Microbial tolerance to lignocellulose-derived inhibitors, such as aromatic acids, is critical for the economical production of biofuels and biochemicals. Here, adaptive laboratory evolution was applied to improve the tolerance of Yarrowia lipolytica to a representative aromatic acid inhibitor vanillic acid. The transcriptome profiling of evolved strain suggested that the tolerance could be related to the up-regulation of RNA processing and multidrug transporting pathways. Further analysis by reverse engineering confirmed that the amplification of YALI0_F13475g coding for transcriptional coactivator and YALI0_E25201g coding for multidrug transporter conferred tolerance not only to vanillic acid but also towards ferulic acid, p-coumaric acid, p-hydroxybenzoic acid and syringic acid. These findings suggested that regulation of RNA processing and multidrug transporting pathways may be important for enhanced aromatic acid tolerance in Y. lipolytica. This study provides valuable genetic information for robust strain construction for lignocellulosic biorefinery.

Aldoxime dehydratases (Oxds) are a unique class of enzymes, which catalyzes the dehydration of aldoximes to nitriles in an aqueous environment. Recently, they gained attention as a catalyst for a green and cyanide-free alternative to established nitrile syntheses, which often require the use of toxic cyanides and harsh reaction conditions. Up to now only thirteen aldoxime dehydratases have been discovered and biochemically characterized. This raised the interest for identifying further Oxds with, e.g., complementary properties in terms of substrate scope. In this study, 16 novel genes, presumably encoding aldoxime dehydratases, were selected by using a commercially available 3DM database based on OxdB, an Oxd from Bacillus sp. OxB-1. Out of 16 proteins, six enzymes with aldoxime dehydratases activity were identified, which differ in their substrate scope and activity. While some novel Oxds showed better performance for aliphatic substrate such as n-octanaloxime compared to the well characterized OxdRE from Rhodococcus sp. N-771, some showed activity for aromatic aldoximes, leading to an overall high usability of these enzymes in organic chemistry. The applicability for organic synthesis was underlined by converting 100 mM n-octanaloxime at a 10 mL scale within 5 h with the novel aldoxime dehydratase OxdHR as whole-cell catalyst (33 mgbww/mL).

d-(-)-Pantolactone (DPL) is a key intermediate for the production of d-(+)-pantothenate (vitamin B5). Deracemization of d,l-pantolactone (D,L-PL) through oxidizing l-(+)-pantolactone (LPL) to ketopantoyl lactone (KPL) and subsequently reducing KPL to DPL is a promising route for synthesizing DPL. Herein, a newly mined l-pantolactone dehydrogenase from Rhodococcus hoagie (RhoLPLDH) was used for the oxidative dehydrogenation of LPL. To alleviate inclusion bodies formed by membrane-bound RhoLPLDH intracellular expression in E. coli, strategies involving chaperone assistance and decreasing induction temperature were used to achieve RhoLPLDH soluble expression. To enhance its activity, directed evolution and hydrophilicity-based engineering yielded increased catalytic activity and thermostability. 1 M LPL was efficiently converted to KPL by engineering strain CM5 co-expressing RhoLPLDHL254I/V241I/I156L/F224Q/N164K and chaperone. A "two stages in one-pot" method was employed in deracemization of 1 M D,L-PL with 91.2% yield. These results demonstrated that CM5 catalyst exhibits great potential in enzyme cascade deracemization for the production of DPL.

Isobutanol is a potential biofuel, and its microbial production systems have demonstrated promising results. In a microbial system, the isobutanol produced is secreted into the media; however, the cells remaining after fermentation cannot be used efficiently during the isobutanol recovery process and are discarded as waste. To address this, we aimed to investigate the strategy of utilizing these remaining cells by combining the isobutanol production system with the indigo production system, wherein the product accumulates intracellularly. Accordingly, we constructed E. coli systems with genes, such as acetolactate synthase gene (alsS), ketol-acid reductoisomerase gene (ilvC), dihydroxyl-acid dehydratase (ilvD), and alpha-ketoisovalerate decarboxylase gene (kivD), for isobutanol production and genes, such as tryptophanase gene (tnaA) and flavin-containing monooxygenase gene (FMO), for indigo production. This system produced isobutanol and indigo simultaneously while accumulating indigo within cells. The production of isobutanol and indigo exhibited a strong linear correlation up to 72 h of production time; however, the pattern of isobutanol and indigo production varied. To our knowledge, this study is the first to simultaneously produce isobutanol and indigo and can potentially enhance the economy of biochemical production.

Microalgae is a potential source of bioproducts, including feedstock to biofuels. Urea has been pointed as potential N source for microalgae growth. Considering that urea metabolism releases HCO3- to the medium, we tested the hypothesis that this carbon source could improve photosynthesis and consequently growth rates of Chlamydomonas reinhardtii. In this sense, the metabolic responses of C. reinhardtii grown with ammonium and urea as nitrogen sources under mixotrophic and autotrophic conditions were investigated. Overall, the mixotrophy led to increased cell growth as well as to a higher accumulation of lipids independent of N source, followed by a decrease in photosynthesis over the growth phases. In mixotrophy, urea stimulates growth in terms of cell number and dry weight. Furthermore, higher photosynthesis was verified in late logarithmic phase compared to ammonium. Under autotrophy conditions, although cell number and biomass were reduced, there was higher production of starch independent of N source. Nonetheless, urea-based autotrophic treatments stimulated biomass production compared to ammonium-based treatment. Under mixotrophy higher input of carbon into the cell from acetate and urea optimized photosynthesis and consequently promoted cell growth. Together, these results suggest urea as alternative source of carbon, improving photosynthesis and cell growth in C. reinhardtii.

Sortase, a bacterial transpeptidase enzyme, is an attractive tool for protein engineering due to its ability to break a peptide bond at a specific site and then reform a new bond with an incoming nucleophile. Here, we present the immobilization of two recombinant proteins, enhanced green fluorescent protein (eGFP) and xylose dehydrogenase (XylB) over triglycine functionalized PEGylated gold nanoparticles (AuNPs) using C. glutamicum sortase E. For the first time, we used a new class of sortase from a non-pathogenic organism for sortagging. The site-specific conjugation of proteins with LAHTG-tagged sequences on AuNPs via covalent cross-linking was successfully detected by surface-enhanced Raman scattering (SERS) and UV-vis spectral analysis. The sortagging was initially validated by an eGFP model protein and later with the xylose dehydrogenase enzyme. The catalytic activity, stability, and reusability of the immobilized XylB were studied with the bioconversion of xylose to xylonic acid. When compared to the free enzyme, the immobilized XylB was able to retain 80% of its initial activity after four sequential cycles and exhibited no significant variations in instability after each cycle for about 72 h. These findings suggest that C. glutamicum sortase could be useful for immobilizing site-specific proteins/enzymes in biotransformation applications for value-added chemical production.

Adeno-associated virus (AAV) based vectors have recently been gaining importance as DNA delivery systems. Efficient downstream processing of AAV remains a major challenge as serotypes differ in physicochemical properties, making it difficult to design uniform purification processes. Clarification of AAV is an especially critical step. Harvesting of AAV, like other viruses, often requires cell lysis, resulting in a difficult-to-filter cell lysate. In this study, we evaluated the applicability of diatomaceous earth (DE) as a filter aid for clarification of AAV crude cell lysates. DE filtration proved to be a viable clarification method for AAV2, AAV5 and AAV8. Based on a design of experiment approach, the DE concentration was identified as the main factor influencing AAV particle loss. The loss of AAV during DE filtration was limited to < 2% by maintaining the DE quantity below 0.181 mg DE/1010 AAV. Use of DE reduced manual handling time 3-fold and increased the filter capacity 3.5-fold compared to filtration combined with a prior centrifugation step. Moreover, we showed that the DE type had only a minor influence on the filtration performance. This study demonstrated that filtration with DE as a filter aid is an efficient clarification method for different AAV serotypes.

In this work, a magnetic nanobiocomposite scaffold based on carboxymethylcellulose (CMC) hydrogel, silk fibroin (SF), and magnetite nanoparticles was fabricated. The structural properties of this new magnetic nanobiocomposite were characterized by various analyses such as FT-IR, XRD, EDX, FE-SEM, TGA and VSM. According to the particle size histogram, most of the particles were between 55 and 77 nm and the value of saturation magnetization of this nanobiocomposite was reported 41.65 emu.g- 1. Hemolysis and MTT tests showed that the designed magnetic nanobiocomposite was compatible with the blood. In addition, the viability percentage of HEK293T normal cells did not change significantly, and the proliferation rate of BT549 cancer cells decreased in its vicinity. EC50 values for HEK293T normal cells after 48 h and 72 h were 3958 and 2566, respectively. Also, these values for BT549 cancer cells after 48 h and 72 h were 0.4545 and 0.9967, respectively. The efficiency of fabricated magnetic nanobiocomposite was appraised in a magnetic fluid hyperthermia manner. The specific absorption rate (SAR) of 69 W/g (for the 1 mg/mL sample at 200 kHz) was measured under the alternating magnetic field (AMF).

CuO Nanoparticles (CuO NPs) retard the plant growth but at appropriate concentration boosts shoot growth and therefore may function as nano-carrier or nano-fertilizer. To overcome the toxic effects, NPs can be capped with plant growth regulators. In this work, CuO-NPs (30 nm) were synthesized as the carrier and capped with indole-3-acetic acid (IAA) to generate CuO-IAA NPs (30.4 nm) as toxicity mitigant molecules. Seedlings of dicots, Lactuca sativa L. (Lettuce) were exposed to 5, 10 mg Kg-1/ of NPs in the soil to analyze shoot length, fresh and dry weight of shoots, phytochemicals, and antioxidant response. Toxicity to shoot length was recorded at higher concentrations of CuO-NPs, however, a reduction in toxicity was observed for CuO-IAA nanocomposite. Concentration-dependent decrease in the biomass of plants was also observed at higher concentrations of CuO-NPs (10 mg/kg). The antioxidative phytochemicals (phenolics and flavonoids) and antioxidative response increased in plants when exposed to CuO-NPs. However, the presence of CuO-IAA NPs combats the toxic response and a significant decrease in non-enzymatic antioxidants and total antioxidative response and total reducing power potential was observed. The results demonstrate that CuO-NPs can be used as a carrier of hormones for the enhancement of plant biomass and IAA on the surface of NPs reduces the toxic effects on NPs.

Raceways are widely used as microalgae culture systems due to their low cost, but they are not the best option for biomass yield. Understanding in situ photosynthetic performance can be a first step to increase their biomass productivity. This study aimed at comparing the real time photosynthetic activity in a greenhouse raceway culture (250 L) with discrete measurements under laboratory conditions. We evaluated the photophysiology and biochemical composition of Chlorella fusca culture up to 120 h. In situ photosynthetic activity was continuously monitored and compared to discrete ex situ measurements; biochemical compounds were measured daily. The results showed a final biomass density of 0.45 g L-1 (5 days - 120 h) and an increase of the electron transport rate (ETR) up to 48 h but decreased thereafter. When the relative ETR was estimated considering the absorption coefficient (a) positive correlations of this parameter with photosynthetic capacity, cell density, biomass, biocompounds and antioxidant activity were obtained, whereas no correlation was detected without considering a. In situ photosynthesis monitoring showed higher absolute maximal ETR (10 - 160 μmol m-3s-1) than discrete ex situ measurements. We demonstrated the importance of considering the light absorption coefficient for expressing photosynthetic capacity and showed that C. fusca can produce, in the short-term, bioactive compounds that are correlated to photosynthetic conditions.

This study investigates the production of the enzyme cocktail by the isolated fungi Aspergillus flavus B2 (GenBank accession number OL655454) using agricultural and industrial (AI) residues as the sole substrate. Of all the AI residues tested, Jew's mallow stalk was the best inducer substrate for enzyme cocktail production without adding any nutrients. Statistical optimization using Response Surface Methodology enhanced the production by 5.45, 5.20, and 3.34-fold, respectively for pectinase, xylanase, and CMCase. Optimum temperature, activation energy (Ea), and activation energy for denaturation (Ed) were determined. Michaelis constant (Km) for CMCase, xylanase, and pectinase enzyme was 1.82, 1.23, and 1.05 mg/mL, respectively. Maximum reaction rate (Vmax) was 4.67, 5.29, and 17.13 U/mL, respectively for CMCase, xylanase, and pectinase. Thermal stability revealed that pectinase, CMCase, and xylanase enzymes retained 64.7%, 61.8%, and 53.2% residual activities after incubation for 1 h at 50 °C. Half-life time (t0.5) of pectinase, CMCase, and xylanase at 50 °C were 189.38, 129.8, and 127.89 min, respectively. Thermodynamics of the produced enzymes enthalpy (ΔH*d), free energy (ΔG*d), and entropy (ΔS*d) were determined at 40, 50, and 60 °C. In the presence of EDTA (5 mM), CMCase, xylanase, and pectinase retained 69.5%, 66.2%, and 41.2%, respectively of their activity. This work is significant for the valorization of AI residues and the production of value-added products.