Promoter trapping in microalgae using the antibiotic paromomycin as selective agent

The lack of highly active endogenous promoters to drive the expression of transgenes is one of the main drawbacks to achieving efficient transformation of many microalgal species. Using the model chlorophyte Chlamydomonas reinhardtii and the paromomycin resistance APHVIII gene from Streptomyces rimosus as a marker, we have demonstrated that random insertion of the promoterless marker gene and subsequent isolation of the most robust transformants allows for the identification of novel strong promoter sequences in microalgae. Digestion of the genomic DNA with an enzyme that has a unique restriction site inside the marker gene and a high number of target sites in the genome of the microalga, followed by inverse PCR, allows for easy determination of the genomic region, which precedes the APHVIII marker gene. In most of the transformants analyzed, the marker gene is inserted in intragenic regions and its expression relies on its adequate insertion in frame with native genes. As an example, one of the new promoters identified was used to direct the expression of the APHVIII marker gene in C. reinhardtii, showing high transformation efficiencies.Junta de Andalucía (P09-CVI-5053)Junta de Andalucía (BIO-214

A novel biocompatible polymer derived from D-mannitol used as a vector in the field of genetic engineering of eukaryotic cells

The design and preparation of new vectors to transport genetic material and increase the transfection efficiency continue being an important research line. Here, a novel biocompatible sugar-based polymer derived from Dmannitol has been synthesized to be used as a gene material nanocarrier in human (gene transfection) and microalga cells (transformation process). Its low toxicity allows its use in processes with both medical and industrial applications. A multidisciplinary study about the formation of polymer/p-DNA polyplexes has been carried out using techniques such as gel electrophoresis, zeta potential, dynamic light scattering, atomic force microscopy, and circular dichroism spectroscopy. The nucleic acids used were the eukaryotic expression plasmid pEGFP-C1 and the microalgal expression plasmid Phyco69, which showed different behaviors. The importance of DNA supercoiling in both transfection and transformation processes was demonstrated. Better results were obtained in microalga cells nuclear transformation than in human cells gene transfection. This was related to the plasmid’s conformational changes, in particular to their superhelical structure. It is noteworthy that the same nanocarrier has been used with eukaryotic cells from both human and microalga.This work was supported by the Consejería de Conocimiento, Innovación y Universidades de la Junta de Andalucía (FQM-206, FQM-274, FQM-135 and P20–01234); VI Plan Propio Universidad de Sevilla (PP2019/00000748), and the European Union (Feder Funds)

Inhibición de la enzima fitoeno desaturasa y acumulación de fitoeno en microalgas : el irna como mecanismo de silenciamiento génico

Los organismos marinos, y en particular las microalgas son una fuente importante de infinidad de productos naturales con potenciales aplicaciones terapéuticas e industriales. Las microalgas son la principal fuente natural de un buen número de carotenoides naturales, entre los que podemos citar β-caroteno, fitoeno, astaxantina o luteína, por lo que su explotación industrial para la producción de estos carotenoides está bastante extendida. En particular, estos carotenoides presentan un especial interés por su capacidad antioxidante, colorante, provitamínica y terapéutica. Este trabajo se basa en el estudio y manipulación de la ruta de síntesis de carotenoides, carotenogénesis, en microalgas. Estudiemos la acumulación del fitoeno en microalgas, mediante mecanismos químicos (con el uso de inhibidores) y mediante mecanismos genéticos (silenciamiento de genes mediante construcciones de ARN interferencia). Además hemos obtenido una pequeña colección de mutantes sensibles a altas intensidades de luz que nos permitan conocer más acerca de su ruta.------------------------------------------------------The marine organisms, and especially the microalgae, are important source of natural products with potencial in therapeutic and industrial applications. Specifically, microalgae are one of the main sources of a wide number of carotenoids, like β-carotene, phytoene, astaxanthin or lutein. These carotenoids are important because of their antioxidant character and can be used as colour additives, provitamins and therapeutic molecules. Nevertheless, their production at industrial scale has yet to be improved, being the molecular biology an important tool to understand and optimize the process. This work has a first approach where the understanding of the carotenogenesis (the route of synthesis of carotenoids) was the main goal. Then, the manipulation of this route was also studied. In this sense, the accumulation of phytoene in microalgae was evaluated by using chemical mechanisms (inhibitors) and genetic mechanisms (genes silencing). By other hand, obtaining sensitives mutants to high light allowed us to know more about the carotenogenesis too

Overexpression of acetyl-CoA synthetase (ACS) enhances the biosynthesis of neutral lipids and starch in the green microalga Chlamydomonas reinhardtii

Genetic engineering can be the solution to achieve the economically feasible production of microalgal based biofuels and other bulk materials. A good number of microalgal species can grow mixotrophically using acetate as carbon source. Moreover, experimental evidence suggests that the biosynthesis of acetyl-CoA could be a limiting step in the complex multifactor-dependent biosynthesis of acylglycerides and point to acetyl-CoA synthetase (ACS) as a key enzyme in the process. In order to test this hypothesis we have engineered the model chlorophyte Chlamydomonas reinhardtii to overexpress the endogenous chloroplastic acetyl-CoA synthetase, ACS2. Expression of the ACS2 encoding gene under the control of the strong constitutive RBCS2 promoter in nitrogen-replete cultures resulted in a 2-fold increase in starch content and 60% higher acyl-CoA pool compared to the parental line. Under nitrogen deprivation, the Cr-acs2 transformant shows 6-fold higher levels of ACS2 transcript and a 2.4-fold higher accumulation of triacylglycerol (TAG) than the untransformed control. Analysis of lipid species and fatty acid profiles in the Cr-acs2 transformant revealed a higher content than the parental strain in the major glycolipids and suggests that the enhanced synthesis of triacylglycerol in the transformant is not achieved at the expense of membrane lipids, but is due to an increase in the carbon flux towards the synthesis of acetyl-CoA in the chloroplast. These data demonstrate the potential of engineering the chloroplastic ACS to increase the carbon flux towards the synthesis of fatty acids as an alternative strategy to enhance the biosynthesis of lipids in microalgae.Part of this work has been supported by research grants from the Spanish (AGL2016-74866-C32R-AEI/FEDER) and European (INTERREG VA POCTEP 2014-20_055 ALGARED_PLUS_5E) governments. The help of CEICAMBIO and CEIMAR University Excellence Campuses is also acknowledged. Rothamsted Research receives grant aided support from the Biotechnology and Biological Research Sciences Council (BBSRC). Haslam, Smith and Sayanova are funded under the BBSRC Institute Strategic Programme grant Tailoring Plant Metabolism (BBS/E/C/000I0420)

Validation of a New Multicistronic Plasmid for the Efficient and Stable Expression of Transgenes in Microalgae

Low stability of transgenes and high variability of their expression levels among the obtained transformants are still pending challenges in the nuclear genetic transformation of microalgae. We have generated a new multicistronic microalgal expression plasmid, called Phyco69, to make easier the large phenotypic screening usually necessary for the selection of high‐expression stable clones. This plasmid contains a polylinker region (PLK) where any gene of interest (GOI) can be inserted and get linked, through a short viral self‐cleaving peptide to the amino terminus of the aminoglycoside 3’‐phosphotransferase (APHVIII) from Streptomyces rimosus, which confers resistance to the antibiotic paromomycin. The plasmid has been validated by expressing a second antibiotic resistance marker, the ShBLE gene, which confers resistance to phleomycin. It has been shown, by RT‐PCR and by phenotypic studies, that the fusion of the GOI to the selective marker gene APHVIII provides a simple method to screen and select the transformants with the highest level of expression of both the APHVIII gene and the GOI among the obtained transformants. Immunodetection studies have shown that the multicistronic transcript generated from Phyco69 is correctly processed, producing independent gene products from a common promoter