Project leader within C4F for: Genetic improvement of oil crops using modern breeding tools, including domestication of field cress (Lepidium campestre), industrial crop crambe (Crambe abyssinica), and important oilseed crop rapeseed (Brassica napus). Plant-based protein production of recombinant heme-binding human proteins for potential medicinal, pharmaceutical and food applications.
Expertise related to the C4F strategic research areas
Extensive research experience in genetic engineering including Agrobacterium transformation and genome editing by TALEN and CRISPR/Cas9 in various crops for improving important traits, including oil quality and quantity, seedcake quality, and insect/disease resistance for sustainable agricultural production and for providing primary products for food, feed and industrial uses. Moreover, research on recombinant protein production in plants with focus on human heme-binding proteins has been recently initiated with the purpose for potential pharmaceutical and food applications.
My expertise relevant to C4F strategic research areas lies thus within i) Improvement of quantities and qualities of oils, starches, proteins, minerals and bioactive compounds and other metabolites or traits in crops for various applications,
ii) Development of novel crops- and bio-based products close to market iii) Development of novel production systems of the abovementioned components such as plant, microbial, and tissue culture based systems.
Brief summary of achievements
In the phase I and II of C4F program, we have been mainly working with genetic improvement of oil qualities and quantities of the oilseed crops crambe (Crambe abyssinica) and field cress (Lepidium campestre) through genetic engineering. In the phase II, we have also worked with production of human heme-binding proteins in plants for potential pharmaceutical, feed and food applications.
Crambe is a dedicated industrial crop for oil production for the chemical industry. The aims of our research in this crop are to increase the erucic acid level and production of wax esters in seed oil. The results obtained include 1) stable transgenic lines with significantly increased erucic acid level through expressing the LdPLAAT gene from Limnanthes douglasii and RNAi-CaFAD2 and RNAi-CaFAE1 genes; 2) stable transgenic lines with significantly increased oleic acid level by expressing RNAi-CaFAD2 and RNAi-CaFAE1 genes; 3) stable transgenic lines with wax ester production in seed oil by overexpressing the jojoba wax biosynthesis genes; 4) hybrids of high oleic and wax ester lines with improved germination and higher levels of unsaturated fatty acids in wax ester, which are more desirable for industrial applications; 5) field trials in Sweden on the high erucic and wax ester lines showed positive results; 6) studies on the roles of diacylglycerol acyltransferases (DGATs) in regulation of oil quantity and quality in crambe have showed that crambe DGATs readily accept 22:1-CoA as acyl donor, but inefficient in utilizing di-22:1DAG as acyl acceptor, a likely limiting factor in further increase in 22:1 level in high erucic lines mentioned above.
Field cress is a wild oilseed species and has been under domestication by SLU researchers for over 30 years, with the aim of developing it into a novel oil and cover crop for cold climate conditions. The species has several good properties, such as very winter hardy, high yield potential and suitable as catch or cover crop. We have been working with genetic engineering for improving the oil content, oil quality and pod shatter of this species. The results obtained so far include: 1) transgenic lines with increased oil content by overexpression the WRI1 gene and hemoglobin genes; 2) transgenic lines with high oleic acid level by expressing the RNAi-LcFAD2 and RNAi-LcFAE1 genes; 3) transgenic lines with wax ester production using the same jojoba genes as for cambe; 4) transgenic lines with reduced pod shatter; 5) development of a highly efficient protoplast regeneration protocol, which is a solid base for producing transgene-free mutants by CRISPR/Cas9. The method is now routinely used for editing target genes controlling important traits in the species in our lab.
In the phase II of C4F, we have explored the possibility of using Nicotiana benthamiana as a production system, with help of Agrobacterium and a viral vector, to produce heme-binding proteins for potential pharmaceutical and food applications. We have successfully produced the human heme-binding proteins in Nicotiana benthamiana, including fetal hemoglobin (HbF), myoglobin, A1M as well as HbF with XTEN-tag. All the purified proteins have showed functions as native ones, confirmed by biophysical, biochemical and preclinical in vivo tests. The results demonstrate a high potential of plant expression systems for producing Hb products for use as blood substitutes.
Recombinant protein production in plants is an attractive method for producing plant-based proteins for feed and food industries. In the phase II of C4F, we have explored the possibility of using Nicotiana benthamiana as a production system, with help of Agrobacterium and a viral vector, to produce heme-binding proteins for potential pharmaceutical and food uses. We have successfully produced the human heme-binding proteins in Nicotiana benthamiana, including fetal hemoglobin (HbF), myoglobin, A1M as well as HbF with XTEN-tag. All the purified proteins have showed functions as native ones, confirmed by biophysical, biochemical and preclinical in vivo tests. The results demonstrate a high potential of plant expression systems for producing heme-binding proteins for various applications.
In the phase III, we will also work with the oilseed crop rapeseed (Brassica napus), the third leading source of vegetable oil in the world. The seedcake or seed meal of rapeseed contains protein of high quality owing to its balanced amino acid composition. However, the seedcake is currently only used as animal feed at a low price and could not be used for human consumption because it contains high levels of anti-nutritional compounds. These compounds need to be removed or reduced before the seedcake can be used as a highly valuable protein source for both feed and food consumption. We aim at eliminating these anti-nutritional compounds through genome editing by CRISPR/Cas9.
Ten selected publications related to C4F
Li X., Guan R., Fan J. and Zhu L.-H. 2019. Development of Industrial Oil Crop Crambe abyssinica for Wax Ester Production through Metabolic Engineering and Cross Breeding. Plant and Cell Physiology. 60 (6): 1274-1283.
Jeppson S., Demski K., Carlsson A.S., Zhu L.-H., Banas A., Stymne S and Lager I. 2019. Crambe hispanica Subsp. abyssinica Diacylglycerol Acyltransferase Specificities Towards Diacylglycerols and Acyl-CoA Reveal Combinatorial Effects That Greatly Affect Enzymatic Activity and Specificity. Front. Plant Sci. 10:1442. doi: 10.3389/fpls.2019.01442
Ivarson, E., Iven, T., Sturtevant, D., Ahlman, A., Cai, Y., Chapman, K., Feussner, I., Zhu, L-H. 2017. Production of wax esters in the wild oil species Lepidium campestre. Industrial Crops & Products 108, 535–542.
Ivarson, E., Leiva-Eriksson, N., Ahlman A., Kanagarajan S., Bülow L., Zhu L-H. 2017. Effects of overexpression of WRI1 and hemoglobin genes on the seed oil content of Lepidium campestre. Frontiers in Plant Science 7, 2032.
Ivarson, E., Ahlman, A., Lager, I., Zhu, L-H. 2016. Significant increase of oleic acid level in the wild species Lepidium campestre through direct gene silencing. Plant Cell Reports. 35(10):2055-63. DOI 10.1007/s00299-016-2016-9.
Li, X., Mei, D., Liu, Q., Fan, J., Singh, S., Green, A., Zhou, X-R., Zhu, L-H. 2016. Down-regulation of crambe fatty acid desaturase and elongase in Arabidopsis and crambe resulted in significantly increased oleic acid content in seed oil. Plant Biotech. J.14: 323–331.
Zhu, L.H., … Li X.,…. et al. 2016. Dedicated industrial oilseed crops as metabolic engineering platforms for sustainable industrial feedstock production. Sci. Rep. 6:22181, DOI: 10.1038/srep22181.
Ivarson E., Ahlman A., Li X.Y. and Zhu L.-H. 2013. Development of an efficient regeneration and transformation method for the new potential oilseed crop Lepidium campestre. BMC Plant Biology 13:115 doi:10.1186/1471-2229-13-115.
Guan R., Lager I., Li XY., Stymne S. and Zhu L.-H. 2013. Bottlenecks in Erucic acid Accumulation in Genetically Engineered Ultra-High Erucic Acid Crambe abyssinica. Plant Biotech. J. 12(2): 193–203.
Li X.Y., Van Loo E.N., Gruber J., Fan J., Guan R., Frentzen M., Stymne S. and Zhu L.-H. 2012. Development of ultra-high erucic acid oil in the industrial oil crop Crambe abyssinica. Plant Biotech. J.10 (7): 862-870.