Creation of potential gene cassette for recombinant protein expression in green microalgal system

Development of efficient strategies and reliable systems for production of recombinant proteins in high quality and quantity to reach the market are gaining more attraction. On the economic side, improvement of the efficiency and cost effectiveness of production process is highly required to be commercially viable. Although many strategies have been theoretically established to increase the level of gene expression in different specific host systems, it is needed to be practically applied. Due to the lack of enough information for expression of recombinant proteins in microalgal systems, the present study aimed at creation of a gene cassette utilizing both upstream (bioinformatics and computational analysis, genetic engineering and molecular biology) and downstream factors (bioprocess engineering) which may have critical roles in gene expression with the emphasis on two microalgae species, Dunaliella salina and Chlamydomonas reinhardtii. As the first step in evaluating the feasibility of molecular farming in microalgae, some of the important upstream factors including matrix attachment regions (MARs), translation initiation sites, signal peptides and gene optimization parameters were studied. An artificial universal MAR sequence was designed and developed considering the MAR specific rules and motifs. To explore the specific translation initiation site, all mRNA sequences in the microalgae D. salina were screened among which the nonredundant mRNAs were selected for bioinformatics analysis. A consensus sequence was finally created to be used in the specific expression vector. For the secretion systems in microalgae D. salina, a screening procedure was carried out to find the specific signal peptides using bioinformatics and in silico tools in DNA, RNA and protein databases. The predicted sequences were then selected for further characterizations and the highest scored predicted signal was embedded in the secretion series of expression vectors. For optimization of the coding sequences, the most important parameters such as the host codon preference, RNA secondary structure, GC and CpG dinucleotides content were taken into consideration. The final expression cassette containing all of the required elements including MAR, signal peptide, translation initiation site, KDEL retention signal, His 6x purification tag, V5 epitope and protease cleavage site was integrated into 25 expression vectors (25 vectors containing different elements). To develop a proprietary series of microalgae specific expression vectors, nine fragments containing the Gateway recombination cassette, optimized genes of interest (2), promoters (2), 5’- UTR, interon, 3’-UTR (2), MARs (2) and pUC18 backbone were amplified using the most high fidelity enzyme available (KAPA). The fragments were successfully assembled in a single recombination reaction through the novel multiple overlap extension PCR (MOE-PCR) technique developed in this study. To assess the efficient transformation techniques for microalgae, three different methods (electroporation, glass beads and PEG-mediated) were used to transform one Dunaliella and three Chlamydomonas strains using the developed expression vectors. Glass bead method with 10 s agitation time, 4% PEG and 300 ng/μL plasmid DNA, as an efficient and simple technique showed the best results despite a drop in viable cells. The use of specific expression vectors resulted in more number and more stable transformants comparing a commercially available vector (pCAMBIA-3301) without the abovementioned optimized elements. The expression cassette containing optimized genetic elements could be delivered into microalgae cells and confer the resistance to the transformants for at least 3 months. For the downstream processes, the culture condition of D. salina was optimized to be used for future studies of the transformants. An experiment was conducted based on response surface methodology (RSM) to realize the reaction of microalgae to different phototrophic conditions including heterotrophic, mixotrophic and phototrophic. It was observed that the higher the amount of supplemented glucose up to 15 g/L, the more glucose utilization, biomass concentration and consequently protein content obtain. The results demonstrated superiority of mixotrophic conditions in constant light in improving the growth of microalgae. This study demonstrated that the upstream factors such as MARs, translation initiation sites and gene optimization along with the optimized transformation systems and downstream processes, could be a potential procedure to be efficiently used in overexpression of recombinant protein production using microalgae as the expression platform. Considering the close relatedness of the studied microalgae and other close species, the proprietary vector series could be generally used in genetic engineering of at least green microalgae

Network Meta-Analysis of Chicken Microarray Data following Avian Influenza Challenge-A Comparison of Highly and Lowly Pathogenic Strains

The current bioinformatics study was undertaken to analyze the transcriptome of chicken (Gallus gallus) after influenza A virus challenge. A meta-analysis was carried out to explore the host expression response after challenge with lowly pathogenic avian influenza (LPAI) (H1N1, H2N3, H5N2, H5N3 and H9N2) and with highly pathogenic avian influenza (HPAI) H5N1 strains. To do so, ten microarray datasets obtained from the Gene Expression Omnibus (GEO) database were normalized and meta-analyzed for the LPAI and HPAI host response individually. Different undirected networks were constructed and their metrics determined e.g., degree centrality, closeness centrality, harmonic centrality, subgraph centrality and eigenvector centrality. The results showed that, based on criteria of centrality, the CMTR1, EPSTI1, RNF213, HERC4L, IFIT5 and LY96 genes were the most significant during HPAI challenge, with PARD6G, HMG20A, PEX14, RNF151 and TLK1L having the lowest values. However, for LPAI challenge, ZDHHC9, IMMP2L, COX7C, RBM18, DCTN3, and NDUFB1 genes had the largest values for aforementioned criteria, with GTF3C5, DROSHA, ATRX, RFWD2, MED23 and SEC23B genes having the lowest values. The results of this study can be used as a basis for future development of treatments/preventions of the effects of avian influenza in chicken

Enhancement of BLIS production by Pediococcus acidilactici kp10 in optimized fermentation conditions using an artificial neural network

The present study was aimed at enhancing the production of bacteriocin-like inhibitory substance (BLIS) produced by Pediococcus acidilactici Kp10 through optimizing the fermentation parameters. M17 was chosen in preliminary study as a culture medium because BLIS production was nine times higher (1427.7 AU mL−1) compared to that produced by MRS (160 AU mL−1). The fermentation parameters such as temperature, inoculum size, buffer strength, concentration of tween 80 and agitation speed were screened using two level half-factorial design. BLIS production is influenced by three most significant factors identified as temperature, inoculum size and agitation speed, which were further optimized using an artificial neural network (ANN). ANN predicted that a maximum activity of 5262.64 AU mL−1 would be obtained at optimum conditions of 120 rpm, 3% and 28.5 °C. The observed BLIS activity at the predicted optimum levels of the tested variables in ANN was 5118.5 AU mL−1, which was close to the predicted BLIS activity. Increased BLIS activity in the final solution, which resulted from the optimized process, would reduce downstream steps such as concentrating the product during purification

CRISPR-mediated genome editing in poplar issued by efficient transformation

BackgroundCRISPR has been increasingly used for plant genetic improvements because of its high efficiency and precision. Recently, the authors have reported the possibility of homology-directed repair (HDR) using CRISPR/Cas9 through woody plants such as poplar. HDR often replaces nucleotides with one donor DNA template (DDT), including homologous sequences.MethodsCRISPR–Cas9 was recruited, and three variables, Agrobacteria inoculator concentration, pDDT/pgRNA ratio, and homologous arm length, were designed to integrate nptII and 2XCamV 35S into the MKK2 promoter zone.ResultsHere, we showed that recovered poplars on kanamycin-supplemented media exhibited enhanced expression of MKK2 affected by the precise integration of 2XcamV 35S and nptII, improving biochemical and phenotypic properties. Our findings confirmed that Agrobacterium inoculator OD600 = 2.5, increased DDT numbers during cell division to 4:1 pDDT/pgRNA, and optimized homologous arms 700 bp caused efficient HDR and increased MKK2 expression.ConclusionEfficient transformations resulted from optimized variables, directly affecting the HDR efficiency through woody plants such as poplar

An Integrated Bioinformatics Approach to Identify Network Derived Hub Genes in Starving Zebrafish

The present study was aimed at identifying causative hub genes within modules formed by co-expression and protein–protein interaction (PPI) networks, followed by Bayesian network (BN) construction in the liver transcriptome of starved zebrafish. To this end, the GSE11107 and GSE112272 datasets from the GEO databases were downloaded and meta-analyzed using the MetaDE package, an add-on R package. Differentially expressed genes (DEGs) were identified based upon expression intensity N(µ = 0.2, σ2 = 0.4). Reconstruction of BNs was performed by the bnlearn R package on genes within modules using STRINGdb and CEMiTool. ndufs5 (shared among PPI, BN and COEX), rps26, rpl10, sdhc (shared between PPI and BN), ndufa6, ndufa10, ndufb8 (shared between PPI and COEX), skp1, atp5h, ndufb10, rpl5b, zgc:193613, zgc:123327, zgc:123178, wu:fc58f10, zgc:111986, wu:fc37b12, taldo1, wu:fb62f08, zgc:64133 and acp5a (shared between COEX and BN) were identified as causative hub genes affecting gene expression in the liver of starving zebrafish. Future work will shed light on using integrative analyses of miRNA and DNA microarrays simultaneously, and performing in silico and experimental validation of these hub-causative (CST) genes affecting starvation in zebrafish

Microtiter miniature shaken bioreactor system as a scale-down model for process development of production of therapeutic alpha-interferon2b by recombinant Escherichia coli

Background: Demand for high-throughput bioprocessing has dramatically increased especially in the biopharmaceutical industry because the technologies are of vital importance to process optimization and media development. This can be efficiently boosted by using microtiter plate (MTP) cultivation setup embedded into an automated liquid-handling system. The objective of this study was to establish an automated microscale method for upstream and downstream bioprocessing of α-IFN2b production by recombinant Escherichia coli. The extraction performance of α-IFN2b by osmotic shock using two different systems, automated microscale platform and manual extraction in MTP was compared. Results: The amount of α-IFN2b extracted using automated microscale platform (49.2 μg/L) was comparable to manual osmotic shock method (48.8 μg/L), but the standard deviation was 2 times lower as compared to manual osmotic shock method. Fermentation parameters in MTP involving inoculum size, agitation speed, working volume and induction profiling revealed that the fermentation conditions for the highest production of α-IFN2b (85.5 μg/L) was attained at inoculum size of 8%, working volume of 40% and agitation speed of 1000 rpm with induction at 4 h after the inoculation. Conclusion: Although the findings at MTP scale did not show perfect scalable results as compared to shake flask culture, but microscale technique development would serve as a convenient and low-cost solution in process optimization for recombinant protein

Kinetic modeling of bacteriocin-like inhibitory substance secretion by Pediococcus acidilactici Kp10 and its stability in food manufacturing conditions

This paper deliberates the modelling and validation of bacteriocin-like inhibitory substance (BLIS) secretion by Pediococcus acidilactici Kp10 at different agitation speeds in a stirred tank bioreactor. A range of models namely the re-parameterised logistic, Luedeking-Piret and maintenance energy were assessed to predict the culture performance of the said bacterium. Growth of P. acidilactici Kp10 was enhanced with increased agitation speed up to 600 rpm while BLIS secretion was maximum at 400 rpm but decreased at higher agitation speed. Growth of P. acidilactici aptly subscribed to the re-parameterised logistic model while BLIS secretion and lactose consumption fitted well with the Luedeking-Piret model. The models revealed a relationship between growth of the bacterium and BLIS secretion. Bacterial growth and BLIS secretion were largely affected by the agitation speed of the stirred tank bioreactor which regulated the oxygen transfer to the culture. BLIS secretion by P. acidilactici Kp10 was however enhanced in oxygen-limited culture. The study also assessed BLIS from the perspective of its stability when subjected to factors such as temperature, pH and detergents. Results showed that BLIS produced by this strain was not affected by heat (at 25-100 °C for 20 min and at 121 °C for 15 min), surfactant (Tween 40, 60 and 80 and urea), detergents (up to 1% SDS), organic solvents (50% each of acetone, methanol and ethanol) and stable in a wide range of pH (2-10). The above information are pertinent with reference to commercial applications of this bacterial product in food manufacturing which invariably involve various sterilization processes and subjected to a wide pH range

Extraction of fresh banana waste juice as non-cellulosic and non-food renewable feedstock for direct lipase production

The bulk availability of banana waste in the world has made a move towards the development of alternative novel renewable sugars in present study as the pressed juice from banana frond and pseudostem was found to contain high level of renewable sugars such as glucose, sucrose and fructose. By using a simple sugarcane press, the glucose content in the banana frond juice was 16.6 g/L, which accounts for 55% of the total fermentable sugars. From the results, the lipase production reached ∼200 U/mL in medium comprising banana frond juice and banana pseudostem juice, comparable to basal medium with glucose (206.3 U/mL) as carbon source in basal media. As banana pseudostem possesses high content of starch, microwave heating pretreatment showed 36% enhancement of glucose content (10.4 g/L) in juice as compared to direct pressed juice (7.78 g/L). This result indicates that banana waste juice can be used as an alternative fermentable carbon source for lipase production and has potential as a fermentable carbon source