Development of a cell surface display system in Chlamydomonas reinhardtii

Cell-surface display systems are biotechnological techniques used to express heterologous proteins on the cell surface. Their application depends directly on the cell system used, as well as on the anchoring point for the surface displayed protein. To meet most application demands an inexpensive, safe, and scalable production platform, that reduces the economic barriers for large scale use is needed. Toward this goal, we screened three possible cell surface anchoring points in the green algae Chlamydomonas by fusing mVenus to prospective anchors moieties. The vectors harboring mVenus:anchor were screened for mVenus fluorescence and tested for cellular localization by confocal laser scanning microscopy. This strategy allowed the identification of two functional anchors, one for the cytoplasmic membrane using the MAW8 GPI-anchor signal, and one for the cell wall using the GP1 protein. We also exploited GP1 chemical and biological traits to release the fused proteins efficiently during cell wall shedding. Our work provides a foundation for surface engineering of C reinhardtii supporting both cell biology studies and biotechnology applications

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

Extraction of adenovirus in aqueous two-phase system

Processos biotecnológicos dependem significativamente das técnicas de separação e purificação utilizadas, para manter boa relação custo-benefício na produção em escala industrial de produtos biotecnológicos com fins comerciais, industriais e terapêuticos. A aplicação do sistema micelar de duas fases aquosas (SMDFA) é proposta como alternativa para purificação de biomoléculas/biopartículas, pois permite sua separação e análise, muitas vezes sem que essas percam sua atividade ou propriedades desejadas. Com essa técnica é possível realizar uma partição seletiva que possibilita altos rendimentos. Esse trabalho destinou-se a estudar o emprego dessa metodologia na extração e purificação de Adenovírus em sistema micelar de duas fases aquosas formado por Triton X-114/Suspensão viral. Os ensaios foram realizados em sistemas de 5 g seguindo um planejamento fatorial completo (23) com 4 pontos centrais. Os fatores estudados foram temperatura de extração, pH da suspensão viral e concentração do tensoativo. Sistemas contendo massas de 3g, 10g e 40g foram avaliados. Foi avaliado o efeito do processo de extração com SMDFA sobre a integridade e infectividade de Adenovírus. Alguns dos parâmetros avaliados no processo foram a recuperação da potência viral (RPv) e a recuperação da potência viral específica (RPvø). Esses dois parâmetros avaliam a inativação do Adenovirus pelo processo de extração e ambos apresentaram melhoras quando comparados com a própria suspensão viral para alguns dos sistemas estudados (i.e RPv:341 % e RPvø 1466 %). Esses resultados indicam que o SMDFA foi capaz de particionar seletivamente as partículas virais infecciosas. De acordo com os resultados do planejamento é possível aumentar ainda mais esses resultados controlando as variáveis concentração de tensoativo, pH da suspensão viral e temperatura de extração.Biotechnological processes depend significantly on separation and purification techniques used to maintain cost-effective industrial-scale production of biotechnological products for commercial, industrial and therapeutic uses. The application of the aqueous two-phase micelar system (ATPMS) is proposed as an alternative for purification, since it allows the separation and analysis of biomolecules /bioparticles, often without loses of activity or their properties. This allows to perform a selective partition that enables high yields. This work aims to study the use of this methodology in the extraction and purification of adenovirus in micelle aqueous two-phase formed by TritonX-114/Viral suspension. All assays were performed in 5 g systems following a full factorial design (23) with four central points. The studied factors were extraction temperature, pH of the viral suspension and concentration of the surfactant. Systems containing masses of 3g, 10g and 40g were evaluated. Extraction procedure effects over integrity and infectivity of adenovirus were also evaluated. Some of the parameters evaluated in the viral recovery process were viral potency (RPv) and recovery of viral specific potency (RPvø). These two parameters measure the inactivation of Adenovirus by the extraction process and both showed improvement when compared with the viral suspension for some of the systems studied (i.e RPv: 341% and RPvø 1466%). These results show that ATPMS selectively partition the infectious viral particles. According to the results of the experimental design is possible to increase, even further, these results controlling the surfactant concentration, viral suspension pH and temperature of extraction

Comparison of secretory signal peptides for heterologous protein expression in microalgae: Expanding the secretion portfolio for <i>Chlamydomonas reinhardtii</i>

<div><p>Efficient protein secretion is a desirable trait for any recombinant protein expression system, together with simple, low-cost, and defined media, such as the typical media used for photosynthetic cultures of microalgae. However, low titers of secreted heterologous proteins are usually obtained, even with the most extensively studied microalga <i>Chlamydomonas reinhardtii</i>, preventing their industrial application. In this study, we aimed to expand and evaluate secretory signal peptides (SP) for heterologous protein secretion in <i>C</i>. <i>reinhardtii</i> by comparing previously described SP with untested sequences. We compared the SPs from arylsulfatase 1 and carbonic anhydrase 1, with those of untried SPs from binding protein 1, an ice-binding protein, and six sequences identified <i>in silico</i>. We identified over 2000 unique SPs using the SignalP 4.0 software. mCherry fluorescence was used to compare the protein secretion of up to 96 colonies for each construct, non-secretion construct, and parental wild-type cc1690 cells. Supernatant fluorescence varied according to the SP used, with a 10-fold difference observed between the highest and lowest secretors. Moreover, two SPs identified <i>in silico</i> secreted the highest amount of mCherry. Our results demonstrate that the SP should be carefully selected and that efficient sequences can be coded in the <i>C</i>. <i>reinhardtii</i> genome. The SPs described here expand the portfolio available for research on heterologous protein secretion and for biomanufacturing applications.</p></div

Comparison of mCherry fluorescence in the supernatant of different constructs.

<p>mCherry fluorescence was measured in supernatant. mCherry fluorescence was measured from 96 individual colonies, grown in a deep-well plate in TAP media for 7 d under constant illumination and agitation. Data presented in the boxplot were collected from colonies where the total fluorescence signal was higher than the auto-fluorescence of the parental wild-type cc1690, above three standard deviations. pAH04 –construct without SP; pJP22 –construct with arylsulfatase 1 SP; pJP26 –construct with binding protein 1 SP; pJP28 –construct with carbonic anhydrase 1 SP; pJP29 –construct with ice-binding protein 1 SP; pJP30-35 –construct with <i>in silico</i> identified SP (iSP). n–number of positive results obtained for each construct. * represents the average result. ○ represents the outliers. No normalization was conducted for mCherry fluorescence.To confirm this hypothesis, a positive colony of each construct with the highest fluorescence was cultivated in 50 mL of TAP media for 7 d, and mCherry fluorescence was determined in both the total cultures and in the supernatant (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192433#pone.0192433.s002" target="_blank">S2 Fig</a>). The supernatant percentage of mCherry fluorescence in the pAH04 strain cultivated in the flask was lower relative to that of the pAH0A strain cultivated on the plate (from 42% on the plate to ~8.5% in the flask), which was consistent with the culture condition hypothesis. Although the test in the flask presented a lower noise, it lacks the throughput to test several colonies, an important feature when comparing different construct designs. Since transformation is based mainly on a random insertion by non-homologous end joining (NHEJ) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192433#pone.0192433.ref039" target="_blank">39</a>], colonies presented a wide range of expression efficiency, from a relative standard deviation of 42.7% to 102.7% (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192433#pone.0192433.s008" target="_blank">S1 Table</a>). Therefore, we chose the 96 well plate assay to compare constructs efficiency since it could prevent sampling bias.</p

mCherry compartmentalization in immunoblotting analysis.

<p>Black arrow–mCherry bands, 27 KDa for unmodified and 29 KDa for post-translational modified; Grey arrow–mCherry band still fused with sh-ble and 2A autocleavage peptide, 46 KDa; Sup.–supernatant; Lys.–lysate, cc1690 –parental wild-type strain; pAH04 –construct without SP; pJP22 –construct with arylsulfatase 1 SP; pJP26 –construct with binding protein 1 SP; pJP28 –construct with carbonic anhydrase 1 SP; pJP29 –construct with ice-binding protein 1 SP; pJP30-35 –construct with <i>in silico</i> identified SP.</p

Comparison of mCherry fluorescence among expression vector constructs in the total culture.

<p>mCherry fluorescence was measured in the total culture sample. mCherry fluorescence was measured from 96 individual colonies, grown in a deep-well plate in TAP media for 7 d under constant illumination and agitation. Data presented in the boxplot were collected from colonies in which the total fluorescence signal was higher than that of the auto fluorescence signal of the parental wild-type cc1690, above three standard deviations. pAH04 –construct without SP; pJP22 –construct with arylsulfatase 1 SP; pJP26 –construct with binding protein 1 SP; pJP28 –construct with carbonic anhydrase 1 SP; pJP29 –construct with ice-binding protein 1 SP; pJP30-35 –construct with <i>in silico</i> identified SP (iSP). n–number of positive results obtained for each construct. * represents the average result. ○ represents the outliers. No normalization was conducted for mCherry fluorescence.</p

Characteristics of the signal peptide tested.

<p>Characteristics of the signal peptide tested.</p

Live-cell fluorescence microscopy of the mCherry-expressing strains.

<p>The mCherry signal from the non-secreting pAH04 construct is distributed in the cytosol, while the secreting pJP transformants present a mCherry signal in vesicles. Live cells were plated on agar pads and images were acquired 0.4 -μm apart in each channel in the z-axis. Then, images were stacked using the Fiji software Z projects function, generating the final images. An argon laser at 543 nm was used to excite mCherry, and a spectral detector set at approximately 610–650 nm was used to detect emitted fluorescence. For chlorophyll, we used a laser at 405 nm for excitation, and a spectral detector set at 680 nm. cc1690 –parental wild-type strain; pAH04 –construct without SP; pJP22 –construct with arylsulfatase 1 SP; pJP26 –construct with binding protein 1 SP; pJP28 –construct with carbonic anhydrase 1 SP; pJP29 –construct with ice-binding protein 1 SP; pJP30-35 –construct with <i>in silico</i> identified SP. All images were processed identically. Scale bar = 5 μm.</p