Advances in Synthetic Biology Approaches for Production of High Value Products in Chloroplasts

Chloroplasts are ideal bioreactors for production of proteins but require synthetic biology approaches to express eukaryotic genes utilizing prokaryotic protein synthetic machinery. A codon optimization algorithm has been developed utilizing highly expressed chloroplast genes from 133 sequenced chloroplast genomes using Java (Plant Physiology 172:62). High level expression of the largest human protein (891 kDa) – pentameric form of human Blood Clotting Factor VIII was demonstrated using this synthetic approach (Plant Biotechnology Journal 16: 1148). In addition, selectable marker genes have been removed to facilitate regulatory approval after expression of therapeutic, food or feed enzymes or proteins in lettuce chloroplasts. Recent advances in this field including commercial scale production of human therapeutic proteins in FDA-approved cGMP facilities (Biomaterials 70: 84-93), development of tags to deliver protein drugs to targeted human cells or tissues (Biomaterials 80: 68-79), methods to quantify in planta drug dose using proteomic quantitation by parallel reaction monitoring analysis (Plant Physiology 172:62-77), long-term stability of proteins/enzymes at ambient temperature (Molecular Therapy, 24: 1342-1350), testing human drug doses in large animals (Molecular Therapy 25: 512-522), toxicology, pharmacokinetic and pharmacodynamics studies to obtain regulatory approval will be presented (Annual Review of Genetics, 50: 595-618; Genome Biology 17:134).

Five newly launched leaf-products will be compared here with 23 commercial microbial-enzyme products for textile, detergent or juice industries. Crude leaf-extract enzymes are functional at low concentrations without protease inhibitors. Contact-angle water droplet absorption by the FAMAS bioscouring videos exceeds 3-second industry requirements. Leaf-lipase/mannanase crude-extracts remove chocolate/mustard oil stains more efficiently at 70°C than commercial enzymes (<10% activity). Endo/exoglucanase crude leaf-extracts remove dye efficiently from denim surface and de-pilled knitted fabric. Leaf-pectinase powder efficiently clarified orange juice pulp. Thus, leaf-production platform offers a novel low-cost approach by elimination of fermentation, purification, concentration, formulation and cold-chain to revolutionize healthcare and enzyme industries.