Abstract

BackgroundThe 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.ResultsHere, 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%.ConclusionsThe 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.

Highlights

  • The global demand for functional proteins is extensive, diverse, and constantly increasing

  • We showed that the response of plant protein metabolism to foreign protein synthesis is largely dependent on environmental factors

  • As we explore in this study, plant age, growth conditions, and fertilization regimes can cause variable recombinant protein yields

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Summary

Introduction

The global demand for functional proteins is extensive, diverse, and constantly increasing. 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. Proteins are used as enzyme additives in many industrial processes, as antibodies and medical peptides for pharmaceuticals, and as nutritional additives to food and animal feed (reviewed in [1]) These high-value proteins are mainly produced for such purposes using large-scale. Schmidt et al Biotechnol Biofuels (2021) 14:42 cultures of bacterial, fungal, or mammalian cells These systems are often expensive to establish, labor-intensive to maintain, prone to contamination, and inflexible to changing market demands [2]. Chloroplast-engineered plants regularly achieve recombinant protein yields averaging 5–20% of total soluble protein (TSP) with exceptional plastid transformants reaching yields of 40–70% of TSP [3, 9,10,11,12,13,14,15,16,17]

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