on filters under a scanning electron microscope. The ΔralA strain formed significantly fewer microcolonies and mBFs than strain OE1‐1. Supplementation of ralfuranones A, B, J and K, but not L, significantly enhanced the development of mBF formation by ΔralA. Furthermore, a phcB‐ and ralA‐deleted mutant (ΔphcB/ralA) exhibited less formation of mBFs than OE1‐1, although a QS‐deficient, phcB‐deleted mutant formed mBFs similar to OE1‐1. Supplementation with 3‐OH MAME significantly reduced the formation of mBFs by ΔphcB/ralA. The application of each ralfuranone significantly increased the formation of mBFs by ΔphcB/ralA supplied with 3‐OH MAME. Together, our findings indicate that ralfuranones are implicated not only in the development of mBFs by strain OE1‐1, but also in the suppression of QS‐mediated negative regulation of mBF formation.     

顺式和反式-AT型聚酮合酶研究进展及应用

聚酮类化合物因广泛应用于医药等方面而被大家所熟知,Ⅰ型聚酮合酶（Polyketide Synthase,PKS）在催化聚酮类化合物的生物合成中起着重要的作用。根据不同的酰基转移酶（Acyltransferase,AT）结构域,I型PKS可分为顺式-AT （cis-Acyltransferase,cis-AT）型PKS和反式-AT （trans-Acyltransferase,trans-AT）型PKS,目前cis-AT型PKS研究得比较透彻,trans-AT型PKS相关研究成为当今热点。本文总结了cis-AT型PKS和trans-AT型PKS的联系与区别、工程进展、相关应用以及目前存在的问题,以期为了解cis-AT型PKS和trans-AT型PKS在聚酮化合物合成中的作用提供参考。     

Current aspects of auxin biosynthesis in plants

Auxin is an important plant hormone essential for many aspects of plant growth and development. Indole-3-acetic acid (IAA) is the most studied auxin in plants, and its biosynthesis pathway has been investigated for over 70 years. Although the complete picture of auxin biosynthesis remains to be elucidated, remarkable progress has been made recently in understanding the mechanism of IAA biosynthesis. Genetic and biochemical studies demonstrate that IAA is mainly synthesized from l-tryptophan (Trp) via indole-3-pyruvate by two-step reactions in Arabidopsis. While IAA is also produced from Trp via indole-3-acetaldoxime in Arabidopsis, this pathway likely plays an auxiliary role in plants of the family Brassicaceae. Recent studies suggest that the Trp-independent pathway is not a major route for IAA biosynthesis, but they reveal an important role for a cytosolic indole synthase in this pathway. In this review, I summarize current views and future prospects of IAA biosynthesis research in plants.     

A genomic approach to isoflavone biosynthesis in kudzu (<Emphasis Type="Italic">Pueraria lobata</Emphasis>)

Roots of kudzu (Pueraria lobata) are a rich source of isoflavone O- and C-glycosides. Although O-glycosylation of (iso)flavonoids has been well characterized at the molecular level, no plant isoflavonoid C-glycosyltransferase genes have yet been isolated. To address the biosynthesis of kudzu isoflavonoids, we generated 6,365 high-quality expressed sequence tags (ESTs) from a subtraction cDNA library constructed using RNA from roots that differentially accumulate puerarin. The ESTs were clustered into 722 TCs and 3,913 singletons, from which 15 family I glycosyltransferases (UGTs) were identified. Hierarchical clustering analysis of the expression patterns of these UGTs with isoflavone synthase (IFS) in a range of tissues identified UGTs with potential functions in isoflavone glycosylation. The open reading frames of these UGTs were expressed in E. coli for functional analysis, and one was shown to preferentially glycosylate isoflavones at the 7-O-position. In addition, ESTs corresponding to chalcone synthase, chalcone reductase, chalcone isomerase (CHI) and 2-hydroxyisoflavanone dehydratase were identified. Recombinant CHI proteins had high activities with both 6′-deoxy- and 6′-hydroxy chalcones, typical of Type II CHIs. Establishment of this EST database and identification of genes associated with kudzu isoflavone biosynthesis and glycosylation provide a new resource for metabolic engineering of bioactive kudzu isoflavones.     

Insect Trypsin Modulating Oostatic Factor (Neb-TMOF) and Its Analogs: Preliminary Structure/Biological Function Relationship Studies

Summary Neb-TMOF, the trypsin modulating oostatic factor of gray fleshflyNeobellieria bullata, is a hexapeptide with the following sequence: H-Asn-Pro-Thr-Asn-Leu-His-OH. It has been isolated from vitellogenic ovaries in 1994. TMOF, the newly discovered insect peptide, inhibits trypsin biosynthesis in the gut, lowers yolk polypeptide concentration in the hemolymph and strongly inhibits ecdysone biosynthesis by larval ring glands. It is interesting that this short non-protected peptide contains in its molecule two Asn residues at positions 1 and 4 and His at its C-terminus. To obtain information about the role of the His-6 and Asn-4 residues we synthesised two series of Neb-TMOF analogs, modified: (1) in position 6 byd-His (I), His(Bzl) (II) and Phe(p-X) derivatives, where X=NH₂ (III), NO₂ (IV), OEt (V) and OH (VI) and (2) in position 4 by such amino acid residues as Ser (VII), Thr (VIII), Gly (IX), Asp (X), Glu (XI) andd-Asn (XII). The influence of these peptides on trypsin biosynthesis inN. bullata was determinedin vivo. In preliminary investigations, we found that Neb-TMOF, [Phe(NH₂)⁶], and [Phe(NO₂)⁶]-Neb-TMOF inhibited trypsin biosynthesis, whereas [d-His)⁶]- and [d-His(Bzl)⁶]-Neb-TMOF were inactive. In further biological studies performedin vitro on heart ofTenebrio molitor were found that-TMOF and [Phe(p-NH₂)⁶]-Neb-TMOF showed weak cardioexcitatory activity, about 30% of the cardioexcitatory activity of proctolin, an insect neuromodulating peptide.     

A cluster of genes for the biosynthesis of spinosyns, novel macrolide insect control agents produced by Saccharopolyspora spinosa

Spinosyns A and D are the active ingredients in a family of insect control agents produced by fermentation of Saccharopolyspora spinosa. Spinosyns are 21–carbon tetracyclic lactones to which are attached two deoxysugars. Most of the genes involved in spinosyn biosynthesis are clustered in an 74 kb region of the S. spinosa genome. This region has been characterized by DNA sequence analysis and by targeted gene disruptions. The spinosyn biosynthetic gene cluster contains five large genes encoding a type I polyketide synthase, and 14 genes involved in modification of the macrolactone, or in the synthesis, modification and attachment of the deoxysugars. Four genes required for rhamnose biosynthesis (two of which are also required for forosamine biosynthesis) are not present in the cluster. A pathway for the biosynthesis of spinosyns is proposed.     

The effect of 6-deoxy-D-fructose on flavour bioformation from strawberry (Fragaria x ananassa, cv. Elsanta) callus cultures

The biosynthesis of 2,5-dimethyl-4-hydroxy-2H-furan-3-one has been investigated in order to improve the flavour of cultivated strawberries. Callus cultures of strawberries have been established. The probable immediate precursor of 2,5-dimethyl-4-hydroxy-2H-furan-3-one (6-deoxy-D-fructose) has been fed to callus cultures and the levels of the product are compared in cultures fed with precursor and control tissues. The increased levels of 2,5-dimethyl-4-hydroxy-2H-furan-3-one-glucoside in the precursor fed cultures suggests that methylpentoses are key compounds for the biosynthesis of this specific furanone.     

Involvements of <Emphasis Type="Italic">S</Emphasis>-nitrosylation and denitrosylation in the production of polyphenols by <Emphasis Type="Italic">Inonotus obliquus</Emphasis>

Nitric oxide (NO) has been evidenced to mediate biosynthesis of polyphenols in Inonotus obliquus. However, it remains unknown how NO regulates their biosynthesis. Here we show that higher cellular NO levels coincided with higher accumulation of S-nitrosothiols (SNO; the products of NO combined with a specific residue in glutathione or proteins) and polyphenols, and higher activity of denitrosylated S-nitrosoglutathione reductase (GSNOR) and thioredoxin reductase (TrxR). This homeostasis was breached by GSNOR or TrxR inhibitors. Inhibiting GSNOR boosted TrxR activity, but reduced SNO formation, coinciding with an enhanced production of polyphenols. Likewise, inhibiting TrxR increased GSNOR activity and SNO production, but downregulated accumulation of polyphenols. Inhibiting GSNOR or TrxR also modified the polyphenolic profiles of I. obliquus. Suppressing GSNOR-enhanced biosynthesis of phelligridins C and H, inoscavin C and methyl inoscavin B, but reduced that of phelligridin D, methyl inoscavin A, davallialactone and methyl davallialactone, the typical polyphenols in I. obliquus. Similarly, downregulating TrxR increased production of phelligridin D, methyl inoscavin A, davallialactone, and methyl davallialactone, but shrinking that of phelligridins C and H, methyl inoscavin B and inoscavin C. Thus, in I. obliquus, the state of S-nitrosylation and denitrosylation affects not only the accumulation of polyphenols, but also their metabolic profiles.     

Trehalose Biosynthesis in Response to Abiotic Stresses

Trehalose is a non‐reducing disaccharide that is present in diverse organisms ranging from bacteria and fungi to invertebrates, in which it serves as an energy source, osmolyte or protein/membrane protectant. The occurrence of trehalose and trehalose biosynthesis pathway in plants has been discovered recently. Multiple studies have revealed regulatory roles of trehalose‐6‐phosphate, a precursor of trehalose, in sugar metabolism, growth and development in plants. Trehalose levels are generally quite low in plants but may alter in response to environmental stresses. Transgenic plants overexpressing microbial trehalose biosynthesis genes have been shown to contain increased levels of trehalose and display drought, salt and cold tolerance. In‐silico expression profiling of all Arabidopsis trehalose‐6‐phosphate synthases (TPSs) and trehalose‐6‐phosphate phosphatases (TPPs) revealed that certain classes of TPS and TPP genes are differentially regulated in response to a variety of abiotic stresses. These studies point to the importance of trehalose biosynthesis in stress responses.     

Identification of three Zwittermicin A biosynthesis-related genes from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> subsp. <Emphasis Type="Italic">kurstaki</Emphasis> strain YBT-1520

Zwittermicin A (ZwA) is a novel, broad-spectrum linear aminopolyol antibiotic produced by some Bacillus cereus and Bacillus thuringiensis. However, only part of its biosynthesis cluster has been identified and characterized from B. cereus UW85. To better understand the biosynthesis cluster of ZwA, a bacterial artificial chromosome (BAC) library of B. thuringiensis subsp. kurstaki strain YBT-1520, a ZwA-producing strain, was constructed. Two BAC clones, 1F8 and 5E2, were obtained by PCR, which overlap the known ZwA biosynthesis cluster of B. cereus UW85. This ZwA biosynthesis cluster is at least 38.6 kb and is located on the chromosome, instead of the plasmid. Partial DNA sequencing revealed both BAC clones carry three new ZwA biosynthesis-related genes, zwa6, zwa5A and zwa5B, which were found at the corresponding location of B. cereus UW85. Putative amino acid sequences of these genes shown that ZWA6 is homologous to a typical carbamoyltransferase from Streptomyces avermitilis, while ZWA5A and ZWA5B are homologs of cysteine synthetase and ornithine cyclodeaminase which jointly synthesize 2,3-diaminopropionate in the viomycin biosynthesis pathway, respectively. The identification of these three genes further supports the hypothesized ZwA biosynthesis pathway.     

Precursor-directed biosynthesis of 6-deoxyerythronolide B analogues is improved by removal of the initial catalytic sites of the polyketide synthase

Precursor-directed biosynthesis has been shown to be a powerful tool for the production of polyketide analogues that would be difficult or cost prohibitive to produce from medicinal chemistry efforts alone. It has been most extensively demonstrated using a KS1 null mutation (KS1⁰) to block the first round of condensation in the biosynthesis of the erythromycin polyketide synthase (DEBS) for the production of analogues of its aglycone, 6-deoxyerythronolide B (6-dEB). Here we show that removing the DEBS loading domain and first module (mod1Δ), rather than using the KS1⁰ system, can lead to an increase in the utilization of some chemical precursors and production of 6-dEB analogues (R-6dEB) in both Streptomyces coelicolor and Saccharopolyspora erythraea. While the difference in utilization of the precursor was diketide specific, in strains fed (2R*, 3S*)-5-fluoro-3-hydroxy-2-methylpentanoate N-propionylcysteamine thioester, twofold increases in both utilization of the diketide and 15-fluoro-6dEB (15F-6dEB) production were observed in S. coelicolor, and S. erythraea exhibited a tenfold increase in production of 15-fluoro-erythromycin when utilizing the mod1Δ rather than the KS1⁰ system.     

Small Cab-like proteins regulating tetrapyrrole biosynthesis in the cyanobacterium Synechocystis sp. PCC 6803

In the cyanobacterium Synechocystis sp. PCC 6803 five open reading frames (scpA–scpE) have been identified that code for single-helix proteins resembling helices I and III of chlorophyll a/b-binding (Cab) antenna proteins from higher plants. They have been named SCPs (small Cab-like proteins). Deletion of a single scp gene in a wild-type or in a photosystem I-less (PS I-less) strain has little effect. However, the effects of functional deletion of scpB or scpE were remarkable under conditions where chlorophyll availability was limited. When cells of a strain lacking PS I and chlL (coding for a polypeptide needed for light-independent protochlorophyllide reduction) were grown in darkness, the phycobilin and protochlorophyllide levels decreased upon deletion of scpB or scpE and the protoheme level was reduced in the strain lacking scpE. Addition of -aminolevulinic acid (ALA) in darkness drastically increased the level of Mg-protoporphyrin IX and Mg-protoporphyrin IX monomethyl ester in the PS I-less/chlL ^–/scpE ^– strain, whereas PChlide accumulated in the PS I-less/chlL ^–/scpB ^– strain. In the PS I-less/chlL ^– control strain ALA supplementation did not lead to large changes in the levels of tetrapyrrole biosynthesis intermediates. We propose that ScpE and ScpB regulate tetrapyrrole biosynthesis as a function of pigment availability. This regulation occurs primarily at an early step of tetrapyrrole biosynthesis, prior to ALA. In view of the conserved nature of chlorophyll-binding sites in these proteins, it seems likely that regulation by SCPs occurs as a function of chlorophyll availability, with SCPs activating chlorophyll biosynthesis steps when they do not have pigments bound.     

Two Alkaline Phosphatases from a Butirosin A Producer Bacillus vitellinus

In low-phosphate medium, a butirosin A producer B. vitellinus produced two alkaline phosphatases. These enzymes were fractionated by DEAE-cellulose column chromatography. One phosphatase (Pho I) was eluted with the lower concentration of NaCl compared with the other phosphatase (Pho II). In the wild type strain, Pho I was completely repressed in the high-phosphate medium, but 30% of the fully-derepressed level of Pho II was still produced.

The phosphatase-negative mutant, P-15, that was shown to accumulate butirosin A-6′-N-diphosphate in our previous study, produced only one phosphatase (Pho I) under the low-phosphate condition. Therefore, P-15 was characteristic of the deficiency in Pho II synthesis.

The partially purified preparations of Pho I and II were characterized. Although both enzymes had a similar molecular weight, they could be differentiated in control of synthesis, heat stability, substrate specificity and other properties. Kinetic properties showed that Pho-II was more specific than Pho I to aminoglycoside-phosphates; butirosin A-3′-phosphate, butirosin A-6′-N-diphosphate and 6′-deamino-6′-hydroxybutirosin A-6′-O-diphosphate. The roles of the two phosphatases in butirosin A biosynthesis were discussed.     

The shikimic acid pathway and polyketide biosynthesis

The shikimic acid pathway, ubiquitous in microorganisms and plants, provides precursors for the biosynthesis of primary metabolites such as the aromatic amino acids and folic acid. Several branchpoints from the primary metabolic pathway also provide aromatic and, in some unusual cases, nonaromatic precursors for the biosynthesis of secondary metabolites. We report herein recent progress in the analysis of two unusual branches of the shikimic acid pathway in streptomycetes; the formation of the cyclohexanecarboxylic acid (CHC)-derived moiety of the antifungal agent ansatrienin and the dihydroxycyclohexanecarboxylic acid (DHCHC) starter unit for the biosynthesis of the immunosuppressant ascomycin. A gene for 1-cyclohexenylcarbonyl-CoA reductase, chcA, which plays a role in catalyzing three of the reductive steps leading from shikimic acid to CHC has been characterized from Streptomyces collinus. A cluster of six open reading frames (ORFs) has been identified by sequencing in both directions from chcA and the putative role of these in CHC biosynthesis is discussed. The individual steps involved in the biosynthesis of DHCHC from shikimic acid in Streptomyces hygroscopicus var ascomyceticus has been delineated and shown to be stereochemically and enzymatically distinct from the CHC pathway. A dehydroquinate dehydratase gene (dhq) likely involved in providing shikimic acid for both DHCHC biosynthesis and primary metabolism has been cloned, sequenced and characterized. Received 17 February 1998/ Accepted in revised form 26 April 1998     

A new method for isolation of S-adenosylmethionine (SAM)-accumulating yeast

S-Adenosylmethionine (SAM) is an important metabolite that participates in many reactions as a methyl group donor in all organisms, and has attracted much interest in clinical research because of its potential to improve many diseases, such as depression, liver disease, and osteoarthritis. Because of these potential applications, a more efficient means is needed to produce SAM. Accordingly, we developed a positive selection method to isolate SAM-accumulating yeast in this study. In Saccharomyces cerevisiae, one of the main reactions consuming SAM is thought to be the methylation reaction in the biosynthesis of ergosterol that is catalyzed by Erg6p. Mutants with deficiencies in ergosterol biosynthesis may accumulate SAM as a result of the reduction of SAM consumption in ergosterol biosynthesis. We have applied this method to isolate SAM-accumulating yeasts with nystatin, which has been used to select mutants with deficiencies in ergosterol biosynthesis. SAM-accumulating mutants from S. cerevisiae K-9 and X2180-1A were efficiently isolated through this method. These mutants accumulated 1.7–5.5 times more SAM than their parental strains. NMR and GC-MS analyses suggested that two mutants from K-9 have a mutation in the erg4 gene, and erg4 disruptants from laboratory strains also accumulated more SAM than their parental strains. These results indicate that mutants having mutations in the genes for enzymes that act downstream of Erg6p in ergosterol biosynthesis are effective in accumulating SAM.     

Recycling of pyridoxine (vitamin B6) by PUP1 in Arabidopsis

Vitamin B6 is a cofactor for more than 140 essential enzymatic reactions and was recently proposed as a potent antioxidant, playing a role in the photoprotection of plants. De novo biosynthesis of the vitamin has been described relatively recently and is derived from simple sugar precursors as well as glutamine. In addition, the vitamin can be taken up from exogenous sources in a broad range of organisms, including plants. However, specific transporters have been identified only in yeast. Here we assess the ability of the family of Arabidopsis purine permeases (PUPs) to transport vitamin B6. Several members of the family complement the growth phenotype of a Saccharomyces cerevisiae mutant strain impaired in both de novo biosynthesis of vitamin B6 as well as its uptake. The strongest activity was observed with PUP1 and was confirmed by direct measurement of uptake in yeast as well as in planta, defining PUP1 as a high affinity transporter for pyridoxine. At the tissue level the protein is localised to hydathodes and here we use confocal microscopy to illustrate that at the cellular level it is targeted to the plasma membrane. Interestingly, we observe alterations in pyridoxine recycling from the guttation sap upon overexpression of PUP1 and in a pup1 mutant, consistent with the role of the protein in retrieval of pyridoxine. Furthermore, combining the pup1 mutant with a vitamin B6 de novo biosynthesis mutant (pdx1.3) corroborates that PUP1 is involved in the uptake of the vitamin.