Generation of Alternate Indole Diterpene Architectures in Two Species of Aspergilli.

The significant structural diversity and potent bioactivity of the fungal indole diterpenes (IDTs) has attracted considerable interest in their biosynthesis. Although substantial skeletal diversity is generated by the action of noncanonical terpene cyclases, comparatively little is known about these enzymes, particularly those involved in the generation of the subgroup containing emindole SA and DA, which show alternate terpenoid skeletons. Here, we describe the IDT biosynthetic machinery generating these unusual IDT architectures from Aspergillus striatus and Aspergillus desertorum. The function of four putative cyclases was interrogated via heterologous expression. Two specific cyclases were identified that catalyze the formation of epimers emindole SA and DA from A. striatus and A. desertorum, respectively. These cyclases are both clustered along with all the elements required for basic IDT biosynthesis yet catalyze an unusual Markovnikov-like cyclization cascade with alternate stereochemical control. Their identification reveals that these alternate architectures are not generated by mechanistically sloppy or promiscuous enzymes, but by cyclases capable of delivering precise regio- and stereospecificities.

Nodulisporic acid E biosynthesis: in vivo characterisation of NodD1, an indole-diterpene prenyltransferase that acts on an emindole SB derived indole-diterpene scaffold.

Prenylation of aromatic compounds is a key tailoring reaction in biosynthesis of bioactive indole-diterpenes. Here, we identify NodD1 as the enzyme responsible for the bisprenylation of nodulisporic acid F. This prenyltransferase showed a preference for its natural indole-diterpene substrate whereas other related enzymes were not able to catalyse this conversion.

Structure of a hyperthermostable dimeric archaeal Rubisco from Hyperthermus butylicus.

The crystal structure of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from the hyperthermophilic archaeon Hyperthermus butylicus is presented at 1.8 Šresolution. Previous structures of archaeal Rubisco have been found to assemble into decamers, and this oligomerization was thought to be required for a highly thermally stable enzyme. In the current study, H. butylicus Rubisco is shown to exist as a dimer in solution, yet has a thermal denaturation midpoint of 114°C, suggesting that high thermal stability can be achieved without an increased oligomeric state. This increased thermal stability appears to be due to an increased number of electrostatic interactions within the monomeric subunit. As such, H. butylicus Rubisco presents a well characterized system in which to investigate the role of assembly and thermal stability in enzyme function.

MIDAS: A Modular DNA Assembly System for Synthetic Biology.

A modular and hierarchical DNA assembly platform for synthetic biology based on Golden Gate (Type IIS restriction enzyme) cloning is described. This enabling technology, termed MIDAS (for Modular Idempotent DNA Assembly System), can be used to precisely assemble multiple DNA fragments in a single reaction using a standardized assembly design. It can be used to build genes from libraries of sequence-verified, reusable parts and to assemble multiple genes in a single vector, with full user control over gene order and orientation, as well as control of the direction of growth (polarity) of the multigene assembly, a feature that allows genes to be nested between other genes or genetic elements. We describe the detailed design and use of MIDAS, exemplified by the reconstruction, in the filamentous fungus Penicillium paxilli, of the metabolic pathway for production of paspaline and paxilline, key intermediates in the biosynthesis of a range of indole diterpenes-a class of secondary metabolites produced by several species of filamentous fungi. MIDAS was used to efficiently assemble a 25.2 kb plasmid from 21 different modules (seven genes, each composed of three basic parts). By using a parts library-based system for construction of complex assemblies, and a unique set of vectors, MIDAS can provide a flexible route to assembling tailored combinations of genes and other genetic elements, thereby supporting synthetic biology applications in a wide range of expression hosts.