Validation of leaf and microbial pectinases: commercial launching of a new platform technology

Abstract

Summary Almost all current genetically modified plant commercial products are derived from seeds. The first protein product made in leaves for commercial use is reported here. Leaf pectinases are validated here with eight liquid commercial microbial enzyme products for textile or juice industry applications. Leaf pectinases are functional in broad pH /temperature ranges as crude leaf extracts, while most commercial enzyme products showed significant loss at alkaline pH or higher temperature, essential for various textile applications. In contrast to commercial liquid enzymes requiring cold storage/transportation, leaf pectinase powder was stored up to 16 months at ambient temperature without loss of enzyme activity. Commercial pectinase products showed much higher enzyme protein PAGE than crude leaf extracts with comparable enzyme activity without protease inhibitors. Natural cotton fibre does not absorb water due to hydrophobic nature of waxes and pectins. After bioscouring with pectinase, measurement of contact‐angle water droplet absorption by the FAMAS videos showed 33 or 63 (leaf pectinase), 61 or 64 (commercial pectinase) milliseconds , well below the 10‐second industry requirements. First marker‐free lettuce plants expressing pectinases were also created by removal of the antibiotic resistance aadA gene. Leaf pectinase powder efficiently clarified orange juice pulp similar to several microbial enzyme products. Commercial pilot scale biomass production of tobacco leaves expressing different pectinases showed that hydroponic growth at Fraunhofer yielded 10 times lower leaf biomass per plant than soil‐grown plants in the greenhouse. Pectinase enzyme yield from the greenhouse plants was double that of Fraunhofer. Thus, this leaf‐production platform offers a novel, low‐cost approach for enzyme production by elimination of fermentation, purification, concentration, formulation and cold chain.

Validation of leaf enzymes in the detergent and textile industries: launching of a new platform technology

“…Detail protocol of transplastomic tobacco biomass production and lyophilization is discussed in (Daniell et al ., ).…”

“…Transplastomic tobacco biomass and lyophilization Detail protocol of transplastomic tobacco biomass production and lyophilization is discussed in (Daniell et al, 2019).…”

In‐depth characterization of Trichoderma reesei cellobiohydrolase TrCel7A produced in Nicotiana benthamiana reveals limitations of cellulase production in plants by host‐specific post‐translational modifications

“…Plant‐based enzyme production offers a highly promising approach, due to low production costs, high attainable expression levels and approved good manufacturing practices (GMPs). Moreover, two plant expression systems have been launched for commercial production of recombinant enzymes, vaccines and biopharmaceuticals (https://www.leafexpressionsystems.com/ and https://www.pennovation.upenn.edu/the-community/innovators/phyllozyme; Daniell et al ., ). Leaf Expression Systems is a transient expression‐based system using Agrobacterium ‐mediated transient expression in leaves, while PhylloZyme is an Agrobacterium ‐free chloroplast genome engineering technology for commercial production of recombinant enzymes (Daniell et al ., ).…”

“…Moreover, two plant expression systems have been launched for commercial production of recombinant enzymes, vaccines and biopharmaceuticals (https://www.leafexpressionsystems.com/ and https://www.pennovation.upenn.edu/the-community/innovators/phyllozyme; Daniell et al ., ). Leaf Expression Systems is a transient expression‐based system using Agrobacterium ‐mediated transient expression in leaves, while PhylloZyme is an Agrobacterium ‐free chloroplast genome engineering technology for commercial production of recombinant enzymes (Daniell et al ., ). These systems can contribute to the cost‐effective, large‐scale production of enzymes for various industrial applications, including the processing of lignocellulosic biomass.…”

“…However, the yield of recombinant protein varies between plant production systems and depends on many factors, including the choice of the host plant, expression method, the protein of interest and its properties, the plant cultivation system and the steps involved in downstream purification. For example, in a leaf‐based production platform (Daniell et al ., ), the yield of leaf biomass was reported to be much higher (approximately 10‐fold) in soil‐grown plants than in hydroponic plants. While some data are available on recombinant protein stability, the catalytic properties and the effects of PTMs on enzyme activity have not been studied in detail and more investigations are needed.…”