Strengthening healthcare providers’ capacity for safe abortion and post-abortion care services in humanitarian settings: lessons learned from the clinical outreach refresher training model (S-CORT) in Uganda, Nigeria, and the Democratic Republic of Congo

Background Fragile and crisis-affected countries account for most maternal deaths worldwide, with unsafe abortion being one of its leading causes. This case study aims to describe the Clinical Outreach Refresher Training strategy for sexual and reproductive health (S-CORT) designed to update health providers’ competencies on uterine evacuation using both medications and manual vacuum aspiration. The paper also explores stakeholders’ experiences, recommendations for improvement, and lessons learned. Methods Using mixed methods, we evaluated three training workshops that piloted the uterine evacuation module in 2019 in humanitarian contexts of Uganda, Nigeria, and the Democratic Republic of Congo. Results Results from the workshops converged to suggest that the module contributed to increasing participants’ theoretical knowledge and possibly technical and counseling skills. Equally noteworthy were their confidence building and positive attitudinal changes promoting a rights-based, fearless, non-judgmental, and non-discriminatory approach toward clients. Participants valued the hands-on, humanistic, and competency-based training methodology, although most regretted the short training duration and lack of practice on real clients. Recommendations to improve the capacity development continuum of uterine evacuation included recruiting the appropriate health cadres for the training; sharing printed pre-reading materials to all participants; sustaining the availability of medication and supplies to offer services to clients after the training; and helping staff through supportive supervision visits to accelerate skills transfer from training to clinic settings. Conclusions When the lack of skilled human resources is a barrier to lifesaving uterine evacuation services in humanitarian settings, the S-CORT strategy could offer a rapid hands-on refresher training opportunity for service providers needing an update in knowledge and skills. Such a capacity-building approach could be useful in humanitarian and fragile settings as well as in development settings with limited resources as part of an overall effort to strengthen other building blocks of the health system

Synthetic biology to access and expand nature's chemical diversity

Bacterial genomes encode the biosynthetic potential to produce hundreds of thousands of complex molecules with diverse applications, from medicine to agriculture and materials. Accessing these natural products promises to reinvigorate drug discovery pipelines and provide novel routes to synthesize complex chemicals. The pathways leading to the production of these molecules often comprise dozens of genes spanning large areas of the genome and are controlled by complex regulatory networks with some of the most interesting molecules being produced by non-model organisms. In this Review, we discuss how advances in synthetic biology — including novel DNA construction technologies, the use of genetic parts for the precise control of expression and for synthetic regulatory circuits — and multiplexed genome engineering can be used to optimize the design and synthesis of pathways that produce natural products

Combinatorialization of Fungal Polyketide Synthase–Peptide Synthetase Hybrid Proteins

The programming of the fungal polyketide synthase (PKS) is quite complex, with a simple domain architecture leading to elaborate products. An additional level of complexity has been found within PKS-based pathways where the PKS is fused to a single module nonribosomal peptide synthetase (NRPS) to synthesize polyketides conjugated to amino acids. Here, we sought to understand the communication between these modules that enable correct formation of polyketide-peptide hybrid products. To do so, we fused together the genes that are responsible for forming five highly chemically diverse fungal natural products in a total of 57 different combinations, comprising 34 distinct module swaps. Gene fusions were formed with the idea of testing the connection and compatibility of the PKS and NRPS modules mediated by the acyl carrier protein (ACP), condensation (C) and ketoreductase (KR) domains. The resulting recombinant gene fusions were analyzed in a high-yielding expression platform to avail six new compounds, including the first successful fusion between a PKS and NRPS that make highly divergent products, and four previously reported molecules. Our results show that C domains are highly selective for a subset of substrates. We discovered that within the highly reducing (hr) PKS class, noncognate ACPs of closely related members complement PKS function. We intercepted a pre-Diels–Alder intermediate in lovastatin synthesis for the first time, shedding light on this canonical fungal biochemical reaction. The results of these experiments provide a set of ground rules for the successful engineering of hr-PKS and PKS-NRPS products in fungi

Biosynthesis of the Tetramic Acids Sch210971 and Sch210972

A biosynthetic pathway to fungal polyketide–nonribosomal peptide natural products, Sch210971 (<b>1a</b>) and Sch210972 (<b>1b</b>) from <i>Hapsidospora irregularis</i>, was characterized by reconstitution and heterologous expression in <i>Fusarium heterosporum</i>. Using genetic, biochemical, and feeding experiments, we show that the incorporated amino acid 4-hydroxyl-4-methyl glutamate (HMG) is synthesized by an aldolase, probably using pyruvate as the precursor

Two Related Pyrrolidinedione Synthetase Loci in <i>Fusarium heterosporum</i> ATCC 74349 Produce Divergent Metabolites

Equisetin synthetase (EqiS), from the filamentous fungus <i>Fusarium heterosporum</i> ATCC 74349, was initially assigned on the basis of genetic knockout and expression analysis. Increasing inconsistencies in experimental results led us to question this assignment. Here, we sequenced the <i>F. heterosporum</i> genome, revealing two hybrid polyketide-peptide proteins that were candidates for the equisetin synthetase. The surrounding genes in both clusters had the needed auxiliary genes that might be responsible for producing equisetin. Genetic mutation, biochemical analysis, and recombinant expression in the fungus enabled us to show that the initially assigned EqiS does not produce equisetin but instead produces a related 2,4-pyrrolidinedione, fusaridione A, that was previously unknown. Fusaridione A is methylated in the 3-position of the pyrrolidinedione, which has not otherwise been found in natural products, leading to spontaneous reverse-Dieckmann reactions. A newly described gene cluster, <i>eqx</i>, is responsible for producing equisetin

Biosynthesis of the Tetramic Acids Sch210971 and Sch210972

A biosynthetic pathway to fungal polyketide–nonribosomal peptide natural products, Sch210971 (<b>1a</b>) and Sch210972 (<b>1b</b>) from <i>Hapsidospora irregularis</i>, was characterized by reconstitution and heterologous expression in <i>Fusarium heterosporum</i>. Using genetic, biochemical, and feeding experiments, we show that the incorporated amino acid 4-hydroxyl-4-methyl glutamate (HMG) is synthesized by an aldolase, probably using pyruvate as the precursor

Biosynthesis of para-Cyclophane-Containing Hirsutellone Family of Fungal Natural Products

Hirsutellones are fungal natural products containing a macrocyclic para-cyclophane connected to a decahydrofluorene ring system. We have elucidated the biosynthetic pathway for pyrrocidine B (3) and GKK1032 A2 (4). Two small hypothetical proteins, an oxidoreductase and a lipocalin-like protein, function cooperatively in the oxidative cyclization of the cyclophane, while an additional hypothetical protein in the pyrrocidine pathway catalyzes the exo-specific cycloaddition to form the cis-fused decahydrofluorene

Native Promoter Strategy for High-Yielding Synthesis and Engineering of Fungal Secondary Metabolites

Strategies are needed for the robust production of cryptic, silenced, or engineered secondary metabolites in fungi. The filamentous fungus <i>Fusarium heterosporum</i> natively synthesizes the polyketide equisetin at >2 g L^–1 in a controllable manner. We hypothesized that this production level was achieved by regulatory elements in the equisetin pathway, leading to the prediction that the same regulatory elements would be useful in producing other secondary metabolites. This was tested by using the native <i>eqxS</i> promoter and <i>eqxR</i> regulator in <i>F. heterosporum</i>, synthesizing heterologous natural products in yields of ∼1 g L^–1. As proof of concept for the practical application, we resurrected an extinct pathway from an endophytic fungus with an initial yield of >800 mg L^–1, leading to the practical synthesis of a selective antituberculosis agent. Finally, the method enabled new insights into the function of polyketide synthases in filamentous fungi. These results demonstrate a strategy for optimally employing native regulators for the robust synthesis of secondary metabolites

An enzymatic Alder-ene reaction.

An ongoing challenge in chemical research is to design catalysts that select the outcomes of the reactions of complex molecules. Chemists rely on organocatalysts or transition metal catalysts to control stereoselectivity, regioselectivity and periselectivity (selectivity among possible pericyclic reactions). Nature achieves these types of selectivity with a variety of enzymes such as the recently discovered pericyclases-a family of enzymes that catalyse pericyclic reactions1. Most characterized enzymatic pericyclic reactions have been cycloadditions, and it has been difficult to rationalize how the observed selectivities are achieved2-13. Here we report the discovery of two homologous groups of pericyclases that catalyse distinct reactions: one group catalyses an Alder-ene reaction that was, to our knowledge, previously unknown in biology; the second catalyses a stereoselective hetero-Diels-Alder reaction. Guided by computational studies, we have rationalized the observed differences in reactivities and designed mutant enzymes that reverse periselectivities from Alder-ene to hetero-Diels-Alder and vice versa. A combination of in vitro biochemical characterizations, computational studies, enzyme co-crystal structures, and mutational studies illustrate how high regioselectivity and periselectivity are achieved in nearly identical active sites

An enzymatic Alder-ene reaction.

An ongoing challenge in chemical research is to design catalysts that select the outcomes of the reactions of complex molecules. Chemists rely on organocatalysts or transition metal catalysts to control stereoselectivity, regioselectivity and periselectivity (selectivity among possible pericyclic reactions). Nature achieves these types of selectivity with a variety of enzymes such as the recently discovered pericyclases-a family of enzymes that catalyse pericyclic reactions1. Most characterized enzymatic pericyclic reactions have been cycloadditions, and it has been difficult to rationalize how the observed selectivities are achieved2-13. Here we report the discovery of two homologous groups of pericyclases that catalyse distinct reactions: one group catalyses an Alder-ene reaction that was, to our knowledge, previously unknown in biology; the second catalyses a stereoselective hetero-Diels-Alder reaction. Guided by computational studies, we have rationalized the observed differences in reactivities and designed mutant enzymes that reverse periselectivities from Alder-ene to hetero-Diels-Alder and vice versa. A combination of in vitro biochemical characterizations, computational studies, enzyme co-crystal structures, and mutational studies illustrate how high regioselectivity and periselectivity are achieved in nearly identical active sites