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Research Highlights

Research Highlights
Genome-Guided Modification of Soybean Meal Composition

By Minviluz G. Stacey, University of Missouri

Of the 20 amino acids, there are a smaller number that humans and animals cannot synthesize, and thus must be obtained from their diet. These are called essential amino acids. All amino acids accumulate in seed storage proteins such as 7S and 11S, whose main function is to sequester nitrogen, carbon and sulfur in the form of proteins, or chains of amino acids. The seed storage proteins found in soybean meal are the major source of amino acids for livestock consumption worldwide, including both essential and non-essential amino acids. Compared to other common plant-based protein feedstocks, soybean meal is superior in terms of both crude protein content and the availability of total and digestible amino acids. However, the levels of the sulfur-containing amino acids, namely methionine and cysteine, in soybean seeds are sub-optimal. Recognizing the need to improve the quality of soybean meal, one of the goals of this project is to increase the content of sulfur-containing amino acids in soybean seeds by at least 1.5-fold.

Figure 1. CRISPR/Cas9-edited soybean growing under greenhouse (A) and field (B) conditions

To achieve this goal, $113,532 was allocated in 2019, and a potentially promising “push-pull” approach was developed. The end goal is to modify the genetic structure in order to alter the seed metabolism to favor the production of seed storage proteins with higher levels of sulfur-containing amino acids. There are three parts of this strategy: production of high levels of free methionine; production of reduced amounts of methionine-poor 7S soybean seed storage protein; and increased amounts of methionine-rich 11S storage protein, along with production of modified 11S (i.e., GY1) storage proteins with an increased number of methionine codons.

To increase free methionine levels in soybean seeds (trait 1, high free-met), the production of enzymes that degrade methionine was knocked out, thereby increasing methionine availability in seeds for use in protein biosynthesis. CRISPR-edited plants were grown in the field, and the free methionine in their seeds was measured. The high free-met seeds indeed contained about a six-fold higher free methionine when compared to wild-type unmodified seeds. Free methionine levels are very low, so a six-fold increase is not enough by itself to reach the goal.

Figure 2. Generation of soybean genotypes lacking 7S proteins. A. SDS-PAGE of seed proteins. B. Methionine content of 7S knock-out mutants.

To obtain genotypes with the second desired trait (reduced 7S and increased 11S production), the six genes encoding the 7S protein subunits (α, α’ and β) were knocked out using CRISPR/Cas9 mutagenesis. By knocking out the methionine-poor 7S proteins, the production of methionine-rich 11S proteins were hypothesized to be increased through a process called protein rebalancing. The CRISPR-edited plants with 7S subunit knockouts were successfully developed (Figure 2A) and were planted in the field. Amino acid analysis showed, on average, 0.50-fold or 0.34-fold increase in total methionine content in seeds lacking α+α’+β or α+β 7S subunit proteins, respectively. Overall protein and oil content did not change in the 7S knockouts. This helps us to reach the goal of a 1.5-fold increase in total methionine, but by itself is probably not enough to reliably reach the target methionine levels.

To generate soybean with the third desired trait (increase in methionine-rich storage protein), three versions of Gly1 gene with increased met codons were designed. While the wild-type GLY1 protein in conventional soybean contains 1.4% methionine, all three modified versions were designed to have higher methionine levels: GLY1 (Ins) with 4.3% methionine, GLY1 (Mod1) with 7.2% methionine and GLY1 (Mod2) with 2.5% methionine. The transgenic plants expressing GLY1 (Mod1) in soybean seeds were obtained and planted in the field. Amino acid analysis of seeds showed up to a 50% increase in total methionine content.

In this project, techniques and reagents for high-efficiency CRISPR/Cas9-mediated gene knockout in soybean were developed, and in two years, eight genes were successfully and simultaneously knocked out, creating plants homozygous for all eight genes. The soybean genotypes that have been generated are all single-trait plants having one of the three characteristics identified as desirable. In the next phase, the three traits will be stacked to obtain further increases in seed methionine content, with the goal of achieving a 1.5-fold increase in seed methionine.

Reprinted with permission from the United Soybean Board FY19 Funded Production Research annual report. Read the full report: https://api.unitedsoybean.org/uploads/files/fy19-funded-production-research.pdf

This project was funded by the soybean checkoff. To find research related to this research highlight or to see other checkoff research projects, please visit the National Soybean Checkoff Research Database.