Putative Diels-Alder-Catalyzed Cyclization during the Biosynthesis of Lovastatin

A Diels-Alder cyclization proposed to occur during polyketide synthase assembly of the bicyclic core of lovastatin (1) (mevinolin) by Aspergillus terreus MF 4845 was examined via the synthesis of the N-acetylcysteamine (NAC) thioester of [2,11-(13)C(2)]-(E,E,E)-(R)-6-methyldodecatri-2,8,10-enoate (5a). In vitro Diels-Alder cyclization of the corresponding unlabeled NAC ester 5b, ethyl ester 18b, and acid 20b yielded two analogous diastereomers in each case, under either thermal or Lewis acid-catalyzed conditions. The reaction of thioester 5 proceeds readily at 22 degrees C in aqueous media. For 18b, one product is trans-fused ethyl (1R,2R,4aS, 6R,8aR)-1,2,4a,5,6,7,8,8a-octahydro-2,6-dimethylnaphthalene-1-carboxylate (30) (endo product), and the other is cis-fused ethyl (1R,2S,4aR,6R,8aR)-1,2,4a,5,6,7,8,8a-octahydro-2,6-dimethylnaphthalene-1-carboxylate (31) (exo product). Isomer 21 with stereochemistry analogous to 4a,5-dihydromonacolin L (2), a precursor of 1, was made by transformation of a tricyclic lactone, (1S,2S,4aR,6S,8S,8aS)-1-(ethoxycarbonyl)-1,2,4a,5,6,7,8,8a-octahydro-2-methyl-6,8-naphthalenecarbolactone (22) using reduction and Barton deoxygenation. Comparison of 21 with 30 and 31 confirmed the structural assignments and showed that the nonenzymatic 4 + 2 cyclizations of 5, 18, and 20 proceed via chairlike exo and endo transition states with the methyl substituent pseudoequatorial. The proposed biosynthetic Diels-Alder leading to lovastatin (1) would require an endo conformation with the methyl substituent pseudoaxial. Intact incorporation of the labeled hexaketide triene 5a into 1 was not achieved because of rapid degradation by A. terreus cells.     

Synthesis of Peptides Potentially Involved in the Biosynthesis of Clavulanic Acid

Two peptides were synthetised by coupling 3-hydroxy propionic acid with L-ornithine and 3-hydroxy-DL-ornithine, to be tested later in order to establish whether they are links in the biosynthesis of clavulanic acid.     

Computational and Synthetic Investigation of Cationic Rearrangement in the Putative Biosynthesis of Justicane Triterpenoids

A biomimetic cationic structural rearrangement of the oleanolic acid framework is reported for the gram‐scale synthesis and structural reassignment of justicioside E aglycone. The mechanism of the putative biosynthetic rearrangement is investigated with kinetic, computational, and synthetic approaches. The precursor to rearrangement was accessed through two strategic advancements: (1) synthesis of a 1,3‐diketone via oxidation of a β‐silyl enone, and (2) diastereoselective 1,3‐diketone reduction to form a syn‐1,3‐diol using SmI₂ with PhSH as a key additive.     

An Atypical Arginine Dihydrolase Involved in the Biosynthesis of Cyclic Hexapeptide Longicatenamides

The incorporation of non-proteinogenic amino acids (NPAAs) enriches the structural diversity of nonribosomal peptides. Recently, four NPAA-containing cyclic hexapeptides, longicatenamides A−D, were isolated using a combined-culture strategy. Based on in silico analysis, we discovered their putative biosynthetic gene cluster (lon) and proposed a possible biosynthetic mechanism. Surprisingly, the lon22 gene encodes an atypical arginine dihydrolase, which can also catalyze the hydrolysis of citrulline to ornithine. Phylogenetic analysis showed that Lon22-like proteins form a novel clade that is separated from other guanidine-modifying enzymes. After rational design, the catalytic efficiencies of a Lon22 Y80F mutant for arginine and citrulline substrates were 2.31- and 4.70-fold that of the wild-type (WT), respectively. In addition, characterization of the Lon20-A4 adenylation domain suggested that it can incorporate both ornithine and lysine into the final products.     

Enhancement of Long-Chain Fatty Acid Production in Escherichia coli by Coexpressing Genes, Including fabF, Involved in the Elongation Cycle of Fatty Acid Biosynthesis

The goal of this study was to increase the production of long-chain fatty acids and to change the composition of fatty acids through the overexpression of genes involved in the fatty acid synthase (FAS) pathway and utilizing characteristics of a specific gene, namely, fabF. The four genes, fabB, fabG, fabZ, and fabI, are Escherichia coli homologues and function in the elongation cycle of fatty acid biosynthesis. FabB (fabB), an activator of FAS, is a β-oxoacyl-ACP synthase, which catalyzes the addition of acyl-ACP to malonyl-ACP to generate β-oxoacyl-ACP. FabF (fabF) participates at the same step as FabB in the elongation cycle and is structurally and functionally similar to FabB. Hence, we attempted to see if FabF was an activator of FAS, like FabB, with the rationale that these two enzymes have striking similarities. FabF exhibits thermal regulation in that enzyme activity increases at lower temperatures. To confirm its role as an activator of FAS, fabF was overexpressed solely or with other genes in the elongation cycle through biochemical engineering. The fabF recombinants were cultured at different temperatures, resulting in increased total and unsaturated fatty acid accumulation in all the recombinants, compared to wild type, at lower temperatures.     

Putative Biosynthesis of Talarodioxadione & Talarooxime from Talaromyces stipitatus

Polyesters containing 2,4-dihydroxy-6-(2-hydroxypropyl)benzoate and 3-hydroxybutyrate moieties have been isolated from many fungal species. Talaromyces stipitatus was previously reported to produce a similar polyester, talapolyester G. The complete genome sequence and the development of bioinformatics tools have enabled the discovery of the biosynthetic potential of this microorganism. Here, a putative biosynthetic gene cluster (BGC) of the polyesters encoding a highly reducing polyketide synthase (HR-PKS) and nonreducing polyketide synthase (NR-PKS), a cytochrome P450 and a regulator, was identified. Although talapolyester G does not require an oxidative step for its biosynthesis, further investigation into the secondary metabolite production of T. stipitatus resulted in isolating two new metabolites called talarodioxadione and talarooxime, in addition to three known compounds, namely 6-hydroxymellein, 15G256α and transtorine that have never been reported from this organism. Interestingly, the biosynthesis of the cyclic polyester 15G256α requires hydroxylation of an inactive methyl group and thus could be a product of the identified gene cluster. The two compounds, talarooxime and transtorine, are probably the catabolic metabolites of tryptophan through the kynurenine pathway. Tryptophan metabolism exists in almost all organisms and has been of interest to many researchers. The biosynthesis of the new oxime is proposed to involve two subsequent N-hydroxylation of 2-aminoacetophenone.     

Heterologous Production of Fungal Maleidrides Reveals the Cryptic Cyclization Involved in their Biosynthesis

Fungal maleidrides are an important family of bioactive secondary metabolites that consist of 7, 8, or 9‐membered carbocycles with one or two fused maleic anhydride moieties. The biosynthesis of byssochlamic acid (a nonadride) and agnestadride A (a heptadride) was investigated through gene disruption and heterologous expression experiments. The results reveal that the precursors for cyclization are formed by an iterative highly reducing fungal polyketide synthase supported by a hydrolase, together with two citrate‐processing enzymes. The enigmatic ring formation is catalyzed by two proteins with homology to ketosteroid isomerases, and assisted by two proteins with homology to phosphatidylethanolamine‐binding proteins.     

Dissection of the Catalytic Mechanisms of Transmembrane Terpene Cyclases Involved in Fungal Meroterpenoid Biosynthesis

Pyr4-family terpene cyclases are noncanonical transmembrane terpene cyclases involved in the biosynthesis of microbial meroterpenoids and catalyze diverse cyclization reactions. Despite the ubiquity of Pyr4-family terpene cyclases in microorganisms, their three-dimensional structures have never been experimentally determined. Herein, we focused on AdrI, the Pyr4-family enzyme for the andrastin A pathway, and its homologues, and performed a series of mutational experiments using their AlphaFold2-generated structures. Intriguingly, we found that AdrI and InsA7, which both accept the same substrate, use different amino acid residues for the initiation of the cyclization cascade. Furthermore, we obtained several AdrI variants with altered product selectivity, one of which dominantly yielded a new meroterpenoid species. Collectively, our study provides important insights into the catalytic functions of Pyr4-family terpene cyclases and will facilitate the engineering of these enzymes.     

Mechanistic Study of Oxidoreductase AprQ Involved in Biosynthesis of Aminoglycoside Antibiotic Apramycin

Main observation and conclusion The aminoglycoside antibiotic apramycin contains a unique bicyclic octose moiety,and biosynthesis of this moiety involves an oxi...     

Identification of Key Candidate Genes Involved in the Progression of Idiopathic Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a lethal, agnogenic interstitial lung disease with limited therapeutic options. To investigate vital genes involved in the development of IPF, we integrated and compared four expression profiles ({"type":"entrez-geo","attrs":{"text":"GSE110147","term_id":"110147"}}GSE110147, {"type":"entrez-geo","attrs":{"text":"GSE53845","term_id":"53845"}}GSE53845, {"type":"entrez-geo","attrs":{"text":"GSE24206","term_id":"24206"}}GSE24206, and {"type":"entrez-geo","attrs":{"text":"GSE10667","term_id":"10667"}}GSE10667), including 87 IPF samples and 40 normal samples. By reanalyzing these datasets, we managed to identify 62 upregulated genes and 20 downregulated genes in IPF samples compared with normal samples. Differentially expressed genes (DEGs) were analyzed by gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis to illustrate relevant pathways of IPF, biological processes, molecular function, and cell components. The DEGs were then subjected to protein–protein interaction (PPI) for network analysis, serving to find 11 key candidate genes (ANXA3, STX11, THBS2, MMP1, MMP9, MMP7, MMP10, SPP1, COL1A1, ITGB8, IGF1). The result of RT-qPCR and immunohistochemical staining verified our finding as well. In summary, we identified 11 key candidate genes related to the process of IPF, which may contribute to novel treatments of IPF.     

Engineering a Metabolic Pathway for Isobutanol Biosynthesis in Bacillus subtilis

Isobutanol can be biosynthesized via ??-ketoisovalerate catalyzed by heterologous keto acid decarboxylase (KDC) and alcohol dehydrogenase (ADH). In this work, isobutanol biosynthesis pathway was designed in Bacillus subtilis, a notable solvent-tolerant host. In order to do that, a plasmid pPKA expressing KDC and ADH under the control of a B. subtilis strong promoter P₄₃ was constructed. Isobutanol was detected in the products of the recombinant B. subtilis harboring pPKA plasmid, whereas none was detected by the wild-type strain. Effects of the medium ingredients such as glucose concentration and valine addition, and operating parameters such as initial pH, inoculation volume, and medium work volume on isobutanol production were also investigated. Isobutanol production reached to the maximum of 0.607?g/L after 35-h cultivation under the conditions: glucose concentration of 3%, valine addition of 2%, initial pH of 7.0, inoculum of 1%, and work volume of 50?mL/250?mL. Though the isobutanol production by the recombinant was low, it was the first successful attempt to produce isobutanol in engineered B. subtilis, and the results showed its great potential as an isobutanol-producing cell factory.     

High-Performance Liquid Chromatography of Derivatives Involved in the Terminal Steps of Tetracycline Biosynthesis in Streptomyces Aureofaciens

Abstract

A simple and reproducible method was developed for the analysis of tetracycline derivatives involved in the last two steps of tetracycline biosynthetic pathway in Streptomyces aureofaciens. The method is based on a gradient liquid chromatographic separation of the compounds using a microbore octadecyl silica column. Beside separation of a mixture of standards, the method was used for separation, detection and quantitation of dehydrotetracycline and tetracycline prepared enzymatically in vitro using anhydrotetracycline oxygenase and tetracycline dehydrogenase, respectively, isolated from S. aureofaciens. The method permits a simple and accurate characterization of kinetics of corresponding enzymatic activities.     

反相离子对色谱分析水稻愈伤组织中的内源生长素

本文用反相离子对色谱技术定量分析了水稻愈伤组织中的内源生长素５０Ｈ－１ＡＡ和１ＡＡ，样品用８０％丙酮－ＢＨＴ－抗坏血酸抽提，经ＤＥＡＥ纤维素柱和ＸＡＤ－７吸附树脂柱净化和富集，在Ｌｉｃｈｒｏｓｏｒｂ ＲＰＣ１８柱上分离，荧光检测。最低检出限：１ＡＡ为８０ｐｇ，５０Ｈ－ＩＡＡ为０．３ｎｇ。回收率为８５．５％～７３．３％，相对标准偏差为２．２％。文中还对ｐＨ值和离子对试剂对保留值的影响，进行了讨论，对