A novel genetic engineering platform for the effective management of biological contaminants for the production of microalgae

Abstract

SummaryMicroalgal cultivation that takes advantage of solar energy is one of the most cost‐effective systems for the biotechnological production of biofuels, and a range of high value products, including pharmaceuticals, fertilizers and feed. However, one of the main constraints for the cultivation of microalgae is the potential contamination with biological pollutants, such as bacteria, fungi, zooplankton or other undesirable microalgae. In closed bioreactors, the control of contamination requires the sterilization of the media, containers and all materials, which increases the cost of production, whereas open pond systems severely limits the number of species that can be cultivated under extreme environmental conditions to prevent contaminations. Here, we report the metabolic engineering of Chlamydomonas reinhardtii to use phosphite as its sole phosphorus source by expressing the ptxD gene from Pseudomonas stutzeri WM88, which encodes a phosphite oxidoreductase able to oxidize phosphite into phosphate using NAD as a cofactor. Engineered C. reinhardtii lines are capable of becoming the dominant species in a mixed culture when fertilized with phosphite as a sole phosphorus source. Our results represent a new platform for the production of microalgae, potentially useful for both closed photobioreactors and open pond systems without the need for using sterile conditions nor antibiotics or herbicides to prevent contamination with biological pollutants.

Assessment of the ptxD gene as a growth and selective marker in Trichoderma atroviride using Pccg6, a novel constitutive promoter

“…The engineered strains were able to grow and outcompete the non-transformed control strains when grown in Phi-based medium [50]. Moreover, we previously reported that expression of PTXD plants and microalgae allowed the creation of a highly selective environment that favors the development of the engineered organisms while compromising the growth of complex mixtures of weedy organisms (plants, microalgae, fungi and bacteria) unable to metabolize Phi [47,48,65,66]. Moreover, the use of Phi was shown to be an efficient strategy to control contaminant organisms (i.e.…”

“…PTXD converts Phi into phosphate allowing organisms that express this enzyme to use Phi as a sole P source, a trait that is not present in most eukaryotic and is present only in few bacterial strains [46,47]. Therefore, PTXD can act as a growth reporter gene, for which enzymatic assays are also available [48,49]. However, there is no information about the functionality of the system in filamentous fungi.…”

Growth and selection of the cyanobacterium Synechococcus sp. PCC 7002 using alternative nitrogen and phosphorus sources

“…The A0935-A0936 site was assembled into the digested pUC19 backbone using the pEASY-Uni Seamless Cloning and Assembly Kit (TransGen Biotech Co., Ltd, China), according to the manufacturer's instructions, and transformed into Stellar E. coli cells, resulting in plasmid pSZT001. Primer pair A0935_UCO_F and A0936_UCO_R were used to reverse-PCR amplify the pSZT001 backbone and pair D98496996 and D100141467 to amplify a synthetic, codonoptimized version (by GenScript, Hong Kong, Ltd) of the Pseudomonas stutzeri WM88 phosphite dehydrogenase (ptxD) gene (Loera-Quezada et al, 2016), driven by the Amaranthus hybridus constitutive psbA promoter (Elhai and Wolk, 1988). Both fragments were assembled using the pEASY-Uni kit, as described above, resulting in plasmid pSJ135.…”

“…Recently, the use of ecologically rare or xenobiotic sources of macronutrients has been explored as a means to generate selective pressure towards the growth of genetically modified organisms without the use of antibiotics (Kanda et al, 2014;Loera-Quezada et al, 2016;Pandeya et al, 2017;Polyviou et al, 2015;Shaw et al, 2016) as well as to allow genetically modified plants to outcompete weeds, while consuming considerably less phosphorus (Lopez-Arredondo and Herrera-Estrella, 2012). To this end, phosphite dehydrogenase (PtxD), an enzyme converting phosphite, an ecologically rare form of phosphorus, into phosphate, has been introduced into a variety of organisms, and in some cases used as a selectable marker (Kanda et al, 2014;Lopez-Arredondo and Herrera-Estrella, 2012;Nahampun et al, 2016;Pandeya et al, 2017).…”

Microalgae‐based Pharmaceuticals and Nutraceuticals: An Emerging Field with Immense Market Potential

“…Engineered strains were able to use phosphite as main phosphorus source, making them a dominant species and reducing the chance of contamination [117] Chlamydomonas reinhardtii Single amino acid mutation L505F in the phytoene desaturase gene 29 % increase in PDS activity, resistance to norflurazon, and increased amount of zeta carotene [118] Chlamydomonas reinhardtii Plastid transformation of the psy gene from D. salina to C. reinhardtii…”