The phosphite oxidoreductase gene, ptxD as a bio-contained chloroplast marker and crop-protection tool for algal biotechnology using Chlamydomonas

Abstract

Edible microalgae have potential as low-cost cell factories for the production and oral delivery of recombinant proteins such as vaccines, anti-bacterials and gut-active enzymes that are beneficial to farmed animals including livestock, poultry and fish. However, a major economic and technical problem associated with large-scale cultivation of microalgae, even in closed photobioreactors, is invasion by contaminating microorganisms. Avoiding this requires costly media sterilisation, aseptic techniques during setup and implementation of 'crop-protection' strategies during cultivation. Here, we report a strain improvement approach in which the chloroplast of Chlamydomonas reinhardtii is engineered to allow oxidation of phosphite to its bioavailable form: phosphate. We have designed a synthetic version of the bacterial gene (ptxD)-encoding phosphite oxidoreductase such that it is highly expressed in the chloroplast but has a Trp→Opal codon reassignment for bio-containment of the transgene. Under mixotrophic conditions, the growth rate of the engineered alga is unaffected when phosphate is replaced with phosphite in the medium. Furthermore, under non-sterile conditions, growth of contaminating microorganisms is severely impeded in phosphite medium. This, therefore, offers the possibility of producing algal biomass under non-sterile conditions. The ptxD gene can also serve as a dominant marker for genetic engineering of any C. reinhardtii strain, thereby avoiding the use of antibiotic resistance genes as markers and allowing the 'retro-fitting' of existing engineered strains. As a proof of concept, we demonstrate the application of our ptxD technology to a strain expressing a subunit vaccine targeting a major viral pathogen of farmed fish.

Metabolic engineering of phosphite metabolism in Synechococcus elongatus PCC 7942 as an effective measure to control biological contaminants in outdoor raceway ponds

“…PCC 7002 [21,22,26]. Furthermore, the system was also re ned to develop biocontainment strategies through more complex molecular strategies [21,22,23]. Previous works with Synechocystis sp.…”

“…Our results suggest that one of the major advantages of using Phimetabolizing strains is the possibility of avoiding all these preventive measures, as the system does not require sterilizing growth media, reactors, tools and piping. Thus, reducing one of the major operating costs for large closed and open bioreactors [20,21,23]. Therefore, the system could potentially be implemented for microalgae and cyanobacteria species other than those capable of growing in extreme environmental conditions.…”

“…Therefore, if an accidental escape occurs, also considering that high concentrations of Phi are required to sustain the normal growth (0.8 to 1.8 mM) of the transgenic strain, the Phi metabolism trait would not represent a competitive advantage in natural conditions and the transgenic strains would display similar requirements as those of the WT counterpart in a natural community. Nevertheless, it is important to consider biocontainment strategies as the ones already developed with the PtxD/Phi system to avoid any risk due accidental releases and of horizontal gene transfer [21,23]. However, as these biocontainment strategies have only been assessed on closed systems and very small scale, their validation in raceway ponds in outdoor conditions is also necessary.…”

“…More recently, the Phi metabolism was also established as a biocontainment strategy, that allows the control of proliferation of genetically modi ed S. elongatus in case an accidental release to the environment, as well as a selectable marker for microalgae, cyanobacteria, and several plant species [21,22,23,24,25]. Biotechnological applications of the selective nutrition based on Phi and the ptxD gene for microalgae/cyanobacteria cultivation have been successful as a proof of concept using small volume closed or semi-closed systems [20,21,23,26]. However, there are genuine concerns regarding the stability and robustness of the Phi metabolism trait for cultivation in larger scale reactors in outdoor conditions.…”

A Phosphite Dehydrogenase Variant with Promiscuous Access to Nicotinamide Cofactor Pools Sustains Fast Phosphite-Dependent Growth of Transplastomic Chlamydomonas reinhardtii

“…Since most parasitic organisms cannot use phosphite in their metabolism, the expression of a PTXD transgene in the nucleus [15] was shown to enable the selective growth of C. reinhardtii in the presence of phosphite as the sole phosphorous source, effectively preventing the risk of culture contamination by other species competing for available resources. More recently, PTXD was successfully expressed as a plastid-targeted transgene in C. reinhardtii [16,17], emerging as a novel selectable marker for the genetic transformation of the plastome [18]. Despite being an attractive system for the chloroplast transformation of C. reinhardtii, the use of PTXD as a selectable marker suffers from some intrinsic limitations that hinder its value.…”