Engineered Crops Could Produce Low Cost Medicines and Industrial Proteins

The results of field trials carried out by scientists at Cornell University and the University of Illinois, Urbana-Champaign, suggest that engineered tobacco plants could feasibly be grown as crops for producing pharmaceutical and industrial enzymes and other proteins. Reporting on the first successful rearing of such plants in the field, the researchers, headed by Cornell University’s Beth Ahner, PhD, a professor in the department of biological and environmental engineering, suggest that it could be cheaper to produce such proteins in transgenic tobacco plants at the commercial scale than using current cell-based methods. “… we estimate that specific proteins could be obtained from field-grown transgenic tobacco plants at costs three orders of magnitude less than current cell culture methods,” they concluded in their published paper, which is titled, “Field-grown tobacco plants maintain robust growth while accumulating large quantities of a bacterial cellulase in chloroplasts.”

The global market for recombinant proteins for pharmaceutical and industrial applications is expected to reach $300 billion in the near future, the authors wrote. It’s a hugely diverse market, encompassing pharmaceuticals, as well as enzymes for consumer goods and industrial applications, and improving the nutritional value of foods and feed products. “Such diversity necessitates versatile and scalable production platforms that can meet varying market demands,” the researchers noted.

Most specialty proteins are produced using mammalian, insect, fungal, or bacterial cell cultures as protein factories, but these methods can be expensive to maintain, are prone to contamination, and can’t easily be modified if demand changes. In contrast, the team suggested, “field-grown transgenic crop plants may offer a safe and cost-effective alternative to augment or replace a large fraction of this large and growing market.”

While foreign gene expression in plants can be achieved by inserting the genes into either the nuclear genome, or into the DNA in the plant’s chloroplasts, commercial efforts have tended to focus on the former, rather than the latter strategy. “Chloroplast engineering is largely untapped by plant biotechnology companies due to a relatively recent shift in research focus from proof-of-concept to commercial applicability.” This may be slightly short-sighted, as the well-studied tobacco plant may offer a commercially viable option for chloroplast engineering, the researchers pointed out. “… plastid engineering is well established in tobacco, providing an agronomically viable platform due to its large yield of leaf material.”

In fact, they suggested, engineering plastid genomes rather than nuclear genomes could have a number of key advantages. In most crop plants chloroplasts are inherited from the female plant, which means any foreign genes wouldn’t be dispersed into the environment via pollen. “One of the advantages of the technology that we’re using is that the chloroplasts in most crop plants are inherited through the maternal line, so the genes are not in the pollen,” Ahner said. “The pollen is one of the main concerns for dispersal to other transgenic crops.”

There are also many thousands of plastid genomes—plastomes—in each cell, so introducing genes into plastid organelles would result in high copy number per cell. Plastids, in addition, exhibit high expression rates, so chloroplast-transformed plants should accumulate large amounts of recombinant proteins.

Although researchers have worked with transgenic plants in the laboratory and in greenhouses, field trials haven’t been carried out. The opportunity arose when University of Illinois plant biology professor Stephen Long, PhD, secured a permit from the U.S. Department of Agriculture to grow the genetically modified tobacco plants out in the field.

This go-ahead meant that the researchers could properly evaluate how well the transgenic plants would grow under variable environmental conditions, and investigate whether—as previous laboratory studies had found—generating transgenic proteins as a very high proportion of total soluble protein (TSP) in plants might come at a price, as that the engineered plans were often severely stunted. “The true test of industrial applicability of chloroplast-engineered plants is to analyze their growth and heterologous protein yield in the high-light, but less predictable environment of open-field cultivation,” the authors commented. “We knew these plants grew well in the greenhouse, but we just never had the opportunity to test them in the field,” Ahner added.

The scientists have now reported on their initial field trials using tobacco plants engineered to produce a bacterial protein, Cel6A, which is one of a group of industrially valuable cellulases that are used in manufacturing detergents, textiles, and in food an animal feed processing. Cheaper cellulases could also help to reduce the end cost of ethanol and other biomass-derived products.

The field trials showed that the transgenic tobacco crops could produce Cel6A as nearly 20% of TSP, without any affects on their overall growth. “Remarkably, Cel6A accumulation in our high yielding line was not drastically reduced when grown in the field compared to the chamber,” the investigators stated. They found that the transformed tobacco plants effectively increased total soluble protein synthesis to meet the demands of producing Cel6A, without affecting production of native proteins. “Even with the highly variable and unpredictable field growth conditions, the TetC-cel6A transformants maintained robust growth and remarkable Cel6A yield,” the team reported.

“When you put plants in the field, they have to face large transitions, in terms of drought or temperature or light, and they’re going to need all the protein that they have,” Ahner pointed out. “But we show that the plant still is able to function perfectly normally in the field [while producing nonnative proteins]. That was really the breakthrough.”

The results did show that Cel6A yields were reduced in the second year, but this may have been at least in part due to how the fields were fertilized. The authors acknowledge that it will be important to understand the exact nutrients needed by the engineered plants. “Fertilizing a field on an as-needed basis is a standard, and economically practical, farm management strategy, but the limitations exhibited by the Year 2 field transformants highlight the importance of carefully evaluating the unique nutrient requirements of chloroplast-engineered tobacco plants for commercial-scale production of a high-value protein.”

Whereas it costs approximately $10 to produce 1 kg of cellulase enzymes via traditional microbial bioreactor methods, the authors calculated that based on an assumed Cel6A yield of 20% TSP, and the ability to make multiple leaf tissue harvests in any one season, producing Cel6A in transgenic tobacco crops could cost as little as $8 per kg, but only if tobacco field cultivation was optimized for maximizing protein production. It’s also necessary to factor in downstream processing costs, which can account for up to 95% of the final production costs for purified medical proteins from plant-based production systems, they pointed out. “Thus, the final manufacturing cost of a pure protein produced from a plastid-engineered, field-grown tobacco plant is expected to be in the order of $0.16–1.20 g-1. For comparison, mammalian cell cultures utilizing Chinese hamster ovary cells have total production costs of $300–3000 g-1 recombinant protein, while production costs from transgenic goat milk are $105 g-1.”

The team acknowledged that its not yet known whether the process of purifying proteins from plant chloroplasts will generate particular challenges, but they state that the significantly lower production costs suggested by the initial field trials suggest that further trials should be undertaken to see if engineered tobacco plants could represent a viable option for producing high-value medical and industrial proteins. “The field trials represent a major step toward commercialization of chloroplast-transformed tobacco plants by demonstrating their profound resilience, even while burdened with remarkable heterologous protein accumulation,” the authors concluded.

In ongoing work, co-author Jennifer Schmidt, a graduate student in the Ahner lab, is investigating how to get plants to consistently produce different types of proteins. “We’re trying to understand the basic biological mechanism that allows any protein to be accumulated” in a genetically modified plant, Ahner said.