High-Level Production of Recombinant Snowdrop Lectin in Sugarcane and Energy Cane

Abstract

Sugarcane and energy cane (Saccharum spp. hybrids) are ideal for plant-based production of recombinant proteins because their high resource-use efficiency, rapid growth and efficient photosynthesis enable extensive biomass production and protein accumulation at a cost-effective scale. Here, we aimed to develop these species as efficient platforms to produce recombinant Galanthus nivalis L. (snowdrop) agglutinin (GNA), a monocot-bulb mannose-specific lectin with potent antiviral, antifungal and antitumor activities. Initially, GNA levels of 0.04% and 0.3% total soluble protein (TSP) (0.3 and 3.8 mg kg −1 tissue) were recovered from the culms and leaves, respectively, of sugarcane lines expressing recombinant GNA under the control of the constitutive maize ubiquitin 1 (Ubi) promoter. Co-expression of recombinant GNA from stacked multiple promoters (pUbi and culm-regulated promoters from sugarcane dirigent5-1 and Sugarcane bacilliform virus) on separate expression vectors increased GNA yields up to 42.3-fold (1.8% TSP or 12.7 mg kg −1 tissue) and 7.7-fold (2.3% TSP or 29.3 mg kg −1 tissue) in sugarcane and energy cane lines, respectively. Moreover, inducing promoter activity in the leaves of GNA transgenic lines with stress-regulated hormones increased GNA accumulation to 2.7% TSP (37.2 mg kg −1 tissue). Purification by mannoseagarose affinity chromatography yielded a functional sugarcane recombinant GNA with binding substrate specificity similar to that of native snowdrop-bulb GNA, as shown by enzyme-linked lectin and mannose-binding inhibition assays. The size and molecular weight of recombinant GNA were identical to those of native GNA, as determined by size-exclusion chromatography and MALDI-TOF mass spectrometry. This work demonstrates the feasibility of producing recombinant GNA at high levels in Saccharum species, with the long-term goal of using it as a broad-spectrum antiviral carrier molecule for hemopurifiers and in related therapeutic applications.

Metabolic engineering of energycane to hyperaccumulate lipids in vegetative biomass

“…However, its recalcitrance in regard to tissue culture and genetic transformation has so far prevented complex metabolic engineering and molecular pharming approaches. To the best of our knowledge, only one earlier report presents energycane as a production platform for recombinant protein [ 39 ].…”

“…Two such reports provided detailed biolistic transformation protocols using neomycin phosphotransferase II ( npt II) [ 37 ] or bar as a selectable marker gene [ 38 ]. A third report described the use of transgenic energycane as a platform for producing a recombinant therapeutic protein via overexpression of a cDNA encoding snowdrop lectin [ 39 ]. Here, we report what is to the best of our knowledge the first metabolic engineering of energycane.…”

Identification of a Novel Anti-HIV-1 Protein from Momordica balsamina Leaf Extract

“…Hevamine A-like proteins are one of several members of a family of plant chitinases and lysozymes produced by plants known as “plant defensins” protective against microbial infections [ 16 ]. There are reports that establish the antitumor, antioxidant, antiviral, and anti-inflammatory properties of chitinases and chitinous materials [ 17 , 18 , 19 , 20 ]. Moreover, chitinases are used in other industries.…”

Plant-based expression platforms to produce high-value metabolites and proteins

“…Combinatorial plant transformation and co-expression of BvLz under the control of various constitutive and culm-regulated promoters yielded high levels of expression (up to 11.5% of total soluble protein) in sugarcane culms (Damaj et al, 2020). Similarly, Padilla et al (2020) successfully enhanced the expression of recombinant Galanthus nivalis L. (snowdrop) agglutinin (GNA) in sugarcane and energy cane. GNA possesses antiviral, antifungal, and antitumor activities.…”

“…Its expression was further increased to 1.8% TSP and 2.3% TSP in sugarcane and energy cane lines, respectively, by co-expressing recombinant GNA under multiple promoters (pUbi-1 and culm-regulated promoters from sugarcane dirigent5-1 and sugarcane bacilliform virus; pUBD5:GNA) from different expression vectors. Moreover, the expression of recombinant GNA in the triple promoter transgenic lines (pUBD5:GNA) was further boosted to 2.7% TSP by inducing promoter activity with salicylic acid ( Padilla et al., 2020 ). These studies demonstrate the great potential of the inducible promoters and combinatorial promoter stacking system to increase the accumulation of high-value therapeutic proteins in plants.…”