Continuous cultivations of a Penicillium chrysogenum strain expressing the expandase gene from Streptomyces clavuligerus: Growth yields and morphological characterization.

The growth stoichiometry of a Penicillium chrysogenum strain expressing the expandase gene from Streptomyces clavuligerus was determined in glucose-limited chemostat cultivations using a chemically defined medium. This strain produces adipoyl-7-aminodeacetoxycephalosporanic acid (ad-7-ADCA) when it is fed with adipic acid. The biomass yield and maintenance coefficients for the strain were similar to those found for penicillin-producing strains of Penicillium chrysogenum. The maximum specific growth rate in the chemostat was found to be 0.11 h(-1). Metabolic degradation of adipate was found to take place in significant amounts only at dilution rates below 0.03 h(-1). After three to five residence times, adipate degradation and ad-7-ADCA production disappeared, and this allowed determination of the biomass yield coefficient on adipate. The morphology was measured at different dilution rates and the mean total hyphal length and mean number of tips both increased with an increase in dilution rate from 0.015 to 0.065 h(-1). Both variables decreased when the dilution rate was increased above 0.065 h(-1). A correlation between mean total hyphal length and productivity of ad-7-ADCA was found.

Continuous cultivations of a Penicillium chrysogenum strain expressing the expandase gene from Streptomyces clavuligerus: Kinetics of adipoyl-7-aminodeacetoxycephalosporanic acid and byproduct formations.

The production kinetics of a transformed strain of Penicillium chrysogenum expressing the expandase gene from Streptomyces clavuligerus was investigated in chemostat cultivations. The recombinant strain produces adipoyl-7-aminodeacetoxycephalosporanic acid (ad-7-ADCA) as the major product; however, during the cultivations, the appearance of a major unknown and poorly secreted product was observed. Investigations using high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectroscopy (LC-MS) showed that this byproduct has a six-membered dihydrothiazine ring, which is characteristic for cephalosporins. The byproduct may be formed via isopenicillin N by as-yet unknown mechanisms, but involving expandase. It is likely that the unknown compound (UC) is deacetoxycephalosporin C (DAOC). Investigation of the instability of the various beta-lactams produced showed higher instability for compounds with a five-membered thiazolidine ring than those with a six-membered dihydrothiazine ring. Furthermore, secretion of products and byproducts was shown to be quite different. The productivity was studied as a function of the dilution rate in the range 0.015 to 0.090 h(-1). The specific productivity of total beta-lactams was compared with that of the penicillin-G-producing host strain, and it was found to be lower at dilution rates of <0.06 h(-1). Quantification of the fluxes through the pathway leading to ad-7-ADCA showed a decrease in flux toward ad-7-ADCA, and an increase in flux toward UC as the dilution rate increased. Northern analysis of the biosynthetic genes showed that expression of the enzymes involved in the ad-7-ADCA pathway decreased as the dilution rate increased.

Influence of the adipate and dissolved oxygen concentrations on the beta-lactam production during continuous cultivations of a Penicillium chrysogenum strain expressing the expandase gene from Streptomyces clavuligerus.

The influence of adipate concentration and dissolved oxygen on production of adipoyl-7-aminodeacetoxycephalosporanic acid (ad-7-ADCA) by a recombinant strain of Penicillium chrysogenum expressing the expandase gene from Streptomyces clavuligerus was studied in glucose-limited continuous cultures. Operating conditions were maintained constant but the adipate and dissolved oxygen concentrations (DOC) were varied separately in a range from 1 to 37.5gl(-1) and from 2% to 125% air saturation (%AS), respectively. The total beta-lactams specific productivity, r(ptotal), was not significantly changed for adipate concentrations from 5 to 25gl(-1), but the flux towards an unknown by-product decreased as the adipate concentration increased. Investigations at different DOC showed that r(ptotal) was stable around 18 micro molgDW(-1)h(-1) for DOC being in the range from 15 to 125%AS. When DOC was decreased from 15 to 7%AS, r(ptotal) increased to 25 micro molgDW(-1)h(-1), mainly due to a two-fold increase in the adipoyl-6-aminopenicillanic acid (ad-6-APA) specific productivity.

Physiological characterisation of Penicillium chrysogenum strains expressing the expandase gene from Streptomyces clavuligerus during batch cultivations. Growth and adipoyl-7-aminodeacetoxycephalosporanic acid production.

The production of adipoyl-7-aminodeacetoxy-cephalosporanic acid (ad-7-ADCA) was studied, using two recombinant strains of Penicillium chrysogenum carrying the expandase gene from Streptomyces clavuligerus. The adipoyl-side chain of this compound may easily be removed using an amidase; and this process therefore represents a new route for the production of 7-ADCA, which serves as a precursor for the production of many semi-synthetic cephalosporins. In this study, one low- and one high-yielding strains were characterised and the specific productivities of ad-7-ADCA and byproducts of the biosynthetic pathway were compared. The fluxes through the biosynthetic pathway were quantified and it was found that there was a 30% higher flux through the expandase in the high-yielding strain. In both strains, there was a significant degradation of adipate. Furthermore, the initial adipate concentration in batch cultures was shown to have a positive effect on the formation of ad-7-ADCA.

Classification, error detection, and reconciliation of process information in complex biochemical systems.

Bioprocess identification starts with collection of process information. Usually there is a variety of information available, consisting of actual measurement data, historical data, empirical kinetic and yield correlations, and general knowledge available from literature. A central problem is to find out how the various pieces of information should be integrated. In addition, one should know how to deal with missing, inconsistent, or too inaccurate data. Recently, a general systematic method for dealing with these problems, based on conservation constraints, was published, and application shown to simple black box systems. In this article, the scope is generalized by including metabolic network data and dispersed process information of diverse type and nature, such as multiple sources of the value of one particular quantity, use of kinetic expressions, analytical problems, cometabolism or mixed substrate utilization, and chemical reactions. The alkalophilic bacterium Acinetobacter calcoaceticus is used as a model organism, growing on acetate and converting xylose into xylonolactone. It is shown that all relevant pieces of information can be straightforwardly and systematically treated, by considering them as constaints. In general, it is illustrated how the search for directed process improvements starts with an optimal selection of information sources, followed by an accurate analysis of possible metabolic bottlenecks. In this particular case it is shown that the yield of A. calcoaceticus on acetate at varying xylose/acetate feed ratios can be accurately predicted using dispersed process information. (c) 1996 John Wiley & Sons, Inc.

Modeling and experimental validation of carbon dioxide evolution in alkalophilic cultures.

The chemical reactions involving carbon dioxide in mineral culture media are considered. A mathematic model is set up, based on published data, which is valid at pH values below 9, and in which the nonideality of the solution is taken into account. The crucial parameter is the constant expressing the equilibrium between carbon dioxide and bicarbonate, K(1).The reactions were studied in three different aqueous solutions: water, mineral salt medium, and a suspension with nongrowing bacterial cells. For each situation, three methods were compared for the determination of the bicarbonate concentration in the solution: equilibrium state total carbon analysis, dynamic monitoring of the rate of acid or alkali addition, and dynamic measurement of the carbon dioxide gas phase mole fraction.In a batch-stirred tank reactor, the equilibrium constant K(1) agreed with the published value, and the three bicarbonate analysis methods give the same results. If the nonideality is not taken into account, the result significantly differed from the published value and is likely to be incorrect.A real alkalophilic process, using Acinetobacter calcoaceticus in a continuous stirred tank reactor at steady state, also gave results that are in accord with the literature. However, the results do not allow validation of the equation expressing the nonideality.The steady state in the batch system and in continuous culture can be well described with the mathematical model. However, in the transient state there are some unexplained differences between simulation and measurement.

Linear relations in microbial reaction systems: a general overview of their origin, form, and use.

In microbial reaction systems, there are a number of linear relations among net conversion rates. These can be very useful in the analysis of experimental data. This article provides a general approach for the formation and application of the linear relations. Two type of system descriptions, one considering the biomass as a black box and the other based on metabolic pathways, are encountered. These are defined in a linear vector and matrix algebra framework. A correct a priori description can be obtained by three useful tests: the independency, consistency, and observability tests. The independency are different. The black box approach provides only conservations relations. They are derived from element, electrical charge, energy, and Gibbs energy balances. The metabolic approach provides, in addition to the conservation relations, metabolic and reaction relations. These result from component, energy, and Gibbs energy balances. Thus it is more attractive to use the metabolic description than the black box approach. A number of different types of linear relations given in the literature are reviewed. They are classified according to the different categories that result from the black box or the metabolic system description. Validation of hypotheses related to metabolic pathways can be supported by experimental validation of the linear metabolic relations. However, definite proof from biochemical evidence remains indispensable.