Global modified-Delphi consensus on comorbidities and prognosis of SCN8A-related epilepsy and/or neurodevelopmental disorders.

OBJECTIVES: We aimed to develop consensus on comorbidities (frequency, severity, and prognosis) and overall outcomes in epilepsy, development, and cognition for the five phenotypes of SCN8A-related disorders. METHODS: A core panel consisting of 13 clinicians, 1 researcher, and 6 caregivers was formed and split into three workgroups. One group focused on comorbidities and prognosis. All groups performed a literature review and developed questions for use in a modified-Delphi process. Twenty-eight clinicians, one researcher, and 13 caregivers from 16 countries participated in three rounds of the modified-Delphi process. Consensus was defined as follows: strong consensus ≥80% fully agree; moderate consensus ≥80% fully or partially agree, <10% disagree; and modest consensus 67%-79% fully or partially agree, <10% disagree. RESULTS: Consensus was reached on the presence of 14 comorbidities in patients with Severe Developmental and Epileptic Encephalopathy (Severe DEE) spanning non-seizure neurological disorders and other organ systems; impacts were mostly severe and unlikely to improve or resolve. Across Mild/Moderate Developmental and Epileptic Encephalopathy (Mild/Moderate DEE), Neurodevelopmental Delay with Generalized Epilepsy (NDDwGE), and NDD without Epilepsy (NDDwoE) phenotypes, cognitive and sleep-related comorbidities as well as fine and gross motor delays may be present but are less severe and more likely to improve compared to Severe DEE. There was no consensus on comorbidities in the SeL(F)IE phenotype but strong conesensus that seizures would largely resolve. Seizure freedom is rare in patients with Severe DEE but may occur in some with Mild/Moderate DEE and NDDwGE. SIGNIFICANCE: Significant comorbidities are present in most phenotypes of SCN8A-related disorders but are most severe and pervasive in the Severe DEE phenotype. We hope that this work will improve recognition, early intervention, and long-term management for patients with these comorbidities and provide the basis for future evidence-based studies on optimal treatments of SCN8A-related disorders. Identifying the prognosis of patients with SCN8A-related disorders will also improve care and quality-of-life for patients and their caregivers.

Global modified Delphi consensus on diagnosis, phenotypes, and treatment of SCN8A-related epilepsy and/or neurodevelopmental disorders.

OBJECTIVE: We aimed to develop consensus for diagnosis/management of SCN8A-related disorders. Utilizing a modified Delphi process, a global cohort of experienced clinicians and caregivers provided input on diagnosis, phenotypes, treatment, and management of SCN8A-related disorders. METHODS: A Core Panel (13 clinicians, one researcher, six caregivers), divided into three subgroups (diagnosis/phenotypes, treatment, comorbidities/prognosis), performed a literature review and developed questions for the modified Delphi process. Twenty-eight expert clinicians, one researcher, and 13 caregivers from 16 countries participated in the subsequent three survey rounds. We defined consensus as follows: strong consensus, ≥80% fully agree; moderate consensus, ≥80% fully/partially agree, <10% disagree; and modest consensus, 67%-79% fully/partially agree, <10% disagree. RESULTS: Early diagnosis is important for long-term clinical outcomes in SCN8A-related disorders. There are five phenotypes: three with early seizure onset (severe developmental and epileptic encephalopathy [DEE], mild/moderate DEE, self-limited (familial) infantile epilepsy [SeL(F)IE]) and two with later/no seizure onset (neurodevelopmental delay with generalized epilepsy [NDDwGE], NDD without epilepsy [NDDwoE]). Caregivers represented six patients with severe DEE, five mild/moderate DEE, one NDDwGE, and one NDDwoE. Phenotypes vary by age at seizures/developmental delay onset, seizure type, electroencephalographic/magnetic resonance imaging findings, and first-line treatment. Gain of function (GOF) versus loss of function (LOF) is valuable for informing treatment. Sodium channel blockers are optimal first-line treatment for GOF, severe DEE, mild/moderate DEE, and SeL(F)IE; levetiracetam is relatively contraindicated in GOF patients. First-line treatment for NDDwGE is valproate, ethosuximide, or lamotrigine; sodium channel blockers are relatively contraindicated in LOF patients. SIGNIFICANCE: This is the first-ever global consensus for the diagnosis and treatment of SCN8A-related disorders. This consensus will reduce knowledge gaps in disease recognition and inform preferred treatment across this heterogeneous disorder. Consensus of this type allows more clinicians to provide evidence-based care and empowers SCN8A families to advocate for their children.

Functional genomics analysis of free fatty acid production under continuous phosphate limiting conditions.

Free fatty acids (FFA) are an attractive platform chemical that serves as a functional intermediate in metabolic pathways for producing oleochemicals. Many groups have established strains of Escherichia coli capable of producing various chain-length mixtures of FFA by heterologous expression of acyl-ACP thioesterases. For example, high levels of dodecanoic acid are produced by an E. coli strain expressing the Umbellularia californica FatB2 thioesterase, BTE. Prior studies achieved high dodecanoic acid yields and productivities under phosphate-limiting media conditions. In an effort to understand the metabolic and physiological changes that led to increased FFA production, the transcriptome of this strain was assessed as a function of nutrient limitation and growth rate. FFA generation under phosphate limitation led to consistent changes in transporter expression, osmoregulation, and central metabolism. Guided by these results, targeted knockouts led to a further ~11 % in yield in FFA.

Nonenzymatic sugar production from biomass using biomass-derived γ-valerolactone.

Widespread production of biomass-derived fuels and chemicals will require cost-effective processes for breaking down cellulose and hemicellulose into their constituent sugars. Here, we report laboratory-scale production of soluble carbohydrates from corn stover, hardwood, and softwood at high yields (70 to 90%) in a solvent mixture of biomass-derived γ-valerolactone (GVL), water, and dilute acid (0.05 weight percent H2SO4). GVL promotes thermocatalytic saccharification through complete solubilization of the biomass, including the lignin fraction. The carbohydrates can be recovered and concentrated (up to 127 grams per liter) by extraction from GVL into an aqueous phase by addition of NaCl or liquid CO2. This strategy is well suited for catalytic upgrading to furans or fermentative upgrading to ethanol at high titers and near theoretical yield. We estimate through preliminary techno-economic modeling that the overall process could be cost-competitive for ethanol production, with biomass pretreatment followed by enzymatic hydrolysis.

Production of medium chain length fatty alcohols from glucose in Escherichia coli.

Metabolic engineering offers the opportunity to produce a wide range of commodity chemicals that are currently derived from petroleum or other non-renewable resources. Microbial synthesis of fatty alcohols is an attractive process because it can control the distribution of chain lengths and utilize low cost fermentation substrates. Specifically, primary alcohols with chain lengths of 12 to 14 carbons have many uses in the production of detergents, surfactants, and personal care products. The current challenge is to produce these compounds at titers and yields that would make them economically competitive. Here, we demonstrate a metabolic engineering strategy for producing fatty alcohols from glucose. To produce a high level of 1-dodecanol and 1-tetradecanol, an acyl-ACP thioesterase (BTE), an acyl-CoA ligase (FadD), and an acyl-CoA/aldehyde reductase (MAACR) were overexpressed in an engineered strain of Escherichia coli. Yields were improved by balancing expression levels of each gene, using a fed-batch cultivation strategy, and adding a solvent to the culture for extracting the product from cells. Using these strategies, a titer of over 1.6 g/L fatty alcohol with a yield of over 0.13 g fatty alcohol/g carbon source was achieved. These are the highest reported yield of fatty alcohols produced from glucose in E. coli.

Free fatty acid production in Escherichia coli under phosphate-limited conditions.

Microbially synthesized fatty acids are an attractive platform for producing renewable alternatives to petrochemically derived transportation fuels and oleochemicals. Free fatty acids (FFA) are a direct precursor to many high-value compounds that can be made via biochemical and ex vivo catalytic pathways. To be competitive with current petrochemicals, flux through these pathways must be optimized to approach theoretical yields. Using a plasmid-free, FFA-producing strain of Escherichia coli, a set of chemostat experiments were conducted to gather data for FFA production under phosphate limitation. A prior study focused on carbon-limited conditions strongly implicated non-carbon limitations as a preferred media formulation for maximizing FFA yield. Here, additional data were collected to expand an established kinetic model of FFA production and identify targets for further metabolic engineering. The updated model was able to successfully predict the strain's behavior and FFA production in a batch culture. The highest yield observed under phosphate-limiting conditions (0.1 g FFA/g glucose) was obtained at a dilution rate of 0.1 h(-1), and the highest biomass-specific productivity (0.068 g FFA/gDCW/h) was observed at a dilution rate of 0.25 h(-1). Phosphate limitation increased yield (∼45 %) and biomass-specific productivity (∼300 %) relative to carbon-limited cultivations using the same strain. FFA production under phosphate limitation also led to a cellular maintenance energy ∼400 % higher (0.28 g/gDCW/h) than that seen under carbon limitation.

Engineering Escherichia coli for production of C12-C14 polyhydroxyalkanoate from glucose.

Demand for sustainable materials motivates the development of microorganisms capable of synthesizing products from renewable substrates. A challenge to commercial production of polyhydroxyalkanoates (PHA), microbially derived polyesters, is engineering metabolic pathways to produce a polymer with the desired monomer composition from an unrelated and renewable source. Here, we demonstrate a metabolic pathway for converting glucose into medium-chain-length (mcl)-PHA composed primarily of 3-hydroxydodecanoate monomers. This pathway combines fatty acid biosynthesis, an acyl-ACP thioesterase to generate desired C12 and C14 fatty acids, ß-oxidation for conversion of fatty acids to (R)-3-hydroxyacyl-CoAs, and a PHA polymerase. A key finding is that Escherichia coli expresses multiple copies of enzymes involved in ß-oxidation under aerobic conditions. To produce polyhydroxydodecanoate, an acyl-ACP thioesterase (BTE), an enoyl-CoA hydratase (phaJ3), and mcl-PHA polymerase (phaC2) were overexpressed in E. coli ΔfadRABIJ. Yields were improved through expression of an acyl-CoA synthetase resulting in production over 15% CDW--the highest reported production of mcl-PHA of a defined composition from an unrelated carbon source.

Kinetic modeling of free fatty acid production in Escherichia coli based on continuous cultivation of a plasmid free strain.

The microbial production of free fatty acids (FFAs) and reduced derivatives is an attractive process for the renewable production of diesel fuels. Toward this goal, a plasmid-free strain of Escherichia coli was engineered to produce FFAs by integrating three copies of a thioesterase gene from Umbellularia californica (BTE) under the control of an inducible promoter onto the chromosome. In batch culture, the resulting strain produced identical titers to a previously reported strain that expressed the thioesterase from a plasmid. The growth rate, glucose consumption rate, and FFA production rate of this strain were studied in continuous cultivation under carbon limitation. The highest yield of FFA on glucose was observed at a dilution rate of 0.05 h(-1) with the highest specific productivity observed at a dilution rate of 0.2 h(-1). The observed yields under the lowest dilution rate were 15% higher than that observed in batch cultures. An increase in both productivity and yield (≈ 40%) was observed when the composition of the nutrients was altered to shift the culture toward non-carbon limitation. A deterministic model of the production strain has been proposed and indicates that maintenance requirements for this strain are significantly higher than wild-type E. coli.

Isolation of improved free fatty acid overproducing strains of Escherichia coli via Nile red based high-throughput screening.

Biological production of hydrocarbons is an attractive strategy to produce drop-in replacement transportation fuels. Several methods for converting microbially-produced fatty acids into reduced compounds compatible with petrodiesel have been reported. For these processes to become economically viable, microorganisms must be engineered to approach the theoretical yield of fatty acid products from renewable feedstocks such as glucose. Strains with increased titers can be obtained through both rational and random approaches. While powerful, random approaches require a genetic selection or facile screen that is amenable to high throughput platforms. Here, we present the use of a high throughput screen for fatty acids based on the hydrophobic dye Nile red. The method was applied to screening a transposon library of a free fatty acid overproducing strain of Escherichia coli in search of high producing mutants. Ten gene targets were identified via primary and secondary screening. A strain comprising a clean knockout of one of the identified genes led to a 20% increase in titer over the baseline strain. A selection strategy that combines these findings and can act in an iterative fashion has been developed and can be used for future strain optimization in hydrocarbon producing strains.