Engineering stringent genetic biocontainment of yeast with a protein stability switch.

Synthetic biology holds immense promise to tackle key problems in resource use, environmental remediation, and human health care. However, comprehensive safety measures are lacking to employ engineered microorganisms in open-environment applications. Genetically encoded biocontainment systems may solve this issue. Here, we describe such a system based on conditional stability of essential proteins. We used a destabilizing domain degron stabilized by estradiol addition (ERdd). We ERdd-tagged 775 essential genes and screened for strains with estradiol dependent growth. Three genes, SPC110, DIS3 and RRP46, were found to be particularly suitable targets. Respective strains showed no growth defect in the presence of estradiol and strong growth inhibition in its absence. SPC110-ERdd offered the most stringent containment, with an escape frequency of <5×10-7. Removal of its C-terminal domain decreased the escape frequency further to <10-8. Being based on conditional protein stability, the presented approach is mechanistically orthogonal to previously reported genetic biocontainment systems.

Heterologous pulcherrimin production in Saccharomyces cerevisiae confers inhibitory activity on Botrytis conidiation.

Pulcherrimin is an iron (III) chelate of pulcherriminic acid that plays a role in antagonistic microbial interactions, iron metabolism, and stress responses. Some bacteria and yeasts produce pulcherriminic acid, but so far, pulcherrimin could not be produced in Saccharomyces cerevisiae. Here, multiple integrations of the Metschnikowia pulcherrima PUL1 and PUL2 genes in the S. cerevisiae genome resulted in red colonies, which indicated pulcherrimin formation. The coloration correlated positively and significantly with the number of PUL1 and PUL2 genes. The presence of pulcherriminic acid was confirmed by mass spectrometry. In vitro competition assays with the plant pathogenic fungus Botrytis caroliana revealed inhibitory activity on conidiation by an engineered, strong pulcherrimin-producing S. cerevisiae strain. We demonstrate that the PUL1 and PUL2 genes from M. pulcherrima, in multiple copies, are sufficient to transfer pulcherrimin production to S. cerevisiae and represent the starting point for engineering and optimizing this biosynthetic pathway in the future.

Safety by design: Biosafety and biosecurity in the age of synthetic genomics.

Technologies to profoundly engineer biology are becoming increasingly affordable, powerful, and accessible to a widening group of actors. While offering tremendous potential to fuel biological research and the bioeconomy, this development also increases the risk of inadvertent or deliberate creation and dissemination of pathogens. Effective regulatory and technological frameworks need to be developed and deployed to manage these emerging biosafety and biosecurity risks. Here, we review digital and biological approaches of a range of technology readiness levels suited to address these challenges. Digital sequence screening technologies already are used to control access to synthetic DNA of concern. We examine the current state of the art of sequence screening, challenges and future directions, and environmental surveillance for the presence of engineered organisms. As biosafety layer on the organism level, we discuss genetic biocontainment systems that can be used to created host organisms with an intrinsic barrier against unchecked environmental proliferation.

New opportunities for genetic code expansion in synthetic yeast.

The synthetic yeast, Sc2.0, is nearing completion as consolidation of all 17 synthetic chromosomes into a single cell advances. This organism will be the first synthetic eukaryote and provides a highly plastic biological chassis built from the bottom-up using principles of biological design. This synthetic approach to genome construction has allowed the genetic code to be re-wired in this background to liberate the amber stop codon as a dedicated triplet for encoding non-canonical amino acids. The availability of an expanded set of amino acid building blocks allows precise control of protein structure and function, providing new opportunities to develop protein-based therapeutics, materials and catalysts. In this article, we review the challenges facing genetic code expansion research in yeast and highlight how the development of Sc2.0 provides new and exciting opportunities to address existing limitations.

Probing eukaryotic genome functions with synthetic chromosomes.

The ability to redesign and reconstruct a cell at whole-genome level provides new platforms for biological study. The international synthetic yeast genome project-Sc2.0, designed by interrogating knowledge amassed by the yeast community to date, exemplifies how a classical synthetic biology "design-build-test-learn" engineering cycle can effectively test hypotheses about various genome fundamentals. The genome reshuffling SCRaMbLE system implemented in synthetic yeast strains also provides unprecedented diversified resources for genotype-phenotype study and yeast metabolic engineering. Further development of genome synthesis technology will shed new lights on complex biological processes in higher eukaryotes.

Characterizing Transcriptional Interference between Converging Genes in Bacteria.

Antisense transcription is common in naturally occurring genomes and is increasingly being used in synthetic genetic circuitry as a tool for gene expression control. Mutual influence on the expression of convergent genes can be mediated by antisense RNA effects and by transcriptional interference (TI). We aimed to quantitatively characterize long-range TI between convergent genes with untranslated intergenic spacers of increasing length. After controlling for antisense RNA-mediated effects, which contributed about half of the observed total expression inhibition, the TI effect was modeled. To achieve model convergence, RNA polymerase processivity and collision resistance were assumed to be modulated by ribosome trailing. The spontaneous transcription termination rate in regions of untranslated DNA was experimentally determined. Our modeling suggests that an elongating RNA polymerase with a trailing ribosome is about 13 times more likely to resume transcription than an opposing RNA polymerase without a trailing ribosome, upon head-on collision of the two.

A user-friendly, low-cost turbidostat with versatile growth rate estimation based on an extended Kalman filter.

For various experimental applications, microbial cultures at defined, constant densities are highly advantageous over simple batch cultures. Due to high costs, however, devices for continuous culture at freely defined densities still experience limited use. We have developed a small-scale turbidostat for research purposes, which is manufactured from inexpensive components and 3D printed parts. A high degree of spatial system integration and a graphical user interface provide user-friendly operability. The used optical density feedback control allows for constant continuous culture at a wide range of densities and offers to vary culture volume and dilution rates without additional parametrization. Further, a recursive algorithm for on-line growth rate estimation has been implemented. The employed Kalman filtering approach based on a very general state model retains the flexibility of the used control type and can be easily adapted to other bioreactor designs. Within several minutes it can converge to robust, accurate growth rate estimates. This is particularly useful for directed evolution experiments or studies on metabolic challenges, as it allows direct monitoring of the population fitness.

Long-range transcriptional interference in E. coli used to construct a dual positive selection system for genetic switches.

We have investigated transcriptional interference between convergent genes in E. coli and demonstrate substantial interference for inter-promoter distances of as far as 3 kb. Interference can be elicited by both strong σ(70) dependent and T7 promoters. In the presented design, a strong promoter driving gene expression of a 'forward' gene interferes with the expression of a 'reverse' gene by a weak promoter. This arrangement allows inversely correlated gene expression without requiring further regulatory components. Thus, modulation of the activity of the strong promoter alters expression of both the forward and the reverse gene. We used this design to develop a dual selection system for conditional operator site binding, allowing positive selection both for binding and for non-binding to DNA. This study demonstrates the utility of this novel system using the Lac repressor as a model protein for conditional DNA binding, and spectinomycin and chloramphenicol resistance genes as positive selection markers in liquid culture. Randomized LacI libraries were created and subjected to subsequent dual selection, but mispairing IPTG and selection cues in respect to the wild-type LacI response, allowing the isolation of a LacI variant with a reversed IPTG response within three rounds of library generation and dual selection.

An Escherichia coli system for evolving improved light-controlled DNA-binding proteins.

Light-switchable proteins offer numerous opportunities as tools for manipulating biological systems with exceptional degrees of spatiotemporal control. Most designed light-switchable proteins currently in use have not been optimised using the randomisation and selection/screening approaches that are widely used in other areas of protein engineering. Here we report an approach for screening light-switchable DNA-binding proteins that relies on light-dependent repression of the transcription of a fluorescent reporter. We demonstrate that the method can be used to recover a known light-switchable DNA-binding protein from a random library.

Serum-free culture of primary human hepatocytes in a miniaturized hollow-fibre membrane bioreactor for pharmacological in vitro studies.

Primary human hepatocytes represent an important cell source for in vitro investigation of hepatic drug metabolism and disposition. In this study, a multi-compartment capillary membrane-based bioreactor technology for three-dimensional (3D) perfusion culture was further developed and miniaturized to a volume of less than 0.5 ml to reduce demand for cells. The miniaturized bioreactor was composed of two capillary layers, each made of alternately arranged oxygen and medium capillaries serving as a 3D culture for the cells. Metabolic activity and stability of primary human hepatocytes was studied in this bioreactor in the presence of 2.5% fetal calf serum (FCS) under serum-free conditions over a culture period of 10 days. The miniaturized bioreactor showed functions comparable to previously reported data for larger variants. Glucose and lactate metabolism, urea production, albumin synthesis and release of intracellular enzymes (AST, ALT, GLDH) showed no significant differences between serum-free and serum-supplemented bioreactors. Activities of human-relevant cytochrome P450 (CYP) isoenzymes (CYP1A2, CYP3A4/5, CYP2C9, CYP2D6, CYP2B6) analyzed by determination of product formation rates from selective probe substrates were also comparable in both groups. Gene expression analysis showed moderately higher expression in the majority of CYP enzymes, transport proteins and enzymes of Phase II metabolism in the serum-free bioreactors compared to those maintained with FCS. In conclusion, the miniaturized bioreactor maintained stable function over the investigated period and thus provides a suitable system for pharmacological studies on primary human hepatocytes under defined serum-free conditions.

Analysis of drug metabolism activities in a miniaturized liver cell bioreactor for use in pharmacological studies.

Based on a hollow fiber perfusion technology with internal oxygenation, a miniaturized bioreactor with a volume of 0.5 mL for in vitro studies was recently developed. Here, the suitability of this novel culture system for pharmacological studies was investigated, focusing on the model drug diclofenac. Primary human liver cells were cultivated in bioreactors and in conventional monolayer cultures in parallel over 10 days. From day 3 on, diclofenac was continuously applied at a therapeutic concentration (6.4 µM) for analysis of its metabolism. In addition, the activity and gene expression of the cytochrome P450 (CYP) isoforms CYP1A2, CYP2B6, CYP2C9, CYP2D6, and CYP3A4 were assessed. Diclofenac was metabolized in bioreactor cultures with an initial conversion rate of 230 ± 57 pmol/h/10(6) cells followed by a period of stable conversion of about 100 pmol/h/10(6) cells. All CYP activities tested were maintained until day 10 of bioreactor culture. The expression of corresponding mRNAs correlated well with the degree of preservation. Immunohistochemical characterization showed the formation of neo-tissue with expression of CYP2C9 and CYP3A4 and the drug transporters breast cancer resistance protein (BCRP) and multidrug resistance protein 2 (MRP2) in the bioreactor. In contrast, monolayer cultures showed a rapid decline of diclofenac conversion and cells had largely lost activity and mRNA expression of the assessed CYP isoforms at the end of the culture period. In conclusion, diclofenac metabolism, CYP activities and gene expression levels were considerably more stable in bioreactor cultures, making the novel bioreactor a useful tool for pharmacological or toxicological investigations requiring a highly physiological in vitro representation of the liver.