Modification of structural components, especially the cell wall, through adequate pretreatment strategies is critical to the bioconversion efficiency of algal biomass to biorefinery products. Over the years, several physical, physicochemical, chemical and green pretreatment methods have been developed to achieve maximum productivity of desirable by-products to sustain a circular bioeconomy. The effectiveness of the pretreatment methods is however, species specific due to diversity in the innate nature of the microalgal cell wall. This review provides a comprehensive overview of the most notable and promising pretreatment strategies for several microalgae species. Methods including the application of stress, ultrasound, electromagnetic fields, pressure, heat as well as chemical solvents (ionic liquids, supercritical fluids, deep eutectic solvents etc.) have been detailed and analyzed. Enzyme and hydrolytic microorganism based green pretreatment methods have also been reviewed. Metabolic engineering of microorganisms for product specificity and lower inhibitors can be a future breakthrough in microalgal pretreatment.

An increase in fish consumption, combined with a decrease in wild fish harvest, is driving the aquaculture industry at rapid pace. Today, farmed seafood accounts for about half of all global seafood demand for human consumption. As the aquaculture industry continues to grow, so does the market for aquafeed. Currently, some of the feed ingredients are coming from low-value forage fishes (fish meal) and terrestrial plants. The production of fish meal can't be increased as it would affect the sustainability and ecosystem of the ocean. Similarly, increasing the production of terrestrial plant-based feed leads to deforestation and increased freshwater use. Hence, alternative and environmentally sustainable sources of feed ingredients need to be developed. Microalgae biomasses represent potential feed source ingredients as the cell metabolites of these microorganisms contain a blend of essential amino acids, healthy triglycerides as fat, vitamins, and pigments. In addition to serving as bulk ingredient in aquafeed, their unique array of bioactive compounds can increase the survivability of farmed species, improve coloration and quality of fillet. Microalgae has the highest areal biomass productivities among photosynthetic organisms, including fodder crops, and thus has a high commercial potential. Also, microalgal production has a low water and arable-land footprint, making microalgal-based feed environmentally sustainable. This review paper will explore the potential of producing microalgae biomass as an ingredient of aquaculture feed.

Phycobiliproteins are colored, active molecules with potential use in different industries. They are the union of proteins and bilins (Chromophores). The primary source of phycobiliproteins is algae; however, the traditional algae culture has production restrictions. The production in bacterial models can be a more efficient alternative to produce these molecules. However, the lack of knowledge in some steps of the phycobiliprotein metabolic pathway limits this alternative. Porphyridium cruentum is a single cell red alga with a high phycobiliprotein content. Its protein sequences were the basis for phycobilin production in this study. In this study, we cloned and characterized enzymes presumably involved in the chromophore production of P. cruentum. Using sequences obtained from its transcriptome, we characterized two cDNA sequences predicted to code respectively for a ferredoxin-dependent bilin reductase and a bilin lyase-isomerase. We expressed these enzymes in Escherichia coli to obtain in vivo evidence of their enzymatic activity on the substrate biliverdin IXα. Lastly, we analyzed them using thin-layer chromatography, spectrophotometry, and fluorescence spectroscopy. These experiments provided evidence of bilin modification. The expressed bilin lyase-isomerase did not show significant activity over the biliverdin molecule. On the contrary, the expressed ferredoxin-dependent bilin reductase showed activity over the biliverdin.

Collaborative research is common practice in modern life sciences. For most projects several researchers from multiple universities collaborate on a specific topic. Frequently, these research projects produce a wealth of data that requires central and secure storage, which should also allow for easy sharing among project participants. Only under best circumstances, this comes with minimal technical overhead for the researchers. Moreover, the need for data to be analyzed in a reproducible way often poses a challenge for researchers without a data science background and thus represents an overly time-consuming process. Here, we report on the integration of CyVerse Austria (CAT), a new cyberinfrastructure for a local community of life science researchers, and provide two examples how it can be used to facilitate FAIR data management and reproducible analytics for teaching and research. In particular, we describe in detail how CAT can be used (i) as a teaching platform with a defined software environment and data management/sharing possibilities, and (ii) to build a data analysis pipeline using the Docker technology tailored to the needs and interests of the researcher.

Industrial production of neuroprotective drug CDP-choline is accomplished via permeabilized or lysed cell biotransformation because of the inefficient penetration of substrates into intact cells. We previously proposed a novel one-step living cell method for CDP-choline production by engineered yeast, but obtained low titer and molar yield. This study develops a high-production strain with improved molar yield by metabolic engineering strategies. The selective markers previously integrated into host cell were recovered for facilitating genetic modification, which however resulted a strain with improved CDP-choline titer and molar yield to CMP. Knockout of 5'-NT or CDA in CMP sinking pathway but not APY in CTP sinking pathway further improved CDP-choline titer and molar yield to CMP. However, overexpression of seven enzymes in CTP synthetic pathway showed no positive functions. Finally, optimization of CMP and choline phosphate levels for the optimized recombinant strains achieved a high-level CDP-choline of ~30 g/L, which was enhanced by 400% compared to the previous work. Also, the molar yield of CDP-choline to CMP increased from 40% to 84.7%. The titer and molar yield are comparable to the reported permeabilized or lysed cell based biotransformation methods. It represents a novel and competitive paradigm for the potential industrial production of CDP-choline.

The chaperone network plays an essential role in cellular protein homeostasis. However, some core components often coaggregate with misfolded proteins for sequestration and dysfunction, leading to abnormal cell proteostasis, aggregation-associated disorders, and poor solubility of overexpressed recombinant proteins. Among them, DnaJ or its ortholog, an obligate co-chaperone in the tripartite DnaK-DnaJ-GrpE system, is of more implications, probably due to its intrinsic propensity for aggregation. Herein, we potentiated the activity of Escherichia coli DnaJ by using hyper-acidified protein fusion strategy. We found DnaJ did possess only a moderate solubility that could be remarkably improved by fusing hyper-acidic minipeptides. Most importantly, we revealed the hyper-acidified DnaJ with a fusion tail could outperform its native form (significantly up to 2.1-fold) to enhance the solubility of target proteins and meanwhile appropriately impart them an elevated activity. These results suggest the hyper-acidified DnaJs can chaperone target proteins with correct folding into a truly soluble and active form. Moreover, we showed these hyper-acidified DnaJ variants could surpass its prototype to confer E. coli or yeast an enhanced heat tolerance, and DnaJ itself could be solubilized by its hyper-acidified fusion cognates. Finally, we discussed the overall mechanism for DnaJ activity potentiation mediated by hyper-acidic tailing fusion.

The environmental impact arising from poly(ethylene terephthalate) (PET) waste is notable worldwide. Enzymatic PET hydrolysis can provide chemicals that serve as intermediates for value-added product synthesis and savings in the resources. In the present work, some reaction parameters were evaluated on the hydrolysis of post-consumer PET (PC-PET) using a cutinase from Humicola insolens (HiC). The increase in PC-PET specific area leads to an 8.5-fold increase of the initial enzymatic hydrolysis rate (from 0.2 to 1.7 mmol L-1 h-1), showing that this parameter plays a crucial role in PET hydrolysis reaction. The effect of HiC concentration was investigated, and the enzymatic PC-PET hydrolysis kinetic parameters were estimated based on three different mathematical models describing heterogeneous biocatalysis. The model that best fits the experimental data (R2 = 0.981) indicated 1.68 mgprotein mL-1 as a maximum value of the enzyme concentration to optimize the reaction rate. The HiC thermal stability was evaluated, considering that it is a key parameter for its efficient use in PET degradation. The enzyme half-life was shown to be 110 h at 70 ºC and pH 7.0, which outperforms most of the known enzymes displaying PET hydrolysis activity. The results evidence that HiC is a very promising biocatalyst for efficient PET depolymerization.

Hyaluronic acid from fermentation (bio-HA) is a high-added-value biopolymer with a wide range of applications in aesthetic and medical areas. Bio-HA is a capsular exopolysaccharide highly hydrophilic with a coiled structure and high negative charge density at physiological pH. Centrifugation, currently used to separate microbial cells after fermentation, should significantly affect the bio-HA interactions with the medium components. Although the literature shows various centrifugation conditions for bio-HA, there is no concern about its recovery and characterization. We examined how centrifugation influences its recovery and characterized interactions with soy peptone (SP), used as a nitrogen source. We collected experimental data according to a statistical central composite design (CCD) planning with replicates at the central point with time (8-22 min) and spin (582xg to 12,402xg) as independent variables. The obtained results show bio-HA recoveries ranged from 45 ± 0.5-100% as dosed in the supernatant. The incorporation of amino acids and peptides from SP increased HA coils' density, causing losses due to sedimentation at higher spins and times. Furthermore, SP incorporation also changed the coils' size regarding HA standards of molar mass 106,105 and 104 Da. We conclude centrifugation should significantly impact downstream processing and the total cost of bio-HA production.

Epoxide hydrolases are ubiquitous in nature and are utilized to catalyze the cofactor-independent hydrolysis of epoxides to their corresponding diols. These enzymes have tremendous potential and have been applied in the synthesis of bulk and fine chemical industry and utilized as chiral building blocks. Herein, we report a green, facile, and economical method for immobilization of epoxide hydrolase based on biomimetic mineralization. The organic-inorganic hybrid nanoflowers have received tremendous attention due to their higher catalytic activity and stability. The nanoflowers were synthesized, with the organic component being enzyme epoxide hydrolase and the inorganic component being Ca2+ ions. A unique hierarchical flower-like spherical structure with hundreds of spiked petals was observed. The synthesized nanoflowers were applied for styrene oxide hydrolysis, producing 1-phenyl-1,2-ethanediol. Further, the factors influencing the morphology, catalytic activity, and stability studies were performed to study the activity recovery of the synthesized organic-inorganic hybrid epoxide hydrolase nanoflowers. The findings will have interesting applications.

Despite the advantages of CRISPR/Cas9 technology in the food industry, controversy over its off-target effects exists. We engineered an industrial Saccharomyces cerevisiae strain isolated from a Korean rice wine starter, Nuruk, using CRISPR/Cas9 to decrease ethyl carbamate (EC) formation. We disrupted the CAR1 gene encoding arginase, which plays a key role in EC formation. Subsequently, we compared the whole genome of the engineered strain to that of the wild type by analyzing heterozygous and homozygous mutations through variant calling. Homozygous mutations in the genome of the engineered strains were identified as the target mutations in CAR1 induced by CRISPR/Cas9, and no other off-target effects were observed. Our findings have critical implications for the use of CRISRP/Cas9 technology in yeasts in the food industry.

Integrative non-viral vectors such as transposons engineered to mediate targeted gene transfer into safe harbor sites in the genome may be a promising approach for the production of therapeutic proteins or for gene therapy in an efficient and secure way. In this context, we designed and evaluated two strategies for targeting the nuclear ribosomal DNA (rDNA) loci. One approach relied on the co-location of the transposase and transposon near transcriptionally active rDNA copies using a nucleolar localization signal (NoLS). Another one consisted of targeting the 18S-coding region in the rDNA loci using a NoLS-FokI-dCas9 endonuclease to perform targeted transgene knock-in. We show that integration into the rDNA of Chinese hamster ovary (CHO) cells can be achieved at a high frequency using the piggyBac transposon system, indicating that the rDNA is highly accessible for transposition. Consistently, rDNA-targeted transposition events were most frequently obtained when both the piggyBac transposon DNA and the transposase were nucleoli-targeted, yielding cells displaying stable and homogeneous expression of the transgene. This approach thus provides an alternative strategy to improve targeted transgene delivery and protein expression using CHO cells.

Hydrogels offer a promising potential for the encapsulation and regulated release of drugs due to their biocompatibility and their tunable properties as materials. Only a limited number of systems and procedures enable the encapsulation of sensitive proteins. N-terminally fmoc-protected phenylalanine has been shown to self-assemble into a transparent, stable hydrogel It can be considered a supergelator due to the low amount of monomers necessary for hydrogelation (0.1% w/v), making it a good candidate for the encapsulation and stabilization of sensitive proteins. However, application options for this hydrogel are rather limited to those of many other fibril-based materials due to its intrinsic lack of mechanical strength and high susceptibility to changes in environmental conditions. Here, we demonstrate that the stability of a fibrillary system and the resulting release of the protein-photosensitizer Azulitox can be increased by combining the hydrogel with a tightly cross-linked BSA hydrogel. Azulitox is known to display cell-penetrating properties, anti-proliferative activity and has a distinctive fluorescence. Confocal microscopy and fluorescence measurements verified the maintenance of all essential functions of the encapsulated protein. In contrast, the combination of fibrillary and protein hydrogel resulted in a significant stabilization of the matrix and an adjustable release pattern for encapsulated protein.

Extractive fermentation has been proven to be efficient in enhancing the secretion and production of secondary metabolites in submerged fermentation by Monascus spp., owing to increased cell membrane permeability and resolved product inhibition. In this study, we investigated the regulation effect of Triton X-100 on cell growth and secondary metabolite biosynthesis in submerged fermentation of M. purpureus DK. The results show that the maximum monascus pigments (MPs), citrinin (CIT) production, and specific growth rate are 136.86 U/mL, 4.57 mg/L, and 0.04 h-1, respectively, when 3 g/L of Triton X-100 is supplemented after fermentation for 10 d, and the extracellular MPs and CIT increased by 127.48% and 288.57%, respectively. RT-qPCR shows that the expression levels of MPs and CIT biosynthesis gene clusters are significantly upregulated, whereas those of glycolysis, tricarboxylic acid cycle, respiratory chains, and ATP synthase are downregulated. This study provides a vital strategy for extractive fermentation under extreme environmental conditions for further enhancing MP production.

Rubia yunnanensis is an important medicinal plant with various bioactive secondary metabolites. In order to reduce the dependence on wild populations of the species, we aim to establish in vitro culture system that can produce Rubiaceae-type cyclopeptides (RAs) and quinones. Agrobacterium rhizogenes-mediated transformation of stem segments of in vitro grown R. yunnanensis plants using four A. rhizogenes strains was studied and transformation conditions were optimized. Hairy roots appeared with the highest frequency (68.89%) when stem segments (with leaves) without pre-culture were immersed in A. rhizogenes A4 strain bacterial suspension for 30 min, co-cultured on Murashige and Skoog (MS) solid medium in the dark for three days, and afterwards incubated in darkness. PCR analysis of rolB and rolC genes confirmed transformed nature of six hairy root clones. The hairy roots grew rapidly, especially showing the highest accumulation of biomass in MS liquid medium compared to in vitro grown plants and calli. Histological observation of hairy root revealed anatomical difference in vascular cylinder, where the cells exhibited high mitotic activity characterized by vigorous growth. The UPLC-MS/MS analysis revealed that the amount of RAs in the hairy roots grown in ½MS liquid medium (4.611 μg g-1 DW) was higher than that in in vitro grown plants (0.331 and 4.096 μg g-1 DW for shoots and roots respectively) and calli (1.082 μg g-1 DW), but still far lower than that in the roots of seed-borne plants (80.296 μg g-1 DW). However, the hairy roots accumulated high level of quinones (2320.923 and 5067.801 μg g-1 DW for MS and ½MS liquid media respectively), of the same order of magnitude as the roots of seed-borne plants (7409.973 μg g-1 DW). Hairy root culture of R. yunnanensis, with high accumulation of biomass and production of quinones, may offer an attractive perspective for the production of the RAs and quinones that could be further optimized for pharmaceutical use.

Haematococcus pluvialis is the prime source of natural astaxanthin for commercial exploitation. The large-scale cultivation of H. pluvialis is one of the key technologies for the development of natural astaxanthin industry. So far, horizontal tubular photobioreactor (HTPBR) circulated by a centrifugal pump has been the main PBR for the large-scale cultivation of H. pluvialis. Shear stress is a negative factor in microalgal cultivation at different scales, particularly for large-scale cultivation. To reduce the adverse impact of shear stress, the tolerance of H. pluvialis to the shear stress during the induction stage was first investigated in this study. H. pluvialis aplanospore was not sensitive to stresses between 19.18 and 27.32 Pa, but was resulted in about 30% cell death under shear stress between 27.32 and 63.84 Pa. Accordingly, two centrifugal pumps with different impellers was selected in 400 L HTPBRs to study the outdoor photoinduction for astaxanthin accumulation. The highest astaxanthin productivity and astaxanthin concentration were obtained in HTPBRs using a centrifugal pump equipped with three unshrouded backward-bladed impellers. The HTPBR was then successfully scaled up to 800 L with a similar performance, showing good scalability.

Bio-desilication of fly ash containing a large amount of crystalline and amorphous silicate compound by silicate bacteria had the advantages of energy-saving, environment-friendly, and controllable cost. In this study, a bacteria strain with desilication ability on fly ash was isolated from the soil and identified as Bacillus amyloliquefaciens by 16sRNA and named ZGW-12. Through the bio-leaching treatment of fly ash by ZGW-12, it was found that the concentration of Si was 306.26 mg/L and the concentration of Al3+ was 0.5 mg/L in the pulp. The mechanism of bio-leaching using ZGW-12 was investigated by XRD, HPLC, SEM, and FTIR. The organic acid and amino acid in the bacteria culture medium and the pulp were detected, and it was found that the content of the acidic metabolites in the pulp was much larger than that of the bacteria culture medium. The ore sample particles adsorbed a large number of bacteria cells and took place obvious corrosion. The surface of the ore sample undergone hydroxylation reaction, and the diffraction peak of the silicate crystal compound was unchanged. ZGW-12 exhibits good performance in retaining aluminum and extracting silicon aspect. AVAILABILITY OF DATA AND MATERIAL (DATA TRANSPARENCY): All data were true and valid.

Light management is important for improving algae cultivation, specifically by enhancing the productivity of biomass and valued bioproducts. In this study, we present evidence that alternating blue and red-orange light can improve the algal growth kinetics and lipid production in a photobioreactor. Blue (430-445, 460-470 nm) and red-orange light (580-660 nm) from a LED were set at the light saturation point (B: 65 μmol/m2s; RO: 155 μmol/m2s) and alternated for the cultivation of the green alga Chlamydomonas reinhardtii. Growth kinetics, lipid, carbohydrate, and protein content were measured as a function of alternating illumination time. Results reveal that the first illumination light and illumination time had a significant impact on the growth kinetics and nutrient composition. When the red-orange light illumination was used at the beginning of cultivation (RO/B alternation), the biomass concentration and productivity increased 8% and 18% on average, respectively; lipid mass fraction and concentration increased 21-27% and 24-26% when 0.25-0.50 h per day of blue light illumination was used; no significant change of carbohydrate and protein content were observed. Relative to blue light alone, the improvement of growth kinetics, lipid mass fraction and concentration, and the carbohydrate concentration was significant. Under B/RO alternation (when the blue light was used first), on average, the protein content was significantly higher than RO/B alternation.

The essential goals of this present study are to elucidate the formation mechanism of ellagic acid rich-blackberry, BBE, (Rubus fruticosus L.) and raspberry, RBE, (Rubus idaeus L.) extracts directed silver nanoparticles and to investigate thier antimicrobial properties towards model dental pathogens E. faecalis and C. albicans compared to BBE, RBE, NaOCl, CHX and EDTA. Both %5 w/w of BBE and RBE reacted with 5 mM Ag + ions at room temperature (25 °C) under mild-stirring, the formation of BBE and RBE directed b@Ag NP and r@Ag NP was monitored over time by using an Uv-vis spectrophotometer. Both b@Ag and r@Ag NPs were also complementarily characterized with SEM and FT-IR. In terms of the antimicrobial studies, b@Ag NP, r@Ag NP, %5 BBE and RBE, 5 mM AgNO3, %5 NaOCl, %1,5 CHX and %15 EDTA were separately incubated with E. faecalis and C. albicans suspensions. The results were evaluated with student t-test using GraphPad Prism 8.0.1 statistical software (P < 0.05). While formation of b@Ag NP was confirmed with characteristic absorbance at ~435 nm in 20 min (min) of incubation, r@Ag NP did not give absorbance till 80 min owing to concentration of ellagic acid acted as a reducing and stabilizng agent for formation of the Ag NPs. Intrestingly, 50 ppm r@Ag NP inactivated ∼89% and ∼99% of E. faecalis and C. albicans cell, respectively, ∼25% and ∼40% cell inactivations for E. faecalis and C. albicans were observed respectively with 50 ppm b@Ag NP. We showed that 50 ppm r@Ag NP has effective antimicrobial property as much as mostly used %5 NaOCl and %1,5 CHX solutions.

Non-coding natural antisense transcripts (ncNATs) are regulatory RNA molecules that are overlapping with as well as complementary to other transcripts. These transcripts are implicated in a broad variety of biological and pathological processes, including tumorigenesis and oncogenic progression. With this complex field still in its infancy, annotations, expression profiling and functional characterisations of ncNATs are far less comprehensive than those for protein-coding genes, pointing out substantial gaps in the analysis and characterisation of these regulatory transcripts. In this review, we discuss ncNATs from an analysis perspective, in particular regarding the use of high-throughput sequencing strategies, such as RNA-sequencing, and summarize the unique challenges of investigating the antisense transcriptome. Finally, we elaborate on their potential as biomarkers and future targets for treatment, focusing on cancer.

(-)-α-Bisabolol is a natural monocyclic sesquiterpene alcohol present in German chamomile and has been used as an ingredient of functional foods, cosmetics and pharmaceuticals. In this study, metabolic engineering strategies were attempted to produce (-)-α-bisabolol in Saccharomyces cerevisiae. The codon-optimized MrBBS gene coding for (-)-α-bisabolol synthase from Matricaria recutita was expressed in S. cerevisiae for (-)-α-bisabolol production. The resulting strain (DM) produced 9.5 mg/L of (-)-α-bisabolol in 24 h of batch culture. Additionally, the mevalonate pathway was intensified by introducing a truncated HMG1 gene coding for HMG-CoA reductase and ERG10 encoding acetyl-CoA thiolase. The resulting strain (DtEM) produced a 2.9-fold increased concentration of (-)-α-bisabolol than the DM strain. To increase the acetyl-CoA pool, the ACS1 gene coding for acetyl-CoA synthetase was also overexpressed in the DtEM strain. Finally, the DtEMA strain produced 124 mg/L of (-)-α-bisabolol with 2.7 mg/L-h of productivity in a fed-batch fermentation, which were 13 and 6.8 times higher than the DM strain in batch culture, respectively. Conclusively, these metabolically-engineered approaches might pave the way for the sustainable production of other sesquiterpenes in engineered S. cerevisiae.

In this work, several immobilization strategies for Gluconobacter oxydans NBRC 14819 (Gox) were tested in the bioconversion of crude glycerol to dihydroxyacetone (DHA). Agar, agarose and polyacrylamide were evaluated as immobilization matrixes. Glutaraldehyde crosslinked versions of the agar and agarose preparations were also tested. Agar immobilized Gox proved to be the best heterogeneous biocatalyst in the bioconversion of crude glycerol reaching a quantitative production of 50 g/L glycerol into DHA solely in water. Immobilization allowed reutilization for at least eight cycles, reaching four times more DHA than the amount obtained by a single batch of free cells which cannot be reutilized. An increase in scale of 34 times had no impact on DHA productivity. The results obtained herein constitute a contribution to the microbiological production of DHA as they not only attain unprecedented productivities for the reaction with immobilized biocatalysts but also proved that it is feasible to do it in a clean background of solely water that alleviates the cost of downstream processing.

A putative diamine oxidase (DAO) from Yarrowia lipolytica PO1f (DAO-1) was homologously recombinantly integrated into the genome of Y. lipolytica PO1f using the CRISPR-Cas9 system for the subsequent DAO production in a bioreactor. Thereby, it was proven that the DAO-1 produced was indeed a functional DAO. The cultivation yielded 2343 ± 98 nkat/Lculture with a specific DAO activity of 1301 ± 54.2 nkat/gprotein, which was a 93-fold increase of specific DAO activity compared to the native Y. lipolytica PO1f DAO-1 production. The DAO-1 showed a broad substrate selectivity with tyramine, histamine, putrescine and cadaverine being the most favored substrates. It was most active at 40 °C, pH 7.2 in Tris-HCl buffer (50 mM) (with histamine as substrate), which is comparable to human and porcine DAOs. The affinity of DAO-1 towards histamine was lower compared to mammalian DAOs (Km = 2.3 ± 0.2 mM). Nevertheless, DAO-1 degraded around 75% of the histamine used in a bioconversion experiment with a food-relevant concentration of 150 mg/L. With its broad selectivity for the most relevant biogenic amines in foods, DAO-1 from Y. lipolytica PO1f is an interesting enzyme for application in the food industry for the degradation of biogenic amines.

Porphyromonas gingivalis (P. gingivalis) is regarded as a keystone pathogen in destructive periodontal diseases. It expresses a variety of virulence factors, amongst them fimbriae that are involved in colonization, invasion, establishment and persistence of the bacteria inside the host cells. The fimbriae also were demonstrated to affect the host immune-response mechanisms. The major fimbriae are able to bind specifically to different host cells, amongst them peripheral blood monocytes. The interaction of these cells with fimbriae induces release of cytokines such as interleukin-1 (IL-1), IL-6, and tumor necrosis factor-α (TNF-α). The aim of this study was to generate recombinant major FimA protein from P. gingivalis W83 fimbriae and to prove its biological activity. FimA of P. gingivalis W83 was amplified from chromosomal DNA, cloned in a vector and transferred into Listeria innocua. (L. innocua).The expressed protein was harvested and purified using FPLC via a His trap HP column. The identity and purity was demonstrated by gel-electrophoresis and mass-spectrometry. The biological activity was assessed by stimulation of human oral epithelial cells and peripheral blood monocytes with the protein and afterwards cytokines in the supernatants were quantified by enzyme linked immunosorbent assay (ELISA) and cytometric bead array. Recombinant FimA could successfully be generated and purified. Gel-electrophoresis and mass-spectrometry confirmed that the detected sequences are identical with FimA. Stimulation of human monocytes induced the release of high concentrations of IL-1β, IL-6, IL-10 and TNF-α by these cells. In conclusion, a recombinant FimA protein was established and its biological activity was proven. This protein may serve as a promising agent for further investigation of its role in periodontitis and possible new therapeutic approaches.

Inflammatory bowel disease (IBD) is a set of immunological disorders which can generate chronic pain and fatigue associated with the inflammatory symptoms. The treatment of IBD remains a significant hurdle with current therapies being only partially effective or having significant side effects, suggesting that new therapies that elicit different modes of action and delivery strategies are required. TGM1 is a TGF-β mimic that was discovered from the intestinal helminth parasite Heligmosomoides polygyrus and is thought to be produced by the parasite to suppress the intestinal inflammation response to help evade host immunity, making it an ideal candidate to be developed as a novel anti-inflammatory bio-therapeutic. Here we utilized the expression system of the edible green algae Chlamydomonas reinhardtii in order to recombinantly produce active TGM1 in a form that could be ingested. C. reinhardtii robustly expressed TGM1, and the resultant recombinant protein is biologically active as measured by regulatory T cell induction. When delivered orally to mice, the algal expressed TGM1 is able to ameliorate weight loss, lymphadenopathy, and disease symptoms in a mouse model of DSS-induced colitis, demonstrating the potential of this biologic as a novel treatment of IBD.

Low production rates are still one limiting factor for the industrial climate-neutral production of biovaluable compounds in cyanobacteria. Next to optimized cultivation conditions, new production strategies are required. Hence, the use of established molecular tools could lead to increased product yields in the cyanobacterial model organism Synechocystis sp. PCC6803. Its main storage compound glycogen was chosen to be increased by the use of these tools. In this study, the three genes glgC, glgA1 and glgA2, which are part of the glycogen synthesis pathway, were combined with the Pcpc560 promoter and the neutral cloning site NSC1. The complete genome integration, protein formation, biomass production and glycogen accumulation were determined to select the most productive transformants. The overexpression of glgA2 did not increase the biomass or glycogen production in short-term trials compared to the other two genes but caused transformants death in long-term trials. The transformants glgA1_11 and glgC_2 showed significantly increased biomass (1.6-fold - 1.7-fold) and glycogen production (3.5-fold - 4-fold) compared to the wild type after 96 h making them a promising energy source for further applications. Those could include for example a two-stage production process, with first energy production (glycogen) and second increased product formation (e.g. ethanol).

2'-Fucosyllactose (2'-FL), a major oligosaccharide of human breast milk, and is currently supplemented into infant formula. For the overproduction of 2'-FL via fucosylation of lactose, conventional approaches have focused on the episomal overexpression of de novo or salvage GDP-L-fucose biosynthetic pathway and α-1,2-fucosyltransferase (FucT2) through T7 RNA polymerase expression system in engineered E. coli. However, these approaches have drawbacks of metabolic burden, plasmid instability, and inclusion body formation. In this study, a deletion mutant of waaF coding for ADP-heptose:LPS heptosyltransferase II was employed for 2'-FL production. As the waaF deletion induces accumulation of colanic acid, additional deletion of wcaJ coding for UDP-glucose-1-phosphate transferase in the waaF deletion mutant resulted in enhanced accumulation of GDP-L-fucose. Besides, 2'-FL yields and titers were drastically improved when T7 promoter was replaced with Trc promoter for α-1,2 fucosyltransferase expressions in the waaF and wcaJ deleted strain. As a result, when FucT2 was expressed under Trc promoter in the E. coli JM109(DE3) ΔwaaFΔwcaJ, 14.7 g/L of 2'-FL was produced with a productivity of 0.31 g/L/h in a fed-batch fermentation. We envision that the deletion-based metabolic design and decreased promoter strength for fucosyltransferase expression can resolve the drawbacks of T7 RNA polymerase-based expression design for 2'-FL production in E. coli.

Biodiesel, as a renewable and eco-friendly energy source that can be produced through algae oil esterification, has recently received much attention. Maximization of algal biomass and lipid content is crucial for commercial biodiesel production. In this study, Chlorella sp. PG96, a microalgal strain isolated from urban wastewater, was identified considering its morphological and molecular characteristics. Fractional factorial design (211-7) was employed to screen medium and environmental factors for achieving high lipid productivity. The effects of eleven factors including light intensity, light spectrum, aeration rate, temperature, salinity, NaHCO3, CO2, NaNO3, NH4Cl, MgSO4.7H2O, and K2HPO4 and their interactions on growth characteristics of Chlorella sp. PG96 (biomass and lipid production) were statistically assessed. Based on the experimental results, lipid productivity was at its maximum (54.19 ± 8.40 mglipid L-1 day-1) under a combination of high levels of all factors. The analysis also showed that physical parameters of light intensity and temperature were more effective on algal growth compared to nutritional parameters. Furthermore, nitrogen source of ammonium and carbon source of bicarbonate played more significant roles in biomass and lipid production, compared with nitrate and CO2, respectively. Although the effect of sulfur limitation on cellular growth was similar to phosphorus deficiency, S-limitation had a greater impact on lipid accumulation. The interaction between NaHCO3 and NH4Cl was the most prominent interaction affecting all responses. It is concluded that Chlorella sp. PG96 at a high level of light intensity and temperature (22500 Lux and 32 °C, respectively) can be a prospective candidate for biodiesel production.

Enterokinase is one of the hydrolases that catalyze hydrolysis to regulate biological processes in intestinal visceral mucosa. Enterokinase plays an essential role in accelerating the process of protein digestion as it converts trypsinogen into active trypsin by accurately recognizing and cleaving a specific peptide sequence, (Asp)4-Lys. Due to its exceptional substrate specificity, enterokinase is widely used as a versatile molecular tool in various bioprocessing, especially in removing fusion tags from recombinant proteins. Despite its biotechnological importance, mass production of soluble enterokinase in bacteria still remains an unsolved challenge. Here, we present an effective production strategy of human enterokinase using tandemly linked solubility enhancers consisting of thioredoxin, phosphoglycerate kinase or maltose-binding protein. The resulting enterokinases exhibited significantly enhanced solubility and bacterial expression level while retaining enzymatic activity, which demonstrates that combinatorial design of fusion proteins has the potential to provide an efficient way to produce recombinant proteins in bacteria.