Chloroplast genome engineering: Recent advances, pathways towards commercialization and future directions

Chloroplasts are ideal biofactories because thousands of transgene copies integrated into each plant cell to produce high levels of recombinant proteins and enzymes. Because chloroplast genomes are predominantly maternally inherited, transgenes are generally not transmitted via pollen. Nuclear genome engineering ushered in the GMO revolution for crop protection that mostly required lower levels of transgene expression. Higher level expression and biosafety through gene containment facilitate new applications of GM crops, including production of high-value biopharmaceuticals and enzymes. This review focuses on recent advances in this field, including the first FDA approval of biopharmaceuticals made in plant cells for evaluation in human clinical trials, topical or oral drug delivery, marker free edible plants by removal of antibiotic resistance genes and novel post-translational modifications required to produce functional human proteins. In addition, new tools are discussed to improve DNA delivery, vector free assembly, episomal or integration methods, enhancement of transgene expression through optimization of regulatory sequences, codons, targeted genome editing and other innovations in biomanufacturing. Topical drug delivery using plant cells incorporated into chewing gums to control oral viruses, pathogenic bacteria, or yeast and oral drug delivery with therapeutic proteins made in plant cells to treat diabetes, obesity and cardiopulmonary diseases currently advanced to human clinical trials are discussed. In addition, high-value industrial enzymes for launching new products or to confer valuable agronomic traits or enhance photosynthesis are discussed. Future perspectives section provides examples of biotechnology products anticipated to be launched in the next 3-5 years, with opportunities and anticipated challenges.