PubMed Health⌕ Search

Biomedical subjects

Bernard Witholt

Publications and source records attributed to Bernard Witholt.

At least 19 recordsLinked to original sources
PubMed2006

The efficiency of recombinant Escherichia coli as biocatalyst for stereospecific epoxidation.

Styrene is efficiently converted into (S)-styrene oxide by growing Escherichia coli expressing the styrene monooxygenase genes styAB of Pseudomonas sp. strain VLB120 in an organic/aqueous emulsion. Now, we investigated factors influencing the epoxidation activity of recombinant E. coli with the aim to improve the process in terms of product concentration and volumetric productivity. The catalytic activity of recombinant E. coli was not stable and decreased with reaction time. Kinetic analyses and the independence of the whole-cell activity on substrate and biocatalyst concentrations indicated that the maximal specific biocatalyst activity was not exploited under process conditions and that substrate mass transfer and enzyme inhibition did not limit bioconversion performance. Elevated styrene oxide concentrations, however, were shown to promote acetic acid formation, membrane permeabilization, and cell lysis, and to reduce growth rate and colony-forming activity. During biotransformations, when cell viability was additionally reduced by styAB overexpression, such effects coincided with decreasing specific epoxidation rates and metabolic activity. This clearly indicated that biocatalyst performance was reduced as a result of product toxicity. The results point to a product toxicity-induced biological energy shortage reducing the biocatalyst activity under process conditions. By reducing exposure time of the biocatalyst to the product and increasing biocatalyst concentrations, volumetric productivities were increased up to 1,800 micromol/min/liter aqueous phase (with an average of 8.4 g/L(aq) x h). This represents the highest productivity reported for oxygenase-based whole-cell biocatalysis involving toxic products.

Biotransformation↗
PubMed2006

Tailor-made olefinic medium-chain-length poly[(R)-3-hydroxyalkanoates] by Pseudomonas putida GPo1: batch versus chemostat production.

Functionalized medium-chain-length polyhydroxyalkanoates (mclPHAs) have gained much interest in research on biopolymers because of their ease of chemical modification. Tailored olefinic mclPHA production from mixtures of octanoic acid and 10-undecenoic acid was investigated in batch and dual (C,N) nutrient limited chemostat cultures of Pseudomonas putida GPo1 (ATCC 29347). In a batch culture, where P. putida GPo1 was grown on a mixture of octanoic acid (58 mol%) and 10-undecenoic acid (42 mol%), it was found that the fraction of aliphatic monomers was slightly lower in mclPHA produced during exponential growth than during late stationary phase. Thus, the total monomeric composition changed over time indicating different kinetics for the two carbon substrates. Chemostat experiments showed that the dual (C,N) nutrient limited growth regime (DNLGR) for 10-undecenoic acid coincided with the one for octanoic acid. Five different chemostats on equimolar mixtures of octanoic acid and 10-undecenoic acid within the DNLGR revealed that the monomeric composition of mclPHA was not a function of the carbon to nitrogen (C(0)/N(0)) ratio in the feed medium but rather of the dilution rate. The fraction of aliphatic monomers in the accumulated mclPHA was slightly lower at high dilution rates and increased towards low dilution rates, again indicating different kinetics for the two carbon substrates in P. putida GPo1.

Bacteriological Techniques↗
PubMed2006

Selection of biocatalysts for chemical synthesis.

To determine whether microbial chemosensors can be used to find new or better biocatalysts, we constructed Escherichia coli hosts that recognize the product of a biocatalytic conversion through the transcriptional activator NahR and respond by expression of a lacZ or tetA reporter gene. Equipped with a benzaldehyde dehydrogenase (XylC from Pseudomonas putida), the lacZ-based host responded to the oxidation of benzaldehyde and 2-hydroxybenzaldehyde to the corresponding benzoic acids by forming blue colonies, whereas XylC- cells did not. Similarly, the tetA-based host was able to grow under selective conditions only when equipped with XylC, enabling selection of biocatalytically active cells in inactive populations at frequencies as low as 10(-6).

Aldehyde Oxidoreductases↗
PubMed2006

Cytochrome P450 alkane hydroxylases of the CYP153 family are common in alkane-degrading eubacteria lacking integral membrane alkane hydroxylases.

Several strains that grow on medium-chain-length alkanes and catalyze interesting hydroxylation and epoxidation reactions do not possess integral membrane nonheme iron alkane hydroxylases. Using PCR, we show that most of these strains possess enzymes related to CYP153A1 and CYP153A6, cytochrome P450 enzymes that were characterized as alkane hydroxylases. A vector for the polycistronic coexpression of individual CYP153 genes with a ferredoxin gene and a ferredoxin reductase gene was constructed. Seven of the 11 CYP153 genes tested allowed Pseudomonas putida GPo12 recombinants to grow well on alkanes, providing evidence that the newly cloned P450s are indeed alkane hydroxylases.

Alkanes↗
PubMed2006

CYP153A6, a soluble P450 oxygenase catalyzing terminal-alkane hydroxylation.

The first and key step in alkane metabolism is the terminal hydroxylation of alkanes to 1-alkanols, a reaction catalyzed by a family of integral-membrane diiron enzymes related to Pseudomonas putida GPo1 AlkB, by a diverse group of methane, propane, and butane monooxygenases and by some membrane-bound cytochrome P450s. Recently, a family of cytoplasmic P450 enzymes was identified in prokaryotes that allow their host to grow on aliphatic alkanes. One member of this family, CYP153A6 from Mycobacterium sp. HXN-1500, hydroxylates medium-chain-length alkanes (C6 to C11) to 1-alkanols with a maximal turnover number of 70 min(-1) and has a regiospecificity of > or =95% for the terminal carbon atom position. Spectroscopic binding studies showed that C6-to-C11 aliphatic alkanes bind in the active site with Kd values varying from approximately 20 nM to 3.7 microM. Longer alkanes bind more strongly than shorter alkanes, while the introduction of sterically hindering groups reduces the affinity. This suggests that the substrate-binding pocket is shaped such that linear alkanes are preferred. Electron paramagnetic resonance spectroscopy in the presence of the substrate showed the formation of an enzyme-substrate complex, which confirmed the binding of substrates observed in optical titrations. To rationalize the experimental observations on a molecular scale, homology modeling of CYP153A6 and docking of substrates were used to provide the first insight into structural features required for terminal alkane hydroxylation.

Alkanes↗
PubMed2005

Poly(3-hydroxyalkanoate) polymerase synthesis and in vitro activity in recombinant Escherichia coli and Pseudomonas putida.

We tested the synthesis and in vitro activity of the poly(3-hydroxyalkanoate) (PHA) polymerase 1 from Pseudomonas putida GPo1 in both P. putida GPp104 and Escherichia coli JMU193. The polymerase encoding gene phaC1 was expressed using the inducible PalkB promoter. It was found that the production of polymerase could be modulated over a wide range of protein levels by varying inducer concentrations. The optimal inducer dicyclopropylketone concentrations for PHA production were at 0.03% (v/v) for P. putida and 0.005% (v/v) for E. coli. Under these concentrations the maximal polymerase level synthesized in the E. coli host (6% of total protein) was about three- to fourfold less than that in P. putida (20%), whereas the maximal level of PHA synthesized in the E. coli host (8% of total cell dry weight) was about fourfold less than that in P. putida (30%). In P. putida, the highest specific activity of polymerase was found in the mid-exponential growth phase with a maximum of 40 U/g polymerase, whereas in E. coli, the maximal specific polymerase activity was found in the early stationary growth phase (2 U/g polymerase). Our results suggest that optimal functioning of the PHA polymerase requires factors or a molecular environment that is available in P. putida but not in E. coli.

Acyltransferases↗
PubMed2005

Expression of PHA polymerase genes of Pseudomonas putida in Escherichia coli and its effect on PHA formation.

Poly-3-hydroxyalkanoates (PHAs) are synthesized by many bacteria as intracellular storage material. The final step in PHA biosynthesis is catalyzed by two PHA polymerases (phaC) in Pseudomonas putida. The expression of these two phaC genes (phaC1 and phaC2)was studied in Escherichia coli, either under control of the native promoter or under control of an external promoter. It was found that the two phaC genes are not expressed in E. coli without an external promoter. During heterologous expression of phaC from Plac on a high copy number plasmid, a rapid reduction of the number of colony forming units was observed, especially for phaC2. It appears that the plasmid instability was partially caused by high-level production of PHA polymerase. Subsequently, tightly regulated phaC2 expression systems on a low copy number vector were applied in E. coli. This resulted in PHA yields of over 20 of total cell dry weight, which was 2 fold higher than that obtained from the system where phaC2 is present on a high copy number vector. In addition, the PHA monomer composition differed when different gene expression systems or different phaC genes were applied.

Acyltransferases↗
PubMed2005

Suitability of recombinant Escherichia coli and Pseudomonas putida strains for selective biotransformation of m-nitrotoluene by xylene monooxygenase.

Escherichia coli JM101(pSPZ3), containing xylene monooxygenase (XMO) from Pseudomonas putida mt-2, catalyzes specific oxidations and reductions of m-nitrotoluene and derivatives thereof. In addition to reactions catalyzed by XMO, we focused on biotransformations by native enzymes of the E. coli host and their effect on overall biocatalyst performance. While m-nitrotoluene was consecutively oxygenated to m-nitrobenzyl alcohol, m-nitrobenzaldehyde, and m-nitrobenzoic acid by XMO, the oxidation was counteracted by an alcohol dehydrogenase(s) from the E. coli host, which reduced m-nitrobenzaldehyde to m-nitrobenzyl alcohol. Furthermore, the enzymatic background of the host reduced the nitro groups of the reactants resulting in the formation of aromatic amines, which were shown to effectively inhibit XMO in a reversible fashion. Host-intrinsic oxidoreductases and their reaction products had a major effect on the activity of XMO during biocatalysis of m-nitrotoluene. P. putida DOT-T1E and P. putida PpS81 were compared to E. coli JM101 as alternative hosts for XMO. These promising strains contained an additional dehydrogenase that oxidized m-nitrobenzaldehyde to the corresponding acid but catalyzed the formation of XMO-inhibiting aromatic amines at a significantly lower level than E. coli JM101.

Biodegradation, Environmental↗
PubMed2005

Biocatalytic production of perillyl alcohol from limonene by using a novel Mycobacterium sp. cytochrome P450 alkane hydroxylase expressed in Pseudomonas putida.

A number of oxygenated monoterpenes present at low concentrations in plant oils have anticarcinogenic properties. One of the most promising compounds in this respect is (-)-perillyl alcohol. Since this natural product is present only at low levels in a few plant oils, an alternative, synthetic source is desirable. Screening of 1,800 bacterial strains showed that many alkane degraders were able to specifically hydroxylate l-limonene in the 7 position to produce enantiopure (-)-perillyl alcohol. The oxygenase responsible for this was purified from the best-performing wild-type strain, Mycobacterium sp. strain HXN-1500. By using N-terminal sequence information, a 6.2-kb ApaI fragment was cloned, which encoded a cytochrome P450, a ferredoxin, and a ferredoxin reductase. The three genes were successfully coexpressed in Pseudomonas putida by using the broad-host-range vector pCom8, and the recombinant converted limonene to perillyl alcohol with a specific activity of 3 U/g (dry weight) of cells. The construct was subsequently used in a 2-liter bioreactor to produce perillyl alcohol on a scale of several grams.

Amino Acid Sequence↗
PubMed2005

Identification of an amino acid position that determines the substrate range of integral membrane alkane hydroxylases.

Selection experiments and protein engineering were used to identify an amino acid position in integral membrane alkane hydroxylases (AHs) that determines whether long-chain-length alkanes can be hydroxylated by these enzymes. First, substrate range mutants of the Pseudomonas putida GPo1 and Alcanivorax borkumensis AP1 medium-chain-length AHs were obtained by selection experiments with a specially constructed host. In all mutants able to oxidize alkanes longer than C13, W55 (in the case of P. putida AlkB) or W58 (in the case of A. borkumensis AlkB1) had changed to a much less bulky amino acid, usually serine or cysteine. The corresponding position in AHs from other bacteria that oxidize alkanes longer than C13 is occupied by a less bulky hydrophobic residue (A, V, L, or I). Site-directed mutagenesis of this position in the Mycobacterium tuberculosis H37Rv AH, which oxidizes C10 to C16 alkanes, to introduce more bulky amino acids changed the substrate range in the opposite direction; L69F and L69W mutants oxidized only C10 and C11 alkanes. Subsequent selection for growth on longer alkanes restored the leucine codon. A structure model of AHs based on these results is discussed.

Alkanes↗
PubMed2004

Dual nutrient limited growth: models, experimental observations, and applications.

Dual nutrient limited growth, the control of the cell growth rate (kinetic aspect) or the restriction of the amount of biomass (stoichiometric aspect) by two nutrients at the same time, is a relatively unknown ability of the microorganisms and consequently, still not mentioned in textbooks to date. Nevertheless, multiple nutrient limited or controlled growth has been reported for different systems; e.g. ecosystems, batch, fed-batch, and chemostat cultures. Generally, dual nutrient limited growth has been observed when the microorganism of interest: (a) showed a variation of the cellular composition, (b) was able to accumulate a storage compound, (c) changed the cell metabolism, or (d) excreted metabolic intermediates. Consequently, stoichiometric models have been developed to estimate the growth conditions leading to dual nutrient limited growth. A general problem of the kinetic aspect is the accurate measurement of the growth controlling nutrients in the culture broth (microg l(-1) range), as the cells may consume residual nutrients during sampling. Nevertheless, most models of dual limited growth deal with the kinetic aspect although the control experiments are difficult to carry out. The aim of this survey is to introduce this special growth feature with respect to basic models, experimental data, and potential applications in bioprocesses.

Bacteria↗
PubMed2004

Expression of poly-3-(R)-hydroxyalkanoate (PHA) polymerase and acyl-CoA-transacylase in plastids of transgenic potato leads to the synthesis of a hydrophobic polymer, presumably medium-chain-length PHAs.

Medium-chain-length poly-3-(R)-hydroxyalkanoates (mcl-PHAs) belong to the group of microbial polyesters. The minimum gene-set for the accumulation of mcl-PHAs from de novo fatty acid biosynthesis has been identified in prokaryotes as consisting of the Pha-C1 polymerase and the ACP-CoA-transacylase. In this paper, the synthesis of mcl-PHAs has been attempted in transgenic potato (Solanum tuberosum L.) using the same set of genes that were introduced into potato by particle bombardment. Polymer contents of transgenic lines were analysed by gas chromatography and by a new simple method employing a size-exclusion filter column. The expression of the Pha-C1 polymerase and the ACP-CoA-transacylase in the plastids of transgenic potato led to the synthesis of a hydrophobic polymer composed of mcl-hydroxy-fatty acids with carbon chain lengths ranging from C-6 to C-12 in leaves of the selected transgenic lines. We strongly suggest that the polymer observed consists of mcl-PHAs and that this report establishes for the first time a possible route for the production of mcl-PHAs from de novo fatty acid biosynthesis in plants.

Acyltransferases↗
PubMed2004

Rapid identification of new bacterial alcohol dehydrogenases for (R)- and (S)-enantioselective reduction of ss-ketoesters.

New bacterial alcohol dehydrogenases with high and complementary enantioselectivity for the reduction of ethyl 3-keto-4,4,4-trifluorobutyrate 1 and methyl 3-keto-3-(3'-pyridyl)-propionate 3 have been rapidly identified by use of a new methodology consisting of preselection of microorganisms based on degradation ability and high-throughput screening with a miniaturized system coupled with fast analysis of enantioselectivities.

Acinetobacter↗
PubMed2004

Characterization of two alkane hydroxylase genes from the marine hydrocarbonoclastic bacterium Alcanivorax borkumensis.

The marine gamma-Proteobacterium Alcanivorax borkumensis is highly specialized in the assimilation of aliphatic hydrocarbons, and makes up a large part of the biomass in oil-polluted marine environments. In addition to the previously identified alkane hydroxylase AlkB1, a second alkane hydroxylase (AlkB2) showing 65% identity to the Pseudomonas aeruginosa AlkB2 alkane hydroxylase was identified. Unlike alkB1, alkB2 is not flanked by genes involved in alkane metabolism. Heterologous expression of the A. borkumensis AP1 alkB1 and alkB2 genes showed that they encode functional alkane hydroxylases with substrate ranges similar to those of their P. putida and P. aeruginosa homologues. The transcription initiation sites and levels of the alkB1, alkB2 and alkS mRNA transcripts were determined. Expression of both alkB1 and alkB2 was induced by alkanes, but transcripts corresponding to alkB1 were much more abundant than those of alkB2. An inverted repeat similar to the binding site for the P. putida GPo1 transcriptional activator AlkS was present upstream of the promoters for alkB1 and alkB2, although that of alkB2 was less well conserved, and only the transcriptional fusion of promoter PalkB1 to the reporter gene lacZ efficiently responded to n-octane. Contrary to what has been found for the P. putida GPo1 alkane degradation pathway, expression of the A. borkumensis AP1 alkS gene was not induced by alkanes, and an AlkS binding site was not present upstream of the promoter for alkS. This indicates that, in spite of the clear similarities, the A. borkumensis alk-genes are regulated by a strategy different from that of the P. putida GPo1 alk genes.

Alkanes↗
PubMed2004

Biochemical characterization of StyAB from Pseudomonas sp. strain VLB120 as a two-component flavin-diffusible monooxygenase.

Pseudomonas sp. VLB120 uses styrene as a sole source of carbon and energy. The first step in this metabolic pathway is catalyzed by an oxygenase (StyA) and a NADH-flavin oxidoreductase (StyB). Both components have been isolated from wild-type Pseudomonas strain VLB120 as well as from recombinant Escherichia coli. StyA from both sources is a dimer, with a subunit size of 47 kDa, and catalyzes the enantioselective epoxidation of CC double bonds. Styrene is exclusively converted to S-styrene oxide with a specific activity of 2.1 U mg(-1) (k(cat) = 1.6 s(-1)) and K(m) values for styrene of 0.45 +/- 0.05 mM (wild type) and 0.38 +/- 0.09 mM (recombinant). The epoxidation reaction depends on the presence of a NADH-flavin adenine dinucleotide (NADH-FAD) oxidoreductase for the supply of reduced FAD. StyB is a dimer with a molecular mass of 18 kDa and a NADH oxidation activity of 200 U mg(-1) (k(cat) [NADH] = 60 s(-1)). Steady-state kinetics determined for StyB indicate a mechanism of sequential binding of NADH and flavin to StyB. This enzyme reduces FAD as well as flavin mononucleotide and riboflavin. The NADH oxidation activity does not depend on the presence of StyA. During the epoxidation reaction, no formation of a complex of StyA and StyB has been observed, suggesting that electron transport between reductase and oxygenase occurs via a diffusing flavin.

Cloning, Molecular↗