Functional and structural characterization of an IclR family transcription factor for the development of dicarboxylic acid biosensors.

Prokaryotic transcription factors (TFs) regulate gene expression in response to small molecules, thus representing promising candidates as versatile small molecule-detecting biosensors valuable for synthetic biology applications. The engineering of such biosensors requires thorough in vitro and in vivo characterization of TF ligand response as well as detailed molecular structure information. In this work, we functionally and structurally characterize the Pca regulon regulatory protein (PcaR) transcription factor belonging to the IclR transcription factor family. Here, we present in vitro functional analysis of the ligand profile of PcaR and the construction of genetic circuits for the characterization of PcaR as an in vivo biosensor in the model eukaryote Saccharomyces cerevisiae. We report the crystal structures of PcaR in the apo state and in complex with one of its ligands, succinate, which suggests the mechanism of dicarboxylic acid recognition by this transcription factor. This work contributes key structural and functional insights enabling the engineering of PcaR for dicarboxylic acid biosensors, in addition to providing more insights into the IclR family of regulators.

Endogenous tagging using split mNeonGreen in human iPSCs for live imaging studies.

Endogenous tags have become invaluable tools to visualize and study native proteins in live cells. However, generating human cell lines carrying endogenous tags is difficult due to the low efficiency of homology-directed repair. Recently, an engineered split mNeonGreen protein was used to generate a large-scale endogenous tag library in HEK293 cells. Using split mNeonGreen for large-scale endogenous tagging in human iPSCs would open the door to studying protein function in healthy cells and across differentiated cell types. We engineered an iPS cell line to express the large fragment of the split mNeonGreen protein (mNG21-10) and showed that it enables fast and efficient endogenous tagging of proteins with the short fragment (mNG211). We also demonstrate that neural network-based image restoration enables live imaging studies of highly dynamic cellular processes such as cytokinesis in iPSCs. This work represents the first step towards a genome-wide endogenous tag library in human stem cells.

The human body contains around 20,000 different proteins that perform a myriad of essential roles. To understand how these proteins work in healthy individuals and during disease, we need to know their precise locations inside cells and how these locations may change in different situations. Genetic tools known as fluorescent proteins are often used as tags to study the location of specific proteins of interest within cells. When exposed to light, the fluorescent proteins emit specific colours of light that can be observed using microscopes. In a fluorescent protein system known as split mNeonGreen, researchers insert the DNA encoding two fragments of a fluorescent protein (one large, one small) separately into cells. The large fragment can be found throughout the cell, while the small fragment is attached to specific host proteins. When the two fragments meet, they assemble into the full mNeonGreen protein and can fluoresce. Researchers can use split mNeonGreen and other similar systems to generate large libraries of cells where the small fragment of a fluorescent protein is attached to thousands of different host proteins. However, so far these libraries are restricted to a handful of different types of cells. To address this challenge, Husser et al. inserted the DNA encoding the large fragment of mNeonGreen into human cells known as induced pluripotent stem cells, which are able to give rise to any other type of human cell. This then enabled the team to quickly and efficiently generate a library of stem cells that express the small fragment of mNeonGreen attached to different host proteins. Further experiments studied the locations of host proteins in the stem cells just before they divided into two cells. This suggested that there are differences between how induced pluripotent stem cells and other types of cells divide. In the future, the cells and the method developed by Husser et al. may be used by other researchers to create atlases showing where human proteins are located in many other types of cells.

Genome sequencing of 15 acid-tolerant yeasts.

We report draft genome sequences for 15 non-conventional Saccharomycotina yeast strains obtained from public culture repositories. Included in our collection are eight strains of Pichia with broad tolerance to dicarboxylic acids. The genome sequences of these strains will enable comparative genomics of acid-tolerant phenotypes and strain engineering of non-conventional hosts.

CRAPS: Chromosomal-Repair-Assisted Pathway Shuffling in Yeast.

A fundamental challenge of metabolic engineering involves assembling and screening vast combinations of orthologous enzymes across a multistep biochemical pathway. Current pathway assembly workflows involve combining genetic parts ex vivo and assembling one pathway configuration per tube or well. Here, we present CRAPS, Chromosomal-Repair-Assisted Pathway Shuffling, an in vivo pathway engineering technique that enables the self-assembly of one pathway configuration per cell. CRAPS leverages the yeast chromosomal repair pathway and utilizes a pool of inactive, chromosomally integrated orthologous gene variants corresponding to a target multistep pathway. Supplying gRNAs to the CRAPS host activates the expression of one gene variant per pathway step, resulting in a unique pathway configuration in each cell. We deployed CRAPS to build more than 1000 theoretical combinations of a four-step carotenoid biosynthesis network. Sampling the CRAPS pathway space yielded strains with distinct color phenotypes and carotenoid product profiles. We anticipate that CRAPS will expedite strain engineering campaigns by enabling the generation and sampling of vast biochemical spaces.

Screening non-conventional yeasts for acid tolerance and engineering Pichia occidentalis for production of muconic acid.

Saccharomyces cerevisiae is a workhorse of industrial biotechnology owing to the organism's prominence in alcohol fermentation and the suite of sophisticated genetic tools available to manipulate its metabolism. However, S. cerevisiae is not suited to overproduce many bulk bioproducts, as toxicity constrains production at high titers. Here, we employ a high-throughput assay to screen 108 publicly accessible yeast strains for tolerance to 20 g L-1 adipic acid (AA), a nylon precursor. We identify 15 tolerant yeasts and select Pichia occidentalis for production of cis,cis-muconic acid (CCM), the precursor to AA. By developing a genome editing toolkit for P. occidentalis, we demonstrate fed-batch production of CCM with a maximum titer (38.8 g L-1), yield (0.134 g g-1 glucose) and productivity (0.511 g L-1 h-1) that surpasses all metrics achieved using S. cerevisiae. This work brings us closer to the industrial bioproduction of AA and underscores the importance of host selection in bioprocessing.

Divergent directed evolution of a TetR-type repressor towards aromatic molecules.

Reprogramming cellular behaviour is one of the hallmarks of synthetic biology. To this end, prokaryotic allosteric transcription factors (aTF) have been repurposed as versatile tools for processing small molecule signals into cellular responses. Expanding the toolbox of aTFs that recognize new inducer molecules is of considerable interest in many applications. Here, we first establish a resorcinol responsive aTF-based biosensor in Escherichia coli using the TetR-family repressor RolR from Corynebacterium glutamicum. We then perform an iterative walk along the fitness landscape of RolR to identify new inducer specificities, namely catechol, methyl catechol, caffeic acid, protocatechuate, L-DOPA, and the tumour biomarker homovanillic acid. Finally, we demonstrate the versatility of these engineered aTFs by transplanting them into the model eukaryote Saccharomyces cerevisiae. This work provides a framework for efficient aTF engineering to expand ligand specificity towards novel molecules on laboratory timescales, which, more broadly, is invaluable across a wide range of applications such as protein and metabolic engineering, as well as point-of-care diagnostics.

Pathway elucidation and microbial synthesis of proaporphine and bis-benzylisoquinoline alkaloids from sacred lotus (Nelumbo nucifera).

Sacred lotus (Nelumbo nucifera) has been utilized as a food, medicine, and spiritual symbol for nearly 3000 years. The medicinal properties of lotus are largely attributed to its unique profile of benzylisoquinoline alkaloids (BIAs), which includes potential anti-cancer, anti-malarial and anti-arrhythmic compounds. BIA biosynthesis in sacred lotus differs markedly from that of opium poppy and other members of the Ranunculales, most notably in an abundance of BIAs possessing the (R)-stereochemical configuration and the absence of reticuline, a major branchpoint intermediate in most BIA producers. Owing to these unique metabolic features and the pharmacological potential of lotus, we set out to elucidate the BIA biosynthesis network in N. nucifera. Here we show that lotus CYP80G (NnCYP80G) and a superior ortholog from Peruvian nutmeg (Laurelia sempervirens; LsCYP80G) stereospecifically convert (R)-N-methylcoclaurine to the proaporphine alkaloid glaziovine, which is subsequently methylated to pronuciferine, the presumed precursor to nuciferine. While sacred lotus employs a dedicated (R)-route to aporphine alkaloids from (R)-norcoclaurine, we implemented an artificial stereochemical inversion approach to flip the stereochemistry of the core BIA pathway. Exploiting the unique substrate specificity of dehydroreticuline synthase from common poppy (Papaver rhoeas) and pairing it with dehydroreticuline reductase enabled de novo synthesis of (R)-N-methylcoclaurine from (S)-norcoclaurine and its subsequent conversion to pronuciferine. We leveraged our stereochemical inversion approach to also elucidate the role of NnCYP80A in sacred lotus metabolism, which we show catalyzes the stereospecific formation of the bis-BIA nelumboferine. Screening our collection of 66 plant O-methyltransferases enabled conversion of nelumboferine to liensinine, a potential anti-cancer bis-BIA from sacred lotus. Our work highlights the unique benzylisoquinoline metabolism of N. nucifera and enables the targeted overproduction of potential lotus pharmaceuticals using engineered microbial systems.

Cytokinetic diversity in mammalian cells is revealed by the characterization of endogenous anillin, Ect2 and RhoA.

Cytokinesis is required to physically separate the daughter cells at the end of mitosis. This crucial process requires the assembly and ingression of an actomyosin ring, which must occur with high fidelity to avoid aneuploidy and cell fate changes. Most of our knowledge of mammalian cytokinesis was generated using over-expressed transgenes in HeLa cells. Over-expression can introduce artefacts, while HeLa are cancerous human cells that have lost their epithelial identity, and the mechanisms controlling cytokinesis in these cells could be vastly different from other cell types. Here, we tagged endogenous anillin, Ect2 and RhoA with mNeonGreen and characterized their localization during cytokinesis for the first time in live human cells. Comparing anillin localization in multiple cell types revealed cytokinetic diversity with differences in the duration and symmetry of ring closure, and the timing of cortical recruitment. Our findings show that the breadth of anillin correlates with the rate of ring closure, and support models where cell size or ploidy affects the cortical organization, and intrinsic mechanisms control the symmetry of ring closure. This work highlights the need to study cytokinesis in more diverse cell types, which will be facilitated by the reagents generated for this study.

The MyLO CRISPR-Cas9 toolkit: a markerless yeast localization and overexpression CRISPR-Cas9 toolkit.

The genetic tractability of the yeast Saccharomyces cerevisiae has made it a key model organism for basic research and a target for metabolic engineering. To streamline the introduction of tagged genes and compartmental markers with powerful Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) - CRISPR-associated protein 9 (Cas9)-based genome editing tools, we constructed a Markerless Yeast Localization and Overexpression (MyLO) CRISPR-Cas9 toolkit with 3 components: (1) a set of optimized Streptococcus pyogenes Cas9-guide RNA expression vectors with 5 selectable markers and the option to either preclone or cotransform the gRNAs; (2) vectors for the one-step construction of integration cassettes expressing an untagged or green fluorescent protein/red fluorescent protein/hemagglutinin-tagged gene of interest at one of 3 levels, supporting localization and overexpression studies; and (3) integration cassettes containing moderately expressed green fluorescent protein- or red fluorescent protein-tagged compartmental markers for colocalization experiments. These components allow rapid, high-efficiency genomic integrations and modifications with only transient selection for the Cas9 vector, resulting in markerless transformations. To demonstrate the ease of use, we applied our complete set of compartmental markers to colabel all target subcellular compartments with green fluorescent protein and red fluorescent protein. Thus, the MyLO toolkit packages CRISPR-Cas9 technology into a flexible, optimized bundle that allows the stable genomic integration of DNA with the ease of use approaching that of transforming plasmids.

Functional expression of opioid receptors and other human GPCRs in yeast engineered to produce human sterols.

The yeast Saccharomyces cerevisiae is powerful for studying human G protein-coupled receptors as they can be coupled to its mating pathway. However, some receptors, including the mu opioid receptor, are non-functional, which may be due to the presence of the fungal sterol ergosterol instead of cholesterol. Here we engineer yeast to produce cholesterol and introduce diverse mu, delta, and kappa opioid receptors to create sensitive opioid biosensors that recapitulate agonist binding profiles and antagonist inhibition. Additionally, human mu opioid receptor variants, including those with clinical relevance, largely display expected phenotypes. By testing mu opioid receptor-based biosensors with systematically adjusted cholesterol biosynthetic intermediates, we relate sterol profiles to biosensor sensitivity. Finally, we apply sterol-modified backgrounds to other human receptors revealing sterol influence in SSTR5, 5-HTR4, FPR1, and NPY1R signaling. This work provides a platform for generating human G protein-coupled receptor-based biosensors, facilitating receptor deorphanization and high-throughput screening of receptors and effectors.

A Versatile Transcription Factor Biosensor System Responsive to Multiple Aromatic and Indole Inducers.

Allosteric transcription factor (aTF) biosensors are valuable tools for engineering microbes toward a multitude of applications in metabolic engineering, biotechnology, and synthetic biology. One of the challenges toward constructing functional and diverse biosensors in engineered microbes is the limited toolbox of identified and characterized aTFs. To overcome this, extensive bioprospecting of aTFs from sequencing databases, as well as aTF ligand-specificity engineering are essential in order to realize their full potential as biosensors for novel applications. In this work, using the TetR-family repressor CmeR from Campylobacter jejuni, we construct aTF genetic circuits that function as salicylate biosensors in the model organisms Escherichia coli and Saccharomyces cerevisiae. In addition to salicylate, we demonstrate the responsiveness of CmeR-regulated promoters to multiple aromatic and indole inducers. This relaxed ligand specificity of CmeR makes it a useful tool for detecting molecules in many metabolic engineering applications, as well as a good target for directed evolution to engineer proteins that are able to detect new and diverse chemistries.

Engineering Yeast for De Novo Synthesis of the Insect Repellent Nepetalactone.

While nepetalactone, the active ingredient in catnip, is a potent insect repellent, its low in planta accumulation limits its commercial viability as an alternative repellent. Here we describe for the first time de novo nepetalactone synthesis in Saccharomyces cerevisiae, enabling sustainable and scalable production. Nepetalactone production required introducing eight exogenous genes including the cytochrome P450 geraniol-8-hydroxylase, the bottleneck of the heterologous pathway. Combinatorial assessment of geraniol-8-hydroxylase and cytochrome P450 reductase variants, and copy-number variations were used to overcome this bottleneck. We found that several reductases improved hydroxylation activity and increasing geraniol-8-hydroxylase gene copy number improved 8-hydroxygeraniol titers. The accumulation of an unwanted metabolite implied inefficient channeling of carbon through the pathway. With the native yeast old yellow enzymes previously shown to use monoterpene intermediates as substrates, both homologues were deleted. These deletions increased 8-hydroxygeraniol yield, resulting in 3.10 mg/L/OD600 of nepetalactone from simple sugar in microtiter plates. This optimized pathway will benefit the development of high yielding strains for the scale up production of nepetalactone.

Developing a Yeast Platform Strain for an Enhanced Taxadiene Biosynthesis by CRISPR/Cas9.

Paclitaxel is an important diterpenoid commonly used as an anticancer drug. Although the paclitaxel biosynthetic pathway has been mostly revealed, some steps remain to be elucidated. The difficulties in plant transformations and the scarcity of the precursor of paclitaxel, (+)-taxa-4(5), 11(12)-diene (taxadiene), have hindered the full comprehension of paclitaxel biochemistry and, therefore, its production by biotechnological approaches. One solution is to use the budding yeast, Saccharomyces cerevisiae, as a platform to elucidate the paclitaxel biosynthesis. As taxadiene is a diterpenoid, its common precursor, geranylgeranyl pyrophosphate (GGPP), needs to be increased in yeast. In this study, we screened various GGPP synthases (GGPPS) to find the most suitable GGPPS for taxadiene production in yeast. We also optimized the taxadiene production by increasing the flux toward the terpenoid pathway. Finally, to remove selection markers, we integrated the required genes using a CRISPR/Cas9 system in the yeast genome. Our result showed that a titer of 2.02 ± 0.40 mg/L (plasmid) and 0.41 ± 0.06 mg/L (integrated) can be achieved using these strategies. This platform strain can be used to readily test the gene candidates for microbial paclitaxel biosynthesis in the future.

CRISPR-Cas tools to study gene function in cytokinesis.

Cytokinesis is the process that separates a cell into two daughter cells at the end of mitosis. Most of our knowledge of cytokinesis comes from overexpression studies, which affects our interpretation of protein function. Gene editing can circumvent this issue by introducing functional mutations or fluorescent probes directly into a gene locus. However, despite its potential, gene editing is just starting to be used in the field of cytokinesis. Here, we discuss the benefits of using gene editing tools for the study of cytokinesis and highlight recent studies that successfully used CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) technology to answer critical questions regarding the function of cytokinesis proteins. We also present methodologies for editing essential genes and discuss how CRISPR interference (CRISPRi) and activation (CRISPRa) can enable precise control of gene expression to answer important questions in the field. Finally, we address the need for gene editing to study cytokinesis in more physiologically relevant contexts. Therefore, this Review provides a roadmap for gene editing to be used in the study of cytokinesis and other cellular processes.

A yeast platform for high-level synthesis of tetrahydroisoquinoline alkaloids.

The tetrahydroisoquinoline (THIQ) moiety is a privileged substructure of many bioactive natural products and semi-synthetic analogs. Plants manufacture more than 3,000 THIQ alkaloids, including the opioids morphine and codeine. While microbial species have been engineered to synthesize a few compounds from the benzylisoquinoline alkaloid (BIA) family of THIQs, low product titers impede industrial viability and limit access to the full chemical space. Here we report a yeast THIQ platform by increasing production of the central BIA intermediate (S)-reticuline to 4.6 g L-1, a 57,000-fold improvement over our first-generation strain. We show that gains in BIA output coincide with the formation of several substituted THIQs derived from amino acid catabolism. We use these insights to repurpose the Ehrlich pathway and synthesize an array of THIQ structures. This work provides a blueprint for building diverse alkaloid scaffolds and enables the targeted overproduction of thousands of THIQ products, including natural and semi-synthetic opioids.

Author Correction: Building a global alliance of biofoundries.

The original version of this Comment contained errors in the legend of Figure 2, in which the locations of the fifteenth and sixteenth GBA members were incorrectly given as '(15) Australian Genome Foundry, Macquarie University; (16) Australian Foundry for Advanced Biomanufacturing, University of Queensland.'. The correct version replaces this with '(15) Australian Foundry for Advanced Biomanufacturing (AusFAB), University of Queensland and (16) Australian Genome Foundry, Macquarie University'. This has been corrected in both the PDF and HTML versions of the Comment.

A Combinatorial Approach To Study Cytochrome P450 Enzymes for De Novo Production of Steviol Glucosides in Baker's Yeast.

Biosynthesis of steviol glycosides in planta proceeds via two cytochrome P450 enzymes (CYPs): kaurene oxidase (KO) and kaurenoic acid hydroxylase (KAH). KO and KAH function in succession with the support of a NADPH-dependent cytochrome P450 reductase (CPR) to convert kaurene to steviol. This work describes a platform for recombinant production of steviol glucosides (SGs) in Saccharomyces cerevisiae, demonstrating the full reconstituted pathway from the simple sugar glucose to the SG precursor steviol. With a focus on optimization of the KO-KAH activities, combinations of functional homologues were tested in batch growth. Among the CYPs, novel KO75 (CYP701) and novel KAH82 (CYP72) outperformed their respective functional homologues from Stevia rebaudiana, SrKO (CYP701A5) and SrKAH (CYP81), in assays where substrate was supplemented to culture broth. With kaurene produced from glucose in the cell, SrCPR1 from S. rebaudiana supported highest turnover for KO-KAH combinations, besting two other CPRs isolated from S. rebaudiana, the Arabidopsis thaliana ATR2, and a new class I CPR12. Some coexpressions of ATR2 with a second CPR were found to diminish KAH activity, showing that coexpression of CPRs can lead to competition for CYPs with possibly adverse effects on catalysis.

A Highly Characterized Synthetic Landing Pad System for Precise Multicopy Gene Integration in Yeast.

A fundamental undertaking of metabolic engineering involves identifying and troubleshooting metabolic bottlenecks that arise from imbalances in pathway flux. To expedite the systematic screening of enzyme orthologs in conjunction with DNA copy number tuning, here we develop a simple and highly characterized CRISPR-Cas9 integration system in Saccharomyces cerevisiae. Our engineering strategy introduces a series of synthetic DNA landing pads (LP) into the S. cerevisiae genome to act as sites for high-level gene integration. LPs facilitate multicopy gene integration of one, two, three, or four DNA copies in a single transformation, thus providing precise control of DNA copy number. We applied our LP system to norcoclaurine synthase (NCS), an enzyme with poor kinetic properties involved in the first committed step of the production of high-value benzylisoquinoline alkaloids. The platform enabled rapid construction of a 40-strain NCS library by integrating ten NCS orthologs in four gene copies each. Six active NCS variants were identified, whereby production of ( S)-norcoclaurine could be further enhanced by increasing NCS copy number. We anticipate the LP system will aid in metabolic engineering efforts by providing strict control of gene copy number and expediting strain and pathway engineering campaigns.