Ensemble Detection of DNA Engineering Signatures.

Synthetic biology is creating genetically engineered organisms at an increasing rate for many potentially valuable applications, but this potential comes with the risk of misuse or accidental release. To begin to address this issue, we have developed a system called GUARDIAN that can automatically detect signatures of engineering in DNA sequencing data, and we have conducted a blinded test of this system using a curated Test and Evaluation (T&E) data set. GUARDIAN uses an ensemble approach based on the guiding principle that no single approach is likely to be able to detect engineering with perfect accuracy. Critically, ensembling enables GUARDIAN to detect sequence inserts in 13 target organisms with a high degree of specificity that requires no subject matter expert (SME) review.

Integration of Yeast Episomal/Integrative Plasmid Causes Genotypic and Phenotypic Diversity and Improved Sesquiterpene Production in Metabolically Engineered Saccharomyces cerevisiae.

The variability in phenotypic outcomes among biological replicates in engineered microbial factories presents a captivating mystery. Establishing the association between phenotypic variability and genetic drivers is important to solve this intricate puzzle. We applied a previously developed auxin-inducible depletion of hexokinase 2 as a metabolic engineering strategy for improved nerolidol production in Saccharomyces cerevisiae, and biological replicates exhibit a dichotomy in nerolidol production of either 3.5 or 2.5 g L-1 nerolidol. Harnessing Oxford Nanopore's long-read genomic sequencing, we reveal a potential genetic cause─the chromosome integration of a 2µ sequence-based yeast episomal plasmid, encoding the expression cassettes for nerolidol synthetic enzymes. This finding was reinforced through chromosome integration revalidation, engineering nerolidol and valencene production strains, and generating a diverse pool of yeast clones, each uniquely fingerprinted by gene copy numbers, plasmid integrations, other genomic rearrangements, protein expression levels, growth rate, and target product productivities. Τhe best clone in two strains produced 3.5 g L-1 nerolidol and ∼0.96 g L-1 valencene. Comparable genotypic and phenotypic variations were also generated through the integration of a yeast integrative plasmid lacking 2µ sequences. Our work shows that multiple factors, including plasmid integration status, subchromosomal location, gene copy number, sesquiterpene synthase expression level, and genome rearrangement, together play a complicated determinant role on the productivities of sesquiterpene product. Integration of yeast episomal/integrative plasmids may be used as a versatile method for increasing the diversity and optimizing the efficiency of yeast cell factories, thereby uncovering metabolic control mechanisms.

Targeted control of supporting pathways in paclitaxel biosynthesis with CRISPR-guided methylation.

Introduction: Plant cell culture biomanufacturing is rapidly becoming an effective strategy for production of high-value plant natural products, such as therapeutic proteins and small molecules, vaccine adjuvants, and nutraceuticals. Many of these plant natural products are synthesized from diverse molecular building blocks sourced from different metabolic pathways. Even so, engineering approaches for increasing plant natural product biosynthesis have typically focused on the core biosynthetic pathway rather than the supporting pathways. Methods: Here, we use both CRISPR-guided DNA methylation and chemical inhibitors to control flux through the phenylpropanoid pathway in Taxus chinensis, which contributes a phenylalanine derivative to the biosynthesis of paclitaxel (Taxol), a potent anticancer drug. To inhibit PAL, the first committed step in phenylpropanoid biosynthesis, we knocked down expression of PAL in Taxus chinensis plant cell cultures using a CRISPR-guided plant DNA methyltransferase (NtDRM). For chemical inhibition of downstream steps in the pathway, we treated Taxus chinensis plant cell cultures with piperonylic acid and caffeic acid, which inhibit the second and third committed steps in phenylpropanoid biosynthesis: cinnamate 4-hydroxylase (C4H) and 4-coumaroyl-CoA ligase (4CL), respectively. Results: Knockdown of PAL through CRISPR-guided DNA methylation resulted in a profound 25-fold increase in paclitaxel accumulation. Further, through the synergistic action of both chemical inhibitors and precursor feeding of exogenous phenylalanine, we achieve a 3.5-fold increase in paclitaxel biosynthesis and a similar reduction in production of total flavonoids and phenolics. We also observed perturbations to both activity and expression of PAL, illustrating the complex transcriptional co-regulation of these first three pathway steps. Discussion: These results highlight the importance of controlling the metabolic flux of supporting pathways in natural product biosynthesis and pioneers CRISPR-guided methylation as an effective method for metabolic engineering in plant cell cultures. Ultimately, this work demonstrates a powerful method for rewiring plant cell culture systems into next-generation chassis for production of societally valuable compounds.

Isolation and sequencing of the spoilage yeast Kregervanrija delftensis.

New sequencing workflows can enable the genomics of microbes isolated from craft beverages. Here, we use hybrid, short, and long-read sequencing to assemble the genome of a yeast isolated from cider, Kregervanrija delftensis NCC-J. The Kregervanrija genus has few genomes available; thus, this contributes to yeast genomics.

Draft genome of the Ogataea polymorpha type strain CBS 4732.

The methylotrophic yeast Ogataea polymorpha is of significant biotechnological interest, particularly in high-temperature fermentations and for recombinant protein production. Here, we present a high-quality genome assembly for the O. polymorpha type strain CBS 4732.

Systematic Part Transfer by Extending a Modular Toolkit to Diverse Bacteria.

It is impractical to develop a new parts collection for every potential host organism. It is well-established that gene expression parts, like genes, are qualitatively transferable, but there is little quantitative information defining transferability. Here, we systematically quantified the behavior of a parts set across multiple hosts. To do this, we developed a broad host range (BHR) plasmid system compatible with the large, modular CIDAR parts collection for E. coli, which we named openCIDAR. This enabled testing of a library of DNA constructs across the Pseudomonadota─Escherichia coli, Pseudomonas putida, Cupriavidus necator, and Komagataeibacter nataicola. Part performance was evaluated with a standardized characterization procedure that quantified expression in terms of molecules of equivalent fluorescein (MEFL), an objective unit of measure. The results showed that the CIDAR parts enable graded gene expression across all organisms─meaning that the same parts can be used to program E. coli, P. putida, C. necator, and K. nataicola. Most parts had a similar expression trend across hosts, although each organism had a different average gene expression level. The variability is enough that to achieve the same MEFL in a different organism, a lookup table is required to translate a design from one host to another. To identify truly divergent parts, we applied linear regression to a combinatorial set of promoters and ribosome binding sites, finding that the promoter J23100 behaves very differently in K. nataicola than in the other hosts. Thus, it is now possible to evaluate any CIDAR compatible part in three other hosts of interest, and the diversity of these hosts implies that the collection will also be compatible with many other Proteobacteria (Pseudomonadota). Furthermore, this work defines an approach to generalize modular synthetic biology parts sets beyond a single host, implying that only a few parts sets may be needed to span the tree of life. This will accelerate current efforts to engineer diverse species for environmental, biotechnological, and health applications.

Genetic basis for probiotic yeast phenotypes revealed by nanopore sequencing.

Probiotic yeasts are emerging as preventative and therapeutic solutions for disease. Often ingested via cultured foods and beverages, they can survive the harsh conditions of the gastrointestinal tract and adhere to it, where they provide nutrients and inhibit pathogens like Candida albicans. Yet, little is known of the genomic determinants of these beneficial traits. To this end, we have sequenced 2 food-derived probiotic yeast isolates that mitigate fungal infections. We find that the first strain, KTP, is a strain of Saccharomyces cerevisiae within a small clade that lacks any apparent ancestry from common European/wine S. cerevisiae strains. Significantly, we show that S. cerevisiae KTP genes involved in general stress, pH tolerance, and adherence are markedly different from S. cerevisiae S288C but are similar to the commercial probiotic yeast species S. boulardii. This suggests that even though S. cerevisiae KTP and S. boulardii are from different clades, they may achieve probiotic effect through similar genetic mechanisms. We find that the second strain, ApC, is a strain of Issatchenkia occidentalis, one of the few of this family of yeasts to be sequenced. Because of the dissimilarity of its genome structure and gene organization, we infer that I. occidentalis ApC likely achieves a probiotic effect through a different mechanism than the Saccharomyces strains. Therefore, this work establishes a strong genetic link among probiotic Saccharomycetes, advances the genomics of Issatchenkia yeasts, and indicates that probiotic activity is not monophyletic and complimentary mixtures of probiotics could enhance health benefits beyond a single species.

Structural properties of optically clear bacterial cellulose produced by Komagataeibacter hansenii using arabitol.

Bacterial cellulose (BC) exhibits beneficial properties for use in biomedical applications but is limited by its lack of tunable transparency capabilities. To overcome this deficiency, a novel method to synthesize transparent BC materials using an alternative carbon source, namely arabitol, was developed. Characterization of the BC pellicles was performed for yield, transparency, surface morphology, and molecular assembly. Transparent BC was produced using mixtures of glucose and arabitol. Zero percent arabitol pellicles exhibited 25% light transmittance, which increased with increasing arabitol concentration through to 75% light transmittance. While transparency increased, overall BC yield was maintained indicating that the altered transparency may be induced on a micro-scale rather than a macro-scale. Significant differences in fiber diameter and the presence of aromatic signatures were observed. Overall, this research outlines methods for producing BC with tunable optical transparency, while also bringing new insight to insoluble components of exopolymers produced by Komagataeibacter hansenii.

Multicolor plate reader fluorescence calibration.

Plate readers are commonly used to measure cell growth and fluorescence, yet the utility and reproducibility of plate reader data is limited by the fact that it is typically reported in arbitrary or relative units. We have previously established a robust serial dilution protocol for calibration of plate reader measurements of absorbance to estimated bacterial cell count and for green fluorescence from proteins expressed in bacterial cells to molecules of equivalent fluorescein. We now extend these protocols to calibration of red fluorescence to the sulforhodamine-101 fluorescent dye and blue fluorescence to Cascade Blue. Evaluating calibration efficacy via an interlaboratory study, we find that these calibrants do indeed provide comparable precision to the prior calibrants and that they enable effective cross-laboratory comparison of measurements of red and blue fluorescence from proteins expressed in bacterial cells.

Discovering Content through Text Mining for a Synthetic Biology Knowledge System.

Scientific articles contain a wealth of information about experimental methods and results describing biological designs. Due to its unstructured nature and multiple sources of ambiguity and variability, extracting this information from text is a difficult task. In this paper, we describe the development of the synthetic biology knowledge system (SBKS) text processing pipeline. The pipeline uses natural language processing techniques to extract and correlate information from the literature for synthetic biology researchers. Specifically, we apply named entity recognition, relation extraction, concept grounding, and topic modeling to extract information from published literature to link articles to elements within our knowledge system. Our results show the efficacy of each of the components on synthetic biology literature and provide future directions for further advancement of the pipeline.

Synthetic Biology Knowledge System.

The Synthetic Biology Knowledge System (SBKS) is an instance of the SynBioHub repository that includes text and data information that has been mined from papers published in ACS Synthetic Biology. This paper describes the SBKS curation framework that is being developed to construct the knowledge stored in this repository. The text mining pipeline performs automatic annotation of the articles using natural language processing techniques to identify salient content such as key terms, relationships between terms, and main topics. The data mining pipeline performs automatic annotation of the sequences extracted from the supplemental documents with the genetic parts used in them. Together these two pipelines link genetic parts to papers describing the context in which they are used. Ultimately, SBKS will reduce the time necessary for synthetic biologists to find the information necessary to complete their designs.

Engineered yeast genomes accurately assembled from pure and mixed samples.

Yeast whole genome sequencing (WGS) lacks end-to-end workflows that identify genetic engineering. Here we present Prymetime, a tool that assembles yeast plasmids and chromosomes and annotates genetic engineering sequences. It is a hybrid workflow-it uses short and long reads as inputs to perform separate linear and circular assembly steps. This structure is necessary to accurately resolve genetic engineering sequences in plasmids and the genome. We show this by assembling diverse engineered yeasts, in some cases revealing unintended deletions and integrations. Furthermore, the resulting whole genomes are high quality, although the underlying assembly software does not consistently resolve highly repetitive genome features. Finally, we assemble plasmids and genome integrations from metagenomic sequencing, even with 1 engineered cell in 1000. This work is a blueprint for building WGS workflows and establishes WGS-based identification of yeast genetic engineering.

Genetic circuit design automation for yeast.

Cells can be programmed to monitor and react to their environment using genetic circuits. Design automation software maps a desired circuit function to a DNA sequence, a process that requires units of gene regulation (gates) that are simple to connect and behave predictably. This poses a challenge for eukaryotes due to their complex mechanisms of transcription and translation. To this end, we have developed gates for yeast (Saccharomyces cerevisiae) that are connected using RNA polymerase flux as the signal carrier and are insulated from each other and host regulation. They are based on minimal constitutive promoters (~120 base pairs), for which rules are developed to insert operators for DNA-binding proteins. Using this approach, we constructed nine NOT/NOR gates with nearly identical response functions and 400-fold dynamic range. In circuits, they are transcriptionally insulated from each other by placing ribozymes downstream of terminators to block nuclear export of messenger RNAs resulting from RNA polymerase readthrough. Based on these gates, Cello 2.0 was used to build circuits with up to 11 regulatory proteins. A simple dynamic model predicts the circuit response over days. Genetic circuit design automation for eukaryotes simplifies the construction of regulatory networks as part of cellular engineering projects, whether it be to stage processes during bioproduction, serve as environmental sentinels or guide living therapeutics.

Compensating for Fingertip Size to Render Tactile Cues More Accurately.

Fingertip haptic feedback offers advantages in many applications, including robotic teleoperation, gaming, and training. However, fingertip size and shape vary significantly across humans, making it difficult to design fingertip interfaces and rendering techniques suitable for everyone. This article starts with an existing data-driven haptic rendering algorithm that ignores fingertip size, and it then develops two software-based approaches to personalize this algorithm for fingertips of different sizes using either additional data or geometry. We evaluate our algorithms in the rendering of pre-recorded tactile sensations onto rubber casts of six different fingertips as well as onto the real fingertips of 13 human participants. Results on the casts show that both approaches significantly improve performance, reducing force error magnitudes by an average of 78% with respect to the standard non-personalized rendering technique. Congruent results were obtained for real fingertips, with subjects rating each of the two personalized rendering techniques significantly better than the standard non-personalized method.

Implementation of a 6-DOF Parallel Continuum Manipulator for Delivering Fingertip Tactile Cues.

Existing fingertip haptic devices can deliver different subsets of tactile cues in a compact package, but we have not yet seen a wearable six-degree-of-freedom (6-DOF) display. This paper presents the Fuppeteer (short for Fingertip Puppeteer), a device that is capable of controlling the position and orientation of a flat platform, such that any combination of normal and shear force can be delivered at any location on any human fingertip. We build on our previous work of designing a parallel continuum manipulator for fingertip haptics by presenting a motorized version in which six flexible Nitinol wires are actuated via independent roller mechanisms and proportional-derivative controllers. We evaluate the settling time and end-effector vibrations observed during system responses to step inputs. After creating a six-dimensional lookup table and adjusting simulated inputs using measured Jacobians, we show that the device can make contact with all parts of the fingertip with a mean error of 1.42 mm. Finally, we present results from a human-subject study. A total of 24 users discerned 9 evenly distributed contact locations with an average accuracy of 80.5%. Translational and rotational shear cues were identified reasonably well near the center of the fingertip and more poorly around the edges.

Iterative algorithm-guided design of massive strain libraries, applied to itaconic acid production in yeast.

Metabolic engineering requires multiple rounds of strain construction to evaluate alternative pathways and enzyme concentrations. Optimizing multigene pathways stepwise or by randomly selecting enzymes and expression levels is inefficient. Here, we apply methods from design of experiments (DOE) to guide the construction of strain libraries from which the maximum information can be extracted without sampling every possible combination. We use Saccharomyces cerevisiae as a host for a novel six-gene pathway to itaconic acid, selected by comparing alternative shunt pathways that bypass the mitochondrial TCA cycle. The pathway is distinctive for the use of acetylating acetaldehyde dehydrogenase to increase cytosolic acetyl-CoA pools, a bacterial enzyme to synthesize citrate in the cytosol, and an itaconic acid exporter. Precise control over the expression of each gene is enabled by a set of promoter-terminator pairs that span a 174-fold range. Two large combinatorial libraries (160 variants, 2.4 Mb and 32 variants, 0.6 Mb) are designed where the expression levels are selected by statistical methods (I-optimal response surface methodology, full factorial, or Plackett-Burman) with the intent of extracting different types of guiding information after the screen. This is applied to the design of a third library (24 variants, 0.5 Mb) intended to alleviate a bottleneck in cis-aconitate decarboxylase (CAD) expression. The top strain produces 815 mg/l itaconic acid, a 4-fold improvement over the initial strain achieved by iteratively balancing pathway expression. Including a methylated product in the total, the strain produces 1.3 g/l combined itaconic acids. Further, a regression analysis of the libraries reveals the optimal expression level of CAD as well as pairwise interdependencies between genes that result in increased titer and purity of itaconic acid. This work demonstrates adapting algorithmic design strategies to guide automated yeast strain construction and learn information after each iteration.

Genetic engineering of host organisms for pharmaceutical synthesis.

Pharmaceutical production hosts may be derived from almost any organism, from Chinese Hamster Ovary (CHO) cell lines to isolated actinomycetes. Each host can be improved, historically only through adaptive evolution. Recently, the maturation of organism engineering has expanded the available models, methods, and tools for altering host phenotypes. New tools like CRISPR-associated endonucleases promise to enable precise cellular reprogramming and to access previously intractable hosts. In this review, we discuss the most recent advances in engineering several types of pharmaceutical production hosts. These include model organisms, potential platform hosts with advantageous metabolism or physiology, specialized producers capable of unique biosynthesis, and CHO, the most widely used recombinant protein production host. To realize improved engineered hosts, an increasing number of approaches involving DNA sequencing and synthesis, host rewriting technologies, computational methods, and organism engineering strategies must be used. Integrative workflows that enable application of the right combination of methods to the right production host could enable economical production solutions for emerging human health treatments.

A Pressure Test to Make 10 Molecules in 90 Days: External Evaluation of Methods to Engineer Biology.

Centralized facilities for genetic engineering, or "biofoundries", offer the potential to design organisms to address emerging needs in medicine, agriculture, industry, and defense. The field has seen rapid advances in technology, but it is difficult to gauge current capabilities or identify gaps across projects. To this end, our foundry was assessed via a timed "pressure test", in which 3 months were given to build organisms to produce 10 molecules unknown to us in advance. By applying a diversity of new approaches, we produced the desired molecule or a closely related one for six out of 10 targets during the performance period and made advances toward production of the others as well. Specifically, we increased the titers of 1-hexadecanol, pyrrolnitrin, and pacidamycin D, found novel routes to the enediyne warhead underlying powerful antimicrobials, established a cell-free system for monoterpene production, produced an intermediate toward vincristine biosynthesis, and encoded 7802 individually retrievable pathways to 540 bisindoles in a DNA pool. Pathways to tetrahydrofuran and barbamide were designed and constructed, but toxicity or analytical tools inhibited further progress. In sum, we constructed 1.2 Mb DNA, built 215 strains spanning five species ( Saccharomyces cerevisiae, Escherichia coli, Streptomyces albidoflavus, Streptomyces coelicolor, and Streptomyces albovinaceus), established two cell-free systems, and performed 690 assays developed in-house for the molecules.

Formation of Nitrogenase NifDK Tetramers in the Mitochondria of Saccharomyces cerevisiae.

Transferring the prokaryotic enzyme nitrogenase into a eukaryotic host with the final aim of developing N2 fixing cereal crops would revolutionize agricultural systems worldwide. Targeting it to mitochondria has potential advantages because of the organelle's high O2 consumption and the presence of bacterial-type iron-sulfur cluster biosynthetic machinery. In this study, we constructed 96 strains of Saccharomyces cerevisiae in which transcriptional units comprising nine Azotobacter vinelandii nif genes (nifHDKUSMBEN) were integrated into the genome. Two combinatorial libraries of nif gene clusters were constructed: a library of mitochondrial leading sequences consisting of 24 clusters within four subsets of nif gene expression strength, and an expression library of 72 clusters with fixed mitochondrial leading sequences and nif expression levels assigned according to factorial design. In total, 29 promoters and 18 terminators were combined to adjust nif gene expression levels. Expression and mitochondrial targeting was confirmed at the protein level as immunoblot analysis showed that Nif proteins could be efficiently accumulated in mitochondria. NifDK tetramer formation, an essential step of nitrogenase assembly, was experimentally proven both in cell-free extracts and in purified NifDK preparations. This work represents a first step toward obtaining functional nitrogenase in the mitochondria of a eukaryotic cell.

Double Dutch: A Tool for Designing Combinatorial Libraries of Biological Systems.

Recently, semirational approaches that rely on combinatorial assembly of characterized DNA components have been used to engineer biosynthetic pathways. In practice, however, it is not practical to assemble and test millions of pathway variants in order to elucidate how different DNA components affect the behavior of a pathway. To address this challenge, we apply a rigorous mathematical approach known as design of experiments (DOE) that can be used to construct empirical models of system behavior without testing all variants. To support this approach, we have developed a tool named Double Dutch, which uses a formal grammar and heuristic algorithms to automate the process of DOE library design. Compared to designing by hand, Double Dutch enables users to more efficiently and scalably design libraries of pathway variants that can be used in a DOE framework and uniquely provides a means to flexibly balance design considerations of statistical analysis, construction cost, and risk of homologous recombination, thereby demonstrating the utility of automating decision making when faced with complex design trade-offs.