Continuous synthesis of E. coli genome sections and Mb-scale human DNA assembly.

Whole-genome synthesis provides a powerful approach for understanding and expanding organism function1-3. To build large genomes rapidly, scalably and in parallel, we need (1) methods for assembling megabases of DNA from shorter precursors and (2) strategies for rapidly and scalably replacing the genomic DNA of organisms with synthetic DNA. Here we develop bacterial artificial chromosome (BAC) stepwise insertion synthesis (BASIS)-a method for megabase-scale assembly of DNA in Escherichia coli episomes. We used BASIS to assemble 1.1 Mb of human DNA containing numerous exons, introns, repetitive sequences, G-quadruplexes, and long and short interspersed nuclear elements (LINEs and SINEs). BASIS provides a powerful platform for building synthetic genomes for diverse organisms. We also developed continuous genome synthesis (CGS)-a method for continuously replacing sequential 100 kb stretches of the E. coli genome with synthetic DNA; CGS minimizes crossovers1,4 between the synthetic DNA and the genome such that the output for each 100 kb replacement provides, without sequencing, the input for the next 100 kb replacement. Using CGS, we synthesized a 0.5 Mb section of the E. coli genome-a key intermediate in its total synthesis1-from five episomes in 10 days. By parallelizing CGS and combining it with rapid oligonucleotide synthesis and episome assembly5,6, along with rapid methods for compiling a single genome from strains bearing distinct synthetic genome sections1,7,8, we anticipate that it will be possible to synthesize entire E. coli genomes from functional designs in less than 2 months.

Genetically programmed cell-based synthesis of non-natural peptide and depsipeptide macrocycles.

The direct genetically encoded cell-based synthesis of non-natural peptide and depsipeptide macrocycles is an outstanding challenge. Here we programme the encoded synthesis of 25 diverse non-natural macrocyclic peptides, each containing two non-canonical amino acids, in Syn61Δ3-derived cells; these cells contain a synthetic Escherichia coli genome in which the annotated occurrences of two sense codons and a stop codon, and the cognate transfer RNAs (tRNAs) and release factor that normally decode these codons, have been removed. We further demonstrate that pyrrolysyl-tRNA synthetase/tRNA pairs from distinct classes can be engineered to direct the co-translational incorporation of diverse alpha hydroxy acids, with both aliphatic and aromatic side chains. We define 49 engineered mutually orthogonal pairs that recognize distinct non-canonical amino acids or alpha hydroxy acids and decode distinct codons. Finally, we combine our advances to programme Syn61Δ3-derived cells for the encoded synthesis of 12 diverse non-natural depsipeptide macrocycles, which contain two non-canonical side chains and either one or two ester bonds.

Metabolically controlled histone H4K5 acylation/acetylation ratio drives BRD4 genomic distribution.

In addition to acetylation, histones are modified by a series of competing longer-chain acylations. Most of these acylation marks are enriched and co-exist with acetylation on active gene regulatory elements. Their seemingly redundant functions hinder our understanding of histone acylations' specific roles. Here, by using an acute lymphoblastic leukemia (ALL) cell model and blasts from individuals with B-precusor ALL (B-ALL), we demonstrate a role of mitochondrial activity in controlling the histone acylation/acetylation ratio, especially at histone H4 lysine 5 (H4K5). An increase in the ratio of non-acetyl acylations (crotonylation or butyrylation) over acetylation on H4K5 weakens bromodomain containing protein 4 (BRD4) bromodomain-dependent chromatin interaction and enhances BRD4 nuclear mobility and availability for binding transcription start site regions of active genes. Our data suggest that the metabolism-driven control of the histone acetylation/longer-chain acylation(s) ratio could be a common mechanism regulating the bromodomain factors' functional genomic distribution.

Sense codon reassignment enables viral resistance and encoded polymer synthesis.

It is widely hypothesized that removing cellular transfer RNAs (tRNAs)-making their cognate codons unreadable-might create a genetic firewall to viral infection and enable sense codon reassignment. However, it has been impossible to test these hypotheses. In this work, following synonymous codon compression and laboratory evolution in Escherichia coli, we deleted the tRNAs and release factor 1, which normally decode two sense codons and a stop codon; the resulting cells could not read the canonical genetic code and were completely resistant to a cocktail of viruses. We reassigned these codons to enable the efficient synthesis of proteins containing three distinct noncanonical amino acids. Notably, we demonstrate the facile reprogramming of our cells for the encoded translation of diverse noncanonical heteropolymers and macrocycles.

Discovery of Dihydro-1,4-Benzoxazine Carboxamides as Potent and Highly Selective Inhibitors of Sirtuin-1.

Sirtuins are signaling hubs orchestrating the cellular response to various stressors with roles in all major civilization diseases. Sirtuins remove acyl groups from lysine residues of proteins, thereby controlling their activity, turnover, and localization. The seven human sirtuins, SirT1-7, are closely related in structure, hindering the development of specific inhibitors. Screening 170,000 compounds, we identify and optimize SirT1-specific benzoxazine inhibitors, Sosbo, which rival the efficiency and surpass the selectivity of selisistat (EX527). The compounds inhibit the deacetylation of p53 in cultured cells, demonstrating their ability to permeate biological membranes. Kinetic analysis of inhibition and docking studies reveal that the inhibitors bind to a complex of SirT1 and nicotinamide adenine dinucleotide, similar to selisistat. These new SirT1 inhibitors are valuable alternatives to selisistat in biochemical and cell biological studies. Their greater selectivity may allow the development of better targeted drugs to combat SirT1 activity in diseases such as cancer, Huntington's chorea, or anorexia.

A Directed Evolution System for Lysine Deacetylases.

Lysine acetylation is a ubiquitous modification permeating the proteomes of organisms from all domains of life. Lysine deacetylases (KDACs) reverse this modification by following two fundamentally different enzymatic mechanisms, which differ mainly by the need for NAD+ as stoichiometric co-substrate. KDACs are often found as catalytic subunit in protein complexes involved in cell cycle regulation, chromatin organization and transcription. Their promiscuity with respect to sequence context and type of lysine acylation convolutes the network of functional and physical connections.Here we present an efficient selection method for KDACs in E. coli, which allows for the creation of acyl-type specific KDAC variants, which greatly facilitate the investigation of their physiological function . The selection system builds on the incorporation of acylated lysines by genetic code expansion in reporter enzymes with essential lysine residues. We describe the creation of KDAC mutant libraries by saturation mutagenesis of active site residues, the isolation of individual mutants from this library using the selection system, and their biochemical characterization with acylated firefly luciferase.

Evolved, Selective Erasers of Distinct Lysine Acylations.

Lysine acylations, a family of diverse protein modifications varying in acyl-group length, charge, and saturation, are linked to many important physiological processes. Only a small set of substrate-promiscuous lysine acetyltransferases and deacetylases (KDACs) install and remove this vast variety of modifications. Engineered KDACs that remove only one type of acylation would help to dissect the different contributions of distinct acylations. We developed a bacterial selection system for the directed evolution of KDACs and identified variants up to 400 times more selective for butyryl-lysine compared to crotonyl-lysine. Structural analyses revealed that the enzyme adopts different conformational states depending on the type of acylation of the bound peptide. We used the butyryl-selective KDAC variant to shift the cellular acylation spectrum towards increased lysine crotonylation. These new enzymes will help in dissecting the roles of different lysine acylations in cell physiology.

Highly Sensitive Lysine Deacetylase Assay Based on Acetylated Firefly Luciferase.

Lysine deacetylases (KDACs) play important roles in many physiological processes and are implicated in many human diseases. Hence, the search for modulators of KDACs is very active, and reliable assays for monitoring their activity are key to success. Here, we describe a new KDAC assay based on Firefly luciferase harboring an acetylation on an essential active site lysine. We show that several KDACs can reverse this modification and hence activate luciferase. This new assay is extremely sensitive, reliable, and fast and can be performed in a continuous format. We used this assay to screen a small library of compounds and identified several novel effectors of SirT2 with low micromolar activity.

Laboratory-Scale Hydroxylation of Steroids by P450BM3 Variants.

This chapter describes the asymmetric hydroxylation of steroids on laboratory preparative scale, using engineered variants of P450BM3 (CYP102A1) as enzyme catalyst. The following protocol covers the creation of an Escherichia coli BL21-Gold (DE3) expression strain, including necessary control experiments like plasmid preparation, test expression, and creation of storage cultures, to verify successful experimental access to recombinant expressed P450BM3 variants. The recombinant expressed P450BM3 variants are obtained as cleared cell lysate and used in a biotransformation setup to hydroxylate 2.8 mg and up to 15 mg testosterone in the presented protocol. Since P450BM3 depends on NADPH as an electron source for the reaction, a glucose and glucose dehydrogenate based recycling system is added to the reaction. The protocol further includes liquid-liquid extraction of hydroxytestosterone and directs the experimenter to compound purification via column chromatography.