Metabolic engineering of astaxanthin production in tobacco flowers

Using metabolic engineering, we have modified the carotenoid biosynthesis pathway in tobacco (Nicotiana tabacum) to produce astaxanthin, a red pigment of considerable economic value. To alter the carotenoid pathway in chromoplasts of higher plants, the cDNA of the gene CrtO from the alga Haematococcus pluvialis, encoding beta-carotene ketolase, was transferred to tobacco under the regulation of the tomato Pds (phytoene desaturase) promoter. The transit peptide of PDS from tomato was used to target the CRTO polypeptide to the plastids. Chromoplasts in the nectary tissue of transgenic plants accumulated (3S,3'S) astaxanthin and other ketocarotenoids, changing the color of the nectary from yellow to red. This accomplishment demonstrates that plants can be used as a source of novel carotenoid pigments such as astaxanthin. The procedures described in this work can serve as a platform technology for future genetic manipulations of pigmentation of fruits and flowers of horticultural and floricultural importance.     

Establishment and characterization of a maize Hi-II endosperm culture

An in vitro continuous endosperm callus culture derived from developing endosperm of transformation-amenable maize Hi-II genotype was obtained. The endosperm callus was composed of cells that differentiated into aleurone-like and starchy endosperm-like cell types. This callus has been maintained for 4?yr. Endosperm callus cells transcribe and produce zein proteins at a level similar to developing endosperm tissue. Starchy endosperm cells of the endosperm callus displayed active starch biosynthetic activity. The dual cell physiology of this culture limited the utility of the cell line for promoter analysis and transient assays of gene expression in the current culture conditions. However, because such cell line can be readily initiated and easily maintained for a long period of time, it provides an alternative tool for analysis of transgene expression in endosperm callus derived from transgenic maize lines in Hi-II background.     

Multigene engineering of starch biosynthesis in maize endosperm increases the total starch content and the proportion of amylose

Maize (Zea mays spp. mays) is a staple crop for more than 900 million people. The seeds or kernels provide a rich source of calories because ~70 % of the weight is carbohydrate, mostly in the form of starch. The content and composition of starch are complex traits controlled by many genes, offering multiple potential targets for intervention. We used a multigene engineering approach combining the overexpression of Bt2, Sh2, Sh1 and GbssIIa (to enhance the activity of sucrose synthase, AGPase and granule-bound starch synthase) with the suppression of SbeI and SbeIIb by RNA interference (to reduce the activity of starch branching enzyme). Maize plants expressing all six genes plus the selectable marker showed a 2.8–7.7 % increase in the endosperm starch content and a 37.8–43.7 % increase in the proportion of amylose, which was significant compared to untransformed control plants. We also observed improvements in other agronomic traits, such as a 20.1–34.7 % increase in 100-grain weight, a 13.9–19.0 % increase in ear weight, and larger kernels with a better appearance, presumably reflecting the modified starch structure within the kernels. Our results confirm that multigene engineering applied to the starch biosynthesis pathway can not only modulate the quality and quantity of starch but can also improve starch-dependent agronomic traits.     

Metabolic engineering of isoflavonoid biosynthesis in alfalfa

The potential health benefits of dietary isoflavones have generated considerable interest in engineering the synthesis of these phytoestrogens into plants. Genistein glucoside production (up to 50 nmol g(-1) fresh weight) was engineered in alfalfa (Medicago sativa) leaves by constitutive expression of isoflavone synthase from Medicago truncatula (MtIFS1). Glucosides of biochanin A (4'-O-methylgenistein) and pratensein (3'-hydroxybiochanin A) also accumulated. Although MtIFS1 was highly expressed in all organs examined, genistein accumulation was limited to leaves. MtIFS1-expressing lines accumulated several additional isoflavones, including formononetin and daidzein, in response to UV-B or Phoma medicaginis, whereas the chalcone and flavanone precursors of these compounds accumulated in control lines. Enhanced accumulation of the phytoalexin medicarpin was observed in P. medicaginis-infected leaves of MtIFS1-expressing plants. Microarray profiling indicated that MtIFS1 expression does not significantly alter global gene expression in the leaves. Our results highlight some of the challenges associated with metabolic engineering of plant natural products, including tissue-specific accumulation, potential for further modification by endogenous enzyme activities (hydroxylation, methylation, and glycosylation), and the differential response of engineered plants to environmental factors.     

Metabolic engineering of carotenoid biosynthesis in plants

Carotenoids are one of the most diverse classes of natural compounds. Plant carotenoids are composed of a C40 isoprenoid skeleton with or without epoxy, hydroxy and keto groups. They have fundamental roles in human nutrition as antioxidants and vitamin A precursors and their consumption is increasingly associated with protection from a range of diseases. They are also used commercially as safe food, feed and cosmetic colorants and they protect plants from photooxidative stress. In the past six years many metabolic engineering efforts have been undertaken in plants aiming to improve the nutritional value of staple crops, to enable the use of plants as 'cell factories' for producing specialty carotenoids and to improve plant resistance to abiotic stress.     

Sugar uptake and starch biosynthesis by slices of developing maize endosperm

¹⁴C-Sugar uptake and incorporation into starch by slices of developing maize (Zea mays L.) endosperm were examined and compared with sugar uptake by maize endosperm-derived suspension cultures. Rates of sucrose, fructose, and d- and l-glucose uptake by slices were similar, whereas uptake rates for these sugars differed greatly in suspension cultures. Concentration dependence of sucrose, fructose, and d-glucose uptake was biphasic (consisting of linear plus saturable components) with suspension cultures but linear with slices. These and other differences suggest that endosperm slices are freely permeable to sugars. After diffusion into the slices, sugars were metabolized and incorporated into starch. Starch synthesis, but not sugar accumulation, was greatly reduced by 2.5 millimolar p-chloromercuribenzenesulfonic acid and 0.1 millimolar carbonyl cyanide m-chlorophenylhydrazone. Starch synthesis was dependent on kernel age and incubation temperature, but not on external pH (5 through 8). Competing sugars generally did not affect the distribution of ¹⁴C among the soluble sugars extracted from endosperm slices incubated in ¹⁴C-sugars. Competing hexoses reduced the incorporation of ¹⁴C into starch, but competing sucrose did not, suggesting that sucrose is not a necessary intermediate in starch biosynthesis. The bidirectional permeability of endosperm slices to sugars makes the characterization of sugar transport into endosperm slices impossible, however the model system is useful for experiments dealing with starch biosynthesis which occurs in the metabolically active tissue.     

Reprogramming microorganisms for the biosynthesis of astaxanthin via metabolic engineering

There is an increasing demand for astaxanthin in food, feed, cosmetics and pharmaceutical applications because of its superior anti-oxidative and coloring properties. However, naturally produced astaxanthin is expensive, mainly due to low productivity and limited sources. Reprogramming of microorganisms for astaxanthin production via metabolic engineering is a promising strategy. We primarily focus on the application of synthetic biology, enzyme engineering and metabolic engineering in enhancing the synthesis and accumulation of astaxanthin in microorganisms in this review. We also discuss the biosynthetic pathways of astaxanthin within natural producers, and summarize the achievements and challenges in reprogramming microorganisms for enhancing astaxanthin production. This review illuminates recent biotechnological advances in microbial production of astaxanthin. Future perspectives on utilization of new technologies for boosting microbial astaxanthin production are also discussed.     

Regulation of starch biosynthesis in normal and opaque-2 maize during endosperm development

The absolute activities of sucrose-UDP glucosyltransferase, glucose-6-phosphate ketoisomerase and soluble and bound ADPG-starch glucosyltransferase have been studied in normal and Opaque-2 maize endosperms during development. In general, the activities of these enzymes except sucrose-UDP glucosyltransferase were higher up to 20 days post-pollination and lower at the 30 day stage in Opaque-2 than in normal maize endosperms. However, sucrose-UDP glucosyltransferase activity was higher in normal maize endosperm up to the 20 day stage while it was lower at subsequent stages than in Opaque-2. It is suggested that the lower level of these enzymes, except sucrose-UDP glucosyltransferase, might be responsible for the reduced accumulation of starch in Opaque-2 endosperm during later stages of endosperm development.     

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 β-carotene by 3-hydroxylation and 4-ketolation at both ionone end groups. These reactions are catalyzed by β-carotene hydroxylase and β-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 β-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 β-carotene ketolases in particular. We also describe examples of ketocarotenoid engineering in plants and we conclude by discussing strategies to efficiently convert β-carotene to astaxanthin in transgenic plants.     

Metabolic engineering of natural product biosynthesis in actinobacteria

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植物类胡萝卜素代谢工程与应用

类胡萝卜素是人类所需要的重要营养成分之一,不仅具有抗氧化、预防肿瘤和心血管等疾病的作用,而且还是人体合成维生素A的前体。全球大约有280万～330万学龄前儿童出现维生素缺乏（vitaminAdeficiency,VAD）的临床症状;近2亿儿童处于半缺乏状态。通过对植物类胡萝卜素生物合成途径的解析,以及对参与这一代谢过程的酶及其调控机制的深入了解,目前已经可以通过基因工程在主要农作物中组织特异性地促进类胡萝卜素的合成与积累。从理论上已经可以利用转基因植物来减少VAD的出现。该文简要回顾近年来这一领域的研究进展。     

Metabolic engineering of anthocyanin biosynthesis in Escherichia coli

Anthocyanins are red, purple, or blue plant pigments that belong to the family of polyphenolic compounds collectively called flavonoids. Their demonstrated antioxidant properties and economic importance to the dye, fruit, and cut-flower industries have driven intensive research into their metabolic biosynthetic pathways. In order to produce stable, glycosylated anthocyanins from colorless flavanones such as naringenin and eriodictyol, a four-step metabolic pathway was constructed that contained plant genes from heterologous origins: flavanone 3beta-hydroxylase from Malus domestica, dihydroflavonol 4-reductase from Anthurium andraeanum, anthocyanidin synthase (ANS) also from M. domestica, and UDP-glucose:flavonoid 3-O-glucosyltransferase from Petunia hybrida. Using two rounds of PCR, each one of the four genes was first placed under the control of the trc promoter and its own bacterial ribosome-binding site and then cloned sequentially into vector pK184. Escherichia coli cells containing the recombinant plant pathway were able to take up either naringenin or eriodictyol and convert it to the corresponding glycosylated anthocyanin, pelargonidin 3-O-glucoside or cyanidin 3-O-glucoside. The produced anthocyanins were present at low concentrations, while most of the metabolites detected corresponded to their dihydroflavonol precursors, as well as the corresponding flavonols. The presence of side product flavonols is at least partly due to an alternate reaction catalyzed by ANS. This is the first time plant-specific anthocyanins have been produced from a microorganism and opens up the possibility of further production improvement by protein and pathway engineering.     

Purification and characterization of soluble starch synthases from maize endosperm

This study identified and characterized the soluble starch synthase of maize endosperm that was initially revealed as the SSII activity peak in anion exchange chromatography (J. L. Ozbun et al. (1971) Plant Physiol. 48, 765-769). At least six different genes coding for starch synthases are expressed in maize, although previously it was not known which of these is responsible for the SSII activity peak. The enzyme activity in the SSII peak was neutralized to a large extent by antibodies raised against the product of the Du1 gene, but was not affected by antibodies specific for the other highly expressed soluble starch synthase, zSSI, or for the zSSIIa or zSSIIb isoforms. These data provide direct evidence that Du1 codes for the starch synthase responsible for the SSII activity peak. This starch synthase was purified approximately 350-fold from endosperm extracts. The following enzymatic properties of the SSII activity were determined: temperature optimum, thermostability, pH effects, K(m) for different glucan primers and the glucosyl unit donor ADPGlc, V(max) using various primers, and stimulation by citrate. These properties were compared to those of zSSI purified over 1600-fold from maize endosperm by a parallel procedure. The major differences between the two enzymes were that the SSII activity displayed higher K(m) values for ADPGlc, a distinct temperature range for maximal activity, and different relative activities toward specific exogenous substrates. The purified SSI and SSII activities both were shown to be capable of elongating maltooligosaccharide primers in vitro.     

Metabolic engineering of fatty acid biosynthesis in plants.

Fatty acids are the most abundant form of reduced carbon chains available from nature and have diverse uses ranging from food to industrial feedstocks. Plants represent a significant renewable source of fatty acids because many species accumulate them in the form of triacylglycerol as major storage components in seeds. With the advent of plant transformation technology, metabolic engineering of oilseed fatty acids has become possible and transgenic plant oils represent some of the first successes in design of modified plant products. Directed gene down-regulation strategies have enabled the specific tailoring of common fatty acids in several oilseed crops. In addition, transfer of novel fatty acid biosynthetic genes from noncommercial plants has allowed the production of novel oil compositions in oilseed crops. These and future endeavors aim to produce seeds higher in oil content as well as new oils that are more stable, are healthier for humans, and can serve as a renewable source of industrial commodities. Large-scale new industrial uses of engineered plant oils are on the horizon but will require a better understanding of factors that limit the accumulation of unusual fatty acid structures in seeds.     

Metabolic engineering for plant natural product biosynthesis in microbes

Plant natural products (NPs) not only serve many functions in an organism's survivability but also demonstrate important pharmacological activities. Isolation of NPs from native sources is frequently limited by low abundance and environmental, seasonal, and regional variation while total chemical synthesis of what are often complex structures is typically commercially infeasible. Reconstruction of biosynthetic pathways in heterologous microorganisms offers significant promise for a scalable means to provide sufficient quantities of a desired NP while using inexpensive renewable resources. To this end, metabolic engineering provides the technological platform for enhancing NP production in these engineered heterologous hosts. Recent advancements in the production of isoprenoids, phenylpropanoids, and alkaloids were made possible by utilizing a variety of techniques including combinatorial biosynthesis, codon optimization, expression of regulatory elements, and protein engineering of P450s.     

Multiple improvement of astaxanthin biosynthesis in Xanthophyllomyces dendrorhous by a combination of conventional mutagenesis and metabolic pathway engineering

Xanthophyllomyces dendrorhous (Phaffia rhodozyma) is the only yeast or fungus that synthesizes the commercially attractive carotenoid astaxanthin. For a suitable bioprocess, the wild type has to be modified for increasing biomass content. To achieve this, a two step strategy has been followed. At first, random mutagenesis was applied leading to colonies with substantially higher astaxanthin content. Then, the resulting strain was genetically engineered by targeting limiting reactions for further enhancement of astaxanthin biosynthesis. This combinatorial approach together with selection of an appropriate growth medium resulted in highest astaxanthin biomass contents reported to date for X. dendrorhous. In a fermenter culture, its maximum content was 9.7 mg/g dry weight.     

Turning a green alga red: engineering astaxanthin biosynthesis by intragenic pseudogene revival in Chlamydomonas reinhardtii

The green alga Chlamydomonas reinhardtii does not synthesize high‐value ketocarotenoids like canthaxanthin and astaxanthin; however, a β‐carotene ketolase (CrBKT) can be found in its genome. CrBKT is poorly expressed, contains a long C‐terminal extension not found in homologues and likely represents a pseudogene in this alga. Here, we used synthetic redesign of this gene to enable its constitutive overexpression from the nuclear genome of C. reinhardtii. Overexpression of the optimized CrBKT extended native carotenoid biosynthesis to generate ketocarotenoids in the algal host causing noticeable changes the green algal colour to reddish‐brown. We found that up to 50% of native carotenoids could be converted into astaxanthin and more than 70% into other ketocarotenoids by robust CrBKT overexpression. Modification of the carotenoid metabolism did not impair growth or biomass productivity of C. reinhardtii, even at high light intensities. Under different growth conditions, the best performing CrBKT overexpression strain was found to reach ketocarotenoid productivities up to 4.3 mg/L/day. Astaxanthin productivity in engineered C. reinhardtii shown here might be competitive with that reported for Haematococcus lacustris (formerly pluvialis) which is currently the main organism cultivated for industrial astaxanthin production. In addition, the extractability and bio‐accessibility of these pigments were much higher in cell wall‐deficient C. reinhardtii than the resting cysts of H. lacustris. Engineered C. reinhardtii strains could thus be a promising alternative to natural astaxanthin producing algal strains and may open the possibility of other tailor‐made pigments from this host.