Bacterial cellulose is a strong and ultrapure form of cellulose produced naturally by several species of the Acetobacteraceae. Its high strength, purity, and biocompatibility make it of great interest to materials science; however, precise control of its biosynthesis has remained a challenge for biotechnology. Here we isolate a strain of Komagataeibacter rhaeticus (K. rhaeticus iGEM) that can produce cellulose at high yields, grow in low-nitrogen conditions, and is highly resistant to toxic chemicals. We achieved external control over its bacterial cellulose production through development of a modular genetic toolkit that enables rational reprogramming of the cell. To further its use as an organism for biotechnology, we sequenced its genome and demonstrate genetic circuits that enable functionalization and patterning of heterologous gene expression within the cellulose matrix. This work lays the foundations for using genetic engineering to produce cellulose-based materials, with numerous applications in basic science, materials engineering, and biotechnology.

Random mutagenesis is a technique used to generate diversity and engineer biological systems. In vivo random mutagenesis generates diversity directly in a host organism, enabling applications such as lineage tracing, continuous evolution, and protein engineering. Here we describe TRIDENT (TaRgeted In vivo Diversification ENabled by T7 RNAP), a platform for targeted, continual, and inducible diversification at genes of interest at mutation rates one-million fold higher than natural genomic error rates. TRIDENT targets mutagenic enzymes to precise genetic loci by fusion to T7 RNA polymerase, resulting in mutation windows following a mutation targeting T7 promoter. Mutational diversity is tuned by DNA repair factors localized to sites of deaminase-driven mutation, enabling sustained mutation of all four DNA nucleotides at rates greater than 10−4 mutations per bp. We show TRIDENT can be applied to routine in vivo mutagenesis applications by evolving a red-shifted fluorescent protein and drug-resistant mutants of an essential enzyme.

Temporal dynamics of gene expression underpin responses to internal and environmental stimuli. In eukaryotes, regulation of gene induction includes changing chromatin states at target genes and recruiting the transcriptional machinery that includes transcription factors. As one of the most potent defense compounds in Arabidopsis thaliana, camalexin can be rapidly induced by bacterial and fungal infections. Though several transcription factors controlling camalexin biosynthesis genes have been characterized, how the rapid activation of genes in this pathway upon a pathogen signal is enabled remains unknown. By combining publicly available epigenomic data with in vivo chromatin modification mapping, we found that camalexin biosynthesis genes are marked with two epigenetic modifications with opposite effects on gene expression, H3K27me3 (repression) and H3K18ac (activation), to form a previously uncharacterized type of bivalent chromatin. Mutants with reduced H3K27m3 or H3K18ac suggested that both modifications were required to determine the timing of gene expression and metabolite accumulation at an early stage of the stress response. Our study indicates that the H3K27me3-H3K18ac bivalent chromatin, which we name a kairostat, plays an important role controlling the timely induction of gene expression upon stimuli in plants.

Chalcone synthase (CHS) canonically catalyzes carbon-carbon bond formation through iterative decarboxylative Claisen condensation. Here, we characterize a previously unidentified biosynthetic capability of SlCHS to catalyze nitrogen-carbon bond formation, leading to the production of a hydroxycinnamic acid amide (HCAA) compound. By expressing a putative tomato (Solanum lycopersicum) gene cluster in yeast (Saccharomyces cerevisiae), we elucidate the activity of a pathway consisting of a carboxyl methyltransferase (SlMT2), which methylates the yeast primary metabolite 3-hydroxyanthranilic acid (3-HAA) to form a methyl ester, and a SlCHS, which catalyzes the condensation of 3-HAA methyl ester and p-coumaroyl-coenzyme A (CoA) through formation of an amide bond. We demonstrate that this aminoacylation activity could be a common secondary activity in plant CHSs by validating the activity in vitro with variants from S. lycopersicum and Arabidopsis thaliana. Our work demonstrates yeast as a platform for characterizing putative plant gene clusters with the potential for compound structure and enzymatic activity discovery.