Engineering of CYP153A33 With Enhanced Ratio of Hydroxylation to Overoxidation Activity in Whole-Cell Biotransformation of Medium-Chain 1-Alkanols

α,ω-Dodecanediol is a versatile material that has been widely used not only as an adhesive and crosslinking reagent, but also as a building block in the pharmaceutical and polymer industries. The biosynthesis of α,ω-dodecanediol from fatty derivatives, such as dodecane and dodecanol, requires an ω-specific hydroxylation step using monooxygenase enzymes. An issue with the whole-cell biotransformation of 1-dodecanol using cytochrome P450 monooxygenase (CYP) with ω-specific hydroxylation activity was the low conversion and production of the over-oxidized product of dodecanoic acid. In this study, CYP153A33 from Marinobacter aquaeolei was engineered to obtain higher ω-specific hydroxylation activity through site-directed mutagenesis. The target residue was mutated to increase flux toward α,ω-dodecanediol synthesis, while reducing the generation of the overoxidation product of dodecanoic acid and α,ω-dodecanedioic acid. Among the evaluated variants, CYP153A33 P136A showed a significant increase in 1-dodecanol conversion, i.e., 71.2% (7.12 mM from 10 mM 1-dodecanol), with an increased hydroxylation to over-oxidation activity ratio, i.e., 32.4. Finally, the applicability of this engineered enzyme for ω-specific hydroxylation against several 1-alkanols, i.e., from C6 to C16, was investigated and discussed based on the structure-activity relationship.

Dietary Amylose/Amylopectin Ratio Modulates Cecal Microbiota and Metabolites in Weaned Goats

Increasing the ratio of amylose in the diet can increase the quantity of starch that flows to the large intestine for microbial fermentation. This leads to the alteration of microbiota and metabolite of the hindgut, where the underlying mechanism is not clearly understood. The present study used a combination of 16S amplicon sequencing technology and metabolomics technique to reveal the effects of increasing ratios of amylose/amylopectin on cecal mucosa- and digesta-associated microbiota and their metabolites in young goats. Twenty-seven Xiangdong black female goats with average body weights (9.00 ± 1.12 kg) were used in this study. The goats were randomly allocated to one of the three diets containing starch with 0% amylose corn (T1), 50% high amylose corn (T2), and 100% high amylose corn (T3) for 35 days. Results showed that cecal valerate concentration was higher (P < 0.05) in the T2 group than those in the T1 and T3 groups. The levels of tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 were decreased (P < 0.05) in cecal tissue while IL-10 was increased (P < 0.05) in the T2 group when compared with T1 or T3 groups. At the phylum level, the proportion of mucosa-associated Spirochaetes was increased (P < 0.05), while Proteobacteria was deceased by feeding high amylose ratios (P < 0.05). The abundance of Verrucomicrobia was decreased (P < 0.05) in the T3 group compared with the T1 and T2 groups. The abundance of digesta-associated Firmicutes was increased (P < 0.05) while Verrucomicrobia and Tenericutes were deceased (P < 0.05) with the increment of amylose/amylopectin ratios. The LEfSe analysis showed that a diet with 50% high amylose enriched the abundance of beneficial bacteria such as Faecalibacterium and Lactobacillus in the digesta and Akkermansia in the mucosa compared with the T1 diet. The metabolomics results revealed that feeding a diet containing 50% high amylose decreased the concentration of fatty acyls-related metabolites, including dodecanedioic acid, heptadecanoic acid, and stearidonic acid ethyl ester compared with the T1 diet. The results suggested that a diet consisting of 50% high amylose could maintain a better cecal microbiota composition and host immune function.

Metabolic Outcomes of Anaplerotic Dodecanedioic Acid Supplementation in Very Long Chain Acyl-CoA Dehydrogenase (VLCAD) Deficient Fibroblasts

Very long-chain acyl-CoA dehydrogenase deficiency (VLCADD, OMIM 609575) is associated with energy deficiency and mitochondrial dysfunction and may lead to rhabdomyolysis and cardiomyopathy. Under physiological conditions, there is a fine balance between the utilization of different carbon nutrients to maintain the Krebs cycle. The maintenance of steady pools of Krebs cycle intermediates is critical formitochondrial energy homeostasis especially in high-energy demanding organs such as muscle and heart. Even-chain dicarboxylic acids are established as alternative energy carbon sources that replenish the Krebs cycle by bypassing a defective β-oxidation pathway. Despite this, even-chain dicarboxylic acids are eliminated in the urine of VLCAD-affected individuals. In this study, we explore dodecanedioic acid (C12; DODA) supplementation and investigate its metabolic effect on Krebs cycle intermediates, glucose uptake, and acylcarnitine profiles in VLCAD-deficient fibroblasts. Our findings indicate that DODA supplementation replenishes the Krebs cycle by increasing the succinate pool, attenuates glycolytic flux, and reduces levels of toxic very long-chain acylcarnitines.

Facile Synthesis of Thermoplastic Polyamide Elastomers Based on Amorphous Polyetheramine with Damping Performance

Novel thermoplastic polyamide elastomers (TPAEs) consisting of long-chain semicrystalline polyamide 1212 (PA1212) and amorphous polyetheramine were synthesized via one-pot melt polycondensation. The method provides accessible routes to prepare TPAEs with a high tolerance of compatibility between polyamide and polyether oligomers compared with the traditional two-step method. These TPAEs with 10 wt % to 76 wt % of soft content were obtained by reaction of dodecanedioic acid, 1,12-dodecanediamine, and poly(propylene glycol) (PPG) diamine. The structure–property relationships of TPAEs were systematically studied. The chemical structure and the morphologic analyses have revealed that microphase separation occurs in the amorphous region. The TPAEs that have long-chain PPG segments consist of a crystalline polyamide domain, amorphous polyamide-rich domain, and amorphous polyetheramine-rich domain, while the ones containing short-chain PPG segments comprise of a crystalline polyamide domain and miscible amorphous polyamide phase and amorphous polyetheramine phase due to the compatibility between short-chain polyetheramine and amorphous polyamide. These novel TPAEs show good damping performance at low temperature, especially the TPAEs that incorporated 76 wt % and 62 wt % of PPG diamine. The TPAEs exhibit high elastic properties and low residual strain at room temperature. They are lightweight with density between 1.01 and 1.03 g/cm3. The long-chain TPAEs have well-balanced properties of low density, high elastic return, and high shock-absorbing ability. This work provides a route to expand TPAEs to damping materials with special application for sports equipment used in extremely cold conditions such as ski boots.

Identification of effector metabolites using exometabolite profiling of diverse microalgae

Dissolved metabolites mediate algal interactions in aquatic ecosystems, but microalgal exometabolomes remain understudied. We conducted an untargeted metabolomic analysis of non-polar exometabolites exuded from four phylogenetically and ecologically diverse eukaryotic microalgal strains grown in the laboratory: freshwater Chlamydomonas reinhardtii, brackish Desmodesmus sp., marine Phaeodactylum tricornutum, and marine Microchloropsis salina, to identify released metabolites based on relative enrichment in the exometabolomes compared to cell pellet metabolomes. Exudates from the different taxa were distinct, but we did not observe clear phylogenetic patterns. We used feature based molecular networking to explore the identities of these metabolites, revealing several distinct di- and tripeptides secreted by each of the algae, lumichrome, a compound that is known to be involved in plant growth and bacterial quorum sensing, and novel prostaglandin-like compounds. We further investigated the impacts of exogenous additions of eight compounds selected based on exometabolome enrichment on algal growth. Of the these, five (lumichrome, 5’-S-methyl-5'-thioadenosine, 17-phenyl trinor prostaglandin A2, dodecanedioic acid, and aleuritic acid) impacted growth in at least one of the algal cultures. Two of these (dodecanedioic acid and aleuritic acid) produced contrasting results, increasing growth in some algae and decreasing growth in others. Together, our results reveal new groups of microalgal exometabolites, some of which could alter algal growth when provided exogenously, suggesting potential roles in allelopathy and algal interactions.

PGMD/curcumin nanoparticles for the treatment of breast cancer

The present study aims at developing PGMD (poly-glycerol-malic acid-dodecanedioic acid)/curcumin nanoparticles based formulation for anticancer activity against breast cancer cells. The nanoparticles were prepared using both the variants of PGMD polymer (PGMD 7:3 and PGMD 6:4) with curcumin (i.e. CUR NP 7:3 and CUR NP 6:4). The size of CUR NP 7:3 and CUR NP 6:4 were found to be ~ 110 and 218 nm with a polydispersity index of 0.174 and 0.36, respectively. Further, the zeta potential of the particles was − 18.9 and − 17.5 mV for CUR NP 7:3 and CUR NP 6:4, respectively. The entrapment efficiency of both the nanoparticles was in the range of 75–81%. In vitro anticancer activity and the scratch assay were conducted on breast cancer cell lines, MCF-7 and MDA-MB-231. The IC50 of the nanoformulations was observed to be 40.2 and 33.6 μM at 48 h for CUR NP 7:3 and CUR NP 6:4, respectively, in MCF-7 cell line; for MDA-MB-231 it was 43.4 and 30.5 μM. Acridine orange/EtBr and DAPI staining assays showed apoptotic features and nuclear anomalies in the treated cells. This was further confirmed by western blot analysis that showed overexpression of caspase 9 indicating curcumin role in apoptosis.

Whole-Cell Biotransformation of 1,12-Dodecanedioic Acid from Coconut Milk Factory Wastewater by Recombinant CYP52A17SS Expressing Saccharomyces cerevisiae

Biotransformation of fatty acids from renewable wastewater as feedstock to value-added chemicals is a fascinating commercial opportunity. α,ω-Dicarboxylic acids (DCAs) are building blocks in many industries, such as polymers, cosmetic intermediates, and pharmaceuticals, and can be obtained by chemical synthesis under extreme conditions. However, biological synthesis can replace the traditional chemical synthesis using cytochrome P450 enzymes to oxidize fatty acids to DCAs. Saccharomyces cerevisiae BY(2R)/pYeDP60-CYP52A17SS (BCM), a transgenic strain expressing the galactose-inducible CYP52A17SS cytochrome P450 enzyme, was able to grow in a coconut milk factory wastewater (CCW) medium and produced 12-hydroxydodecanoic acid (HDDA) and 1,12-dodecanedioic acid (DDA). The supplementation of CCW with 10 g/L yeast extract and 20 g/L peptone (YPCCW) markedly increased the yeast growth rate and the yields of 12-HDDA and 1,12-DDA, with the highest levels of approximately 60 and 38 µg/L, respectively, obtained at 30 °C and pH 5. The incubation temperature and medium pH strongly influenced the yeast growth and 1,12-DDA yield, with the highest 1,12-DDA formation at 30 °C and pH 5–5.5. Hence, the S. cerevisiae BCM strain can potentially be used for producing value-added products from CCW.