Abstract
BackgroundThe use of cyanobacteria and microalgae as cell factories to produce biofuels and added-value bioproducts has received great attention during the last two decades. Important investments have been made by public and private sectors to develop this field. However, it has been a challenge to develop a viable and cost-effective platform for cultivation of cyanobacteria and microalgae under outdoor conditions. Dealing with contamination caused by bacteria, weedy algae/cyanobacteria and other organisms is a major constraint to establish effective cultivation processes.ResultsHere, we describe the implementation in the cyanobacterium Synechococcus elongatus PCC 7942 of a phosphorus selective nutrition system to control biological contamination during cultivation. The system is based on metabolic engineering of S. elongatus to metabolize phosphite, a phosphorus source not normally metabolized by most organisms, by expressing a bacterial phosphite oxidoreductase (PtxD). Engineered S. elongatus strains expressing PtxD grow at a similar rate on media supplemented with phosphite as the non-transformed control supplemented with phosphate. We show that when grown in media containing phosphite as the sole phosphorus source in glass flasks, the engineered strain was able to grow and outcompete biological contaminants even when the system was intentionally inoculated with natural competitors isolated from an irrigation canal. The PtxD/phosphite system was successfully used for outdoor cultivation of engineered S. elongatus in 100-L cylindrical reactors and 1000-L raceway ponds, under non-axenic conditions and without the need of sterilizing containers and media. Finally, we also show that the PtxD/phosphite system can be used as selectable marker for S. elongatus PCC 7942 transgenic strains selection, eliminating the need of antibiotic resistance genes.ConclusionsOur results suggest that the PtxD/phosphite system is a stable and sufficiently robust strategy to control biological contaminants without the need of sterilization or other complex aseptic procedures. Our data show that the PtxD/phosphite system can be used as selectable marker and allows production of the cyanobacterium S. elongatus PCC 7942 in non-axenic outdoor reactors at lower cost, which in principle should be applicable to other cyanobacteria and microalgae engineered to metabolize phosphite.
Highlights
The use of cyanobacteria and microalgae as cell factories to produce biofuels and added-value bio‐ products has received great attention during the last two decades
González‐Morales et al Biotechnol Biofuels (2020) 13:119 elongatus PCC 7942 in non-axenic outdoor reactors at lower cost, which in principle should be applicable to other cyanobacteria and microalgae engineered to metabolize phosphite
We show that the phosphite oxidoreductase (PtxD)/Phi system can be used as a selectable marker in S. elongatus PCC 7942
Summary
The use of cyanobacteria and microalgae as cell factories to produce biofuels and added-value bio‐ products has received great attention during the last two decades. Cyanobacteria are emerging as promising systems for biotechnological applications They offer a number of advantages over other microorganisms, including rapid reproduction with conversion rates into biomass much higher than that of plants, as well as the possibility of photoautotrophic cultivation harvesting environmental CO2 and harnessing solar light energy [1]. Progress in developing genetic and genomic tools has been slow compared to that for bacteria or even plants, genetic manipulation of some cyanobacterial strains by classical genetic approaches, gene transfer, and genome editing-based techniques is possible. Model cyanobacteria such as Synechococcus elongatus PCC 7942 and UTEX 2973, and Synechocystis sp. Production costs become a major factor in selecting cyanobacterial versus bacterial as production systems
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