Gerhard Sandmann

The potential for the improvement of carotenoid levels in foods and the likely systemic effects

Carotenoids form one of the most important classes of plant pigments and play a crucial role in defining the quality parameters of fruit and vegetables. Their role in the plant is to act as accessory pigments for light harvesting and in the prevention of photo-oxidative damage, as well as acting as attractants for pollinators. Their function as antioxidants in the plant shows interesting parallels with their potential role as antioxidants in foods and humans. Carotenoids are products of the isoprenoid biosynthetic pathway. The enzymes leading to carotenoid biosynthesis have all been characterised, and more recently the genes encoding these enzymes have been cloned from bacteria, fungi and plants. New information on enzyme activities and the factors leading to the regulation of the pathway is reviewed. Vitamin A deficiency is a widespread problem in the developing world, causing blindness, particularly in the young. This has driven research into finding ways of introducing provitamin A carotenoids into staple crops, and this has recently been achieved in rice and canola through genetic manipulation. The fact that carotenoids show protective activity in vitro and in vivo against a variety of degenerative disease end points has also give impetus to studying whether increasing intakes of the commonly consumed carotenoids would have public health benefits in the developed world. Human intervention studies have been undertaken using supplements of β-carotene rather than utilising foods with enhanced carotenoid levels, but no potential benefit has been shown. Indeed, there is evidence of an increased health risk from the consumption of β-carotene supplements. These observations suggest that the threshold between the beneficial and adverse effects of some carotenoids is low and provides a strong stimulus to further understanding the functional effects of specific carotenoids. Specific needs for future research are identified in the review. © 2000 Society of Chemical Industry

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.

Mutations in the Arabidopsis Gene IMMUTANS Cause a Variegated Phenotype by Inactivating a Chloroplast Terminal Oxidase Associated with Phytoene Desaturation

The immutans (im) mutant of Arabidopsis shows a variegated phenotype comprising albino and green somatic sectors. We have cloned the IM gene by transposon tagging and show that even stable null alleles give rise to a variegated phenotype. The gene product has amino acid similarity to the mitochondrial alternative oxidase. We show that the IM protein is synthesized as a precursor polypeptide that is imported into chloroplasts and inserted into the thylakoid membrane. The albino sectors of im plants contain reduced levels of carotenoids and increased levels of the carotenoid precursor phytoene. The data presented here are consistent with a role for the IM protein as a cofactor for carotenoid desaturation. The suggested terminal oxidase function of IM appears to be essential to prevent photooxidative damage during early steps of chloroplast formation. We propose a model in which IM function is linked to phytoene desaturation and, possibly, to the respiratory activity of the chloroplast.

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.

High-Level Production of Beta-Carotene in Saccharomyces cerevisiae by Successive Transformation with Carotenogenic Genes from Xanthophyllomyces dendrorhous

ABSTRACT To determine whether Saccharomyces cerevisiae can serve as a host for efficient carotenoid and especially β-carotene production, carotenogenic genes from the carotenoid-producing yeast Xanthophyllomyces dendrorhous were introduced and overexpressed in S. cerevisiae. Because overexpression of these genes from an episomal expression vector resulted in unstable strains, the genes were integrated into genomic DNA to yield stable, carotenoid-producing S. cerevisiae cells. Furthermore, carotenoid production levels were higher in strains containing integrated carotenogenic genes. Overexpression of crtYB (which encodes a bifunctional phytoene synthase and lycopene cyclase) and crtI (phytoene desaturase) from X. dendrorhous was sufficient to enable carotenoid production. Carotenoid production levels were increased by additional overexpression of a homologous geranylgeranyl diphosphate (GGPP) synthase from S. cerevisiae that is encoded by BTS1. Combined overexpression of crtE (heterologous GGPP synthase) from X. dendrorhous with crtYB and crtI and introduction of an additional copy of a truncated 3-hydroxy-3-methylglutaryl-coenzyme A reductase gene (tHMG1) into carotenoid-producing cells resulted in a successive increase in carotenoid production levels. The strains mentioned produced high levels of intermediates of the carotenogenic pathway and comparable low levels of the preferred end product β-carotene, as determined by high-performance liquid chromatography. We finally succeeded in constructing an S. cerevisiae strain capable of producing high levels of β-carotene, up to 5.9 mg/g (dry weight), which was accomplished by the introduction of an additional copy of crtI and tHMG1 into carotenoid-producing yeast cells. This transformant is promising for further development toward the biotechnological production of β-carotene by S. cerevisiae.

Transformation of the Green Alga Haematococcus pluvialis with a Phytoene Desaturase for Accelerated Astaxanthin Biosynthesis

ABSTRACT Astaxanthin is a high-value carotenoid which is used as a pigmentation source in fish aquaculture. Additionally, a beneficial role of astaxanthin as a food supplement for humans has been suggested. The unicellular alga Haematococcus pluvialis is a suitable biological source for astaxanthin production. In the context of the strong biotechnological relevance of H. pluvialis, we developed a genetic transformation protocol for metabolic engineering of this green alga. First, the gene coding for the carotenoid biosynthesis enzyme phytoene desaturase was isolated from H. pluvialis and modified by site-directed mutagenesis, changing the leucine codon at position 504 to an arginine codon. In an in vitro assay, the modified phytoene desaturase was still active in conversion of phytoene to ζ-carotene and exhibited 43-fold-higher resistance to the bleaching herbicide norflurazon. Upon biolistic transformation using the modified phytoene desaturase gene as a reporter and selection with norflurazon, integration into the nuclear genome of H. pluvialis and phytoene desaturase gene and protein expression were demonstrated by Southern, Northern, and Western blotting, respectively, in 11 transformants. Some of the transformants had a higher carotenoid content in the green state, which correlated with increased nonphotochemical quenching. This measurement of chlorophyll fluorescence can be used as a screening procedure for stable transformants. Stress induction of astaxanthin biosynthesis by high light showed that there was accelerated accumulation of astaxanthin in one of the transformants compared to the accumulation in the wild type. Our results strongly indicate that the modified phytoene desaturase gene is a useful tool for genetic engineering of carotenoid biosynthesis in H. pluvialis.

Isolation and functional characterisation of a novel type of carotenoid biosynthetic gene from Xanthophyllomyces dendrorhous

Abstract The red heterobasidiomycetous yeast Xanthophyllomyces dendrorhous (perfect state of Phaffia rhodozyma) contains a novel type of carotenoid biosynthetic enzyme. Its structural gene, designated crtYB, was isolated by functional complementation in a genetically modified, carotenogenic Escherichia coli strain. Expression studies in different carotenogenic E. coli strains demonstrated that the crtYB gene encodes a bifunctional protein involved both in synthesis of phytoene from geranylgeranyl diphosphate and in cyclisation of lycopene to β-carotene. By sequence comparison with other phytoene synthases and complementation studies in E. coli with various deletion derivatives of the crtYB gene, the regions responsible for phytoene synthesis and lycopene cyclisation were localised within the protein.

Novel hydroxycarotenoids with improved antioxidative properties produced by gene combination in Escherichia coli.

We have used combinatorial biosynthesis to synthesize novel lipophilic carotenoids that are powerful cellular antioxidants. By co-expressing three different carotenoid desaturases in combination with a carotenoid hydratase, a cyclase, and a hydroxylase on compatible plasmids in Escherichia coli, we synthesized four novel carotenoids not previously detected in biological material or chemically synthesized. Their identification was based on their relative retention times on HPLC, spectroscopic properties, molecular weights, number of hydroxy groups, and 1H-NMR spectra. The carotenoids were designated as 1-HO-3′, 4′-didehydrolycopene, 3, 1′-(HO)2-γ-carotene, 1,1′-(HO)2-3, 4, 3′, 4′-tetradehydrolycopene, and 1, 1′-(HO)2-3, 4-didehydrolycopene. These novel acyclic derivatives differ from structurally related compounds by extension of the conjugated polyene chain as well as additional hydroxy groups at position C-1′. We determined their antioxidative activity in a liposome-membrane model system, which showed that their ability to protect against photooxidation and radical-mediated peroxidation reactions was linked to the length of the conjugated double-bond system and the presence of a single hydroxy group. The protection of membrane degradation was superior to the related 1-HO and 1, 1′-(HO)2 lycopene derivatives, making them interesting pharmaceutical candidates.

Genetic manipulation of carotenoid biosynthesis: strategies, problems and achievements

Carotenoids, some of which are provitamin A, have a range of diverse biological functions and actions, especially in relation to human health. For example, carotenoids are known to be crucial for normal vision and have been associated with reducing the risk of several degenerative diseases including cancer. The putative advantage of modifying and engineering the carotenoid biosynthetic pathways is obvious: to provide sources for the isolation of desired carotenoids or to generate food plants with increased carotenoid content. This article reviews the studies of carotenoid production in heterologous microorganisms and the engineering of crop plants using manipulated carotenoid biosynthesis.

Biotechnological production of astaxanthin with Phaffia rhodozyma/Xanthophyllomyces dendrorhous

The oxygenated β-carotene derivative astaxanthin exhibits outstanding colouring, antioxidative and health-promoting properties and is mainly found in the marine environment. To satisfy the growing demand for this ketocarotenoid in the feed, food and cosmetics industries, there are strong efforts to develop economically viable bioprocesses alternative to the current chemical synthesis. However, up to now, natural astaxanthin from Haematococcus pluvialis, Phaffia rhodozyma or Paracoccus carotinifaciens has not been cost competitive with chemically synthesized astaxanthin, thus only serving niche applications. This review illuminates recent advances made in elucidating astaxanthin biosynthesis in P. rhodozyma. It intensely focuses on strategies to increase astaxanthin titers in the heterobasidiomycetous yeast by genetic engineering of the astaxanthin pathway, random mutagenesis and optimization of fermentation processes. This review emphasizes the potential of P. rhodozyma for the biotechnological production of astaxanthin in comparison to other natural sources such as the microalga H. pluvialis, other fungi and transgenic plants and to chemical synthesis.