Identification and Analysis of the Biosynthetic Gene Cluster for the Hydrazide-Containing Aryl Polyene Spinamycin.

Natural products containing nitrogen-nitrogen (N-N) bonds have attracted much attention because of their bioactivities and chemical features. Several recent studies have revealed the nitrous acid-dependent N-N bond-forming machinery. However, the catalytic mechanisms of hydrazide synthesis using nitrous acid remain unknown. Herein, we focused on spinamycin, a hydrazide-containing aryl polyene produced by Streptomyces albospinus JCM3399. In the S. albospinus genome, we discovered a putative spinamycin biosynthetic gene (spi) cluster containing genes that encode a type II polyketide synthase and genes for the secondary metabolism-specific nitrous acid biosynthesis pathway. A gene inactivation experiment showed that this cluster was responsible for spinamycin biosynthesis. A feeding experiment using stable isotope-labeled sodium nitrite and analysis of nitrous acid-synthesizing enzymes in vitro strongly indicated that one of the nitrogen atoms of the hydrazide group was derived from nitrous acid. In vitro substrate specificity analysis of SpiA3, which is responsible for loading a starter substrate onto polyketide synthase, indicated that N-N bond formation occurs after starter substrate loading. In vitro analysis showed that the AMP-dependent ligase SpiA7 catalyzes the diazotization of an amino group on a benzene ring without a hydroxy group, resulting in a highly reactive diazo intermediate, which may be the key step in hydrazide group formation. Therefore, we propose the overall biosynthetic pathway of spinamycin. This study expands our knowledge of N-N bond formation in microbial secondary metabolism.

A spatiotemporal transcriptomic network dynamically modulates stalk development in maize.

Maize (Zea mays) is an important cereal crop with suitable stalk formation which is beneficial for acquiring an ideal agronomic trait to resist lodging and higher planting density. The elongation pattern of stalks arises from the variable growth of individual internodes driven by cell division and cell expansion comprising the maize stalk. However, the spatiotemporal dynamics and regulatory network of the maize stalk development and differentiation process remain unclear. Here, we report spatiotemporally resolved transcriptomes using all internodes of the whole stalks from developing maize at the elongation and maturation stages. We identified four distinct groups corresponding to four developmental zones and nine specific clusters with diverse spatiotemporal expression patterns among individual internodes of the stalk. Through weighted gene coexpression network analysis, we constructed transcriptional regulatory networks at a fine spatiotemporal resolution and uncovered key modules and candidate genes involved in internode maintenance, elongation, and division that determine stalk length and thickness in maize. Further CRISPR/Cas9-mediated knockout validated the function of a cytochrome P450 gene, ZmD1, in the regulation of stalk length and thickness as predicted by the WGCN. Collectively, these results provide insights into the high genetic complexity of stalk development and the potentially valuable resources with ideal stalk lengths and widths for genetic improvements in maize.

Enantioselective synthesis and stereochemical determination of the highly reduced polyketide ishigamide.

Ishigamide was isolated as a metabolite of a recombinant strain of Streptomyces sp. MSC090213JE08 and its unsaturated fatty acid moiety has been confirmed in vitro to be synthesized by a type II PKS. Biosynthesis of such a highly reduced polyketide by a type II PKS is worthy of note. However, absolute configuration of ishigamide remained unknown. (R)-Ishigamide was synthesized enantioselectively employing Stille coupling and Wittig reaction between three units, vinyl iodide, stannyldienal, and Wittig salt. Stereochemistry of natural ishigamide was determined to be R by chiral HPLC analysis comparing with the synthesized standard.

Structural basis for selectivity in a highly reducing type II polyketide synthase.

In type II polyketide synthases (PKSs), the ketosynthase-chain length factor (KS-CLF) complex catalyzes polyketide chain elongation with the acyl carrier protein (ACP). Highly reducing type II PKSs, represented by IgaPKS, produce polyene structures instead of the well-known aromatic skeletons. Here, we report the crystal structures of the Iga11-Iga12 (KS-CLF) heterodimer and the covalently cross-linked Iga10=Iga11-Iga12 (ACP=KS-CLF) tripartite complex. The latter structure revealed the molecular basis of the interaction between Iga10 and Iga11-Iga12, which differs from that between the ACP and KS of Escherichia coli fatty acid synthase. Furthermore, the reaction pocket structure and site-directed mutagenesis revealed that the negative charge of Asp 113 of Iga11 prevents further condensation using a ß-ketoacyl product as a substrate, which distinguishes IgaPKS from typical type II PKSs. This work will facilitate the future rational design of PKSs.

Overexpression of a maize BR transcription factor ZmBZR1 in Arabidopsis enlarges organ and seed size of the transgenic plants.

In plants, the organ size is one of the most important features and regulated by an elaborate developmental program involving both internal and external signals. The steroidal hormone brassinosteroid (BR) plays an important role in regulating the organ size. BRASSINAZOLE RESISTANT 1 (BZR1) is one of important transcription factors that regulate organ size in BR signal pathway in Arabidopsis. The function of BZR1 on organ size is well characterized in Arabidopsis, but poorly understood in maize (Zea mays). To understand the mechanism of intrinsic organ size regulated by BZR1 during organogenesis, we identified the maize BZR1 and examined its function in Arabidopsis. Overexpression of ZmBZR1 displayed phenotypes of enlarged cotyledons, rosette leaves, floral organ and seed size in Arabidopsis. The cells in rosette leaves as well as other organs in transgenic ZmBZR1 lines were dramatically larger and longer than those in Col-0. ChIP and RNA-seq analysis showed ZmBZR1 can directly bind to the promoter region of organ size related genes, Germination Repression and Cell Expansion receptor-like kinase (GRACE) and KIP-RELATED PROTEIN6 (KRP6) to regulate their expression, suggesting ZmBZR1 is required for the progressive increase in cells during Arabidopsis development. Collectively, our findings provide significant insights into the mechanisms underlying regulation of organ size mediated by maize BZR1.

Reconstitution of a Type II Polyketide Synthase that Catalyzes Polyene Formation.

While type II polyketide synthases (PKSs) are known for producing aromatic compounds, a phylogenetically new subfamily of type II PKSs have been recently proposed to synthesize polyene structures. Here we report in vitro analysis of such a type II PKS, IgaPKS for ishigamide biosynthesis. The ketoreductase (Iga13) and dehydratase (Iga16) were shown to catalyze the reduction of a ß-keto group and dehydration of a ß-hydroxy group, respectively, to form a trans double bond. Incubation of the acyl carrier protein (Iga10), the ketosynthase/chain length factor complex (Iga11-Iga12), Iga13 and Iga16 with malonyl and hexanoyl-CoAs and NADPH followed by KOH hydrolysis resulted in the formation of four unsaturated carboxylic acids (C8 , C10 , C12 , and C14 ), indicating that IgaPKS catalyzes tetraene formation by repeating the cycle of condensation, keto-reduction and dehydration with strict stereo-specificity. We propose "highly reducing type II PKS subfamily" for the polyene-producing type II PKSs.

Production of a Novel Amide-Containing Polyene by Activating a Cryptic Biosynthetic Gene Cluster in Streptomyces sp. MSC090213JE08.

Streptomyces sp. MSC090213JE08 seems to have more than 20 cryptic biosynthetic gene clusters for secondary metabolites. We aimed to activate some of them by forced production of Streptomyces antibiotic regulatory protein (SARP) family transcriptional activators. We constructed seven recombinant strains, each of which contained a SARP gene under the control of a constitutive promoter, and subjected them to comparative metabolic profiling analysis. Four of the seven strains produced nine metabolites that were hardly detected in the control strains. We isolated a new metabolite (named ishigamide) from the SARP-7-expressing strain and determined its structure as 3-((2E,4E,6E,8E)-13-hydroxytetradeca-2,4,6,8-tetraenamido)propanoic acid. Genome scanning and gene disruption studies identified the ishigamide biosynthetic gene cluster adjacent to the SARP-7 gene. We think that a new subfamily of type II polyketide synthase is involved in the biosynthesis of the polyene structure of ishigamide.

Systematic Analysis of the Functions of Lysine Acetylation in the Regulation of Tat Activity.

The Tat protein of HIV-1 has several well-known properties, such as nucleocytoplasmic trafficking, transactivation of transcription, interaction with tubulin, regulation of mitotic progression, and induction of apoptosis. Previous studies have identified a couple of lysine residues in Tat that are essential for its functions. In order to analyze the functions of all the lysine residues in Tat, we mutated them individually to alanine, glutamine, and arginine. Through systematic analysis of the lysine mutants, we discovered several previously unidentified characteristics of Tat. We found that lysine acetylation could modulate the subcellular localization of Tat, in addition to the regulation of its transactivation activity. Our data also revealed that lysine mutations had distinct effects on microtubule assembly and Tat binding to bromodomain proteins. By correlation analysis, we further found that the effects of Tat on apoptosis and mitotic progression were not entirely attributed to its effect on microtubule assembly. Our findings suggest that Tat may regulate diverse cellular activities through binding to different proteins and that the acetylation of distinct lysine residues in Tat may modulate its interaction with various partners.