Synthesis of the food flavoring methyl benzoate by genetically engineered Saccharomyces cerevisiae

Current means of production for plant-derived aroma compounds include chemical synthesis and extraction from plant material. Both methods are environmentally detrimental and relatively expensive: plant material is only seasonally available and only a small subset of the plant biomass produces the desired aroma compounds, while organic synthesis inevitably involves waste byproducts with a negative ecological impact. Benzenoids are a class of plant metabolites that includes a number of aroma compounds. This paper explores, for the first time, the feasibility of producing benzenoids in yeast. We present a method for the production of the phenylpropanoid methyl benzoate in Saccharomyces cerevisiae using benzoic acid as a substrate, by heterologous expression of Antirrhinum majus benzoic acid methyl transferase. Production was pH dependent with a maximal yield of approximately 50 microg of methyl benzoate per liter of culture per hour, and with linear kinetics over at least 24 h. In addition, we have analyzed two alternative expression vectors for the production of benzoic acid methyl transferase in S. cerevisiae: a constitutive triosephosphate isomerase promoter-based system was compared with a copper-inducible CUP1 promoter system. We find major differences in the amounts of methylbenzoate produced by these respective systems. Potential applications are discussed.     

Computing competition for light in the GREENLAB model of plant growth: a contribution to the study of the effects of density on resource acquisition and architectural development

BACKGROUND AND AIMS: The dynamical system of plant growth GREENLAB was originally developed for individual plants, without explicitly taking into account interplant competition for light. Inspired by the competition models developed in the context of forest science for mono-specific stands, we propose to adapt the method of crown projection onto the x-y plane to GREENLAB, in order to study the effects of density on resource acquisition and on architectural development. METHODS: The empirical production equation of GREENLAB is extrapolated to stands by computing the exposed photosynthetic foliage area of each plant. The computation is based on the combination of Poisson models of leaf distribution for all the neighbouring plants whose crown projection surfaces overlap. To study the effects of density on architectural development, we link the proposed competition model to the model of interaction between functional growth and structural development introduced by Mathieu (2006, PhD Thesis, Ecole Centrale de Paris, France). KEY RESULTS AND CONCLUSIONS: The model is applied to mono-specific field crops and forest stands. For high-density crops at full cover, the model is shown to be equivalent to the classical equation of field crop production (Howell and Musick, 1985, in Les besoins en eau des cultures; Paris: INRA Editions). However, our method is more accurate at the early stages of growth (before cover) or in the case of intermediate densities. It may potentially account for local effects, such as uneven spacing, variation in the time of plant emergence or variation in seed biomass. The application of the model to trees illustrates the expression of plant plasticity in response to competition for light. Density strongly impacts on tree architectural development through interactions with the source-sink balances during growth. The effects of density on tree height and radial growth that are commonly observed in real stands appear as emerging properties of the model.     

Optimal distribution of herbivory and localized compensatory responses within a plant

Because the impact of induced resistance on herbivores is often too small to have a considerable impact on herbivore populations, it has been suggested that the function of defenses is not necessarily to affect herbivore densities per se, but to spread the damage evenly throughout a plant. Some observations suggest that evenly-distributed herbivory results in a smaller decrease in growht and reproduction than the same level of herbivory concentrated on only one part of the canopy. One possible reason for this is that plant parts are able to compensate for small amounts of local damage spread all over the plant but not for larger concentrated damage of the same extent because of the of resource distribution patterns in a plant.Models were constructed to analyze how to optimally distribute damage among modules so that the total performance (growth, subsequent reproduction) of a plant, measured as the sum of the module performances, is maximized. According to the models, spreading damage is profitable when the performance of all or most modules decreases slowly with small damage levels and then faster as damage increases. In addition, the pattern of resource movement between modules must be tolerant to damage. It is proposed a hypothesis that the resource distribution system is often tolerant to at least small or moderate total damage levels, since there are several alternative pathways of movement for assimilates and water through the plant's structure.It has been suggested that defense and compensatory growth are alternative strategies for a plant in the struggle against herbivores. The present models suggest that they can also work together to increase fitness under herbivore pressure.     

Gas composition strategies for the successful scale-up of Catharanthus roseus cell cultures for the production of ajmalicine

Ajmalicine, serpentine, catharanthine, and vindoline are monoterpenoid indole alkaloids (MIAs) of commercial interest which are produced by the Catharanthus roseus plant. Cultures of C. roseus have been investigated as a potential source of these pharmaceutically important compounds since the early 1960s. In addition, their production from C. roseus cultures has served as a model system for investigating secondary metabolism and for evaluating production-enhancing strategies. Initially, this review will survey (1) the MIAs of interest for large-scale production from plant cell cultures and (2) the volumetric productivities of a specific MIA, ajmalicine, achieved and projected using plant cell cultures. To meet the need for these valuable compounds, the production of these MIAs from plant cell cultures must be successfully reproduced in large-scale aerated and agitated reactors. While the large-scale cultivation of plant cell cultures is currently feasible, initial attempts at scale-up may yield results that differ from that optimized in flasks. To bridge the jump between production in flasks and production in large-scale bioreactors, changes introduced with scale-up such as gas composition must be identified and rationally manipulated to reproduce or even improve growth and secondary metabolite production. Hence, this review will (1) identify the effects of gas composition (i.e., O₂, CO₂, ethylene, or other endogenous volatile compounds) on growth and secondary metabolism and (2) draw operating strategies for optimizing the gas composition for growth of C. roseus cultures and the production of ajmalicine.     

Reproductive response of generalist and specialist aphid morphs with the same genotype to plant secondary compounds and amino acids

Many studies have paid particular attention to the role of either secondary plant compounds or amino acids as determinants of host-plant range in phytophagous insects. Here we examine the relative importance of both of these classes of compound in host acceptance by generalist and specialist morphs of the black bean aphid, Aphis fabae, that are morphologically similar and genetically identical. Eleven secondary plant compounds and six amino acids with known biological activity in aphids are presented to insects within an artificial membrane system as single compounds, mixtures of same-class compounds and combined mixtures of the two classes of compound. It is found that 1) when specific single secondary compounds and amino acids are presented to generalist and specialist morphs of A. fabae, differential responses are exclusively consistent with plant-use strategy for amino acids but not for secondary compounds, 2) neither secondary compound nor amino acid mixtures give reproductive responses entirely consistent with plant range, but the response to secondary compounds is broadly consistent with plant range whereas the response to amino acids is not, 3) when secondary compounds and amino acid mixtures are combined, the response to secondary compounds generally dominates that to amino acids. Some scenarios of plant-range determination by secondary plant compounds and amino acids, suggested by results, are discussed.     

Propagule vs. niche limitation: untangling the mechanisms behind plant species' distributions

Distinguishing the roles of propagule limitation and niche requirements in controlling plant species distributions is important for understanding community structure, invasion, and restoration. We used species distribution models based on plant and environmental survey data to assess the strength of species' affinities for particular environmental conditions. We hypothesized that species with statistically detectable environmental requirements were primarily niche-limited, while species with weak habitat affinities were primarily propagule-limited. We tested this hypothesis via a seeding experiment in which we compared species' reproductive fitness in occupied and unoccupied sites. Species that appeared to be niche-limited based on distribution models had lower fitness when planted in unoccupied sites, while species that models suggested were propagule-limited had equivalent fitness when planted in occupied and unoccupied sites. Our results demonstrate that within a single community, both species limited primarily by niche availability or primarily by propagule availability can be identified using observational data.     

Assessing the distribution of the Argentine ant using physiological data

To address the lack of physiological approaches in current models assessing the potential distribution of the Argentine ant, we used data on brood development from distinct sources to evaluate a series of degree-day models for Catalonia (NE Iberian Peninsula), and data on the brood survival and oviposition rates to develop a worker production model. The degree-day model generated using data from Newell and Barber (1913) and Benois (1973) indicated that the number of degree-days required for the complete development from egg to adult worker was 445.4 degree-days above a threshold of 15.9 °C, while the model calibrated using data from Abril et al. (2008, in press) suggested 599.5 degree-days above 18.4 °C. Comparisons between the degree-day model predictions and the currently known distribution of the Argentine ant suggested that the one generated using data from Newell and Barber (1913) and Benois (1973) overestimated the presence of the species, while the one calibrated using data from Abril et al. (2008; in press) underestimated it. On the other hand, the predicted daily net production of Argentine ant workers generated by the worker production model predicted more accurately the distribution of the Argentine ant than the degree-day models. Our results show the utility of incorporating physiological data in models to assess the distribution limits of the Argentine ant, which up to date have taken little account of the physiological needs of the species in terms of its establishment and dispersion in its introduced ranges.     

Tailoring of microbes for the production of high value plant-derived compounds: From pathway engineering to fermentative production

Plant natural products have been an attracting platform for the isolation of various active drugs and other bioactives. However large-scale extraction of these compounds is affected by the difficulty in mass cultivation of these plants and absence of strategies for successful extraction. Even though, synthesis by chemical method is an alternative method; it is less efficient as their chemical structure is highly complex which involve enantio-selectivity. Thus an alternate bio-system for heterologous production of plant natural products using microbes has emerged. Advent of various omics, synthetic and metabolic engineering strategies revolutionised the field of heterologous plant metabolite production. In this context, various engineering methods taken to synthesise plant natural products are described with an additional focus to fermentation strategies.     

Monte carlo studies of plant mating system estimation models: the one-pollen parent and mixed mating models

Estimation of mating system parameters in plant populations typically employs family-structured samples of progeny genotypes. These estimation models postulate a mixture of self-fertilization and random outcrossing. One assumption of such models concerns the distribution of pollen genotypes among eggs within single maternal families. Previous applications of the mixed mating model to mating system estimation have assumed that pollen genotypes are sampled randomly from the total population in forming outcrossed progeny within families. In contrast, the one-pollen parent model assumes that outcrossed progeny within a family share a single-pollen parent genotype. Monte Carlo simulations of family-structured sampling were carried out to examine the consequences of violations of the different assumptions of the two models regarding the distribution of pollen genotypes among eggs. When these assumptions are violated, estimates of mating system parameters may be significantly different from their true values and may exhibit distributions which depart from normality. Monte Carlo methods were also used to examine the utility of the bootstrap resampling algorithm for estimating the variances of mating system parameters. The bootstrap method gives variance estimates that approximate empirically determined values. When applied to data from two plant populations which differ in pollen genotype distributions within families, the two estimation procedures exhibit the same behavior as that seen with the simulated data.     

Use of a semicontinuous culture system as a model for determining the role of human intestinal microflora in the metabolism of xenobiotics

The use of semicontinuous culture system for determining the role of the human intestinal microflora int he biotransformation of xenobiotics is discussed. This model system, which stimulates the lumen of the large intestine, has been used to investigate human intestinal microflora metabolism of compounds representative of three different classes of chemicals; a benzidine-based azo dye, Direct Black 38, a nitropolycyclic aromatic hydrocarbon, 1-nitropyrene and a substituted pyrimidine, 5-fluorocytosine. Metabolites of each of the test compounds were identified, and the kinetics of production and biological activity of the metabolites determined. Metabolic adaptation was observed with 1-nitropyrene and 5-fluorocytosine incubations. This microbial culture system could be quite useful, especially in concert with other in vitro models and animal studies, for determining the pharmacological and toxicological role of the human intestinal microflora in the transformation of xenobiotics. Particular emphasis on the application of this method for toxicological studies in elucidating the role of the intestinal microflora in the etiology of cancer is described.     

The Adaptation of the Immunofluorescence Technique for Use in Bacteriological Investigations of Plant Tissue

Summary: The preparation of plant tissue for examination by the fluorescent antibody technique is described. The procedures suggested are intended to overcome some of the major obstacles encountered and the efficacy of the method for plant investigations is upheld by its successful application in a simple test system.     

Influence of polyphenols on bacterial biofilm formation and quorum-sensing

Many bacteria utilize sophisticated regulatory systems to ensure that some functions are only expressed when a particular population density has been reached. The term 'quorum-sensing' has been coined to describe this form of density-dependent gene regulation which relies on the production and perception of small signal molecules by bacterial cells. As in many pathogenic bacteria the production of virulence factors is quorum-sensing regulated, it has been suggested that this form of gene regulation allows the bacteria to remain invisible to the defence systems of the host until the population is sufficiently large to successfully establish the infection. Here we present first evidence that polyphenolic compounds can interfere with bacterial quorum-sensing. Since polyphenols are widely distributed in the plant kingdom, they may be important for promoting plant fitness.     

The perception of gravity by a plant

Ever since geotropism was first studied in plants, attempts have been made to create model systems which might simulate the perception by a plant of a gravitational change. The most resilient of these models, the so-called statolith theory, has now enjoyed a run of over 75 years and demonstrates its viability by reappearing in many different forms. It has shown its value by anticipating the now well understood graviperception mechanism in the Chara rhizoid and this will be described. However, it is unlikely that many features of this relatively simple system can be translated to the higher plant. We now know precisely, at least in many primary roots, the distribution and approximate numbers of the cells that perceive gravity. There is no reason to assume that an identical system operates in shoots, since it is now clear that the fundamental hormonal bases of these two systems are different. We also know much about the ultrastructure of many geoperceptive cells, but apart from speculative models a satisfactory explanation of this very rapid and flexible system eludes us. A possible model system is proposed and ways of testing it in zero gravity are suggested.     

Production of plant growth modulating volatiles is widespread among rhizosphere bacteria and strongly depends on culture conditions

Recent studies have suggested that bacterial volatiles play an important role in bacterial-plant interactions. However, few reports of bacterial species that produce plant growth modulating volatiles have been published, raising the question whether this is just an anecdotal phenomenon. To address this question, we performed a large screen of strains originating from the soil for volatile-mediated effects on Arabidopsis thaliana. All of the 42 strains tested showed significant volatile-mediated plant growth modulation, with effects ranging from plant death to a sixfold increase in plant biomass. The effects of bacterial volatiles were highly dependent on the cultivation medium and the inoculum quantity. GC-MS analysis of the tested strains revealed over 130 bacterial volatile compounds. Indole, 1-hexanol and pentadecane were selected for further studies because they appeared to promote plant growth. None of these compounds triggered a typical defence response, using production of ethylene and of reactive oxygen species (ROS) as read-outs. However, when plants were challenged with the flg-22 epitope of bacterial flagellin, a prototypical elicitor of defence responses, additional exposure to the volatiles reduced the flg-22-induced production of ethylene and ROS in a dose-dependent manner, suggesting that bacterial volatiles may act as effectors to inhibit the plant's defence response.     

Interpreting phenotypic variation in plant allelochemistry: problems with the use of concentrations

Ecologists often use concentrations of defensive compounds as measures of plant allocation to defence and/or allelochemical production. I demonstrate that this practice may lead to erroneous conclusions because plants produce and allocate molecules (quantities) of compounds whereas concentrations reflect the distribution of these quantities in plant tissues and are, therefore, functions of plant biomass. As a tool for distinguishing between shifts in allelochemical production versus changes in plant biomass in determining allelochemical concentrations, I suggest using a technique known as graphical vector analysis (GVA) which has been developed for diagnosing nutrient limitations in forest stands, but has seldom been applied by researchers studying plant allelochemicals. I used data from several published studies to demonstrate how GVA can be applied to interpret ontogenetic and environmental effects on allelochemical levels and to compare the results obtained for different allelochemical types, plant species, treatments and experiments. These examples show that changes in plant biomass per se are an important source of variation in allelochemical concentrations and, therefore, concentration data can be easily misinterpreted if changes in absolute content and plant biomass are not considered simultaneously. Because studies reporting variation in allelochemical concentrations have been considered as tests for general theories of plant chemical defence, evidence in support of or against these theories should be re-examined using multivariate techniques such as analysis of covariance, allometric analysis and GVA. Received: 16 September 1998 / Accepted: 4 March 1999     

Dynamic process,spatial pattern and species coexistence in plants

The significance of dynamic processes of individual genets/ramets for the spatial pattern of plant species and community structure is discussed. It is suggested that under a different mode of competition (symmetric vs. asymmetric), spatial distribution of individuals, initial size distribution at the establishment stage and boundary conditions as recruitment influence differently the species coexistence pattern. It is therefore important to consider the mode of competition for the study of community structure. To know the mode and degree of intra- and interspecific competition, the dynamic processes of individual genets/ramets must be studied by following the growth, mortality and recruitment of each genet/ramet of each component species in a plant community. The models and methods of plant population ecology are therefore useful also for plant community ecology.     

Production of the siderophore enterobactin: use of four different fermentation systems and identification of the compound by HPLC

The article describes four different fermentation procedures for Escherichia coli AN311, a producer of enterobactin. A regular rotary shaker culture with a biphasic system consisting of an agar layer (as a reservoir for feeding processes) and a layer of liquid medium, 2.4 L and 10 L batch cultures, and a novel dialysis membrane fermentor were used. With the use of this latter fermentor type, the production of enterobactin could be increased by a factor of about 9.5, while growth increased by a factor of 12 compared to the other systems. For the rapid and reliable quantification of the concentration and purity of enterobactin an analytical and preparative high-performance liquid chromatography (HPLC) method was established. The degradation compounds of this siderophore were detected by diodearray and bioassays. A comparison of total catechol production as well as the distribution between enterobactin and its degradation compounds is given. (c) 1993 John Wiley & Sons, Inc.     

Computer simulation: The imaginary friend of auxin transport biology

Regulated transport of the plant hormone auxin is central to many aspects of plant development. Directional transport, mediated by membrane transporters, produces patterns of auxin distribution in tissues that trigger developmental processes, such as vascular patterning or leaf formation. Experimentation has produced many, largely qualitative, data providing strong evidence for multiple feedback systems between auxin and its transport. However, the exact mechanisms concerned remain elusive and the experiments required to evaluate alternative hypotheses are challenging. Because of this, computational modelling now plays an important role in auxin transport research. Here we review some current approaches and underlying assumptions of computational auxin transport models. We focus on self‐organising models for polar auxin transport and on recent attempts to unify conflicting mechanistic explanations. In addition, we discuss in general how these computer simulations are proving to be increasingly effective in hypothesis generation and testing, and how simulation can be used to direct future experiments. Editor's suggested further reading in BioEssays Local auxin production: a small contribution to a big field Abstract     

The dynamics of root meristem distribution in the soil

Plants must develop efficient root architectures to secure access to nutrients and water in soil. This is achieved during plant development through a series of expansion and branching processes, mostly in the proximity of root apical meristems, where the plant senses the environment and explores immediate regions of the soil. We have developed a new approach to study the dynamics of root meristem distribution in soil, using the relationship between the increase in root length density and the root meristem density. Initiated at the seed, the location of root meristems in barley seedlings was shown to propagate, wave‐like, through the soil, leaving behind a permanent network of roots for the plant to acquire water and nutrients. Data from observations on barley roots were used to construct mathematical models to describe the density of root meristems in space. These models suggested that the morphology of the waves of meristems was a function of specific root developmental processes. The waves of meristems observed in root systems of barley seedlings exploring the soil might represent a more general and fundamental aspect of plant rooting strategies for securing soil resources.     

Metabolic engineering of plant secondary metabolite pathways for the production of fine chemicals

The technology of large-scale plant cell culture is feasible for the industrial production of plant-derived fine chemicals. Due to low or no productivity of the desired compounds the economy is only in a few cases favorable. Various approaches are studied to increase yields, these encompass screening and selection of high producing cell lines, media optimization, elicitation, culturing of differentiated cells (organ cultures), immobilization. In recent years metabolic engineering has opened a new promising perspectives for improved production in a plant or plant cell culture.