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FAIR-CT96-3140
Application of novel inhibitors from microorganisms: a new bioprocess for the production of 'green' agrochemicals |
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Proposal No: | FAIR-CT96-3140 |
| Date Prepared: | July 2001, May 1999 | |
| Source: |
Final Report
Abstract and
Executive Summary
First Annual Progress Report |
Final Report Abstract
Source: Final Report, September 2000
Consortium: This project was co-ordinated by the Agrotechnological Research Institute (ATO-DLO), Wageningen (The Netherlands) in partnership with Universidad de Leon, Facultad de Biologia, Area de Microbiologia, Leon (Spain), Vioryl S.A. Chemical and Agricultural Industry, Kifissia (Greece), Horticulture Research Institute, West Malling (UK), GAIKER, Zamudio (Spain)
Abstract
At present, the detrimental effects of the senescence-hormone ethylene on horticultural produce are combated by pre-treatment of ornamental produce or by spraying fruit trees with various chemicals. This way, senescence is suppressed and the life of the produce is increased creating extended opportunities for storage, transport and processing. The aim of this study was to replace these, often persistent, chemicals by alternative, environmentally friendly agents. Vinylglycines, produced by different micro-organisms, have been shown to suppress ethylene biosynthesis and to possess antimicrobial activity. These compounds and various analogues, which are not commercially available in large amounts, will be produced on a large scale and tested for economical exploitation in the fruit and ornamental horticultural industry.
The approaches employed to increase the production of vinylglycines and design a large scale bioprocess were improving fermentation based on physiological parameters, strain improvement by genetic engineering and technical adjustments of the fermentation system to enhance product recovery. For the development of novel vinylglycine analogues and other inhibitors a combination of biochemical and analytical techniques to elucidate the biosynthetic pathways, and chemical modifications have been employed. To study the mode of action of natural vinylglycines and other inhibitors various model systems were developed, such as in vitro enzyme assays, plant model systems and in plants assays.
Application parameters of vinylglycines on horticultural produce included determination of biological activity, formulation conditions, and large scale storage experiments. Preliminary studies were performed on the toxicological and mutagenic properties of the various compounds. Microbial production and purification of a few gram of a vinylglycine, rhizobitoxine, has been achieved by optimising fermentation conditions and technology and by development and scaling up of downstream processes. Rtx production was stimulated 4-fold and the fermentation process has been optimised resulting in higher production rates and yields.
The process has been scaled-up to the sale of a 10 l fermentation without any adverse effects on production efficiency. Cost calculations showed that the production needs to be increased 10 to 100 fold to attain an economically viable process. Further increases in vinylglycine production are expected to be obtained by genetic modification of vinylglycine producing bacteria, more specifically by over-expression and deregulation of genes involved in vinylglycine biosynthesis. For this purpose relevant genes involved in AVG biosynthesis have been cloned and sequenced and a new transformation system for the bacterial strain involved has been developed.
The first recombinant bacterial strains showed an increased AVG production. Novel ACC synthase inhibitors have been designed and chemically synthesised. The biological activity of these new inhibitors and the natural vinylglycines have been tested in newly developed model systems, amongst others living yeast cells expressing ACS enzyme and an in 'vitro ACS assay. The order of potency of the various inhibitors was determined ' and structure function relationships were established. Results showed that vinylglycines are more potent than e.g. AOA, a well known ACC synthase inhibitor widely used in commercial floriculture. In turn, Rtx and AVG appeared to be equally effective in inhibiting ethylene production, but Rtx was far more active in inhibition of P-cystathionase. Novel ACC synthase inhibitor have been developed based on S-adenosylmethionine, the natural substrate of ACC synthase. These inhibitors showed an inhibition of ACC synthase activity in vitro. Additionally, ACC structural analogues have been synthesised and tested on flower longevity. Furthermore, indication of production of n fermentation have been obtained.
Application parameters have been tested in field trials which were carried out during two seasons. Experiments started in the late summer by spraying AVG on apple trees of different cultivars, because the degree of effect depends on the cultivar, and also on the time of application. The quality and other characteristics of the fruits have been followed at time of harvest and during the storage period which ended in spring. In general, pre-harvest application of AVG reduced the concentration of internal ethylene and so delayed harvest maturity. However, once the internal ethylene concentration exceeds a threshold value (at late picking dates) control of ethylene production during storage is difficult to achieve. Major benefits of AVG treatment are extension of the harvesting period and secondly, reduction of the amount of ethylene scrubbing from the storage environment and thus running-costs because of lower ethylene production rates of AVG-treated apples during storage. Other positive effects have been obtained on fruit quality, firmness of ex-store apples is better directly after storage and after a shelf-life period, and yields which are improved. Commercial strategies for the application of AVG on apple cultivars (time of application and dose) in combination with storage in Controlled Atmospheres will be developed. A prerequisite for the use of vinylglycines in the fruit and ornamental horticultural industry is the safety of these compounds in relation to human health. From the current available information, a toxicological database and our own preliminary studies, it is concluded that AVG is considered as non-cytotoxic and non- mutagenic. This will facilitate the introduction and acceptance of AVG and possibly other natural and novel vinylglycines as a 'green' chemical for the horticultural industry.
Introduction
At present, the detrimental effects of the senescence-hormone ethylene on horticultural produce are combated by spraying fruit trees with various chemicals. These measures are taken to increase the ability to keep the produce in good condition in order to extend opportunities for storage, transport and processing. Environmental concerns related to the persistence of these chemicals have prompted an intensive search for alternatives for guarding or increasing product quality in horticulture. Previously, three vinyl-lycine compounds, i.e. rhizobitoxine produced by Burkholderia andropogonis and Bradyrhizobium japonicum, AVG produced by an unidentified species of Streptomyces and MVG isolated from a culture broth of Pseudomonas aeruginosa were shown to suppress ethylene biosynthesis with increasing- effectiveness from MVG, AVG to rhizobitoxine, and to possess antimicrobial activity. The mode of action of vinylglycine compounds is partially resolved. In general, they inhibit pyridoxal phosphate-linked enzymes such as the plant aminocyclopropane-l-carboxylic acid synthase (ACC synthase) which is the key enzyme in ethylene biosynthesis.
Former fermentation data allow a rough estimation of the production costs of AVG and MVG, ranging from approximately 100 to 15000 ECU per gram, respectively. As the final users will only take an interest when prices drop below 1 ECU per gram, the productivity of a new bioprocess has to be considerably increased. This scaling-up is similar to the production of antibiotics and vitamins, where 400 to 50.000-fold overproduction were attained by combining mutations, screening, manupilation of the environment and improvements in fermentation technology. The main objective of this project is to design a large scale bioprocess aimed at the production of 'green" ethylene inhibiting and antimicrobial agents for economical exploitation in the fruit and ornamental horticultural industry. Other objectives are:
Results
Task 1: Production increase The aim of this task is to increase the microbial production of natural vinylglycines, The research was focused on production of Rtx by Burkholderia andropogonis and AVG by a Streptomyces sp. NRRL 5331. Rtx is also produced by Bradyrhizobium spp. but to significant lesser amounts than by B. andropogonis . In this first period no attemps were made to produce MVG, which is the natural vinylglycine with the lowest activity. Production of Rtx and AVG will be stimulated by combining microbial physiology and fermentation technology (Rtx) and by genetic modification of the Streptomyces strain (AVG).
In this first year, Rtx production was 4-fold increased by optimising the production medium (nature and concentration of the carbon and nitrogen sources) and by improving the fermentation technology (from shake flask to 2 liter (fed) batch fermentations). Further increases in Rtx production could be hampered by inhibition of bacterial growth, as vinylglycines in general inhibit pyridoxal phospate-linked enzymes, or by feedback inhibition whereby Rtx blocks its own biosynthesis. No indications for growth inhibition were obtained in an experiment with high concentrations of AVG present during- growth of Burkholderia andropogonis. This type of experiment should be repeated with Rtx when larger amounts are available.
In the next period attention will be paid to feedback inhibition, experiments and when necessary proper action will be taken to overcome this problem. AVG will be overproduced by genetic engineering of Streptomyces sp. 5331. Starting from the proposed biosynthetic route for Rtx appropriate genes will be overexpressed or deregulated in Streptomyces sp. 5331 to manipulate the AVG production. As in the case of Rtx, aspartate is most probably the building block of the amino acid moiety in AVG with homoserine and 2-hydroxythreonine as intermediates. Much knowledge is available on the genes and their regulation of this primary metabolic pathway from aspartate to threonine in various bacterial strains.
During the first period the first two genes of the homoserine pathway in Streptomyces sp. 5331, ask and asd, were cloned and partially sequenced. These genes will be overexpressed in this Streptomyces strain using a newly developed protoplast transformation system for this bacterium. This will result in an increased flux through the aspartic acid pathway. A second approach is to block the conversion of homoserine to threonine. For this purpose research was started on the identification of the hom, thrC and thrB genes of Streptomyces sp. 5331 using heterologous probes of various bacterial strains.
A method for the downstream processing of bacterially produced vinylclycines will be developed. Rtx was secreted into the medium and recovered from the culture broth by standard procedures of centrifugation and filtration followed by a 2-step ion exchange chromatography procedure. In this way a partially purified sample was obtained with a total recovery of more than 80% or 100 mg Rtx was isolated per liter culture broth. Batches of not more than 1 liter at the time were processed. In the next period the system will be scaled-up for processing of larger volumes taken into account the economic constraints of the process and the degree of purity of the active fraction with respect to its future applications.
Task 2: Structure function analysis The aim of this task is to elucidate the working mechanism of vinylglycines and to get insight into their structure function relationship in order to adjust natural vinylglycines to improve their biological activity. Additionally, uptake and transport parameters will be determined. To study the mode of action of vinylglycines various model systems were used. Potential targets of vinylglycines are all pyridoxal phosphate-linked enzymes, such as ACC synthase in plants as well as enzymes involved in amino acid biosynthesis (such as beta-cystathionase as part of the methionine biosynthesis route).
First, various inhibitors, the vinylglycines Rtx and AVG, and other known inhibitors of ACC synthase (AOA, canaline and sinefungin) were tested in three different systems:
The order of activity of the inhibitors was established. In the plant assays Rtx was as effective as AVG, AOA and the other inhibitors were less effective. However,Rtx was the most effective on bacterial beta-cystathionase. Experiments, using another model system of apple discs, showed comparable results: Rtx and AVG were as effective in inhibition of ethylene biosynthesis, AOA was less effective. Thus, in plants Rtx and AVG are equally effective in inhibition of ACC synthase activity, while Rtx is a much better inhibitor than AVG on bacterial beta-cystathionase activity. More detailed studies on inhibition of ACC synthase by vinylglycines will be carried out in the next period. For that purpose a more defined system was developed, which consist of yeast cells expressing ACC synthase of tomato. Yeast itself does not contain an endogenous copy of ACC synthase. Inhibition studies will be done in vivo or on the isolated enzyme.
Besides the enzyme inhibitory activity of vinylclycines, their uptake and transport in the products is of importance. The effect of vinyglycines on flowers and fruits have been determined. Rtx and AVG have an positive effect on Cymbidium flowers (increased vase life), Delphinium (delay of flower abscission) and Aconitum, (increased vase life), while their was no effect on carnations. In the latter case transport of vinyl-lycines might be hampered by the long- stems, the use of detergents to improve uptake will be tested. Rtx and AVG reduced the ethylene production of treated apples compared to controls, while AOA was less active. Both vinylglycines were able to reduce ethylene production in the inner cortex of the apples, indicating that vinylglycines were taken.up through the peel and transported in the cortex. Quantities and possible metabolites of vinylglycines in apples will be determined by GC-MS.
New compounds have been developed based on S-adenosylmethionine, the natural substrate of ACC synthase. The methylthio region of the new inhibitors was conserved while the carbonyl and amino group of.methionine were modified. By testing the inhibitory activity of these new compounds in various model systems, the structure function relationships will be determined.
Task 3: Applications The aim of this task is the study of the application parameters of vinylglycines in fruit and ornamental horticulture, mainly in view of ethylene suppression in plants, but also in view of inhibition of bacterial growth. Additionally, the determination of the safety, with respect to toxic or mutagenic properties of the produced compounds, will be included. The effects on nutritional quality and factors affecting long-term stability will be verified.
New analogues based on the structure of S-adenosylmethionine were initially tested for their inhibitory activity on apple ACC synthase in an in vitro assay.
Most new compounds inhibited ACC synthase, but high concentrations were needed. The functionality of the various introduced groups can be determined. In the next period these compounds need to be tested on a range of commercially important fruits. Formulations of vinylglycine compounds and other inhibitors will be developed to preserve their stability during storage for extended periods. Further-more, research on coupling to or entrapment in large molecules is needed to facilitate uptake and transport of vinylglycine in various products.
A field study with AVG was conducted to determine the optimum concentration and time of application. Trees of two apple cultivars were sprayed with AVG at two concentrations, 6, 4 and 2 weeks before harvest (September 1998). At harvest weight of the fruits, soluble solids as a measure for maturity, starch, firmness and background colour were determined. The internal ethylene concentration in the core cavity of the fruits was determined. Dependent on the cultivar and the time of application preharvest treatment of apples with AVG maintained fruit firmness. The internal ethylene concentration in preharvest AVG-treated apples was significantly less compared to untreated controls at harvest.
A large scale storage experiment with AVG sprayed fruits has been set up. Fruits will be monitored and regular measurements of ethylene production and respiration rates will be made. Apples will be stored until June 1999 when assessments of fruit quality (external and internal) will be carried out. Trees sprayed in the summer of 1998 will be monitored and the time of flowering in spring 1999 will be recorded.
Future field trials with Rtx and AVG are planned for 1999. In the next period preliminary studies of the toxicological and mutagenic properties of natural vinylglycines (Rtx and as a control AVG) and novel analogues will be done using in vitro tests. From toxicological databases information on AVG was obtained: lowest- and no-observed-effect levels (LOEL and NOEL) were determined in various toxicity tests. From diverse tests it appeared that AVG was not mutagenic or cytotoxic.
Discussion
A major development affecting the project is the commercial availability of one of the natural vinylglycines, AVG. A commercial formulation, called Retain™, has been developed by Abbott Laboratories, USA. The active compound is produced by fermentation and its commercial use on apples and pears was approved in the USA in 1997 and in California in 1998. In a first reaction this development seems to be a disadvantage for the progress of the ANIMO project and European competitiveness. However, this is not necessarily the case. The commercial exploitation of AVG proves the feasibility and possibilities of using vinylglycines in fruit horticulture and possibly also for ornamentals. It will be a challenge to produce and develop a better product and find new applications for the vinylglycines.
The ANIMO project can be devided in three major areas:
Production increase Research was started on the increase in microbial production of Rtx (by Burkholderia andropogonis) and AVG (by Streptomyces sp. NRRL 5331). Two strategies are being used. Overproduction of Rtx should be attained by optimizing the fermentation medium and by improving the fermentation technology. In the first year a 4-fold stimulation of Rtx production was achieved using both approaches. Much higher Rtx concentrations are not expected, but by scaling-up the fermentation system higher productions of Rtx could be achieved. The other strategy to improve microbial vinylglycine production is by genetic modification, more specifically, production will be increased by overexpression or deregulation of genes involved in AVG biosynthesis. This direct approach will probably be more successful and knowledge obtained on the stimulation of AVG production by Streptomyces can be applied to other vinylglycine-producing bacteria in the future.
The commercial product, Retain, is produced by fermentation, but the strain and whether modified bacteria are used is not known. Apparently, production by fermentation is cheaper than by chemical synthesis. Therefore, we will continue our research on production of vinylglycines by fermentation.
Novel vinylglycine analogues and other inhibitors The development of new vinylglycine analogues and other inhibitors will be an important part of the project the next two years. The activity tests in the various model systems with the available natural vinylglycines and other inhibitors showed that the vinylglycines are more potent than e g. AOA, a well known ACC synthase inhibitor widely used in commercial floriculture. In turn, Rtx and AVG appeared to be equally effective in inhibiting ethylene production, but Rtx was far more active in inhibition of beta-cystathionase. Since AVG is already on the market, there is a need for production of' other natural vinylglycines or new analogues with better qualities. These could be inhibitors with higher potency (thereby lowering the amounts necessary to obtained the desired effect), or compounds with a higher specific activity towards the target enzymes thereby showing less side effects or inhibitors which show better uptake, transport and stability characteristics. New inhibitors will be designed by applying molecular modelling, to the relevant enzymes (comparison of sequences and molecular models of ACC synthase, cystathionase and other available aminotransferases). With this information the structural parameters of the inhibitor compounds for binding to and inhibition of the enzymes will be studied. New compounds will be synthesized chemically or by fermentation and tested in the various model systems and when available in sufficient amounts on fruits and flowers.
By chemical synthesis analogues of the natural substrate of ACC synthase, S-adenosylmethionine, are produced. First test with these inhibitors showed a inhibition of ACC synthase activity in vitro, future experiments will be done with a range of commercially important fruits. Also in the next period research on the production of new vinylglycine analogues (by fermentation of chemical modification of existing vinylglycines) will be started.
Application of inhibitors The commercial product Retain is used in the USA on apples and pears, mainly to prevent premature fruit abscission. However, there appear to be other benefits, fruit maturation is delayed on the tree and during storage. Similar field trials are now performed with Retain in England as part of the ANIMO project. Experiments were started in the late summer of 1998 by spraying apple trees of different cultivars, because the degree of effect depends on the cultivar, and also on the time of application. The quality and other characteristics of the fruits will be followed at time of harvest and during the, storage period which last until spring 1999. Experiments will be repeated in next seasons and will be carried out again with AVG and, when available in sufficient quantities, with other inhibitors.
During the time of the project other horticultural produce, like cut flowers and potted plants, will be treated with vinylglycine compounds. The available quantities of the compounds will determine the scale of the treatments. AVG will not be produced in large quantities as part of this project, but is commercially availble (Sigma) and is available from Abbott Laboratories as Retain. Until now batches of 50-100 mg of Rtx have been produced and Rtx was used only in model systems and for toxicology studies. By scaling up the fermentation systems and downstream processing, probably larger quantities will become available in the next period.
Future actions
The project will proceed as proposed and discussed above. The availability of the commercial product Retain underlines the importance of the development of novel vinylglycine analogues and the establishment of new application areas.
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