Acetogenic bacteria produce acetate following the fixation of CO2 via the Wood-Ljungdahl pathway. As such, they represent excellent process organisms for the production of novel chemicals and fuels from this waste greenhouse gas. Acetobacterium woodii is the model acetogen and numerous studies have been conducted investigating its biochemistry, gas consumption and use as a production chassis. However, there are a dearth of available tools for A. woodii gene modification which limits the research options available for genetic studies. Here, the previously proposed Clostridia Roadmap is implemented in A. woodii leading to the derivation of a knockout system for the generation of clean, in-frame deletions. The replicon of the Gram-positive plasmid pCD6 that originated in Clostridioides difficile was identified as being replication-defective in A. woodii, a property that was exploited to construct a pseudo-suicide knockout plasmid which was used to generate an auxotrophic, pyrE mutant. This allowed the subsequent use of a heterologous pyrE gene (from Clostridium acetobutylicum) as a counter selection marker and the deletion of a number of genes by allelic exchange. Specific mutants generated were affected in growth on glucose, fructose and ethanol as a consequence of deletion of fruA, pstG and adhE, respectively.

The design of mesoporous silica nanoparticles (MSNs) for drug delivery is attracting increasing interest. Controlled release of their cargo is usually mediated by diffusion and erosion mechanisms, which might not reach the expected therapeutic effects. Here, we report the development and characterization of MSNs which modulate the cargo release in different cell models: fibroblasts and embryonic stem cells. Based on a double strategy: the presence of multimodal pore channels and a chitosan coating, we demonstrated a modulated release. Our results show that MSNs could be used for controlled drug delivery in different cell types, showing the potential of improving pluripotent stem cells differentiation and reprogramming protocols with promising applications in biomedicine.

ATP plays an essential role in the substrate/product transmembrane transportation during whole-cell bioconversion. This study aimed to address the impact of ATP upon cadaverine synthesis by whole-cell biocatalysts. The results showed no significant change in the ATP content (P = 0.625), and the specific cadaverine yield (P = 0.374) was observed in enzyme-catalyzed cadaverine synthesis with exogenous addition of ATP, indicating that the enzyme-catalyzed process does not require the participation of ATP. Furthermore, a whole-cell biocatalyst co-overexpressed methionine adenosyltransferase (MetK), lysine decarboxylase (CadA), and lysine/cadaverine antiporter (CadB) was constructed and used to investigate the effect of ATP deficiency on the cadaverine production by conversion of L-methionine and L-lysine, simultaneously. The results showed no significant difference (P = 0.585) in the specific cadaverine content between high and low levels of intracellular ATP. In addition, the intra- and extracellular cadaverine concentration and the ratio of ATP/ADP of whole-cell biocatalyst were determined. Results showed that the extracellular cadaverine concentration was much higher than the intracellular concentration, and no significant changes in ATP/ADP ratio during cadaverine synthesis. In contrast, an inhibition effect of the proton motive force (PMF) inhibitor carbonyl cyanide m-chlorophenylhydrazone (CCCP) on cadaverine production was detected. These findings strongly suggest that cadaverine transport in whole-cell biocatalysts was energized by PMF rather than ATP. Finally, a model was proposed to describe cadaverine's PMF-driven transport under different external pHs during whole-cell biocatalysis. This study is the first to experimentally confirm that the cadaverine production by Escherichia coli whole-cell bioconversion is independent of intracellular ATP, which helps guide the subsequent construction of biocatalysts and optimize transformation conditions.

Cytochrome P450 enzyme CYP68J5 from filamentous fungus Aspergillus ochraceus is industrially used for selective C11α-hydroxylation of canrenone and progesterone. To improve its selectivity of C11α-hydroxylation for relevant steroid substrates, a sequence-based targeted mutagenesis combined with saturation mutagenesis was conducted to search for variants with improved hydroxylation reaction specificity toward progesterone and D-ethylgonendione. Recombinant yeast expressing triple mutant V64F/E65G/N66T showed significantly increased C11α-hydroxylation selectivity (85 % VS WT 69.7 %). Saturation mutagenesis of V64, E65 and N66 resulted in the identification of single mutant V64K with greatly enhanced 11α-hydroxylation specificity toward progesterone (90.6 % VS WT 69.7 %). Furthermore, mutant N66D showed significant enhanced selectivity of C11α-hydroxylation toward D-ethylgonendione (70.8 % VS WT 58 %). Evaluation of recombinant yeast over-expressing V64K for progesterone transformation in 50 mL scale resulted in product 11α-OH progesterone concentrations of 432.5 mg/L, a 30.2 % increase compared with the CYP68J5 control. Our results also reveal that V64, E65 and N66 are key residues of CYP68J5 influencing its selectivity of C11α-hydroxylation, thus offering opportunities for further engineering of CYP68J5 for expanded industrial applications.

Adhatoda vasica is used in the treatment of cold, cough, chronic bronchitis, asthma, diarrhea, and dysentery. The biological activities of this species are attributed with the presence of alkaloids, triterpenoids, and flavonoids. Agrobacterium rhizogenes-mediated transformation of A. vasica, produces pyrroloquinazoline alkaloids, was achieved by infecting leaf discs with strain ATCC15834. The bacterial strain infected 82.7% leaf discs and 5-7 hairy root initials were developed from the cut edges of leaf discs. In this study, seven strains of Azotobacter chroococcum and five strains of Pseudomonas putida were used for the biotization of hairy roots. Plant growth-promoting rhizobacteria (PGPR) develops symbiotic association with roots of plants and increases the growth parameters of plants. PGPR (A. chroococcum and P. putida) increased the profiles of nitrogenase and acid phosphatase enzymes, biomass, dry matter contents, anthranilate synthase activity and accumulation of pyrroloquizoline alkaloids in the biotized hairy roots. Both enzymes (nitrogenase and acid phosphatase) maintain sufficient supply of nitrogen and dissolved phosphorus to the cells of hairy roots therefore, the levels of anthranilate synthase activity and pyrroloquinazoline alkaloids are increased. Total seven pyrroloquinazoline alkaloids (vasicine, vasicinone, vasicine acetate, 2-acetyl benzyl amine, vasicinolone, deoxyvasicine and vasicol) were identified from the biotized hairy roots of A. vasica. In our study, biotization increased the profiles of pyrroloquinazoline alkaloids therefore, this strategy may be used in increasing the production of medicinally important secondary metabolites in other plant species also. Our hypothetical model demonstrates that P. putida cell surface receptors receive root exudates by attaching on hairy roots. After attachment, the bacterial strain penetrates in the biotized hairy roots. This endophytic interaction stimulates acid phosphatase activity in the cells of biotized hairy roots. The P. putida plasmid gene (ppp1) expression led to the synthesis of acid phosphatase in cytosol. The enzyme enhances phosphorus availability as well as induces the formation of phosphoribosyl diphosphate. Later, phosphoribosyl diphosphate metabolizes to tryptophan and finally tryptophan converts to anthranilic acid. The synthesized anthranilic acid used in the synthesis of alkaloids in A. vasica.

Dilute acid hydrolysis of lignocellulose biomass had been used for production of cellulosic ethanol since 1940 s. The major technical barrier is the acid catalyzed dehydration of monosaccharides to furan aldehydes (furfural and 5-hydroxymethylfurfural), resulting in the high loss of fermentable sugars and significant inhibition on the fermentability of ethanologenic strains. This study re-examined the dilute acid hydrolysis of corn stover and cellulosic ethanol fermentation after a novel biodetoxification approach was introduced to de-bottleneck the inhibitor barrier. The cocktail of sulfuric acid, phosphoric acid and oxalic acid hydrolyzed corn stover to the 51.1 g/L of glucose (0.50 g/g cellulose) and 18.1 g/L of xylose (0.22 g/g xylan). The furfural, 5-hydroxymethylfurfural and acetic acid in the corn stover hydrolysate were completely removed by Paecilomyces variotii FN89, leading to the successful ethanol fermentation of 24.2 g/L, corresponding to 72.6 kg per metric ton of dry corn stover. No wastewater streams, solid wastes and toxic compounds were generated in hydrolysis, biodetoxification and fermentation. The techno-economic evaluations suggest that the cost reduction of replacing cellulase enzyme with cheap acid catalysts compensated the partial ethanol loss of sugar conversion to inhibitors (21.5-89.1%). The re-examination of acid hydrolysis process reveals that a substantial breakthrough in highly active and selective acid catalyst is required for acid hydrolysis to compete with enzymic hydrolysis for cellulosic ethanol fermentation.

Sericin, a silk-derived non-immunogenic protein, has been used to improve cell culture performance by increasing viability, cell concentration, and promoting adherence of several cell lines. Here, we hypothesized that the properties of sericin can enhance the amplification of flaviviruses in cell cultures. The propagation of flavivirus is inefficient and limits scientific research. Zika virus (ZIKV) is an important human pathogen that has been widely studied because of its high impact on public health. There is a need to amplify Zika virus both for research and vaccine development. In this work, we show that sericin improves ZIKV amplification in insect (C6/36) and mammalian (Vero) cell cultures, and that it has a cryoprotectant capacity. Supplementation of cell culture media with sericin at 80 µg/mL resulted in a significant increase of 1 log in the concentration of ZIKV infectious particles produced from both cell lines. Furthermore, final virus yields increased between 5 and 10-fold in Vero cells and between 7 and 23-fold in C6/36 cells when sericin was supplemented, compared to control conditions. These results show that sericin is an effective supplement to increase ZIKV production by Vero and C6/36 cells. Additionally, sericin was a suitable cryoprotective agent, and hence an alternative to FBS and DMSO, for the cryopreservation of C6/36 cells but not for Vero cells.