Characterization the response of Chlamydomonas reinhardtii serine/threonine protein kinase mutant to blue light / 生物工程学报

In order to investigate the molecular mechanism of silk/threonine protein kinase (STK)-mediated blue light response in the algal Chlamydomonas reinhardtii, phenotype identification and transcriptome analysis were conducted for C. reinhardtii STK mutant strain crstk11 (with an AphvIII box reverse insertion in stk11 gene coding region) under blue light stress. Phenotypic examination showed that under normal light (white light), there was a slight difference in growth and pigment contents between the wild-type strain CC5325 and the mutant strain crstk11. Blue light inhibited the growth and chlorophyll synthesis in crstk11 cells, but significantly promoted the accumulation of carotenoids in crstk11. Transcriptome analysis showed that 860 differential expression genes (DEG) (559 up-regulated and 301 down-regulated) were detected in mutant (STK4) vs. wild type (WT4) upon treatment under high intensity blue light for 4 days. After being treated under high intensity blue light for 8 days, a total of 1 088 DEGs (468 upregulated and 620 downregulated) were obtained in STK8 vs. WT8. KEGG enrichment analysis revealed that compared to CC5325, the crstk11 blue light responsive genes were mainly involved in catalytic activity of intracellular photosynthesis, carbon metabolism, and pigment synthesis. Among them, upregulated genes included psaA, psaB, and psaC, psbA, psbB, psbC, psbD, psbH, and L, petA, petB, and petD, as well as genes encoding ATP synthase α, β and c subunits. Downregulated genes included petF and petJ. The present study uncovered that the protein kinase CrSTK11 of C. reinhardtii may participate in the blue light response of algal cells by mediating photosynthesis as well as pigment and carbon metabolism, providing new knowledge for in-depth analysis of the mechanism of light stress resistance in the algae.

Tactic movement of microalgae and its application in targeted transport: a review / 生物工程学报

Microalgae are a group of photosynthetic microorganisms, which have the general characteristics of plants such as photosynthesis, and some species have the ability of movement which resembles animals. Recently, it was reported that microalgae cells can be engineered to precisely deliver medicine-particles and other goods in microfluidic chips. These studies showed great application potential in biomedical treatment and pharmacodynamic analysis, which have become one of the current research hotspots. However, these developments have been rarely reviewed. Here, we summarized the advances in manageable movement exemplified by a model microalgae Chlamydomonas reinhardtii based on its characteristics of chemotaxis, phototaxis, and magnetotaxis. The bottlenecks and prospects in the application of microalgae-based tactic movement were also discussed. This review might be useful for rational design and modification of microalgal manageable movement to achieve targeted transport in medical and other fields.

Bardet-Biedl syndrome protein-8 is involved in flagellar membrane protein transport in Chlamydomonas reinhardtii / 生物工程学报

Cilia and flagella on eukaryotic cells are polarized organelles extending from the surfaces of cells, which participate not only in cell motility, but also in signal transduction and other processes. Structural or functional abnormalities of cilia can cause various human diseases, termed ciliopathies. Bardet-Biedl syndrome (BBS) is a ciliopathic human genetic disorder, and the pathogenesis is that mutated BBS genes result in abnormal cilia function. In order to study the pathogenic genes BBS8, we screened bbs8 mutant in Chlamydomonas reinhardtii and did a lot of physiology and biochemistry experiments. We affirmed that BBS8 protein was a cilia protein and had specific localization in the basal body by immunofluorescence (IF). The bbs8 mutant lost photokinesis, and it was defective in flagella shortening with drug induction. The results of silver staining and mass spectrometric analysis showed aberrant accumulation of flagellar proteins in the mutant flagella. We concluded that the BBS8 protein plays a significant role in flagellar membrane proteins transport, and the BBS8 protein might mediate retrograde transport to exert physiological function in the process.

Prokaryotic expression, purification and polyclonal antibody preparation of chlamydomonas reinhardtii bbsome component protein bbs4 / 国际药学研究杂志

Objective: To investigate the role of BBS protein in ciliary signal transduction by studying the pro- karyotic expression, purification and polyclonal antibody preparation of Chlamydomonas reinhardtii protein BBS4. Methods: Prokaryotic Expression Vector pET-28a(+)-bbs4 and pMAL-c2X-bbs4 were constructed by the cDNA sequence of bbs4 Gene from C. reinhardtii, and then transformed into Escherichia coli BL21(DE3)for protein expression. The fusion protein with maltose binding protein(MBP)and 6×His tag was obtained by inducing expression. The purified fusion protein 6×His-BBS4 were used to immunize New Zealand white rabbits and the antiserum was isolated from the blood collected from the ear vein. The titer of the antiserum was measured by indirect ELISA essay, the specificity of the antibody was tested by Western blotting method and immunofluorescence test. Results: Prokaryotic expression plasmids pET-28a(+)-bbs4 and pMAL-c2X-bbs4 were successfully constructed. The relative molecular weights of 6×His-BBS4 and MBP-BBS4 fusion proteins were 45 kDa and 85 kDa, respectively. The purity of the fusion proteins was more than 85%, and the concentration of the fusion proteins was more than 0.5 mg/ml. The proteins were used for immunization. The titer of the fusion proteins was 51 200. Western blotting showed a high specificity for the detection of C. reinhardtii CC-125. Prokaryotic expression of BBS4 protein of C. reinhardtii and preparation of polyclonal antibody were realized. Conclu- sion: The polyclonal antibody against BBS4 of C. reinhardtii was prepared successfully, which laid a foundation for further study on the role of BBS4 in ciliopathies.

Produção de proteínas heterólogas em microalga / Production of heterologous protein in microalga

O objetivo desta tese foi explorar o sistema de produção de proteínas heterólogas em microalga com ênfase em Chlamydomonas reinhardtii por meio de: (1) desenvolvimento de um fotobiorreator tubular fechado de escala laboratorial, utilizando técnicas de manufatura digital; (2) avaliação de 7 diferentes proteínas fluorescentes (mTagBFP, Cerulean, Emerald, crGFP, cOFP, tdTomato e mCherry), como sistema reporter de secreção de proteínas em microalga; (3) avaliação do fotobiorreator desenvolvido utilizando cultivo de cepas recombinantes; (4) desenvolvimento de novos peptídeos sinais para secreção de proteínas em C. reinhardtii; (5) avaliação da produção de um biofármaco (hialuronidase) em microalgas, por meio da expressão de duas isoenzimas codificadas pelos genes HYA1 e SPAM1 em C. reinhardtii. O fotobiorreator tubular foi avaliado quanto a sua capacidade de resistir ao processo de esterilização por autoclavação e seu desempenho por meio do cultivo de cepa recombinante secretando mCherry. A fluorescência das proteínas fluorescentes foi medida por leitor de placas de fluorescência e visualizada intracelularmente por microscopia confocal de fluorescência. A atividade de hialuronidase foi determinada através de um ensaio enzimático turbidimétrico. O desenvolvimento do fotobiorreator resultou em um sistema fechado resistente a autoclavação, com capacidade de cultivo de cepas recombinantes de C. reinhardtii. Esse fotobiorreator proporcionou uma produtividade máxima de 10 mg/L.d de mCherry da cepa recombinante em sistema fechado, com velocidade específica de crescimento máxima de 1,27 d-1 para a cepa recombinante testada. Todas as proteínas fluorescentes avaliadas apresentaram capacidade de secreção por C. reinhardtii, com diferentes níveis de interferências em sua medição, permitindo a escolha da mCherry como proteína reporter. Entre os peptídeos sinais avaliados (quatro descritos na literatura - BiP, ARS1, CAH1 e IBP1 - e seis preditos), o peptídeo predito "SP5" foi o que apresentou maior capacidade de secreção, determinado por níveis de fluorescência no sobrenadante. A avaliação dos peptídeos sinais constatou a necessidade de explorar o desenvolvimento de sistemas de expressão (e.g. vetores de expressão) aliados a análises computacionais, como o SignalP 4.0. Por último, os dados desse estudo mostram que C. reinhardtii transformadas com o vetor de expressão foi capaz de produzir as duas isoformas de hialuronidase em sua forma ativa, evidenciando a capacidade desse sistema para a produção de biofármacos. Portanto, nesta tese o sistema de expressão de proteínas heterólogas baseado em microalgas foi explorado, atingindo os objetivos propostos. O fotobiorreator desenvolvido tem a capacidade de esterilização em escala laboratorial (1) e em cultivo com cepa recombinante propiciou elevada produtividade (3). As proteínas vermelhas fluorescentes apresentaram-se como as proteínas com menores interferências para estudos de secreção em C. reinhardtii (2). Além disso, o peptídeo predito SP5 apresentou o melhor desempenho na secreção de proteínas (4) e o vetor de expressão empregado permitiu a identificação de cepas produtoras de biofármaco hialuronidase (5). Portanto, o sistema de produção de proteínas heterólogas por microalgas é um sistema promissor e poderá permitir, utilizando sistemas de secreção, obter proteínas de alto valor comercial a baixos custos, empregando a secreção e técnicas de cultivo como a fermentação extrativa

In this thesis, the heterologous protein production in microalgae with emphasis on Chlamydomonas reinhardtii was explored through: (1) development of a laboratory scale closed tubular photobioreactor using digital manufacturing techniques; (2) evaluation of different fluorescent proteins (mTagBFP, Cerulean, Emerald, crGFP, cOFP, tdTomato and mCherry) as a reporter system for protein secretion in microalgae (3) evaluation of photobioreactor developed using recombinant strains culture; (4) development of new signals peptides for protein secretion in C. reinhardtii (5) expression evaluation of a biopharmaceutical (Hyaluronidase) in microalgae, through the expression of two isoenzymes encoded by the HYA1 and SPAM1 genes in C. reinhardtii. The tubular photobioreactor was evaluated for its ability to resist sterilization process by autoclaving and its performance by culturing recombinant strain secreting mCherry. Fluorescence of fluorescent proteins was measured by fluorescence plate reader and observed intracellularly by confocal fluorescence microscopy. The hyaluronidase activity was determined by a turbidimetric enzymatic assay. The development of the photobioreactor resulted in a closed system resistant to autoclaving, capable of culturing recombinant strains of C. reinhardtii. This recombinant strain achieved a maximum productivity of 10 mg/L.day of mcherry in the closed system, with a maximum growth rate of 1.27 d-1 for the recombinant strain tested. All the fluorescent proteins evaluated had C. reinhardtii secretion capacity, with different interference levels in their measurement, allowing the selection of mCherry as a reporter protein. Among the evaluated peptides (four described in the literature - BiP, ARS1, CAH1 and IBP1 - and six predicted), the predicted peptide "SP5" was the one that presented greater capacity of secretion, determined by levels of fluorescence in the supernatant. The results of this study point out the need to explore the development of biological systems (i.e., expression vectors) allied to computational analysis. Finally, the data from this study showed that C. reinhardtii could produce the two isoforms of hyaluronidase in its active form, evidencing the capacity of this system to produce biopharmaceuticals. Therefore, in this thesis the heterologous protein expression system based on microalgae was explored, reaching the proposed objectives. The developed photobioreator has sterilization capabilityin laboratorial scale (1) and in culture with recombinant strain had high productivity (3). The red fluorescent proteins was found as the most suitable proteins for studies of secretion in C. reinhardtii with lower interference levels(2). In addition, the predicted SP5 peptide showed the best performance in protein secretion (4) and the expression vector employed allowed the identification of strains producing biopharmaceutical hyaluronidase (5). Therefore, the system of heterologous proteins production by microalgae is promising and will allow, using secretion systems, to obtain proteins of high commercial value at low costs, using secretion and cultivation techniques such as extractive fermentation

Processos de produção da proteína heteróloga mCherry por Chlamydomonas reinhardtii / Production processes of heterologous mCherry protein by Chlamydomonas reinhardtii

Este trabalho tem como finalidade estudar as melhores condições do cultivo da microalga Chlamydomonas reindhartii geneticamente modificada para a produção da proteína fluorescente mCherry a partir do estudo dos macronutrientes contidos no meio de cultivo. A proteína mCherry possui a vantagem de ser facilmente detectada no meio de cultivo por espectofotometria convencional, convertendo-se, desta forma, em uma molécula interessante para o estudo como modelo de expressão. Inicialmente, foram estudadas três diferentes fontes de nitrogênio para avaliar a expressão da proteína recombinante. Os resultados indicaram que a melhor fonte de nitrogênio para a produção da mCherry foi o NH4NO3. Em seguida, para avaliar os efeitos gerados pelos macronutrientes (acetato, cloreto de cálcio, sultato de magnésio, nitrato de amônio e fostato total) contidos no meio de cultivo TAP, foi realizado um planejamento composto central 25, em cultivos em microplacas, sendo os resultados avaliados por regressão multivariada. Além disso, a análise realizada por regressão multivariada indicou que, dos níveis avaliados das variáveis, as condições que melhor atendem à otimização da produção de mCherry e crescimento celular são: acetato, 33,35 mM; cloreto de cálcio, 0,45 mM; sulfato de magnésio, 0,83 mM; nitrato de amônio, 10,31 mM; fosfato total, 1,96 mM. Essas condições foram escolhidas para cultivo em fotobiorreator tubular, onde foi obtido título de fluorescência de mCherry a 608 nm de 59209 UF, correspondendo a um aumento de 118,5% maior que o título de fluorescência obtido com uso de meio TAP padrão. Com a finalidade de seguir com os processos de produção foi disenhado um biorreator tipo coluna e foi reaizado um estudio de produção em sistema semicontinuo. Os resultados obtidos demostraram que o sistema semicontinuo aumento 2,6 veces a produtividade da biomassa

This work aims to study the best conditions of the cultivation of the microalgae Chlamydomonas reindhartii genetically modified for the production of the fluorescent protein mCherry from the study of the macronutrients contained in the culture medium. The mCherry protein has the advantage of being easily detected in the culture medium by conventional spectrophotometry, thus becoming an interesting molecule for the study as an expression model. Initially, three different nitrogen sources were studied to evaluate the expression of the recombinant protein. The results indicated that the best source of nitrogen for the production of mCherry was NH4NO3. Then, to evaluate the effects generated by macronutrients (acetate, calcium chloride, magnesium sulphate, ammonium nitrate and total phosphate) contained in the TAP culture medium, a central composite 25 was carried out in cultures on microplates, Results evaluated by multivariate regression. In addition, multivariate regression analysis indicated that, from the evaluated levels of the variables, the conditions that best serve the optimization of mCherry production and cell growth are: acetate, 33.35 mM; Calcium chloride, 0.45 mM; Magnesium sulfate, 0.83 mM; 10.31 mM ammonium nitrate; Total phosphate, 1.96 mM. These conditions were chosen for cultivation in tubular photobioreactor where fluorescence titre of mCherry at 608 nm of 59209 UF was obtained, corresponding to an increase of 118.5% greater than the titer of fluorescence obtained using standard TAP medium. In order to follow the production processes, a column type bioreactor was designed and a production study was carried out in a semicontinuous system. The results showed that the semicontinuous system increased 2.6 times the productivity of the biomass

Prokaryotic expression and purification of Chlamydomonas reinhardtii intraflagellar transport protein 46(IFT46) and preparation of polyclonal antibody / 生物工程学报

IFT46 is one of the important components of intraflagellar transport complex B in Chlamydomonas reinhardtii, and plays important roles in the assembly, movement and perception of ciliary. To study its functional mechanism, a GST-tagged and an MBP-tagged prokaryotic expression plasmid, pGEX-2T-ift46 and pMAL-C2X-ift46 were constructed, respectively, by inserting ift46 into the pGEX-2T and pMAL-C2X vector, and then transformed into Escherichia coli BL21 (DE3) for protein expression. SDS-PAGE (15%) analysis results showed that the molecular weights of the fusion protein GST-IFT46 and MBP-IFT46 were 70 kDa and 86 kDa, respectively. We used the fusion protein GST-IFT46 purified by affinity adsorption purification (more than 95% purity) for immunity to New Zealand white rabbits. The 5th immune serum was collected and the antibody titer was determined to be 256 000 by ELISA. The antiserum was purified by Protein A affinity adsorption purification and immobilized MBP-IFT46 purification, and the specificity of polyclonal antibodies was evaluated by Western blotting and immunofluorescence. Results showed that the polyclonal antibody prepared could specifically and precisely bind IFT46 in C. reinhardtii, and IFT46 was mainly concentrated at basal body regions and few localized along the entire length of the flagellum as punctuated dots, which will make a foundation to further study the mechanism of IFT46 in cilia related diseases such as obesity, diabetes and polycystic kidney disease.

Production and characterization of algae extract from Chlamydomonas reinhardtii

Background: Algae offer many advantages as biofuel sources including: high growth rates, high lipid content, the ability to grow on non-agricultural land, and the genetic versatility to improve strains rapidly and produce co-products. Research is ongoing to make algae biofuels a more financially attractive energy option; however, it is becoming evident that the economic viability of algae-based fuels may hinge upon high-value co-products. This work evaluated the feasibility of using a co-product, algae extract, as a nutrient source in cell culture media. Results: Algae extract prepared from autolysed Chlamydomonas reinhardtii was found to contain 3.0% protein, 9.2% total carbohydrate, and 3.9% free α-amino acid which is similar to the nutrient content of commercially available yeast extract. The effects of algae extract on the growth and metabolism of laboratory strains of Escherichia coli and Saccharomyces cerevisiae were tested by substituting algae extract for yeast extract in LB and YPAD growth media recipes. Complex laboratory media supplemented with algae extract instead of yeast extract showed markedly improved effects on the growth and metabolism of common laboratory microorganisms in all cases except ethanol production rates in yeast. Conclusions: This study showed that algae extract derived from C. reinhardtii is similar, if not superior, to commercially available yeast extract in nutrient content and effects on the growth and metabolism of E. coli and S. cerevisiae. Bacto™ yeast extract is valued at USD $0.15-0.35 per gram, if algae extract was sold at similar prices, it would serve as a high-value co-product in algae-based fuel processes.

Cloning and Transformation of lba Gene of Glycine max into Chloroplast of Chlamydomonas reinhardtii / 中国生物工程杂志

To decrease the oxygen content in the cell is a key method to improve hydrogen production in Chlamydomonas reinhardtii.A new approach was developed by transforming the leghemoglobin gene lba,which has high affinity to oxygen,into the chloroplast of C.reinhardtii to get a low dissolved oxygen in the cell and result into improvement of H2 ase activity and H2 yield. The results showed that lba was successfully transformed into the chloroplast of C.reinahrdtii strain 849 and did not affect its growth significantly. The work paved the road for further regulation of lba expression in the chloroplast to improve of hydrogen production of C.reinahrdtii.