Tailored polyhydroxyalkanoate production from renewable non-fatty acid carbon sources using engineered Cupriavidus necator H16.

As thermoplastic, nontoxic, and biocompatible polyesters, polyhydroxyalkanoates (PHAs) are considered promising biodegradable plastic candidates for diverse applications. Short-chain-length/medium-chain-length (SCL/MCL) PHA copolymers are flexible and versatile PHAs that are typically produced from fatty acids, which are expensive and toxic. Therefore, to achieve the sustainable biosynthesis of SCL/MCL-PHAs from renewable non-fatty acid carbon sources (e.g., sugar or CO2), we used the lithoautotrophic bacterium Cupriavidus necator H16 as a microbial platform. Specifically, we synthesized tailored PHA copolymers with varying MCL-3-hydroxyalkanoate (3HA) compositions (10-70 mol%) from fructose by rewiring the MCL-3HA biosynthetic pathways, including (i) the thioesterase-mediated free fatty acid biosynthetic pathway coupled with the beta-oxidation cycle and (ii) the hydroxyacyl transferase-mediated fatty acid de novo biosynthetic pathway. In addition to sugar-based feedstocks, engineered strains are also promising platforms for the lithoautotrophic production of SCL/MCL-PHAs from CO2. The set of engineered C. necator strains developed in this study provides greater opportunities to produce customized polymers with controllable monomer compositions from renewable resources.

Enhanced production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) with modulated 3-hydroxyvalerate fraction by overexpressing acetolactate synthase in Cupriavidus necator H16.

The elastomeric properties of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), a biodegradable copolymer, strongly depend on the molar composition of 3-hydroxyvalerate (3HV). This paper reports an improved artificial pathway for enhancing the 3HV component during PHBV biosynthesis from a structurally unrelated carbon source by Cupriavidus necator H16. To increase the intracellular accumulation of propionyl-CoA, a key precursor of the 3HV monomer, we developed a recombinant strain by genetically manipulating the branched-chain amino acid (e.g., valine, isoleucine) pathways. Overexpression of the heterologous feedback-resistant acetolactate synthase (alsS), (R)-citramalate synthase (leuA), homologous 3-ketothiolase (bktB), and the deletion of 2-methylcitrate synthase (prpC) resulted in biosynthesis of 42.5 % (g PHBV/g dry cell weight) PHBV with 64.9 mol% 3HV monomer from fructose as the sole carbon source. This recombinant strain also accumulated the highest PHBV content of 54.5 % dry cell weight (DCW) with 24 mol% 3HV monomer from CO2 ever reported. The lithoautotrophic cell growth and PHBV production by the recombinant C. necator were promoted by oxygen stress. The thermal properties of PHBV showed a decreasing trend of the glass transition and melting temperatures with increasing 3HV fraction. The average molecular weights of PHBV with modulated 3HV fractions were between 20 and 26 × 104 g/mol.

Assessing the cytotoxicity of aerosolized carbon black and benzo[a]pyrene with controlled physical and chemical properties on human lung epithelial cells.

Atmospheric particulate matter (PM) is a complex mixture of hazardous particles containing hundreds of inorganic and organic species. Organic components, such as carbon black (CB) and benzo[a]pyrene (BaP), are known to exhibit diverse genotoxic and carcinogenic effects. The toxicity of CB and polycyclic aromatic hydrocarbons has been well studied, however the combined toxicity is much less understood. A spray-drying system was used to control the size and chemical composition of PMs. PMs were prepared by loading BaP on three different sized CBs (0.1 µm, 2.5 µm, and 10 µm) to obtain BaP-unloaded CB (CB0.1, CB2.5, and CB10) and BaP-loaded CB (CB0.1-BaP, CB2.5-BaP, and CB10-BaP). We analyzed cell viability, levels of oxidative stress, and pro-inflammatory cytokines using human lung cells (A549 epithelial cells). Cell viability decreased when exposed to all PMs (PM0.1, PM2.5, and PM10), regardless of the presence of BaP. The increase in PM size due to BaP-adsorption to CB resulted in insufficient toxic effects on human lung cells compared to CB alone. Smaller CBs reduced cell viability, leading to reactive oxygen species formation, which can cause damage to cellular structures deliver more harmful substances. Additionally, small CBs were predominant in inducing the expression of pro-inflammatory cytokines in A549 epithelial cells. These results indicate that the size of CB is a key factor that immediately affects the inflammation of lung cells, compared to the presence of BaP.

The effect of PhIP precursors on the generation of particulate matter in cooking oil fumes at high cooking temperatures and the inflammation response in human lung cells.

Cooking Oil Fumes (COFs) contain carcinogenic organic substances such as polycyclic aromatic hydrocarbons (PAHs) and heterocyclic amines (HCAs), of which 2-Amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP) is known as mainly meat-borne carcinogens. In this work, to identify the mechanisms to induce the inflammation response in human lung cells (A549) exposed to COFs, we investigated the physicochemical and biological characteristics of COFs generated with PhIP precursors (L-phenylalanine, creatinine, and glucose) at high cooking temperatures (300 °C and 600 °C). Interestingly, we found that PhIP was not formed both at 300 °C and 600 °C, while a large number of carbon nanoparticles were generated from soybean oil containing the PhIP precursors at 600 °C. From the biological analysis, COFs generated with the PhIP precursors at 600 °C induced the most significant pro-inflammatory cytokine (IL-6). This result indicates that the particulate matter in COFs generated with the PhIP precursors above the smoke temperature is the primary factor directly affecting the lung inflammatory response rather than PhIP. This study demonstrates for the first time a novel principle of the inflammatory response that the PhIP precursors can aggravate lung injury by affecting the physical properties of COFs depending on cooking temperature. Therefore, our finding is a significant result of overcoming the bias in previous studies focusing only on the chemical toxicity of PhIP in the inflammatory response of COFs.