Natural rubber biosynthesis in plants,the rubber transferase complex,and metabolic engineering progress and prospects

Natural rubber (NR) is a nonfungible and valuable biopolymer, used to manufacture ~50 000 rubber products, including tires and medical gloves. Current production of NR is derived entirely from the para rubber tree (Hevea brasiliensis). The increasing demand for NR, coupled with limitations and vulnerability of H. brasiliensis production systems, has induced increasing interest among scientists and companies in potential alternative NR crops. Genetic/metabolic pathway engineering approaches, to generate NR‐enriched genotypes of alternative NR plants, are of great importance. However, although our knowledge of rubber biochemistry has significantly advanced, our current understanding of NR biosynthesis, the biosynthetic machinery and the molecular mechanisms involved remains incomplete. Two spatially separated metabolic pathways provide precursors for NR biosynthesis in plants and their genes and enzymes/complexes are quite well understood. In contrast, understanding of the proteins and genes involved in the final step(s)—the synthesis of the high molecular weight rubber polymer itself—is only now beginning to emerge. In this review, we provide a critical evaluation of recent research developments in NR biosynthesis, in vitro reconstitution, and the genetic and metabolic pathway engineering advances intended to improve NR content in plants, including H. brasiliensis, two other prospective alternative rubber crops, namely the rubber dandelion and guayule, and model species, such as lettuce. We describe a new model of the rubber transferase complex, which integrates these developments. In addition, we highlight the current challenges in NR biosynthesis research and future perspectives on metabolic pathway engineering of NR to speed alternative rubber crop commercial development.     

Guayule and Russian Dandelion as Alternative Sources of Natural Rubber

ABSTRACT

Natural rubber, obtained almost exclusively from the Para rubber tree (Hevea brasiliensis), is a unique biopolymer of strategic importance that, in many of its most significant applications, cannot be replaced by synthetic rubber alternatives. Several pressing motives lead to the search for alternative sources of natural rubber. These include increased evidence of allergenic reactions to Hevea rubber, the danger that the fungal pathogen Microcyclus ulei, causative agent of South American Leaf Blight (SALB), might spread to Southeast Asia, which would severely disrupt rubber production, potential shortages of supply due to increasing demand and changes in land use, and a general trend towards the replacement of petroleum-derived chemicals with renewables. Two plant species have received considerable attention as potential alternative sources of natural rubber: the Mexican shrub Guayule (Parthenium argentatum Gray) and the Russian dandelion (Taraxacum koksaghyz). This review will summarize the current production methods and applications of natural rubber (dry rubber and latex), the threats to the production of natural rubber from the rubber tree, and describe the current knowledge of the production of natural rubber from guayule and Russian dandelion.     

Initiator-independent and initiator-dependent rubber biosynthesis in Ficus elastica

The rubber-producing tree, Ficus elastica (the Indian rubber tree), requires the same substrates for rubber production as other rubber-producing plants, such as Hevea brasiliensis (the Brazilian or Para rubber tree), the major source of commercial natural rubber in the world, and Parthenium argentatum (guayule), a widely studied alternative for natural rubber production currently under commercial development. Rubber biosynthesis can be studied, in vitro, using purified, enzymatically active rubber particles, an initiator such as FPP, IPP as the source of monomer, and a metal cofactor such as Mg2+. However, unlike H. brasiliensis and P. argentatum, we show that enzymatically active rubber particles purified from F. elastica are able to synthesize rubber, in vitro, in the absence of added initiator. In this paper, we characterize, for the first time, the kinetic differences between initiator-dependent rubber biosynthesis, and initiator-independent rubber biosynthesis, and the effect of cofactor concentration on both of these processes.     

Guayule and Russian dandelion as alternative sources of natural rubber

Natural rubber, obtained almost exclusively from the Para rubber tree (Hevea brasiliensis), is a unique biopolymer of strategic importance that, in many of its most significant applications, cannot be replaced by synthetic rubber alternatives. Several pressing motives lead to the search for alternative sources of natural rubber. These include increased evidence of allergenic reactions to Hevea rubber, the danger that the fungal pathogen Microcyclus ulei, causative agent of South American Leaf Blight (SALB), might spread to Southeast Asia, which would severely disrupt rubber production, potential shortages of supply due to increasing demand and changes in land use, and a general trend towards the replacement of petroleum-derived chemicals with renewables. Two plant species have received considerable attention as potential alternative sources of natural rubber: the Mexican shrub Guayule (Parthenium argentatum Gray) and the Russian dandelion (Taraxacum koksaghyz). This review will summarize the current production methods and applications of natural rubber (dry rubber and latex), the threats to the production of natural rubber from the rubber tree, and describe the current knowledge of the production of natural rubber from guayule and Russian dandelion.     

TRICHOME MORPHOLOGY AS AN INDICATOR OF HIGH RUBBER BEARING GUAYULE (PARTHENIUM ARGENTATUM GRAY) PLANTS IN NATIVE POPULATIONS

Parthenium argentatum Gray (guayule) and P. incanum (mariola) grow in close associations in their native habitat of Mexico. Variation in rubber concentration and morphology of guayule has been known for a long time. Studies of over 75 native guayule plants indicated the presence of at least three prominent forms of guayule plants. Group I guayule plants have oblanceolate leaves and leaf margins are entire to two-toothed. T-shaped leaf trichomes are with centrally attached stalk and cap cell with two blunt ends. Plants in this group are high in rubber, containing on the average 17% rubber. Group II guayule plants have narrow elliptic leaves and leaf margins are entire to four-toothed. T-shaped trichomes have an acentrally attached stalk and a cap cell with short end blunt, long end pointed and straight. Rubber content in these plants averages 10%. Group III guayule plants have ovate leaves and leaf margins are four- to eight-toothed. T-trichomes have an acentrally attached stalk and a cap cell with short end blunt, long end pointed and wavy or curved. These plants on the average contain 6% rubber. Morphological as well as biochemical data indicate the presence of mariola genes in the last two groups of plants and this has resulted in an increase in trichome length and a decrease in the rubber content. Group II guayule plants are of more common occurrence than the others. Based on the data presented here, high rubber bearing guayule plants in native stands can be easily selected by analyzing trichome morphology.     

Establishment of new crops for the production of natural rubber

Natural rubber is a unique biopolymer of strategic importance that, in many of its most significant applications, cannot be replaced by synthetic alternatives. The rubber tree Hevea brasiliensis is the almost exclusive commercial source of natural rubber currently and alternative crops should be developed for several reasons, including: a disease risk to the rubber tree that could potentially decimate current production, a predicted shortage of natural rubber supply, increasing allergic reactions to rubber obtained from the Brazilian rubber tree and a general shift towards renewables. This review summarizes our knowledge of plants that can serve as alternative sources of natural rubber, of rubber biosynthesis and the scientific gaps that must be filled to bring the alternative crops into production.     

Performance of USDA guayule lines in the northern Negev of Israel

The rubber yield of the guayule plant (Tarthenium argentatum) is determined by its rubber concentration and biomass production, both of which are a function of genetic constituents and environmental conditions. With the aim of selecting best performing lines of guayule under the climatic conditions of the northern Negev of Israel, two-yr-old guayule plants from 13 USDA lines were evaluated for height, spread, canopy fresh and dry weight, branch and leaf dry weight, rubber and resin concentration, and rubber and resin yield per plant. Significant differences were found among the lines for all the characteristics tested. Lines 11600 and 11604 excelled in rubber and biomass production and were taller and more uniform than the other lines; whereas line N593, conventionally used as a standard, performed poorly and was less uniform. The correlation between rubber yield per plant and rubber concentration was poor, rubber yield being related mainly to biomass production and to a lesser degree to height and spread of the plants. Taken together with the fact that biomass production was more variable than rubber concentration, this finding suggests that breeding and selection of guayule for high rubber yield should be directed primarily towards improvement of biomass production rather than rubber concentration. In the northern Negev of Israel, USDA lines 11600 and 11604 performed well.     

Application of size-exclusion chromatography to polymers of ultra-high molar mass

This article describes the experimental conditions that should be applied to avoid molecular degradation in size-exclusion chromatography of polymers of ultra-high molar mass (weight-average molar mass M(w)>5000 kg/mol). The applicability of the optimized experimental conditions is demonstrated using polystyrene as a model substance, but also by using polymers of biochemical and biophysical interest, such as polyethylene of ultra-high molar mass, suitable, e.g., for articulating surfaces in joint endoprotheses, and natural rubber, the most important commercial source for products like septa or medical gloves.     

Magnesium ion regulation of in vitro rubber biosynthesis by Parthenium argentatum Gray

Natural rubber is produced by a rubber transferase (a cis-prenyltransferase). Rubber transferase uses allylic pyrophosphate to initiate the rubber molecule and isopentenyl pyrophosphate (IPP) to form the polymer. Rubber biosynthesis also requires a divalent metal cation. Understanding how molecular weight is regulated is important because high molecular weight is required for high quality rubber. We characterized the in vitro effects of Mg(2+) on the biosynthetic rate of rubber produced by an alternative natural rubber crop, Parthenium argentatum (guayule). The affinity of the rubber transferase from P. argentatum for IPP.Mg was shown to depend on the Mg(2+) concentration in a similar fashion to the H. brasiliensis rubber transferase, although to a less extreme degree. Also, in vitro Mg(2+) concentration significantly affects rubber molecular weight of both species, but molecular weight is less sensitive to Mg(2+) concentration in P. argentatum than in H. brasiliensis.     

Rubber content in diploid guayuleParthenium argentatum: chromosomes,rubber variation,and implications for economic use

Guayule,Parthenium argentatum, is a well-known source of high quality natural rubber. Despite the fact that a wealth of information exists in the literature on polyploidy, aneuploidy, and apomixis of guayule, little is understood of the basis for rubber variation in the native shrub. Selective breeding continues in an attempt to enhance the human use of this economic plant against a background of disappointingly low rubber content in experimental germplasm. Successful economic development of the guayule resource depends, in part, upon the introduction of high rubber-containing diploid plants into the selection process. Other factors contribute to problems relating to human use of guayule. This paper reports high rubber-containing diploid plants from native stands in Mexico. Since our data indicate that ploidy is not related to rubber content per se, we suggest that observed rubber variation among naturally occurring plants may be a consequence of ecological factors, possibly mineral nutrition, water relations, geologic substrate, and clone longevity.     

Photosynthesis and assimilate partitioning between carbohydrates and isoprenoid products in vegetatively active and dormant guayule: physiological and environmental constraints on rubber accumulation in a semiarid shrub

The stems and roots of the semiarid shrub guayule, Parthenium argentatum, contain a significant amount of natural rubber. Rubber accumulates in guayule when plants are vegetatively and reproductively dormant, complicating the relationship between growth/reproduction and product synthesis. To evaluate the factors regulating the partitioning of carbon to rubber, carbon assimilation and partitioning were measured in guayule plants that were grown under simulated summer‐ and winter‐like conditions and under winter‐like conditions with CO₂ enrichment. These conditions were used to induce vegetatively active and dormant states and to increase the source strength of vegetatively dormant plants, respectively. Rates of CO₂ assimilation, measured under growth temperatures and CO₂, were similar for plants grown under summer‐ and winter‐like conditions, but were higher with elevated CO₂. After 5 months, plants grown under summer‐like conditions had the greatest aboveground biomass, but the lowest levels of non‐structural carbohydrates and rubber. In contrast, the amount of resin in the stems was similar under all growth conditions. Emission of biogenic volatile compounds was more than three‐fold higher in plants grown under summer‐ compared with winter‐like conditions. Taken together, the results show that guayule plants maintain a high rate of photosynthesis and accumulate non‐structural carbohydrates and rubber in the vegetatively dormant state, but emit volatile compounds at a lower rate when compared with more vegetatively active plants. Enrichment with CO₂ in the vegetatively dormant state increased carbohydrate content but not the amount of rubber, suggesting that partitioning of assimilate to rubber is limited by sink strength in guayule.     

A novel cDNA from Parthenium argentatum Gray enhances the rubber biosynthetic activity in vitro

Natural rubber (cis-1,4-polyisoprene) is an isoprenoid compound produced exclusively in plants by the action of rubber transferase. Despite a keen interest in revealing the mechanisms of rubber chain elongation and chain length determination, the molecular nature of rubber transferase has not yet been identified. A recent report has revealed that a 24 kDa protein tightly associated with the small rubber particles of Hevea brasiliensis, therefore designated small rubber particle protein (SRPP), plays a positive role in rubber biosynthesis. Since guayule (Parthenium argentatum Gray) produces natural rubber similar in size to H. brasiliensis, it is of critical interest to investigate whether guayule contains a similar protein to the SRPP. A cDNA clone has been isolated in guayule that shares a sequence homology with the SRPP, thus designated guayule homologue of SRPP (GHS), and the catalytic function of the protein was characterized. Sequence analysis revealed that the GHS is highly homologous in several conserved regions to the SRPP (50% identity). In vitro functional analysis of the recombinant protein overexpressed in E. coli revealed that the GHS plays a positive role in isopentenyl diphosphate incorporation into high molecular weight rubbers as SRPP does. These results indicate that guayule and Hevea rubber trees contain a protein that is similar in its amino acid sequence and plays a role in isopentenyl diphosphate incorporation in vitro, implying that it contributes to the enhancement of rubber biosynthetic activity in rubber trees.     

Quantitation of latex allergens

Minimizing allergen concentration in latex goods to prevent sensitization to natural rubber latex (NRL) and thereby the development of clinical allergy is acknowledged as of mutual interest for rubber manufacturers and regulatory health authorities. However, measuring total protein, the principal currently available method, cannot be deemed a satisfactory regulatory measure to control allergen content. Specific methods based on human IgE-containing reagents, such as radioallergosorbent test (RAST) inhibition, have been available in certain laboratories for demonstrating NRL allergens in rubber products but the methods lack standardization. Currently, one commercial test has become available for measuring individual NRL allergens by capture ELISA-based assays using monoclonal antibodies and purified or recombinant allergens. Such methods are specific, they can be properly standardized, and they are of sufficient sensitivity and reproducibility. Results from medical gloves collected in two national market surveys in Finland in 1995 and 1999, respectively, show that Hev b 6.02 and Hev b 5, the two major allergens for NRL-allergic adults, are the most abundant allergens regularly detectable in high- and moderate-allergen gloves. In addition, Hev b 3 and Hev b 1, the two major allergens for children with spina bifida, are also commonly found. In general, when the sum of the four allergens exceeded 1 microg/g, most NRL-allergic patients showed positive skin prick test reactions against them. Using these new methods assessment of threshold levels that could in due course become guidelines for the rubber industry and regulatory health authorities is becoming possible. Eventually, this progress is expected to lead to a declining incidence of latex allergy.     

Low Night Temperature-Induced Changes in Photosynthesis and Rubber Accumulation in Guayule (Parthenium Argentatum Gray)

Three-year-old plants of Parthenium argentatum Gray cv. 11591 grown under natural photoperiod were exposed for 60 d to low night temperature (LNT) of 15 °C (daily from 18:00 to 06:00). Effects of the treatment on net photosynthetic rates (P _N), rubber accumulation, and associated biochemical traits were examined. LNT initially reduced P _N with a parallel decline in the activities of ribulose-1,5-bisphosphate carboxylase, fructose bisphosphatase, and sucrose phosphate synthase for 20–30 d. Later, LNT enhanced P _N and the activities of photosynthetic enzymes. Associated with high P _N in LNT-treated guayule plants was a two-fold increase in rubber content and rubber transferase activity per unit of protein. The initial decrease in P _N in LNT-treated guayule was associated with low content of chlorophyll (a+b), large starch accumulation, and higher ratio of glucose-6-phosphate/fructose-6-phosphate. Photosystem 2 activity in isolated chloroplasts was initially decreased, but increased after 30 d. There was a significant increase in the leaf soluble protein content in LNT-treated plants. Hence the photosynthetic performance of plants grown at 15 °C night temperature for 50 d was superior to those grown under natural photoperiod in all parameters studied. The high photosynthetic capacity may contribute to superior rubber yields under LNT. This revised version was published online in August 2006 with corrections to the Cover Date.     

Morphological,chemical, and cytogenetic characters of F1 hybrids betweenParthenium argentatum (Guayule) andP. fruticosum var.fruticosum (Asteraceae) and their potential in rubber improvement

Interspecific hybrids betweenParthenium argentatum, the guayule rubber plant, andP. fruticosum var. fruticosum were evaluated for their potential rubber content and quality. Fifteen-mo-old field-grown plants ofP. fruticosum var.fruticosum measured four times more in height and spread than those ofP. argentatum, but contained less than 0.05% rubber of low mol wt.Parthenium argentatum showed 2% rubber content, with a mol wt of about one million. Resin contents varied little among parents or hybrids. The same age F₁ hybrids were intermediate in height and spread and had low rubber content, but showed presence of high mol wt rubber like the guayule parent. This indicates that high mol wt rubber is expressed over the low mol wt rubber in F₁ hybrids. Despite low rubber content but favorable biomass production, F₁ hybrids revealed irregular meiotic chromosome behavior and low pollen and seed germination. These results suggest that interspecific F₁ hybrids may be used in backcross programs to increase biomass and rubber content in guayule.     

The role of the alternative respiratory pathway in the stimulation of cephalosporin C formation by soybean oil in Acremonium chrysogenum

Addition of soybean oil to Acremonium chrysogenum cultures growing on sugars doubled the specific production of cephalosporin C during the idiophase of growth. While the addition of soybean oil had no effect on the total rate of respiration, the respiration that proceeded via the alternative, cyanide-insensitive pathway exhibited a more than twofold increase. Addition of soybean oil also stimulated the formation of isocitrate lyase activities. Inhibition of oxidative metabolism of one of the products of isocitrate lyase (succinate) by thenoyltrifluoroacetone completely inhibited the alternative respiratory pathway. The role of soybean-oil-stimulated alternative respiration in the stimulation of cephalosporin C production and the role of isocitrate lyase are discussed. Received: 13 October 1998 / Revised revision: 14 January 1999 / Accepted: 22 January 1999     

橡胶草的研究进展

天然橡胶是一种不可替代的重要战略工业原料, 用途广泛。巴西橡胶树作为天然橡胶的主要来源, 受种植面积限制, 难以满足全球日益增长的对天然橡胶的需求。而南美叶疫病也是巴西橡胶树(Hevea brasiliensis)安全的潜在威胁。蒲公英属产胶植物(橡胶草)最早发现于20世纪30年代, 可产生高质量的天然橡胶, 具有生长周期短、地理适应范围广、适合机械化生产等特点, 被认为是一种理想的产胶备选作物。该文从橡胶草种质资源、遗传改良、栽培技术及产胶生物学机制等方面综述了国内外橡胶草的研究进展和存在的问题, 并为我国开展橡胶草相关研究提出建议。     

橡胶草90年来主要研究成果及最新研究进展

天然橡胶是重要的国防战略物质,巴西橡胶树（Hevea brasiliensis（Willd.ex A.Juss.） Muell.Arg）是天然橡胶的唯一来源,天然橡胶商业化形式极为单一,潜在的供给不足问题亟待解决。因此,寻找可替代巴西橡胶树的产胶植物一直受到全世界高度重视。蒲公英属橡胶草（Taraxacum kok-saghyz Rodin）根部含有与橡胶树橡胶类似的天然橡胶分子,该植物主要分布在温带和寒带地区,具有易于机械化收获、生长周期短、遗传转化相对容易等特点,是最具开发潜力的产胶植物。本文对橡胶草90年（1931-2018）来的研究历史和主要成果进行了概括,对近10年取得的最新成果进行了深度分析,并预测橡胶草在未来天然橡胶产业中的作用,期望为开展橡胶草商业化生产和橡胶生物合成相关基础研究提供一定的参考。     

橡胶草90年来主要研究成果及最新研究进展

天然橡胶是重要的国防战略物质,巴西橡胶树( Hevea brasiliensis (Willd. ex A. Juss.) Muell. Arg)是天然橡胶的唯一来源,天然橡胶商业化形式极为单一,潜在的供给不足问题亟待解决。因此,寻找可替代巴西橡胶树的产胶植物一直受到全世界高度重视。蒲公英属橡胶草( Taraxacum kok-saghyz Rodin)根部含有与橡胶树橡胶类似的天然橡胶分子,该植物主要分布在温带和寒带地区,具有易于机械化收获、生长周期短、遗传转化相对容易等特点,是最具开发潜力的产胶植物。本文对橡胶草90年(1931 - 2018)来的研究历史和主要成果进行了概括,对近10年取得的最新成果进行了深度分析,并预测橡胶草在未来天然橡胶产业中的作用,期望为开展橡胶草商业化生产和橡胶生物合成相关基础研究提供一定的参考。     

Seasonal Variations in Rubber Biosynthesis, 3-Hydroxy-3-Methylglutaryl-Coenzyme A Reductase,and Rubber Transferase Activities in Parthenium argentatum in the Chihuahuan Desert

The rubber content and the activities of enzymes in the polyisoprenoid pathway in Parthenium argentatum (guayule) were examined throughout the growing season in field plots in the Chihuahuan Desert. The rubber content of the plants was low in July and August and slowly increased until October. From October to December there was a rapid increase in rubber formation (per plant) from 589.0 mg to 4438.0 mg. The percentage of rubber in the plants increased from 0.7% (mg/g dry weight) in August and 1.27% in October to 5.5% in December. The rapid increase in rubber formation may result from exposing the plants to low temperatures of 5 to 7[deg]C. The activity of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) was 21.1 nmol mevalonic acid (MVA) h-1 g-1 fresh weight in the bark of the lower stems in June during seedling growth and decreased to 5.1 nmol MVA h-1g-1 fresh weight in July and 2.9 nmol MVA h-1 g-1 fresh weight in September. From October to December, the activity increased from 5.0 to 29.9 nmol MVA h-1 g-1 fresh weight. The activity of rubber transferase was 65.5 nmol isopentenyl pyrophosphate (IPP) h-1 g-1fresh weight in the bark in September and increased to 357.5 nmol IPP h-1 g-1 fresh weight in December. The rapid increase in the activities of HMGR and rubber transferase coincided with the rapid increase in rubber formation. The activities of MVA kinase and IPP isomerase did not significantly increase in the fall and winter. A tomato HMGR-1 cDNA probe containing a highly conserved C-terminal region of HMGR genes hybridized at low stringency with several bands on blots of HindIII-digested genomic DNA from guayule. In northern blots with the HMGR-1 cDNA probe at low stringency, HMGR mRNA was high in June and November, corresponding to periods of high HMGR activity during seedling growth and rapid increase in rubber formation. The seasonal variations in rubber formation and HMGR mRNA, HMGR activity, and rubber transferase activity may be due to low temperature stimulation in the fall and winter months.