Dye-sensitized solar cells have been of great interest in photovoltaic technology due to their capacity to convert energy at a low cost. The use of natural pigments means replacing expensive chemical synthesis processes by easily extractable pigments that are non-toxic and environmentally friendly. Although most of the pigments used for this purpose are obtained from higher plants, there are potential alternative sources that have been underexploited and have shown encouraging results, since pigments can also be obtained from organisms like bacteria, cyanobacteria, microalgae, yeast, and molds, which have the potential of being cultivated in bioreactors or optimized by biotechnological processes. The aforementioned organisms are sources of diverse sensitizers like photosynthetic pigments, accessory pigments, and secondary metabolites such as chlorophylls, bacteriochlorophylls, carotenoids, and phycobiliproteins. Moreover, retinal proteins, photosystems, and reaction centers from these organisms can also act as sensitizers. In this review, the use of natural sensitizers extracted from algae, cyanobacteria, bacteria, archaea, and fungi is assessed. The reported photoconversion efficiencies vary from 0.001 % to 4.6 % for sensitizers extracted from algae and microalgae, 0.004 to 1.67 % for bacterial sensitizers, 0.07-0.23 % for cyanobacteria, 0.09 to 0.049 % for archaea and 0.26-2.3 % for pigments from fungi.

To explore the suitability of Corynebacterium glutamicum as a chassis for diacetyl production from glucose, diacetyl metabolic pathway and the respiratory chain were linked to achieve redox balance. The carbon flux was redirected from pyruvate to diacetyl by overexpressing the α-acetolactate synthase, in combination with disruption the biosynthetic pathways of lactate, acetoin, 2,3-butanediol and acetate in C. glutamicum ATCC 13032. These modifications resulted in a sharp increase of the NADH/NAD+ ratio from 0.53 to 1.10, and produced 0.58 g/L diacetyl under aerobic conditions, representing a 58-fold increase over the wild type. Although the modification of the by-product pathways is an effective strategy, these disruption led to intracellular cofactor imbalance. NADH re-oxidization was further successfully solved by overexpressing of cytochrome bd oxidase. We constructed an efficient respiration-dependent cell factory by modification of the respiratory chain, improving diacetyl titer to 1.29 g/L in CGC11, decreased NADH/NAD+ ratio to 0.45, increased the ATP concentration from 8.51 to 10.64 μM/gDCW. To our best knowledge, this is the first report of diacetyl synthesis in C. glutamicum. Intracellular cofactor imbalance can be reduced by modification of the respiratory chain for production of diacetyl as well as other bio-based products with cofactor imbalance in C. glutamicum.

We evaluated several intein-based self-cleaving affinity tags for expression and single-step affinity chromatography purification of recombinant proteins produced in Escherichia coli. We used human growth hormone (hGH) as target protein that contains two internal disulfide bridges and an N-terminal phenylalanine. Use of N-terminal thiol-induced Sce VMA1 intein affinity tag resulted in purified hGH deficient in disulfide bonds. Inteins with self-cleavage inducible by pH and/or temperature shift were analyzed. N-terminal Ssp DnaX intein affinity tag resulted in a completely cleaved cytosolic protein, whereas N-terminal Ssp DnaB intein affinity tag resulted in a cytosolic fusion protein incapable of releasing hGH. Periplasmic expression of target protein was analyzed using an N-terminal signal peptide and C-terminal Ssp DnaX pH-inducible self-cleaving affinity tag. The fusion protein was properly expressed in pH 8 buffered culture medium. Fusion of a periplasmic signal peptide to the N-terminus of the POI allowed secretion to the periplasmic region and presence of the natural N-terminal amino acid of the POI following cleavage. Periplasmic expression of hGH fused to this novel C-terminal DnaX intein-based self-cleaving affinity tag made possible expression and purification of hGH protein containing disulfide bonds and free of extra amino acids.

Plastic waste can serve as a feedstock for microbial bioconversion using a chemical/biological hybrid strategy. We developed a polypropylene (PP) upcycling process that coupled pyrolysis with bioconversion by the oleaginous yeast Yarrowia lipolytica. Using virgin PP, we optimized pH, inoculum density, C/N ratio, and osmolarity and increased the fatty acid titer nearly four-fold to 1.9 g L-1, with 41 percent cellular fatty acid content, the highest content reported to date for plastic-to-lipid microbial bioconversion. The highest fatty acid titer was achieved with an inoculum density of 3 (OD 600 nm), pH = 6.0 and C/N ratio of 80:1. Increasing the medium osmolarity by adding sodium chloride adversely affected cell growth and did not improve the fatty acid titer. The maximum fatty acid titer occurred under conditions that balanced cell growth versus lipogenesis. Using postconsumer PP, the fatty acid titer was significantly lower (0.13 g L-1). Overall, the work demonstrates the potential and the challenges associated with microbial bioconversion of plastics.

Glucaric acid has been successfully produced in Escherichia coli and fungus. Here, we first analyzed the effects of different metal ions on glucaric acid production in the engineered Saccharomyces cerevisiae Bga-3 strain harboring the glucaric acid synthesis pathway. We found that magnesium ions could promote the growth rate of yeast cells, and thus, increase the glucaric acid production by elevating the glucose and myo-inositol utilization of Bga-3 strain. RNA-Seq transcriptome analysis results showed that the upregulation of genes involved in the gluconeogenesis pathway, as well as the downregulation of genes associated with the glycolysis pathway and pentose phosphate pathway in response to MgCl2 were all benefit for the enhancement of the glucose-6-phosphate flux, which was the precursor for myo-inositol and glucaric acid. In addition, we found that MgCl2 could also increase the activity of MIOX4, which was also crucial for glucaric acid synthesis. At last, a final glucaric acid titer of 10.6 g/L, the highest reported titer, was achieved in the fed-batch fermentation using a 5-L bioreactor by adding 100 mM MgCl2. Our findings will provide a new way of promoting the production of other chemicals in the engineered yeast cells.

Protein kinases are involved in the transfer of phosphate group to serine, threonine, and tyrosine residues of a target protein. With No Lysine (WNK) kinase is a member of the serine/threonine protein kinase family, which has conserved catalytic lysine (K) residue in subdomain I instead of being in subdomain II.The WNKs family members in plants are stress inducible and have been validated for their role in abiotic stress tolerance. In the present study, we have characterized Arabidopsis overexpressed lines of OsWNK9 regulated by the constitutive promoter under arsenite stress. Moreover, we have performed In silico expression analysis of OsWNK9 under nutrient deficiency and heavy metal stress. Three independent transgenic Arabidopsis (OsWNK9-OX T11, T12,andT13) lines showed tolerance to arsenite stress compared to wild-type (WT) plants. Under arsenite stress, transgenic lines T11, T12 and T13 showed 56.46, 57.8 and 51.66 % increased biomass respectively, as compared to WT plants. All three ArabidopsisOsWNK9-OX lines exhibited higher proline content, increased antioxidant enzyme activities and lower hydrogen peroxide levels under arsenite stress. Besides, the total antioxidant capacity in terms of DPPH (2, 2-diphenyl-1-picrylhydrazyl) free radical scavenging percentage was increased by 8-15 % in three independent OsWNK9-OX lines compared with those of WT plants. Protein-protein interaction analysis of OsWNK9 predicted interaction partners with protein kinase and oxidative stress-responsive protein. Co-expression analysis of OsWNK9 in phosphate deficiency and arsenate stress condition predicted various proteins including membrane transporter and transcription factors. Taken together, our results, for the first time, provide evidence that OsWNK9 could positively mediate arsenite stress tolerance in plants.

Real-time quantitative polymerase chain reaction (RT-qPCR) is the most common approach to quantify changes in gene expression. Appropriate internal reference genes are essential for normalization of data of RT-qPCR. In the present study, we identified suitable reference genes for analysis of gene expression in rice seedlings subjected to different heavy metal stresses such as deficiencies of iron and zinc and toxicities of cobalt, cadmium and nickel. First, from publically available RNA-Seq data we identified 10 candidate genes having stable expression. We also included commonly used house-keeping gene OsUBQ5 (Ubiquitin 5) in our analysis. Expression stability of all the 11 genes was determined by two independent tools, NormFinder and geNorm. Our results show that selected candidate reference genes have higher stability in their expression compared to that of OsUBQ5. Genes with locus ID LOC_Os03g16690, encoding an oxysterol-binding protein (OsOBP) and LOC_Os01g56580, encoding Casein Kinase_1a.3 (OsCK1a.3) were identified to be the most stably expressed reference genes under most of the conditions tested. Finally, the study reveals that it is better to use a specific reference gene for a specific heavy metal stress condition rather than using a common reference gene for multiple heavy metal stress conditions. The reference genes identified here would be very useful for gene expression studies under heavy metal stresses in rice.

Automation, parallelization and autonomous operation of standard lab equipment, usually applied for manual bioprocess development, is considered as the key for reduction of bioprocess development time and costs. An automated bioreactor system with 4 stirred-tank bioreactors on a L-scale was combined with a custom-made biomass transfer system to distribute the cell suspensions produced on the L-scale into 48 parallel stirred-tank bioreactors on a mL-scale. Afterwards parallel protein expression studies automated by a liquid handling system with integrated fluorescence reader were performed. Isopropyl β-D-1-thiogalactopyranoside-induced (IPTG) expression of the red fluorescence protein mCherry was studied as an example of using fed-batch processes with recombinant Escherichia coli. In a first automated study, IPTG concentrations were varied in 48 parallel fed-batch processes with E. coli cells produced at a growth rate of 0.1 h-1 on an L-scale and transferred automatically to the mL-scale. The mCherry expression rate increased with increasing inducer concentration until the highest protein expression rate was observed at > 9 μM IPTG. In a second automated study, the growth rate of E. coli was varied between 0.1-0.2 h-1 in parallelly-operated stirred-tank bioreactors on a L-scale. The cells were automatically transferred and distributed into the stirred-tank bioreactors on a mL-scale and the concentration of the inducer IPTG was varied as before in parallel fed-batch processes. An increased growth rate during the production of the recombinant E. coli cells and/or higher cell densities during protein expression resulted in the increased IPTG concentrations necessary to achieve identical expression rates compared to a growth rate of 0.1 h-1 with the exception of very low inducer concentrations and inducer concentrations in excess. The new automated multi-scale cascade of parallel stirred-tank bioreactors should easily be applicable for performing fast optimisation studies with other microbial production systems and will have the potential to reduce bioprocess development time and staff assignment considerably.

Antibody-drug conjugates offers many advantages as a drug delivery platform that allows for highly specific targeting of cell types and genes. Ideally, testing the efficacy of these systems requires two cell types to be different only in the gene targeted by the drug, with the rest of the cellular machinery unchanged, in order to minimize other potential differences from obscuring the effects of the drug. In this study, we created multiple variants of U87MG cells with targeted mutation in the TP53 gene using the CRISPR-Cas9 system, and determined that their major transcriptional differences stem from the loss of p53 function. Using the transcriptome data, we predicted which mutant clones would have less divergent phenotypes from the wild type and thereby serve as the best candidates to be used as drug delivery testing platforms. Further in vitro and in vivo assays of cell morphology, proliferation rate and target antigen-mediated uptake supported our predictions. Based on the combined analysis results, we successfully selected the best qualifying mutant clone. This study serves as proof-of-principle of the approach and paves the way for extending to additional cell types and target genes.

Carotenoids and triacylglycerols from yeasts are important bioproducts that can be utilized for the nutraceutical and biodiesel industries respectively. Rhodotorula diobovata is capable of producing these bioproducts under varied culture conditions. These productions have been linked to the early stationary growth phase and their levels only start to decline at the late stationary phase when carbon becomes limiting. While nitrogen-limitation influences the onset of lipogenesis, continuous synthesis and accumulation of neutral lipids (triacylglycerides) may be dependent on other culture conditions such as aeration. Proteomic analyses were conducted to enhance our understanding of changes in gene product expression under culture conditions with nitrogen-limitation, coupled with insufficient aeration, and revealed a correlation between the upregulation of proteins in the lipolysis pathways and the reduced synthesis of fatty acids at the early stationary phase. Upregulation of glycolytic pathway enzymes suggested that glucose was quickly converted into pyruvate and then acetyl-CoA. However, acetyl-CoA flux favoured carotenoids biosynthesis over fatty acid synthesis, as cells transitioned into the stationary phase. This work provides insights into how culture conditions influence gene product expression levels, pathway utilization, and end-product synthesis patterns.

Nanocellulose has many promising applications such as a green ingredient for Pickering emulsion. Traditional strategies to produce nanocellulose, which are acid or enzymatic hydrolysis and mechanical methods on natural complicated cellulose, are hard to control and can result in significant pollutants during the processes. Herein, we demonstrated a facile and sustainable method for the biocatalytic production of insoluble synthetic crystalline cellulose nanoribbon (CCNR) from cheap maltodextrin by coupling α-glucan phosphorylase (αGP) and cellodextrin phosphorylase (CDP) using cellobiose as a primer. And by optimizing the combination of different αGP and CDP, it turned out that the optimal enzyme combination is αGP from Thermotoga maritime and CDP from Clostridium thermocellum, in which CDP was attached to a family 9 cellulose-binding module. The product yield and degree of polymerization (DP) of insoluble synthetic CCNR was affected by the primer concentration at a fixed concentration of maltodextrin. After optimization of reaction conditions, the highest product yield of insoluble synthetic CCNR was 44.92 % and the highest DP of the insoluble synthetic CCNR was 24 from 50 g 1-1 maltodextrin. This insoluble synthetic CCNR can be used as a Pickering emulsions stabilizer, showing excellent emulsifiability. This study provides a promising alternative for cost-efficient production of insoluble synthetic CCNR which was used as a green emulsion stabilizer.

A stirred tank bioreactor (STR) coupled with two column bioreactors (CRs) was used for ethanol production from sweet sorghum stem juice by Saccharomyces cerevisiae SSJ01KKU in a very high gravity fermentation. The effects of the medium circulation rate between the STR and CRs (2.6 and 5.2 mL/min, corresponding to 25 and 50 % of the S. cerevisiae specific growth rate), the starting time of medium circulation (0 and 4 h) and cell inoculation were investigated. The results showed that a medium circulation rate of 5.2 mL/min, starting the medium circulation at the beginning of fermentation (0 h) with cell inoculation into the STR only were appropriate conditions for ethanol production. This yielded an average ethanol concentration (PE) of 120.96 g/L and ethanol productivity (QP) of 2.52 g/L⋅h. When a repeated-batch (RB) ethanol fermentation in the STR coupled with CR was carried out using a drain and fill technique at different volumes (75 and 90 %, referenced as RB1 and RB2, respectively), it was found that at least eight successive cycles could be operated under both RB1 and RB2. The average PE and QP for RB1 and RB2 were not significantly different. However, the average total ethanol production rate in RB2 (3.25 g/h) over the eight cycles was significantly higher than that of RB1 (2.60 g/h).