Dick Schipper

Physiological Characterization of the ARO10-Dependent, Broad-Substrate-Specificity 2-Oxo Acid Decarboxylase Activity of Saccharomyces cerevisiae

ABSTRACT Aerobic, glucose-limited chemostat cultures of Saccharomyces cerevisiae CEN.PK113-7D were grown with different nitrogen sources. Cultures grown with phenylalanine, leucine, or methionine as a nitrogen source contained high levels of the corresponding fusel alcohols and organic acids, indicating activity of the Ehrlich pathway. Also, fusel alcohols derived from the other two amino acids were detected in the supernatant, suggesting the involvement of a common enzyme activity. Transcript level analysis revealed that among the five thiamine-pyrophospate-dependent decarboxylases (PDC1, PDC5, PDC6, ARO10, and THI3), only ARO10 was transcriptionally up-regulated when phenylalanine, leucine, or methionine was used as a nitrogen source compared to growth on ammonia, proline, and asparagine. Moreover, 2-oxo acid decarboxylase activity measured in cell extract from CEN.PK113-7D grown with phenylalanine, methionine, or leucine displayed similar broad-substrate 2-oxo acid decarboxylase activity. Constitutive expression of ARO10 in ethanol-limited chemostat cultures in a strain lacking the five thiamine-pyrophosphate-dependent decarboxylases, grown with ammonia as a nitrogen source, led to a measurable decarboxylase activity with phenylalanine-, leucine-, and methionine-derived 2-oxo acids. Moreover, even with ammonia as the nitrogen source, these cultures produced significant amounts of the corresponding fusel alcohols. Nonetheless, the constitutive expression of ARO10 in an isogenic wild-type strain grown in a glucose-limited chemostat with ammonia did not lead to any 2-oxo acid decarboxylase activity. Furthermore, even when ARO10 was constitutively expressed, growth with phenylalanine as the nitrogen source led to increased decarboxylase activities in cell extracts. The results reported here indicate the involvement of posttranscriptional regulation and/or a second protein in the ARO10-dependent, broad-substrate-specificity decarboxylase activity.

Asymmetric reduction of ketones via whole cell bioconversions and transfer hydrogenation: complementary approaches

Abstract Prochiral aryl and dialkyl ketones were enantioselectively reduced to the corresponding alcohols using whole cells of the white-rot fungus Merulius tremellosus ono991 as a biocatalytic reduction system and ruthenium(II)–amino alcohol and iridium(I)–amino sulfide complexes as metal catalysts in asymmetric transfer hydrogenation. Comparison of the results showed that the corresponding chiral alcohols could be obtained with moderate to high enantioselectivities (e.e.s of up to 98%). The biocatalytic and transfer hydrogenation approaches appear to be complementary. The biocatalytic approach is the most suitable for the enantioselective reduction of chloro-substituted (aryl) ketones, whereas in the reduction of α,β-unsaturated compounds excellent results were obtained using the catalytic hydrogenation protocol.

Engineering of Penicillium chrysogenum for fermentative production of a novel carbamoylated cephem antibiotic precursor

Penicillium chrysogenum was successfully engineered to produce a novel carbamoylated cephalosporin that can be used as a synthon for semi-synthetic cephalosporins. To this end, genes for Acremonium chrysogenum expandase/hydroxylase and Streptomyces clavuligerus carbamoyltransferase were expressed in a penicillinG high-producing strain of P.chrysogenum. Growth of the engineered strain in the presence of adipic acid resulted in production of adipoyl-7-amino-3-carbamoyloxymethyl-3-cephem-4-carboxylic acid (ad7-ACCCA) and of several adipoylated pathway intermediates. A combinatorial chemostat-based transcriptome study, in which the ad7-ACCCA-producing strain and a strain lacking key genes in beta-lactam synthesis were grown in the presence and absence of adipic acid, enabled the dissection of transcriptional responses to adipic acid per se and to ad7-ACCCA production. Transcriptome analysis revealed that adipate catabolism in P.chrysogenum occurs via beta-oxidation and enabled the identification of putative genes for enzymes involved in mitochondrial and peroxisomal beta-oxidation pathways. Several of the genes that showed a specifically altered transcript level in ad7-ACCCA-producing cultures were previously implicated in oxidative stress responses.

13C-Labeled Gluconate Tracing as a Direct and Accurate Method for Determining the Pentose Phosphate Pathway Split Ratio in Penicillium chrysogenum

ABSTRACT In this study we developed a new method for accurately determining the pentose phosphate pathway (PPP) split ratio, an important metabolic parameter in the primary metabolism of a cell. This method is based on simultaneous feeding of unlabeled glucose and trace amounts of [U-13C]gluconate, followed by measurement of the mass isotopomers of the intracellular metabolites surrounding the 6-phosphogluconate node. The gluconate tracer method was used with a penicillin G-producing chemostat culture of the filamentous fungus Penicillium chrysogenum. For comparison, a 13C-labeling-based metabolic flux analysis (MFA) was performed for glycolysis and the PPP of P. chrysogenum. For the first time mass isotopomer measurements of 13C-labeled primary metabolites are reported for P. chrysogenum and used for a 13C-based MFA. Estimation of the PPP split ratio of P. chrysogenum at a growth rate of 0.02 h−1 yielded comparable values for the gluconate tracer method and the 13C-based MFA method, 51.8% and 51.1%, respectively. A sensitivity analysis of the estimated PPP split ratios showed that the 95% confidence interval was almost threefold smaller for the gluconate tracer method than for the 13C-based MFA method (40.0 to 63.5% and 46.0 to 56.5%, respectively). From these results we concluded that the gluconate tracer method permits accurate determination of the PPP split ratio but provides no information about the remaining cellular metabolism, while the 13C-based MFA method permits estimation of multiple fluxes but provides a less accurate estimate of the PPP split ratio.

Serial 13C-Based Flux Analysis of an L-Phenylalanine-Producing E. coli Strain Using the Sensor Reactor

With the aid of the recently developed Sensor reactor system, a series of three subsequent 13C labeling experiments was performed mirroring the l‐phenylalanine (l‐Phe) production phase of a recombinant E. coli strain that was cultivated under industry‐like conditions in a 300 L bioreactor. On the basis of the data from NMR labeling analysis, three subsequent flux patterns were successfully derived monitoring the l‐Phe formation during an observation window from 14 to 23.3 h process time. Linear programming was performed to identify optimal flux patterns for l‐Phe formation. Additionally, flux sensitivity analysis was used to identify the most promising metabolic engineering target. As a result, high rates of phosphoenolpyruvate (PEP) to pyruvate (PYR) conversion were identified as the most important reason for deterioration of the l‐Phe/glucose yield from 20 to finally 11 mol %. Considering the characteristics of the enzyme kinetics involved, the working hypothesis was formulated that phosphoenolpyruvate synthase activity was increasingly hampered by rising oxaloacetate and 2‐oxoglutarate concentrations, while at the same time pyruvate kinase activity arose due to activation by fructose 1,6‐diphosphate. Hence, pps overexpression should be performed to optimize the existing production strain.

Kinetic studies on the carboxylation of 6-amino-penicillanic acid to 8-hydroxy-penillic acid

Abstract The carboxylation in aqueous solution of 6-amino-penicillanic acid (6-APA) to 8-hydroxy-penillic acid (8-HPA) was studied at 25°C and pH 6·5. During sparging with either a citrate buffer or a chemically defined cultivation medium containing 6-APA with mixtures of carbon dioxide and air (2·7–41% (v/v) CO 2 ), the kinetics for conversion of 6-APA was followed by HPLC. In the citrate buffer 6-APA was converted by two competitive reactions each following first order kinetics with respect to the concentration of 6-APA: 1. carboxylation into 8-HPA; and 2. slow conversion into an unknown compound. Formation of the unknown compound was not observed in the cultivation medium. The carboxylation of 6-APA was also found to be first order with respect to the concentration of dissolved carbon dioxide. The rate constant for formation of 8-HPA did not differ significantly in the cultivation medium compared to the citrate buffer. The kinetic was found to be the same at pH 5·0 and 6·5, and it is therefore concluded that it is dissolved carbon dioxide and not bicarbonate (or carbonate) that is involved in the carboxylation of 6-APA. Attempts to identify the unknown compound in an incubation mixture of citrate and 6-APA by both NMR spectroscopy and HPLC revealed the presence of three new citrate-containing penicillins substituted at the amino position. Upon treatment with β-lactamase one of the citrate-containing penicillins coeluted with the unknown compound. The unknown compound is most likely formed by hydrolysis of the β-lactam ring and eventually further chemical modifications of a citrate-containing penicillin.

Influence of the adipate and dissolved oxygen concentrations on the β-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.

Synthesis of Penicillin N and Isopenicillin N

Abstract Enantiomeric 2-(N-allyloxycarbonyl)aminoadipic acid 1-allyl esters were obtained from the corresponding 2-(N-trityl)aminoadipic acid diallyl esters following selective hydrolysis of the allyl 6-ester group and subsequent exchange of the trityl group by the allyloxycarbonyl function. The resulting monoacids were used to acylate 6-aminopenicillanic acid allyl ester using a carbodiimide-mediated coupling. The products, the l - and d -isomers of 6-[6-(2-(N-allyloxycarbonyl) aminoadipyl)]aminopenicillanic acid diallyl ester were deprotected in one step by catalytic allyl transfer using tetrakis-(triphenylphosphine)palladium(0), to afford isopenicillin N and penicillin N, respectively. The presented straightforward route to penicillin N and isopenicillin N is uniquely compatible with the sensitive nature of the condensation products and gives entry to a new and high yielding procedure that is superior to existing approaches.