Overexpression of Hydroxynitrile Lyase in Cassava Roots Elevates Protein and Free Amino Acids while Reducing Residual Cyanogen Levels

Abstract

Cassava is the major source of calories for more than 250 million Sub-Saharan Africans, however, it has the lowest protein-to-energy ratio of any major staple food crop in the world. A cassava-based diet provides less than 30% of the minimum daily requirement for protein. Moreover, both leaves and roots contain potentially toxic levels of cyanogenic glucosides. The major cyanogen in cassava is linamarin which is stored in the vacuole. Upon tissue disruption linamarin is deglycosylated by the apolplastic enzyme, linamarase, producing acetone cyanohydrin. Acetone cyanohydrin can spontaneously decompose at pHs >5.0 or temperatures >35°C, or is enzymatically broken down by hydroxynitrile lyase (HNL) to produce acetone and free cyanide which is then volatilized. Unlike leaves, cassava roots have little HNL activity. The lack of HNL activity in roots is associated with the accumulation of potentially toxic levels of acetone cyanohydrin in poorly processed roots. We hypothesized that the over-expression of HNL in cassava roots under the control of a root-specific, patatin promoter would not only accelerate cyanogenesis during food processing, resulting in a safer food product, but lead to increased root protein levels since HNL is sequestered in the cell wall. Transgenic lines expressing a patatin-driven HNL gene construct exhibited a 2–20 fold increase in relative HNL mRNA levels in roots when compared with wild type resulting in a threefold increase in total root protein in 7 month old plants. After food processing, HNL overexpressing lines had substantially reduced acetone cyanohydrin and cyanide levels in roots relative to wild-type roots. Furthermore, steady state linamarin levels in intact tissues were reduced by 80% in transgenic cassava roots. These results suggest that enhanced linamarin metabolism contributed to the elevated root protein levels.

Structure, Expression, and Functional Analysis of the Hexokinase Gene Family in Cassava

“…The cassava tuber root is rich in starch (30% starch/g fresh weight) [26]. HXKs play a key role in cassava starch synthesis [19].…”

Recent Biotechnological Advances in the Improvement of Cassava

“…Consumption of residual cyanogens (linamarin and lotaustralin) in incompletely processed cassava roots can cause various health disorders that render a person unsteady and uncoordinated [12]. Hydroxynitrile lyase (HNL) catalyzes the conversion of acetone cyanohydrin to cyanide and is expressed predominantly in the cell walls and laticifers of leaves, compared with tuberous roots, which exhibit very low [10].…”

“…Hydroxynitrile lyase (HNL) catalyzes the conversion of acetone cyanohydrin to cyanide and is expressed predominantly in the cell walls and laticifers of leaves, compared with tuberous roots, which exhibit very low [10]. Transgenic cassava over-expressing HNL was shown to display significantly reduced acetone cyanohydrin levels and exhibited increased cyanide volatilization in processed or homogenized roots [12]. It has been shown that the genomic region surrounding the cytochrome P450, CYP79D3, contains all genes required for cyanogenic glucoside biosynthesis in cassava [13].…”

Transgenic technologies in cassava for nutritional improvement and viral disease resistance: a key strategy for food security in Africa

“…It has been demonstrated that cyanogens provide reduced nitrogen substrates for amino acid synthesis in cassava roots and, therefore, manipulation of linamarin metabolism in cassava might elevate root amino acid pools and promote total protein biosynthesis [6] . Overexpression of hydroxynitrile lyase, the enzyme that catalyzes the conversion of acetone cyanohydrin to cyanide, in cassava roots was reported to increase overall protein content [18] . In fact, efforts to develop high protein cassava by conventional breeding have also been made by the investigation of crude protein content in cassava roots Fig.…”