INFORM January 2024

BIOENGINEERING

inform January 2024, Vol. 35 (1) • 25

The major source of HFA for the industry is castor seed oil, which contains 80-90% ricinoleic acid. Castor, belonging to the Euphorbiacea family, is traditionally cultivated in the tropical, semi-arid regions of the Asia-Pacific, including India, Vietnam, Cambodia, Thailand, Indonesia, China, and South Korea. North America and Europe are the largest consumers of castor seed oil, with the majority imported from India and China. Owing to the increased demand for castor oil for bio based products, the global market value of castor oil and its derivatives is expected to increase from $1.21 billion in 2021 to $1.9 billion in 2030, with a compounded annual growth rate of 5.8% ( https://tinyurl.com/44s6nbnj ). CHALLENGES Castor cultivation is burdened with multiple, undesirable agro nomic and economic challenges. Climate change disrupts productivity leading to oscillating prices during monsoons. Mechanical harvesting is difficult due to the crop being peren nial and invasive, with unpredictable growth habits and a ten dency to displace native vegetation (https://doi.org/10.2134/ agronj2011.0210 ). In addition, castor accumulates a neurotoxic protein called ricin in its seeds which according to the Centers for Disease Control is “one of the most toxic biological agents known: a Category B bioterrorism agent and a Schedule 1 chemical warfare agent.” Hence, castor cultivation is banned in many countries including the US (https://tinyurl.com/4ffjt6e6). These undesirable features of the castor have led to genetic engineering of HFA traits into non-toxic traditional oilseed crops that could be grown in the US. Most of the bioengineering strategies over the past two decades focused on over-expressing the castor or Physaria fen dleri fatty acid hydroxylase gene together with HFA-selective acyltransferase genes in the model plant Arabidopsis thaliana or crop and Camelina sativa as proof-of-concept. However,

Physaria fendleri is a species of flowering plant in the family Brassicaceae.

HFA levels in the engineered seeds were less than the levels of HFA in castor (~80-90%) and P. fendleri (~60%) oils. Arabidopsis produced less than 40% and Camelina seeds less than 24%. Researchers have determined that these crops are hin dered by inefficient flux of HFA from the site of synthesis within the membrane lipid phosphatidylcholine (PC) to accu mulation in triacylglycerol (TAG), reducing fatty acid synthesis and oil accumulation. In addition, poor germination rates of the engineered plants hamper their commercial exploitation. One of the major bottlenecks affecting successful bio engineering of the HFA trait is a lack of knowledge on how different plant species use different branches of the lipid met abolic network (Figure 2) to accumulate various oil composi tions. For instance, castor assembles diacylglycerol (DAG) and then triacylglycerol containing ricinoleate de novo through the Kennedy pathway (Figure 2, orange). Whereas, model and crop Brassicaceae species ( Arabidopsis and Camelina ) use DAG derived from the membrane lipid PC as a substrate to produce TAG (Figure 2, blue).

FIG. 2. Different TAG biosynthetic routes via lipid metabolic networks in different crop species. (DAG = diacylglycerol, DGAT = diacylglycerol acyltransferase, F.A.S = fatty acid synthesis, FA = fatty acid, G3P = glycerol-3-phosphate, GPAT = glycerol-3-phosphate acyltransferase, HFA = hydroxy fatty acid, LPA = lysophosphatidic acid, LPAT = lysophosphatidic acid acyltransferase, PA = phosphatidic acid, PC = phosphatidyl choline, ROD1 = reduced oleate desaturation 1, TAG = triacylglycerol, X denotes number of double bonds.) Source: Bates Lab.