Changfu Zhu

Transgenic multivitamin corn through biofortification of endosperm with three vitamins representing three distinct metabolic pathways

Vitamin deficiency affects up to 50% of the worlds population, disproportionately impacting on developing countries where populations endure monotonous, cereal-rich diets. Transgenic plants offer an effective way to increase the vitamin content of staple crops, but thus far it has only been possible to enhance individual vitamins. We created elite inbred South African transgenic corn plants in which the levels of 3 vitamins were increased specifically in the endosperm through the simultaneous modification of 3 separate metabolic pathways. The transgenic kernels contained 169-fold the normal amount of β-carotene, 6-fold the normal amount of ascorbate, and double the normal amount of folate. Levels of engineered vitamins remained stable at least through to the T3 homozygous generation. This achievement, which vastly exceeds any realized thus far by conventional breeding alone, opens the way for the development of nutritionally complete cereals to benefit the worlds poorest people.

Combinatorial genetic transformation generates a library of metabolic phenotypes for the carotenoid pathway in maize

Combinatorial nuclear transformation is a novel method for the rapid production of multiplex-transgenic plants, which we have used to dissect and modify a complex metabolic pathway. To demonstrate the principle, we transferred 5 carotenogenic genes controlled by different endosperm-specific promoters into a white maize variety deficient for endosperm carotenoid synthesis. We recovered a diverse population of transgenic plants expressing different enzyme combinations and showing distinct metabolic phenotypes that allowed us to identify and complement rate-limiting steps in the pathway and to demonstrate competition between β-carotene hydroxylase and bacterial β-carotene ketolase for substrates in 4 sequential steps of the extended pathway. Importantly, this process allowed us to generate plants with extraordinary levels of β-carotene and other carotenoids, including complex mixtures of hydroxycarotenoids and ketocarotenoids. Combinatorial transformation is a versatile approach that could be used to modify any metabolic pathway and pathways controlling other biochemical, physiological, or developmental processes.

When more is better: multigene engineering in plants

The genomics revolution has taught us that a great deal of information can be derived from studying many genes or proteins at the same time. We are beginning to see this approach blossoming in applied research. Instead of attempting to generate useful transgenic plants by introducing single genes, we now see an increasing number of researchers embracing multigene transfer (MGT) as an approach to generate plants with more ambitious phenotypes. MGT allows researchers to achieve goals that were once impossible - the import of entire metabolic pathways, the expression of entire protein complexes, the development of transgenic crops simultaneously engineered to produce a spectrum of added-value compounds. The potential appears limitless.

Critical evaluation of strategies for mineral fortification of staple food crops.

Staple food crops, in particular cereal grains, are poor sources of key mineral nutrients. As a result, the world’s poorest people, generally those subsisting on a monotonous cereal diet, are also those most vulnerable to mineral deficiency diseases. Various strategies have been proposed to deal with micronutrient deficiencies including the provision of mineral supplements, the fortification of processed food, the biofortification of crop plants at source with mineral-rich fertilizers and the implementation of breeding programs and genetic engineering approaches to generate mineral-rich varieties of staple crops. This review provides a critical comparison of the strategies that have been developed to address deficiencies in five key mineral nutrients—iodine, iron, zinc, calcium and selenium—and discusses the most recent advances in genetic engineering to increase mineral levels and bioavailability in our most important staple food crops.

The regulation of carotenoid pigmentation in flowers

Carotenoids fulfill many processes that are essential for normal growth and development in plants, but they are also responsible for the breathtaking variety of red-to-yellow colors we see in flowers and fruits. Although such visual diversity helps to attract pollinators and encourages herbivores to distribute seeds, humans also benefit from the aesthetic properties of flowers and an entire floriculture industry has developed on the basis that new and attractive varieties can be produced. Over the last decade, much has been learned about the impact of carotenoid metabolism on flower color development and the molecular basis of flower color. A number of different regulatory mechanisms have been described ranging from the transcriptional regulation of genes involved in carotenoid synthesis to the control of carotenoid storage in sink organs. This means we can now explain many of the natural colorful varieties we see around us and also engineer plants to produce flowers with novel and exciting varieties that are not provided by nature.

Promoter diversity in multigene transformation

Multigene transformation (MGT) is becoming routine in plant biotechnology as researchers seek to generate more complex and ambitious phenotypes in transgenic plants. Every nuclear transgene requires its own promoter, so when coordinated expression is required, the introduction of multiple genes leads inevitably to two opposing strategies: different promoters may be used for each transgene, or the same promoter may be used over and over again. In the former case, there may be a shortage of different promoters with matching activities, but repetitious promoter use may in some cases have a negative impact on transgene stability and expression. Using illustrative case studies, we discuss promoter deployment strategies in transgenic plants that increase the likelihood of successful and stable multiple transgene expression.

Nutritious crops producing multiple carotenoids – a metabolic balancing act

Plants and microbes produce multiple carotenoid pigments with important nutritional roles in animals. By unraveling the basis of carotenoid biosynthesis it has become possible to modulate the key metabolic steps in plants and thus increase the nutritional value of staple crops, such as rice (Oryza sativa), maize (Zea mays) and potato (Solanum tuberosum). Multigene engineering has been used to modify three different metabolic pathways simultaneously, producing maize seeds with higher levels of carotenoids, folate and ascorbate. This strategy may allow the development of nutritionally enhanced staples providing adequate amounts of several unrelated nutrients. By focusing on different steps in the carotenoid biosynthesis pathway, it is also possible to generate plants with enhanced levels of several nutritionally-beneficial carotenoid molecules simultaneously.

Light-dark regulation of carotenoid biosynthesis in pepper (Capsicum annuum) leaves

The carotenoid content in photosynthetic plant tissue reflects a steady state value resulting from permanent biosynthesis and concurrent photo-oxidation. The contributions of both reactions were determined in illuminated pepper leaves. The amount of carotenoids provided by biosynthesis were quantified by the accumulation of the colourless carotenoid phytoene in the presence of the inhibitor norflurazon. When applied, substantial amounts of this rather photo-stable intermediate were formed in the light. However, carotenoid biosynthesis was completely stalled in darkness. This switch off in the absence of light is related to the presence of very low messenger levels of the phytoene synthase gene, psy and the phytoene desaturase gene, pds. Other carotenogenic genes, such as zds, ptox and Icy-b also were shown to be down-regulated to some extent. By comparison of the carotenoid concentration before and after transfer of plants to increasing light intensities and accounting for the contribution of biosynthesis, the rate of photo-oxidation was estimated for pepper leaves. It could be demonstrated that light-independent degradation or conversion of carotenoids e.g. to abscisic acid is a minor process.

A golden era—pro-vitamin A enhancement in diverse crops

Numerous crops have been bred or engineered to increase carotenoid levels in an effort to develop novel strategies that address vitamin A deficiency in the developing world. The pioneering work in rice (not covered in this review) has been followed up in many additional crops, some of which are staples like rice whereas others are luxury products whose impact on food security is likely to be marginal. This review surveys the progress that has been made in carotenoid breeding and metabolic engineering, focusing on β-carotene enhancement in crops other than rice. We ask if these efforts have the potential to address vitamin A deficiency in developing countries by comparing bioavailable pro-vitamin A levels in wild type and enhanced crops to determine whether nutritional requirements can be met without the consumption of unrealistic amounts of food. The potential impact of carotenoid enhancement should therefore be judged against benchmarks that include the importance of particular crops in terms of global food security, the amount of bioavailable β-carotene, and the amount of food that must be consumed to achieve the reference daily intake of vitamin A.

Metabolic engineering of ketocarotenoid biosynthesis in higher plants.

Ketocarotenoids such as astaxanthin and canthaxanthin have important applications in the nutraceutical, cosmetic, food and feed industries. Astaxanthin is derived from beta-carotene by 3-hydroxylation and 4-ketolation at both ionone end groups. These reactions are catalyzed by beta-carotene hydroxylase and beta-carotene ketolase, respectively. The hydroxylation reaction is widespread in higher plants, but ketolation is restricted to a few bacteria, fungi, and some unicellular green algae. The recent cloning and characterization of beta-carotene ketolase genes in conjunction with the development of effective co-transformation strategies permitting facile co-integration of multiple transgenes in target plants provided essential resources and tools to produce ketocarotenoids in planta by genetic engineering. In this review, we discuss ketocarotenoid biosynthesis in general, and characteristics and functional properties of beta-carotene ketolases in particular. We also describe examples of ketocarotenoid engineering in plants and we conclude by discussing strategies to efficiently convert beta-carotene to astaxanthin in transgenic plants.