Distribution and diversity of 'Tectomicrobia', a deep-branching uncultivated bacterial lineage harboring rich producers of bioactive metabolites.

Genomic and functional analyses of bacterial sponge symbionts belonging to the uncultivated candidate genus 'Entotheonella' has revealed them as the prolific producers of bioactive compounds previously identified from their invertebrate hosts. These studies also suggested 'Entotheonella' as the first members of a new candidate phylum, 'Tectomicrobia'. Here we analyzed the phylogenetic structure and environmental distribution of this as-yet sparsely populated phylum-like lineage.

Marine Sponge Endosymbionts: Structural and Functional Specificity of the Microbiome within Cells.

Sponge microbiomes are typically profiled by analyzing the community DNA of whole tissues, which does not distinguish the taxa residing within sponge cells from extracellular microbes. To uncover the endosymbiotic microbiome, we separated the sponge cells to enrich the intracellular microbes. The intracellular bacterial community of sponge was initially assessed by amplicon sequencing, which indicated that it hosts three unique phyla not found in the extracellular and bulk tissue microbiomes.

A monodomain class II terpene cyclase assembles complex isoprenoid scaffolds.

Class II terpene cyclases, such as oxidosqualene and squalene-hopene cyclases, catalyse some of the most complex polycyclization reactions. They minimally exhibit a β,γ-didomain architecture that has been evolutionarily repurposed in a wide range of terpene-processing enzymes and likely resulted from a fusion of unidentified monodomain proteins. Although single domain class I terpene cyclases have already been identified, the corresponding class II counterparts have not been previously reported.

Characterization of an Orphan Type III Polyketide Synthase Conserved in Uncultivated "Entotheonella" Sponge Symbionts.

Uncultivated bacterial symbionts from the candidate genus "Entotheonella" have been shown to produce diverse natural products previously attributed to their sponge hosts. In addition to these known compounds, "Entotheonella" genomes contain rich sets of biosynthetic gene clusters that lack identified natural products. Among these is a small type III polyketide synthase (PKS) cluster, one of only three clusters present in all known "Entotheonella" genomes.

Opening up the Single-Cell Toolbox for Microbial Natural Products Research.

The diverse microbes that produce natural products represent an important source of novel therapeutics, drug leads, and scientific tools. However, the vast majority have not been grown in axenic culture and are members of complex communities. While meta-'omic methods such as metagenomics, -transcriptomics, and -proteomics reveal collective molecular features of this "microbial dark matter", the study of individual microbiome members can be challenging.

The impact of cryosolution thermal contraction on proteins and protein crystals: volumes, conformation and order.

Cryocooling of macromolecular crystals is commonly employed to limit radiation damage during X-ray diffraction data collection. However, cooling itself affects macromolecular conformation and often damages crystals via poorly understood processes. Here, the effects of cryosolution thermal contraction on macromolecular conformation and crystal order in crystals ranging from 32 to 67% solvent content are systematically investigated.

Directed Evolution Mimics Allosteric Activation by Stepwise Tuning of the Conformational Ensemble.

Allosteric enzymes contain a wealth of catalytic diversity that remains distinctly underutilized for biocatalysis. Tryptophan synthase is a model allosteric system and a valuable enzyme for the synthesis of noncanonical amino acids (ncAA). Previously, we evolved the β-subunit from Pyrococcus furiosus, PfTrpB, for ncAA synthase activity in the absence of its native partner protein PfTrpA.

Single-bacterial genomics validates rich and varied specialized metabolism of uncultivated sponge symbionts.

Marine sponges are prolific sources of unique bioactive natural products. The sponge is represented by several distinct variants with largely nonoverlapping chemistry. For the Japanese chemotype Y harboring diverse complex polyketides and peptides, we previously provided genomic and functional evidence that a single symbiont, the filamentous, multicellular organism " Entotheonella factor," produces almost all of these compounds.

Enzyme Nicotinamide Cofactor Specificity Reversal Guided by Automated Structural Analysis and Library Design.

The specificity of enzymes for nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP) as redox carriers can pose a significant hurdle for metabolic engineering and synthetic biology applications, where switching the specificity might be beneficial. We have developed an easy-to-use computational tool (CSR-SALAD) for the design of mutant libraries to simplify the process of reversing the cofactor specificity of an enzyme. Here, we describe the optimal use of this tool and present methods for its application in a laboratory setting.

4S-Hydroxylation of Insulin at ProB28 Accelerates Hexamer Dissociation and Delays Fibrillation.

Daily injections of insulin provide lifesaving benefits to millions of diabetics. But currently available prandial insulins are suboptimal: The onset of action is delayed by slow dissociation of the insulin hexamer in the subcutaneous space, and insulin forms amyloid fibrils upon storage in solution. Here we show, through the use of noncanonical amino acid mutagenesis, that replacement of the proline residue at position 28 of the insulin B-chain (ProB28) by (4S)-hydroxyproline (Hzp) yields an active form of insulin that dissociates more rapidly, and fibrillates more slowly, than the wild-type protein.

Directed Evolution of a Bright Near-Infrared Fluorescent Rhodopsin Using a Synthetic Chromophore.

By engineering a microbial rhodopsin, Archaerhodopsin-3 (Arch), to bind a synthetic chromophore, merocyanine retinal, in place of the natural chromophore all-trans-retinal (ATR), we generated a protein with exceptionally bright and unprecedentedly red-shifted near-infrared (NIR) fluorescence. We show that chromophore substitution generates a fluorescent Arch complex with a 200-nm bathochromic excitation shift relative to ATR-bound wild-type Arch and an emission maximum at 772 nm. Directed evolution of this complex produced variants with pH-sensitive NIR fluorescence and molecular brightness 8.

A General Tool for Engineering the NAD/NADP Cofactor Preference of Oxidoreductases.

The ability to control enzymatic nicotinamide cofactor utilization is critical for engineering efficient metabolic pathways. However, the complex interactions that determine cofactor-binding preference render this engineering particularly challenging. Physics-based models have been insufficiently accurate and blind directed evolution methods too inefficient to be widely adopted.

Discovery of a regioselectivity switch in nitrating P450s guided by molecular dynamics simulations and Markov models.

The dynamic motions of protein structural elements, particularly flexible loops, are intimately linked with diverse aspects of enzyme catalysis. Engineering of these loop regions can alter protein stability, substrate binding and even dramatically impact enzyme function. When these flexible regions are unresolvable structurally, computational reconstruction in combination with large-scale molecular dynamics simulations can be used to guide the engineering strategy.

Artificial domain duplication replicates evolutionary history of ketol-acid reductoisomerases.

The duplication of protein structural domains has been proposed as a common mechanism for the generation of new protein folds. A particularly interesting case is the class II ketol-acid reductoisomerase (KARI), which putatively arose from an ancestral class I KARI by duplication of the C-terminal domain and corresponding loss of obligate dimerization. As a result, the class II enzymes acquired a deeply embedded figure-of-eight knot.

Cofactor specificity motifs and the induced fit mechanism in class I ketol-acid reductoisomerases.

Although most sequenced members of the industrially important ketol-acid reductoisomerase (KARI) family are class I enzymes, structural studies to date have focused primarily on the class II KARIs, which arose through domain duplication. In the present study, we present five new crystal structures of class I KARIs. These include the first structure of a KARI with a six-residue β2αB (cofactor specificity determining) loop and an NADPH phosphate-binding geometry distinct from that of the seven- and 12-residue loops.

Structural, functional, and spectroscopic characterization of the substrate scope of the novel nitrating cytochrome P450 TxtE.

A novel cytochrome P450 enzyme, TxtE, was recently shown to catalyze the direct aromatic nitration of L-tryptophan. This unique chemistry inspired us to ask whether TxtE could serve as a platform for engineering new nitration biocatalysts to replace current harsh synthetic methods. As a first step toward this goal, and to better understand the wild-type enzyme, we obtained high-resolution structures of TxtE in its substrate-free and substrate-bound forms.

General approach to reversing ketol-acid reductoisomerase cofactor dependence from NADPH to NADH.

To date, efforts to switch the cofactor specificity of oxidoreductases from nicotinamide adenine dinucleotide phosphate (NADPH) to nicotinamide adenine dinucleotide (NADH) have been made on a case-by-case basis with varying degrees of success. Here we present a straightforward recipe for altering the cofactor specificity of a class of NADPH-dependent oxidoreductases, the ketol-acid reductoisomerases (KARIs). Combining previous results for an engineered NADH-dependent variant of Escherichia coli KARI with available KARI crystal structures and a comprehensive KARI-sequence alignment, we identified key cofactor specificity determinants and used this information to construct five KARIs with reversed cofactor preference.