Prototyping of microbial chassis for the biomanufacturing of high-value chemical targets.

Metabolic engineering technologies have been employed with increasing success over the last three decades for the engineering and optimization of industrial host strains to competitively produce high-value chemical targets. To this end, continued reductions in the time taken from concept, to development, to scale-up are essential. Design-Build-Test-Learn pipelines that are able to rapidly deliver diverse chemical targets through iterative optimization of microbial production strains have been established. Biofoundries are employing in silico tools for the design of genetic parts, alongside combinatorial design of experiments approaches to optimize selection from within the potential design space of biological circuits based on multi-criteria objectives. These genetic constructs can then be built and tested through automated laboratory workflows, with performance data analysed in the learn phase to inform further design. Successful examples of rapid prototyping processes for microbially produced compounds reveal the potential role of biofoundries in leading the sustainable production of next-generation bio-based chemicals.

Rapid prototyping of microbial production strains for the biomanufacture of potential materials monomers.

Bio-based production of industrial chemicals using synthetic biology can provide alternative green routes from renewable resources, allowing for cleaner production processes. To efficiently produce chemicals on-demand through microbial strain engineering, biomanufacturing foundries have developed automated pipelines that are largely compound agnostic in their time to delivery. Here we benchmark the capabilities of a biomanufacturing pipeline to enable rapid prototyping of microbial cell factories for the production of chemically diverse industrially relevant material building blocks. Over 85 days the pipeline was able to produce 17 potential material monomers and key intermediates by combining 160 genetic parts into 115 unique biosynthetic pathways. To explore the scale-up potential of our prototype production strains, we optimized the enantioselective production of mandelic acid and hydroxymandelic acid, achieving gram-scale production in fed-batch fermenters. The high success rate in the rapid design and prototyping of microbially-produced material building blocks reveals the potential role of biofoundries in leading the transition to sustainable materials production.

Coordination Chemistry of a Molecular Pentafoil Knot.

The binding of Zn(II) cations to a pentafoil (51) knotted ligand allows the synthesis of otherwise inaccessible metalated molecular pentafoil knots via transmetalation, affording the corresponding "first-sphere" coordination Co(II), Ni(II), and Cu(II) pentanuclear knots in good yields (≥85%). Each of the knot complexes was characterized by mass spectrometry, the diamagnetic (zinc) knot complex was characterized by 1H and 13C NMR spectroscopy, and the zinc, cobalt, and nickel pentafoil knots afforded single crystals whose structures were determined by X-ray crystallography. Lehn-type circular helicates generally only form with tris-bipy ligand strands and Fe(II) (and, in some cases, Ni(II) and Zn(II)) salts, so such architectures become accessible for other metal cations only through the use of knotted ligands. The different metalated knots all exhibit "second-sphere" coordination of a single chloride ion within the central cavity of the knot through CH···Cl- hydrogen bonding and electrostatic interactions. The chloride binding affinities were determined in MeCN by isothermal titration calorimetry, and the strength of binding was shown to vary over 3 orders of magnitude for the different metal-ion-knotted-ligand second-sphere coordination complexes.

PartsGenie: an integrated tool for optimizing and sharing synthetic biology parts.

Motivation: Synthetic biology is typified by developing novel genetic constructs from the assembly of reusable synthetic DNA parts, which contain one or more features such as promoters, ribosome binding sites, coding sequences and terminators. PartsGenie is introduced to facilitate the computational design of such synthetic biology parts, bridging the gap between optimization tools for the design of novel parts, the representation of such parts in community-developed data standards such as Synthetic Biology Open Language, and their sharing in journal-recommended data repositories. Consisting of a drag-and-drop web interface, a number of DNA optimization algorithms, and an interface to the well-used data repository JBEI ICE, PartsGenie facilitates the design, optimization and dissemination of reusable synthetic biology parts through an integrated application. Availability and implementation: PartsGenie is freely available at https://parts.synbiochem.co.uk.

Dual transcriptional-translational cascade permits cellular level tuneable expression control.

The ability to induce gene expression in a small molecule dependent manner has led to many applications in target discovery, functional elucidation and bio-production. To date these applications have relied on a limited set of protein-based control mechanisms operating at the level of transcription initiation. The discovery, design and reengineering of riboswitches offer an alternative means by which to control gene expression. Here we report the development and characterization of a novel tunable recombinant expression system, termed RiboTite, which operates at both the transcriptional and translational level. Using standard inducible promoters and orthogonal riboswitches, a multi-layered modular genetic control circuit was developed to control the expression of both bacteriophage T7 RNA polymerase and recombinant gene(s) of interest. The system was benchmarked against a number of commonly used E. coli expression systems, and shows tight basal control, precise analogue tunability of gene expression at the cellular level, dose-dependent regulation of protein production rates over extended growth periods and enhanced cell viability. This novel system expands the number of E. coli expression systems for use in recombinant protein production and represents a major performance enhancement over and above the most widely used expression systems.

SYNBIOCHEM-a SynBio foundry for the biosynthesis and sustainable production of fine and speciality chemicals.

The Manchester Synthetic Biology Research Centre (SYNBIOCHEM) is a foundry for the biosynthesis and sustainable production of fine and speciality chemicals. The Centre's integrated technology platforms provide a unique capability to facilitate predictable engineering of microbial bio-factories for chemicals production. An overview of these capabilities is described.

Rational Re-engineering of a Transcriptional Silencing PreQ1 Riboswitch.

Re-engineered riboswitches that no longer respond to cellular metabolites, but that instead can be controlled by synthetic molecules, are potentially useful gene regulatory tools for use in synthetic biology and biotechnology fields. Previously, extensive genetic selection and screening approaches were employed to re-engineer a natural adenine riboswitch to create orthogonal ON-switches, enabling translational control of target gene expression in response to synthetic ligands. Here, we describe how a rational targeted approach was used to re-engineer the PreQ1 riboswitch from Bacillus subtilis into an orthogonal OFF-switch. In this case, the evaluation of just six synthetic compounds with seven riboswitch mutants led to the identification of an orthogonal riboswitch-ligand pairing that effectively repressed the transcription of selected genes in B. subtilis. The streamlining of the re-engineering approach, and its extension to a second class of riboswitches, provides a methodological platform for the creation of new orthogonal regulatory components for biotechnological applications including gene functional analysis and antimicrobial target validation and screening.

Modular riboswitch toolsets for synthetic genetic control in diverse bacterial species.

Ligand-dependent control of gene expression is essential for gene functional analysis, target validation, protein production, and metabolic engineering. However, the expression tools currently available are difficult to transfer between species and exhibit limited mechanistic diversity. Here we demonstrate how the modular architecture of purine riboswitches can be exploited to develop orthogonal and chimeric switches that are transferable across diverse bacterial species, modulating either transcription or translation, to provide tunable activation or repression of target gene expression, in response to synthetic non-natural effector molecules. Our novel riboswitch-ligand pairings are shown to regulate physiologically important genes required for bacterial motility in Escherichia coli and cell morphology in Bacillus subtilis. These findings are relevant for future gene function studies and antimicrobial target validation, while providing new modular and orthogonal regulatory components for deployment in synthetic biology regimes.

Macrophage Colony Stimulating Factor (M-CSF) and Interleukin-34 (IL-34) Expression in Canine Osteosarcoma in the Context of the Tumour Immune Microenvironment.

Canine osteosarcoma (OSA) is a malignancy that has been shown to modulate the host immune system. Macrophage colony-stimulating factor (M-CSF; CSF1) and interleukin-34 (IL-34; IL34) are both ligands of colony stimulating factor 1 receptor (CSF-1R), and may play a role in the pathogenesis of a variety of human cancers, including OSA. This study aimed to, (1) assess M-CSF and IL-34 expression in canine OSA cell lines and tissue samples, and (2) determine any correlations between M-CSF and IL-34 expression and immune cell infiltrates within canine OSA tissues. Four canine OSA cell lines and canine osteoblasts were treated with control media, TNFα (10 ng/mL) or IL-1ß (10 ng/mL) and analysed with RT-qPCR and ELISA. IL-34 and M-CSF mRNA and protein were detectable in all cell lines, however upregulation following TNFα or IL-1ß exposure was only consistently observed for transcript expression. Baseline expression of CSF1 and IL34 mRNA in OSA cell lines was equal to or higher than that of canine osteoblasts. All 10 OSA tissue samples expressed IL34 and CSF1 transcripts to varying degrees. Furthermore, CSF1 and IL34 expression both showed a moderate to high degree of correlation with M1 macrophage lineage-associated transcripts (CD80 and IL15RA). There was a moderate degree of correlation between CSF1 and CD163, but no correlation between IL34 and either M2 macrophage-associated transcripts (CD163 and CCL24). In summary, IL-34 and M-CSF are expressed in canine OSA cell lines and tissues, and expression positively correlates with a wide range of immune-related transcripts.

Cooperative heparin-mediated oligomerization of fibroblast growth factor-1 (FGF1) precedes recruitment of FGFR2 to ternary complexes.

Fibroblast growth factors (FGFs) utilize cell surface heparan sulfate as a coreceptor in the assembly of signaling complexes with FGF-receptors on the plasma membrane. Here we undertake a complete thermodynamic characterization of the assembly of the FGF signaling complex using isothermal titration calorimetry. Heparin fragments of defined length are used as chemical analogs of the sulfated domains of heparan sulfate and examined for their ability to oligomerize FGF1. Binding is modeled using the McGhee-von Hippel formalism for the cooperative binding of ligands to a monodimensional lattice. Oligomerization of FGFs on heparin is shown to be mediated by positive cooperativity (α = 6). Heparin octasaccharide is the shortest length capable of dimerizing FGF1 and on longer heparin chains FGF1 binds with a minimal footprint of 4.2 saccharide units. The thermodynamics and stoichiometry of the ternary complex suggest that in solution FGF1 binds to heparin in a trans-dimeric manner before FGFR recruitment.

Digoxin antibody fragment, antigen binding (Fab), treatment of preeclampsia in women with endogenous digitalis-like factor: a secondary analysis of the DEEP Trial.

OBJECTIVE: Endogenous digitalis-like factors (EDLFs) are elevated in women with preeclampsia, and the use of an anti-digoxin antibody Fab (DIF) in women with preeclampsia who were remote from term reduced maternal blood pressure and preserved renal function. The objective was to determine whether DIF treatment in women with severe preeclampsia in association with positive EDLFs in maternal serum improves maternal-perinatal outcomes. STUDY DESIGN: This was a planned secondary analysis from a randomized, placebo-controlled, double-blind study of DIF in women with severe preeclampsia with positive EDLF status that was managed expectantly between 23 weeks 5 days and 34 weeks' gestation (19 women received placebo, and 17 women received DIF). Primary outcome variables were a change in creatinine clearance and the use of antihypertensives. Secondary outcomes were maternal and perinatal complications. RESULTS: Women with positive EDLFs who received DIF had an attenuated decline in creatinine clearance from baseline compared with placebo (-4.5 ± 12.9 vs -53.2 ± 12.6 mL/min; P = .005). In this same group, the use of antihypertensives (the other primary outcome) was lower but not significantly so (41% vs 63%; P = .12). However, women who were treated with DIF had a lower rate of pulmonary edema (1/17 vs 6/19 women; P = .035) and lower rates of neonatal intraventricular hemorrhage (DIF: 0/17 women vs placebo: 5/19 women; P = .015). CONCLUSION: In women with severe preeclampsia who were remote from term who were EDLF positive, the use of DIF was associated with improved maternal and neonatal outcome. These findings suggest the need for a large multicenter trial that would evaluate the benefits of DIF in the treatment of women with severe preeclampsia who are remote from term and with positive EDLF status.

Association of maternal vitamin D and placenta growth factor with the diagnosis of early onset severe preeclampsia.

OBJECTIVE: Decreased maternal 25-hydroxyvitamin D (25-OH-D) and placenta growth factor (PlGF) have both been associated with the diagnosis of early onset severe preeclampsia (EOSPE). This investigation aimed to define the association of these biomarkers with EOSPE. STUDY DESIGN: Patients with EOSPE (n = 40) and healthy controls (n = 40) were recruited and information on demographics, outcomes, and plasma was collected at diagnosis of EOSPE or gestational age-matched controls. 25-OH-D was assessed by radioimmunoassay and reported in nanogram per milliliter. PlGF was assessed by enzyme-linked immunosorbent assay and reported in picogram per milliliter. Kruskal-Wallis test was used to compare biomarkers between groups. Multivariable logistic regression was used to determine associations between 25-OH-D or PlGF and the diagnosis of EOSPE. RESULTS: In EOSPE, both 25-OH-D and PlGF were decreased significantly compared with controls. After controlling for age, race, body mass index, and gestational age at sample collection, both 25-OH-D (adjusted odds ratio 0.14 [0.05, 0.36]) and PlGF (adjusted odds ratio 0.03 [0.01, 0.24] were significantly associated with diagnosis of ESOPE (p < 0.001 for both markers). CONCLUSION: PlGF and 25-OH-D are both associated with the diagnosis of EOSPE. These biomarkers may be helpful in development of novel rapid diagnostic tests for preeclampsia.

Maternal vitamin D and fetal growth in early-onset severe preeclampsia.

OBJECTIVE: Recently, vitamin D deficiency has been associated with increased risks for preeclampsia and diagnosis of early-onset, severe preeclampsia (EOSPE). The purpose of this investigation was to examine the association between vitamin D levels and small-for-gestational age (SGA) in patients with EOSPE. STUDY DESIGN: Patients with EOSPE were recruited, and demographics, outcomes, and plasma were collected. We assessed 25-hydroxyvitamin D (25[OH]D) by radioimmunoassay and reported our findings in nanograms per milliliter. Results were analyzed by Mann-Whitney U test and Spearman correlation and were reported as median (Q1-Q3). RESULTS: In patients with EOSPE (n = 56), 25(OH)D was lower in patients with SGA (16.8 ng/mL; range, 8.9-23 ng/mL) vs normal fetal growth (25.3 ng/mL; range, 16-33 ng/mL; P = .02). 25(OH)D was correlated significantly with percentile growth at delivery (ρ = 0.31; P = .02). CONCLUSION: Vitamin D is lower among patients with SGA in EOSPE than those without growth retardation. We suspect that vitamin D may impact fetal growth through placental mechanisms.

The evolving art of creating genetic diversity: From directed evolution to synthetic biology.

The ability to engineer biological systems, whether to introduce novel functionality or improved performance, is a cornerstone of biotechnology and synthetic biology. Typically, this requires the generation of genetic diversity to explore variations in phenotype, a process that can be performed at many levels, from single molecule targets (i.e., in directed evolution of enzymes) to whole organisms (e.g., in chassis engineering). Recent advances in DNA synthesis technology and automation have enhanced our ability to create variant libraries with greater control and throughput. This review highlights the latest developments in approaches to create such a hierarchy of diversity from the enzyme level to entire pathways in vitro, with a focus on the creation of combinatorial libraries that are required to navigate a target's vast design space successfully to uncover significant improvements in function.

A plasmid toolset for CRISPR-mediated genome editing and CRISPRi gene regulation in Escherichia coli.

CRISPR technologies have become standard laboratory tools for genetic manipulations across all kingdoms of life. Despite their origins in bacteria, the development of CRISPR tools for engineering bacteria has been slower than for eukaryotes; nevertheless, their function and application for genome engineering and gene regulation via CRISPR interference (CRISPRi) has been demonstrated in various bacteria, and adoption has become more widespread. Here, we provide simple plasmid-based systems for genome editing (gene knockouts/knock-ins, and genome integration of large DNA fragments) and CRISPRi in E. coli using a CRISPR-Cas12a system. The described genome engineering protocols allow markerless deletion or genome integration in just seven working days with high efficiency (> 80% and 50%, respectively), and the CRISPRi protocols allow robust transcriptional repression of target genes (> 90%) with a single cloning step. The presented minimized plasmids and their associated design and experimental protocols provide efficient and effective CRISPR-Cas12 genome editing, genome integration and CRISPRi implementation. These simple-to-use systems and protocols will allow the easy adoption of CRISPR technology by any laboratory.

Timing of elective repeat cesarean delivery at term and neonatal outcomes: a cost analysis.

OBJECTIVE: The purpose of this investigation was to examine the economic impact of performing elective repeat cesarean during 37 or 38 weeks of gestation relative to the American College of Obstetricians and Gynecologists recommendation of a 39-week delivery. STUDY DESIGN: Decision analysis modeling was used to estimate economic outcomes for a hypothetical cohort of neonates using data from the Eunice Kennedy Shriver National Institute of Child Health and Human Development Maternal-Fetal Medicine Units Network study of "Timing of Elective Repeat Cesarean Delivery at Term and Neonatal Outcomes." Costs and charges were estimated using the Florida Healthcare Cost and Utilization Project. RESULTS: A total of 82,541 deliveries occurring between 37-39 completed weeks of gestation were analyzed for the incidence of adverse outcomes and their hospital costs and charges. The model demonstrated increased costs through increasing adverse outcomes among elective repeat cesarean deliveries performed <39 weeks of gestation. CONCLUSION: Our findings suggest that there are benefits to waiting until 39 weeks of gestation to perform an elective repeat cesarean delivery.

Plasma 25-hydroxyvitamin D levels in early-onset severe preeclampsia.

OBJECTIVE: Vitamin D deficiency has been linked to adverse pregnancy outcomes. The purpose of this investigation was to assess total 25-hydroxyvitamin D (25-OH-D) levels at diagnosis of early-onset severe preeclampsia (EOSPE). STUDY DESIGN: After institutional review board approval, we enrolled subjects with EOSPE (<34 weeks' gestation with severe preeclampsia) in this case-control investigation in a 1:2 ratio with gestation-matched, contemporaneous control subjects. Demographic and outcome information was collected for each subject. Plasma total 25-OH-D levels were determined by radioimmunoassay and reported in nanograms per milliliter. Results were analyzed by Mann-Whitney U and multivariable regression. RESULTS: Subjects with EOSPE (n = 50) were noted to have decreased total 25-OH-D levels relative to healthy control subjects (n = 100; P < .001). This difference in total 25-OH-D remained significant after control for potential confounders. CONCLUSION: Total 25-OH-D is decreased at diagnosis of EOSPE. Further study is needed to understand the impact of vitamin D deficiency on pregnancy outcomes.

Complications of cesarean delivery in the massively obese parturient.

OBJECTIVE: The objective of the study was to determine predictors of cesarean delivery morbidity associated with massive obesity. STUDY DESIGN: This was an institutional review board-approved retrospective study of massively obese women (body mass index, > or = 50 kg/m(2)) undergoing cesarean delivery. Bivariable and multivariable analyses were used to assess the strength of association between wound complication and various predictors. RESULTS: Fifty-eight of 194 patients (30%) had a wound complication. Most (90%) were wound disruptions, and 86% were diagnosed after hospital discharge (median postoperative day, 8.5; interquartile range, 6-12). Subcutaneous drains and smoking, but not labor or ruptured membranes, were independently associated with wound complication after controlling for various confounders. Vertical abdominal incisions were associated with increased operative time, blood loss, and vertical hysterotomy. CONCLUSION: Women with a body mass index > or = 50 kg/m(2) have a much greater risk for cesarean wound complications than previously reported. Avoidance of subcutaneous drains and increased use of transverse abdominal wall incisions should be considered in massively obese parturients to reduce operative morbidity.

Maternal super-obesity (body mass index > or = 50) and adverse pregnancy outcomes.

OBJECTIVE: To determine if pregnancy complications are increased in super-obese (a body mass index (BMI) of 50 or more) compared to other, less obese parturients. DESIGN: Cross-sectional study. SETTING AND POPULATION: All 19,700 eligible women, including 425 (2.2%) super-obese women with singleton births between 1996 and 2007 delivering at a tertiary referral center, identified using a perinatal research database. METHODS: Bivariate and trend analyses were used to assess the relation between super-obesity and various pregnancy complications compared to other well-established BMI categories. Adjusted odds ratios (ORs) were calculated using multivariable logistic regression techniques. MAIN OUTCOME MEASURES: Outcomes for adjusted and unadjusted analyses were small-for-gestational age (SGA) birth, large-for-gestational age (LGA) birth, preeclampsia, gestational diabetes mellitus (GDM), fetal death, preterm birth, placental abruption, cesarean delivery, and Apgar scores < 7. RESULTS: Compared to all other obese and non-obese women, super-obese women had the highest rates of preeclampsia, GDM, LGA, and cesarean delivery (all p < 0.05 for trend test). Super-obesity was also associated with a 44% reduction in SGA compared to all other women (OR 0.55, 95% confidence interval (CI) 0.40-0.76) and a 25% reduction compared to other, less obese women (OR 0.75, 95% CI 0.54-1.03). Super-obesity was positively associated with LGA, GDM, preeclampsia, cesarean delivery, and a 5-minute Apgar score < 7 compared to all other women after controlling for important confounders. CONCLUSION: Super-obesity is associated with higher rates of pregnancy complications compared to women of all other BMI classes, including other obese women.

Engineering Escherichia coli towards de novo production of gatekeeper (2S)-flavanones: naringenin, pinocembrin, eriodictyol and homoeriodictyol.

Natural plant-based flavonoids have drawn significant attention as dietary supplements due to their potential health benefits, including anti-cancer, anti-oxidant and anti-asthmatic activities. Naringenin, pinocembrin, eriodictyol and homoeriodictyol are classified as (2S)-flavanones, an important sub-group of naturally occurring flavonoids, with wide-reaching applications in human health and nutrition. These four compounds occupy a central position as branch point intermediates towards a broad spectrum of naturally occurring flavonoids. Here, we report the development of Escherichia coli production chassis for each of these key gatekeeper flavonoids. Selection of key enzymes, genetic construct design and the optimization of process conditions resulted in the highest reported titers for naringenin (484 mg/l), improved production of pinocembrin (198 mg/l) and eriodictyol (55 mg/l from caffeic acid), and provided the first example of in vivo production of homoeriodictyol directly from glycerol (17 mg/l). This work provides a springboard for future production of diverse downstream natural and non-natural flavonoid targets.