Metabolic engineering of plant L-ascorbic acid biosynthesis: recent trends and applications

Vitamin C (L-ascorbic acid; AsA) is the major soluble antioxidant found in plants and is also an essential component of human nutrition. Although numerous biotechnological methods have been exploited to increase its yield, pressures such as commercial competition and environmental concerns make it urgent to find a new way for industrial production of plant-derived AsA. Engineering plant AsA has now become feasible because of our increased understanding of its biosynthetic pathway. Several possible strategies could be followed to increase AsA production, such as overcoming the rate limiting steps in the biosynthetic pathway, promoting recycling, and reducing catabolism. For these purposes, genes of plant, microbial and animal origins have been successfully used. Several examples will be given to illustrate these various approaches. The existing and potential achievements in increasing AsA production would provide the opportunity for enhancing nutritional quality and stress tolerance of crop plants.     

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 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.     

Metabolic engineering of Escherichia coli for biosynthesis of hyaluronic acid

Engineering of hyaluronic acid (HA) biosynthetic pathway in recombinant Escherichia coli as production host is reported in this work. A hyaluronic acid synthase (HAS) gene, sphasA, from Sreptococcus pyogenes with the start codon gtg to atg mutant, was expressed in recombinant E. coli with or without the genes ugd, galF and glmU, which are analogs of hasB, hasC and hasD from Streptococcus, respectively, encoding UDP-glucose 6-dehygrogenase, Glucose-1-P uridyltransferase, and N-acetyl glucosamine uridyltransferase enzymes in the HA biosynthetic pathway. The single, double and triple organized artificial operons of sphasA, ugd, galF and glmU were designed and constructed using the inducible plasmid backbone of pMBAD. Only the triple expression recombinant, Top10/pMBAD-spABC, generated a relatively high titer of HA (approximately 48 mg/l at 48 h), indicating that both of the enzymes encoded by ugd and galF are essential for HA biosynthesis. A new gene of ssehasA with identical protein sequence of seHAS from Streptococcus equisimilis, was artificially synthesized after substituting all of the rare codons in the natural sehasA. The HA titer at 24 h flask culture increased to approximately 190 mg/l in sseAB and 160 mg/l in sseABC, respectively. Sorbitol could be used as another carbon source for HA accumulation, and the metabolic pathway for HA synthesis in a recombinant E. coli was presented. The concentration of Mg(2+) cofactor of HA synthase was optimized and a cell growth inhibition phenomenon was observed during HA accumulation. Molecular weight (MW) measurements revealed that the mean MW of HA produced from the recombinant E. coli under different conditions ranges from approximately 3.5x10(5) to 1.9x10(6)Da, indicating that the recombinant E. coli can be used as a potential host candidate for industrial production of HA.     

Heterologous biosynthesis of artemisinic acid in Saccharomyces cerevisiae

Artemisinic acid is a precursor of antimalarial compound artemisinin. The titre of biosynthesis of artemisinic acid using Saccharomyces cerevisiae platform has been achieved up to 25 g l^?1; however, the performance of platform cells is still industrial unsatisfied. Many strategies have been proposed to improve the titre of artemisinic acid. The traditional strategies mainly focused on partial target sites, simple up‐regulation key genes or repression competing pathways in the total synthesis route. However, this may result in unbalance of carbon fluxes and dysfunction of metabolism. In this review, the recent advances on the promising methods in silico and in vivo for biosynthesis of artemisinic acid have been discussed. The bioinformatics and omics techniques have brought a great prospect for improving production of artemisinin and other pharmacal compounds in heterologous platform.     

Metabolic engineering of an alternative pathway for ascorbic acid biosynthesis in plants

Plants and most animals can synthesize their own L-ascorbic acid (vitamin C), but a mutation in the L-gulono--lactone oxidase gene in the primate lineage makes it necessary for humans to acquire this vital compound from their diet. Despite the fact that plants and animals synthesize ascorbic acid via different pathways, transgenic tobacco and lettuce plants expressing a rat cDNA encoding L-gulono--lactone oxidase accumulated up to seven times more ascorbic acid than untransformed plants. These results demonstrate that basal levels of ascorbic acid in plants can be significantly increased by expressing a single gene from the animal pathway.     

Metabolic engineering of plant volatiles

Metabolic engineering of the volatile spectrum offers enormous potential for plant improvement because of the great contribution of volatile secondary metabolites to reproduction, defense and food quality. Recent advances in the identification of the genes and enzymes responsible for the biosynthesis of volatile compounds have made this metabolic engineering highly feasible. Notable successes have been reported in enhancing plant defenses and improving scent and aroma quality of flowers and fruits. These studies have also revealed challenges and limitations which will be likely surmounted as our understanding of plant volatile network improves.     

Metabolic engineering of Escherichia coli for biosynthesis of d-galactonate

d-galactose is an attractive substrate for bioconversion. Herein, Escherichia coli was metabolically engineered to convert d-galactose into d-galactonate, a valuable compound in the polymer and cosmetic industries. d-galactonate productions by engineered E. coli strains were observed in shake flask cultivations containing 2 g L^?1 d-galactose. Engineered E. coli expressing gld coding for galactose dehydrogenase from Pseudomonas syringae was able to produce 0.17 g L^?1 d-galactonate. Inherent metabolic pathways for assimilating both d-galactose and d-galactonate were blocked to enhance the production of d-galactonate. This approach finally led to a 7.3-fold increase with d-galactonate concentration of 1.24 g L^?1 and yield of 62.0 %. Batch fermentation in 20 g L^?1 d-galactose of E. coli ?galK?dgoK mutant expressing the gld resulted in 17.6 g L^?1 of d-galactonate accumulation and highest yield of 88.1 %. Metabolic engineering strategy developed in this study could be useful for industrial production of d-galactonate.     

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.     

Metabolic engineering of plant secondary products

Plants interact with their environment by producing a diverse array of secondary metabolites. Many of these compounds are valued for their medicinal, industrial or agricultural properties. Other secondary products are toxic or otherwise undesirable and can reduce the commercial value of crops. Gene transfer technology offers new opportunities to modify directly plant secondary product synthesis through metabolic engineering. This article reviews some of the strategies which have been used to increase or decrease the synthesis of specific plant metabolites, as well as methods for expanding the biosynthetic capabilities of individual species.     

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 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.     

Metabolic pathway engineering for complex polyketide biosynthesis in Saccharomyces cerevisiae

Polyketides are a diverse group of natural products with significance in human and veterinary medicine. Because polyketides are structurally complex molecules and fermentation is the most commercially viable route of production, a generic heterologous host system for high-level polyketide production is desirable. Saccharomyces cerevisiae has been shown to be an excellent production host for a simple polyketide, yielding 1.7 g of 6-methylsalicylic acid per liter of culture in un-optimized shake-flask fermentations. However, a barrier to the heterologous production of more complex 'modular' polyketides in S. cerevisiae is the lack of required polyketide precursor pathways. In this work, we describe the introduction into S. cerevisiae of pathways for the production of methylmalonyl-coenzyme A (CoA), a precursor for complex polyketides, by both propionyl-CoA-dependent and propionyl-CoA-independent routes. Furthermore, we demonstrate that the methylmalonyl-CoA produced in the engineered yeast strains is used in vivo for the production of a polyketide product, a triketide lactone.     

Metabolic engineering for microbial production of shikimic acid

Shikimic acid is a high valued compound used as a key starting material for the synthesis of the neuramidase inhibitor GS4104, which was developed under the name Tamiflu for treatment of antiviral infections. An excellent alternative to the isolation of shikimic acid from fruits of the Illicium plant is the fermentative production by metabolic engineered microorganisms. Fermentative production of shikimic acid was most successfully carried out by rational designed Escherichia coli strains by blocking the aromatic amino acid pathway after the production of shikimic acid. An alternative is to produce shikimic acid as a result of dephosphorylation of shikimate-3-phosphate. Engineering the uptake of carbon, the regulatory circuits, central metabolism and the common aromatic pathway including shikimic acid import that have all been targeted to effect higher productivities and lower by-product formation are discussed.     

Metabolic engineering of edible plant oils]

Plant seed oil is the major source of many fatty acids for human nutrition, and also one of industrial feedstocks. Recent advances in understanding of the basic biochemistry of seed oil biosynthesis, coupled with cloning of the genes encoding the enzymes involved in fatty acid modification and oil accumulation, have set the stage for the metabolic engineering of oilseed crops that produce "designer" plant seed oils with the improved nutritional values for human being. In this review we provide an overview of seed oil biosynthesis/regulation and highlight the key enzymatic steps that are targets for gene manipulation. The strategies of metabolic engineering of fatty acids in oilseeds, including overexpression or suppression of genes encoding single or multi-step biosynthetic pathways and assembling the complete pathway for the synthesis of long-chain polyunsaturated fatty acids (e.g. arachidonic acid, eicosapentaenoic acid and docosahexaenoic acid) are described in detail. The current "bottlenecks" in using common oilseeds as "bioreactors" for commercial production of high-value fatty acids are analyzed. It is also discussed that the future research focuses of oilseed metabolic engineering and the prospects in creating renewable sources and promoting the sustainable development of human society and economy.     

Metabolic engineering of the chloroplast genome using the Echerichia coli ubiC gene reveals that chorismate is a readily abundant plant precursor for p-hydroxybenzoic acid biosynthesis

p-Hydroxybenzoic acid (pHBA) is the major monomer in liquid crystal polymers. In this study, the Escherichia coli ubiC gene that codes for chorismate pyruvate-lyase (CPL) was integrated into the tobacco (Nicotiana tabacum) chloroplast genome under the control of the light-regulated psbA 5' untranslated region. CPL catalyzes the direct conversion of chorismate, an important branch point intermediate in the shikimate pathway that is exclusively synthesized in plastids, to pHBA and pyruvate. The leaf content of pHBA glucose conjugates in fully mature T1 plants exposed to continuous light (total pooled material) varied between 13% and 18% dry weight, while the oldest leaves had levels as high as 26.5% dry weight. The latter value is 50-fold higher than the best value reported for nuclear-transformed tobacco plants expressing a chloroplast-targeted version of CPL. Despite the massive diversion of chorismate to pHBA, the plastid-transformed plants and control plants were indistinguishable. The highest CPL enzyme activity in pooled leaf material from adult T1 plants was 50,783 pkat/mg of protein, which is equivalent to approximately 35% of the total soluble protein and approximately 250 times higher than the highest reported value for nuclear transformation. These experiments demonstrate that the current limitation for pHBA production in nuclear-transformed plants is CPL enzyme activity, and that the process becomes substrate-limited only when the enzyme is present at very high levels in the compartment of interest, such as the case with plastid transformation. Integration of CPL into the chloroplast genome provides a dramatic demonstration of the high-flux potential of the shikimate pathway for chorismate biosynthesis, and could prove to be a cost-effective route to pHBA. Moreover, exploiting this strategy to create an artificial metabolic sink for chorismate could provide new insight on regulation of the plant shikimate pathway and its complex interactions with downstream branches of secondary metabolism, which is currently poorly understood.     

Metabolic engineering of Rhizopus oryzae: Effects of overexpressing pyc and pepc genes on fumaric acid biosynthesis from glucose

Fumaric acid, a dicarboxylic acid used as a food acidulant and in manufacturing synthetic resins, can be produced from glucose in fermentation by Rhizopus oryzae. However, the fumaric acid yield is limited by the co-production of ethanol and other byproducts. To increase fumaric acid production, overexpressing endogenous pyruvate carboxylase (PYC) and exogenous phosphoenolpyruvate carboxylase (PEPC) to increase the carbon flux toward oxaloacetate were investigated. Compared to the wild type, the PYC activity in the pyc transformants increased 56%-83%, whereas pepc transformants exhibited significant PEPC activity (3-6mU/mg) that was absent in the wild type. Fumaric acid production by the pepc transformant increased 26% (0.78g/g glucose vs. 0.62g/g for the wild type). However, the pyc transformants grew poorly and had low fumaric acid yields (<0.05g/g glucose) due to the formation of large cell pellets that limited oxygen supply and resulted in the accumulation of ethanol with a high yield of 0.13-0.36g/g glucose. This study is the first attempt to use metabolic engineering to modify the fumaric acid biosynthesis pathway to increase fumaric acid production in R. oryzae.