Conversion of CO2 into organic acids by engineered autotrophic yeast

The increase of CO₂ emissions due to human activity is one of the preeminent reasons for the present climate crisis. In addition, considering the increasing demand for renewable resources, the upcycling of CO₂ as a feedstock gains an extensive importance to establish CO₂-neutral or CO₂-negative industrial processes independent of agricultural resources. Here we assess whether synthetic autotrophic Komagataella phaffii (Pichia pastoris) can be used as a platform for value-added chemicals using CO₂ as a feedstock by integrating the heterologous genes for lactic and itaconic acid synthesis. ¹³C labeling experiments proved that the resulting strains are able to produce organic acids via the assimilation of CO₂ as a sole carbon source. Further engineering attempts to prevent the lactic acid consumption increased the titers to 600 mg L⁻¹, while balancing the expression of key genes and modifying screening conditions led to 2 g L⁻¹ itaconic acid. Bioreactor cultivations suggest that a fine-tuning on CO₂ uptake and oxygen demand of the cells is essential to reach a higher productivity. We believe that through further metabolic and process engineering, the resulting engineered strain can become a promising host for the production of value-added bulk chemicals by microbial assimilation of CO₂, to support sustainability of industrial bioprocesses.

Between 2011 and 2020 the annual average CO₂ emission due to human activity exceeded 38 gigatons, of which around 22 gigatons are removed again from the atmosphere to terrestrial and ocean CO₂ sinks. This imbalance, mostly due to combustion of fossil fuels, causes the steady increase in CO₂ levels in the atmosphere which is one of the primary reasons for the climate crisis our planet is facing today (1).Biologically produced fuels and commodity chemicals bear the potential to counteract this deleterious development, but the most common feedstocks used for bioproduction, such as glucose, sucrose and starch rely on agricultural production and are bearing the risk to threaten food security. Using autotrophic microorganisms as production platforms exploits the potential of CO₂ itself as an alternative carbon source. In nature, autotrophic microorganisms play a major role in CO₂ fixation by fixing 200 gigatons of CO₂ every year (2). However, the rates of most natural microbial CO₂ fixing pathways are low: the photosynthetic efficiency in cyanobacteria is limited to 1 to 2% which makes industrial processes using photoautotrophs economically less feasible (3). Chemoautotrophs may overcome this barrier as their energy harvesting processes are more efficient than light harvesting of photoautotrophs.One well-known autotroph that is being developed for biological production of materials is Cupriavidus necator (formerly known as Ralstonia eutropha). By harvesting energy with a controlled Knallgas reaction these bacteria assimilate CO₂ via the Calvin-Benson-Bessham (CBB) cycle. Besides naturally produced polyhydroxyalkanoates (PHA), metabolic pathway engineering enabled the production of several other chemicals (4–7).Several chemolithotrophic bacteria were demonstrated as production hosts for various chemicals such as ethanol, 2,3-butanediol, butanol, isopropanol, acetone, or isobutyric acid via natural carbon assimilation pathways (8–11). In addition to natural CO₂ fixing microorganisms, implementation of heterologous CO₂ fixing pathways to heterotrophic microorganisms like Myceliophthora thermophila provided a mixotrophic strain that is able to produce malic acid with a higher yield compared to the parent strain in which only the reductive tricarboxylic acid (TCA) cycle is used for the production (12).Natural chemoautotrophs have a large potential to convert CO₂ to chemicals. However, they are often recalcitrant to genetic editing, have complex nutrient demands, or may require complex process technological solutions like the transfer of gaseous substrates and energy sources. To circumvent some of these limitations well established prokaryotic and eukaryotic production hosts have been engineered to assimilate CO₂. The bacterial workhorse Escherichia coli and the yeast Komagataella phaffii (Pichia pastoris) were provided with the CBB cycle, enabling them to assimilate CO₂ by using formate or methanol, respectively, as energy sources. Both engineered microorganisms can grow sustainably with CO₂ as carbon source (13, 14). Conceptually formate and methanol are regarded as sustainable feedstocks for biotechnology when they are derived from CO₂ by hydrogenation or electrochemical reduction (15).To make an impact on the global CO₂ household such autotrophic processes need to convert CO₂ into bulk products. Besides ethanol, short chain organic acids are the second largest group of chemicals manufactured by industrial biotechnology. The market of biologically produced organic acids is expected to reach more than $36 billion by 2026 (16). The annual bioproduction of some of the key organic acids (citric, acetic, lactic, succinic acid) is more than 12 million metric tons (17–20). Recently, itaconic acid has also gained attention as a promising chemical building block with an estimated market increase to 170 kilotons per year and $260 million in 2025 (21). Lactic and itaconic acid are feedstocks for polymer production so that they, and other biobased commodity chemicals compete for the annual polymer production of more than 300 million tons, a volume that denotes a significant impact on the global CO₂ balance.Here, we set out to evaluate if the autotrophic K. phaffii strain can be used as a platform for organic acid production. Synthetic autotrophy was introduced to K. phaffii by converting the native peroxisomal methanol assimilation pathway, the xylulose monophosphate (XuMP) cycle, into the CBB cycle (13). To achieve that, the formaldehyde assimilating enzyme dihydroxyacetone synthase (DAS) was replaced by a bacterial ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO), and phosphoribulose kinase (PRK) from spinach was added to supply ribulose bisphosphate from XuMP precursors. Four yeast glycolytic enzymes were targeted to peroxisomes to close the CBB cycle to glyceraldehyde 3-phosphate (G3P), both as an intermediate and the product of the assimilation cycle (Fig. 1 A and B). In the present work, using modular synthetic biology tools, we implemented the genes for lactic and itaconic acid synthesis plus accessory genes into the K. phaffii genome and demonstrate that the autotrophic K. phaffii strain is capable of producing organic acids solely from CO₂ as carbon source.Open in a separate windowFig. 1.Expression of cadA and ldhL enables organic acid production in synthetic autotrophic K. phaffii. (A–D) Schematic pathways. (A) In wild-type K. phaffii methanol is oxidized to formaldehyde (black arrow) and assimilated in the XuMP cycle (orange arrows) or dissimilated to CO₂, respectively (purple arrow). (B) synthetic autotrophy in K. phaffii: the native assimilatory branch of methanol utilization was interrupted by deleting DAS1 and DAS2 (dashed gray line). AOX1 was knocked out to reduce the rate of formaldehyde formation which could be toxic to the cells. RuBisCO and PRK were integrated to complete a functional CBB cycle (green arrows). Additionally, two bacterial chaperones, groEL and groES, were overexpressed to assist the folding of RuBisCO. TDH3, PGK1, TKL1, TPI1 carrying each a peroxisomal targeting signal were overexpressed to assure the localization of the entire CBB cycle in peroxisomes. More details about the engineering strategy can be found in ref. (13). (C) Itaconic acid (red) and (D) lactic acid production (blue), (E) growth profiles, and (F) organic acid production profiles of the producing strains and the control. Time axis corresponds to the production phase under autotrophic conditions. At least three biological replicates were used in the screening to monitor the producing strains. Shades represent the SDs (±). 3PG: 3-phosphoglycerate, AcCoA: acetyl-coenzyme A, AOX1 and AOX2: alcohol oxidase 1 and 2, cadA: cis-aconitate decarboxylase, CBB cycle: Calvin-Benson-Bassham cycle, CISA_c: cytosolic cis-aconitate, CISA_m: mitochondrial cis-aconitate, DAS1 and DAS2: dihydroxyacetone synthase 1 and 2, DHA: dihydroxyacetone, FAL: formaldehyde, G3P: glyceraldehyde 3-phosphate, ITA: itaconic acid, LA: lactic acid, ldhL: L-lactate dehydrogenase, MeOH: methanol, mttA: mitochondrial tricarboxylic acid transporter, NAD⁺/NADH: nicotinamide adenine dinucleotide, PRK: phosphoribulokinase, PYR: pyruvate, RuBP: ribulose 1,5-bisphosphate, RuBisCO: ribulose 1,5-bisphosphate carboxylase/oxygenase, Xu5P: xylulose 5-phosphate, XuMP cycle: xylulose monophosphate cycle.     

Genotype phenotype correlation in a pediatric population with antithrombin deficiency

Inherited antithrombin (AT) deficiency is a rare autosomal dominant disorder, caused by mutations in the AT gene (SERPINC1). Considering that the genotype phenotype relationship in AT deficiency patients remains unclear, especially in pediatric patients, the aim of our study was to evaluate genotype phenotype correlation in a Serbian pediatric population. A retrospective cohort study included 19 children younger than 18 years, from 15 Serbian families, with newly diagnosed AT deficiency. In 21% of the recruited families, mutations affecting exon 4, 5, and 6 of the SERPINC1 gene that causes type I AT deficiency were detected. In the remaining families, the mutation in exon 2 causing type II HBS (AT Budapest 3) was found. Thrombosis events were observed in 1 (33%) of those with type I, 11 (85%) of those with AT Budapest 3 in the homozygous respectively, and 1(33%) in the heterozygous form. Recurrent thrombosis was observed only in AT Budapest 3 in the homozygous form, in 27% during initial treatment of the first thrombotic event. Abdominal venous thrombosis and arterial ischemic stroke, observed in almost half of the children from the group with AT Budapest 3 in the homozygous form, were unprovoked in all cases.

Conclusion: Type II HBS (AT Budapest 3) in the homozygous form is a strong risk factor for arterial and venous thrombosis in pediatric patients.

Alcohol's role in the deaths of BC children and youth

OBJECTIVE: To determine the prevalence and context of alcohol use in the deaths of children and youth reviewed by the BC Children's Commission. METHODS: In 489 case reviews of BC children and youth, we examined the role that alcohol may have had at the time of death or whether there was a history of alcohol use either by the deceased child or another person in the child's life. RESULTS: Alcohol is most prevalent in the lives of 15-18 year olds. It is present at the time of death in two fifths of Motor Vehicle Incidents (MVI) and one third of suicides and drownings. INTERPRETATION: Alcohol has a profound presence in the lives and deaths of children reviewed by the Children's Commission. Enhancing deterrence laws and alcohol control policies, and increasing public awareness are warranted.     

Transmembrane proteins in the tight junction barrier.

Three types of transmembrane proteins have been identified within the tight junction, but it remains to be determined how they provide the molecular basis for regulating the paracellular permeability for water, solutes, and immune cells. Several of these proteins localize specifically within the continuous cell-to-cell contacts of the tight junction. One of these, occludin, is a cell adhesion molecule that has been demonstrated to influence ion and solute permeability. The claudins are a family of four-membrane spanning proteins; unexpectedly, other members of this family have already been characterized without recognizing their relationship to tight junctions. Junction adhesion molecule, the most recently identified tight junction component, is a member of the Ig superfamily and influences the paracellular transmigration of immune cells. A plaque of cytoplasmic proteins under the junction may be responsible for scaffolding the transmembrane proteins, creating a link to the perijunctional actin cytoskeleton and transducing regulatory signals that control the paracellular barrier.     

The oncology treatment of patients who use oral anticoagulants is connected with high risk of bleeding complications

The data about risk for bleeding complications during anticoagulation in cancer patients with different oncology treatment are conflicting. To investigate the rate of bleeding in the course of oral anticoagulants, during treatment of malignant diseases, we conducted a retrospective study including 75 patients on stable anticoagulation prior to commencing their different oncology treatment. All patients were treated according to the consiliar decision, made based on the localization and pathohistological findings of the malignant disease. During their treatment the regular laboratory monitoring of INR was done. Every dose of oral anticoagulants, INR changes, as well as the size and localization of bleeding were recorded. During all the malignancy treatment 22 (30%) of patients were overanticoagulated. In 15 (20%) patients it was associated with bleeding, while 3 (4%) of them had to be transfused with fresh frozen plasma to stop the major bleeding. Most bleeding complications occurred in the group of patients treated with chemotherapy or with analgesics in the group with advanced disease. None of the bleeding complications were observed in patients treated with irradiation and surgery alone, where the bridging of oral anticoagulants with low molecular weight heparin was done before surgery. The oncology treatment of patients who take oral anticoagulants was connected with high risk for bleeding especially if chemotherapy as a therapeutic options was used. Therefore physicians should be aware of this risk and carefully monitor the intensity of anticoagulant therapy, especially during the first treatment weeks when the risk of bleeding is greatest.     

Heterogeneity in expression and subcellular localization of claudins 2, 3, 4, and 5 in the rat liver, pancreas, and gut

BACKGROUND & AIMS: Paracellular transport varies widely among epithelia of the gastrointestinal tract. We determined whether members of the claudin family of tight junction proteins are differentially expressed consistent with a potential role in creating these variable properties. METHODS: Rabbit polyclonal antibodies were produced against peptides from claudins 2 through 5. The distribution of individual claudins was detected by immunoblotting, and their cell type and subcellular localization were determined by immunofluorescence on cryosections of rat liver, pancreas, stomach, and small and large intestine. RESULTS: All antibodies detected single bands of the expected size on immunoblots and were monospecific based on peptide competition studies. Immunoblotting detected strong differences among tissues in the expression level of each claudin. Immunolocalization confirmed these differences and revealed striking variations in expression patterns. In the liver, claudin 2 shows a lobular gradient increasing from periportal to pericentral hepatocytes, claudin 3 is uniformly expressed, claudin 4 is absent, and claudin 5 is only expressed in endothelial junctions. In the pancreas, claudin 2 is only detected in junctions of the duct epithelia, claudin 5 only in junctions of acinar cells, whereas claudin 3 and 4 are in both. Among differences in the gut are a crypt-to-villus decrease in claudin 2, a highly restricted expression of claudin 4 to colonic surface cells, and the finding that some claudins can be junctional, lateral, or show a gradient in junctional vs. lateral localization along the crypt-to-villus surface axis. CONCLUSIONS: Claudins have very different expression patterns among and within gastrointestinal tissues. We propose these patterns underlie differences in paracellular permeability properties, such as electrical resistance and ion selectivity that would complement known differences in transcellular transport.     

Miconazole and nystatin used as topical antifungal drugs interact equally strongly with warfarin

What is known and Objective: Medline search disclosed 10 case reports of interactions between oral anticoagulants and miconazole oral gel, but none so far between nystatin solution and anticoagulants. We report on change in anticoagulant activity with use of different topical antifungal drugs, miconazole oral gel and vaginal suppositories, and nystatin solution. Methods: We conducted a retrospective study that included 43 patients on stable anticoagulation before the introduction of topical antifungal drugs. Miconazole oral gel was prescribed for 32 patients, nystatin solution for eight patients and miconazole vaginal suppositories for three patients. Results and Discussion: Nineteen (44·2%) of the patients reported bleeding complications and some of these were severe. Fifteen of 32 who used miconazole oral gel and four of 8 of those who used nystatin solution were affected. Before use of the antifungal drugs, the mean weekly warfarin dose in the nystatin group was 14·5 mg, and after antifungal drugs, 9 mg, P = 0·038, while the mean international normalized ratio (INR) before antifungal drugs was 2·5 (range 1·9–3·5) and afterwards it was 10·6 (range 4·5–19·3), P = 0·0001. In the miconazole oral gel group the mean weekly warfarin dose was 15·7 mg, and after 10·8 mg, P = 0·008, while the mean INR before antifungal drugs was 2·44 (range 1·92–3·18) and afterwards it was 8·8 (range 4·9–16·9), P < 0·0001. What is new and Conclusion: Miconazole oral gel and topically applied nystatin solution have equally strong effects on warfarin activity and can provoke major bleeding. Prospective evaluation of this effect is called for. However, based on our results the warfarin dose adjustment appears necessary when the anticoagulant is used concomitantly with those topical antifungals.