Mitigation of deleterious phenotypes in chloroplast-engineered plants accumulating high levels of foreign proteins

Abstract

Background The global demand for functional proteins is extensive, diverse, and constantly increasing. Medicine, agriculture, and industrial manufacturing all rely on high-quality proteins as major active components or process additives. Historically, these demands have been met by microbial bioreactors that are expensive to operate and maintain, prone to contamination, and relatively inflexible to changing market demands. Well-established crop cultivation techniques coupled with new advancements in genetic engineering may offer a cheaper and more versatile protein production platform. Chloroplast-engineered plants, like tobacco, have the potential to produce large quantities of high-value proteins, but often result in engineered plants with mutant phenotypes. This technology needs to be fine-tuned for commercial applications to maximize target protein yield while maintaining robust plant growth. Results Here, we show that a previously developed Nicotiana tabacum line, TetC-cel6A, can produce an industrial cellulase at levels of up to 28% of total soluble protein (TSP) with a slight dwarf phenotype but no loss in biomass. In seedlings, the dwarf phenotype is recovered by exogenous application of gibberellic acid. We also demonstrate that accumulating foreign protein represents an added burden to the plants’ metabolism that can make them more sensitive to limiting growth conditions such as low nitrogen. The biomass of nitrogen-limited TetC-cel6A plants was found to be as much as 40% lower than wildtype (WT) tobacco, although heterologous cellulase production was not greatly reduced compared to well-fertilized TetC-cel6A plants. Furthermore, cultivation at elevated carbon dioxide (1600 ppm CO2) restored biomass accumulation in TetC-cel6A plants to that of WT, while also increasing total heterologous protein yield (mg Cel6A plant−1) by 50–70%. Conclusions The work reported here demonstrates that well-fertilized tobacco plants have a substantial degree of flexibility in protein metabolism and can accommodate considerable levels of some recombinant proteins without exhibiting deleterious mutant phenotypes. Furthermore, we show that the alterations to protein expression triggered by growth at elevated CO2 can help rebalance endogenous protein expression and/or increase foreign protein production in chloroplast-engineered tobacco.

Developing climate‐resilient crops: improving plant tolerance to stress combination

“…This is another aspect of high CO 2 and stress combination that needs to be addressed, particularly in light of the potential negative effects of stress combinations on NUE. Because the breath of high CO 2 effects on crops is too vast to cover here, the reader is directed to several excellent reviews on the subject (Ainsworth and Long, 2021;Bright and Lund, 2021;Delabre et al, 2021;Green et al, 2020;Kurganskiy et al, 2021;Lamichaney and Maity, 2021;Lu et al, 2020;Penuelas et al, 2020;Schmidt et al, 2021;Smith, 2021;Soh et al, 2019;Tausz-Posch et al, 2020;Toreti et al, 2020;Wang and Liu, 2021;Wang et al, 2020;Xu et al, 2020).…”

Efficient expression of fusion human epidermal growth factor in tobacco chloroplasts

“…Details on these advantages have been illustrated by Lössl and Waheed [ 50 ] and other reviews [ 51 , 52 , 53 , 54 ]. Many proteins have been successfully expressed in plant plastids and summarized in previous reviews [ 41 , 55 , 56 , 57 , 58 , 59 , 60 , 61 , 62 , 63 , 64 ]. Up to date, there have been three reports that presented the expression of EGF in plant chloroplasts.…”