Microparticle-enhanced cultivation (MPEC) was applied as a novel method for improved biomass and product formation during cultivation of filamentous microorganisms. Exemplarily, chloroperoxidase (CPO) formation by Caldariomyces fumago was analyzed in the presence and absence of microparticles of different size. Particles of $500 mm in diameter had no effect on growth morphology or productivity of CPO formation by C. fumago. In contrast particles of 42 mm in diameter led to the dispersion of the C. fumago mycelia up to the level of single hyphae. Under these conditions the maximum specific productivity of CPO formation was enhanced about fivefold and an accumulated CPO activity in the culture supernatant of more than 1,000 U mL À1 was achieved after 10-12 days of cultivation. In addition, the novel cultivation method also showed a positive effect on growth characteristics of other filamentous microorganisms proven by the stimulation of single hyphae/ cell formation.
A whole-cell biotransformation system for the reduction of prochiral carbonyl compounds, such as methyl acetoacetate, to chiral hydroxy acid derivatives [methyl (R)-3-hydroxy butanoate] was developed in Escherichia coli by construction of a recombinant oxidation/reduction cycle. Alcohol dehydrogenase from Lactobacillus brevis catalyzes a highly regioselective and enantioselective reduction of several ketones or keto acid derivatives to chiral alcohols or hydroxy acid esters. The adh gene encoding for the alcohol dehydrogenase of L. brevis was expressed in E. coli. As expected, whole cells of the recombinant strain produced only low quantities of methyl (R)-3-hydroxy butanoate from the substrate methyl acetoacetate. Therefore, the fdh gene from Mycobacterium vaccae N10, encoding NAD+-dependent formate dehydrogenase, was functionally coexpressed. The resulting two-fold recombinant strain exhibited an in vitro catalytic alcohol dehydrogenase activity of 6.5 units mg-1 protein in reducing methyl acetoacetate to methyl (R)-3-hydroxy butanoate with NADPH as the cofactor and 0.7 units mg-1 protein with NADH. The in vitro formate dehydrogenase activity was 1.3 units mg-1 protein. Whole resting cells of this strain catalyzed the formation of 40 mM methyl (R)-3-hydroxy butanoate from methyl acetoacetate. The product yield was 100 mol% at a productivity of 200 micromol g-1 (cell dry weight) min-1. In the presence of formate, the intracellular [NADH]/[NAD+] ratio of the cells increased seven-fold. Thus, the functional overexpression of alcohol dehydrogenase in the presence of formate dehydrogenase was sufficient to enable and sustain the desired reduction reaction via the relatively low specific activity of alcohol dehydrogenase with NADH, instead of NADPH, as a cofactor.
Escherichia coli BL21, expressing a quintuple mutant of P450(BM-3), oxyfunctionalizes alpha-pinene in an NADPH-dependent reaction to alpha-pinene oxide, verbenol, and myrtenol. We optimized the whole-cell biocatalyst by integrating a recombinant intracellular NADPH regeneration system through co-expression of a glucose facilitator from Zymomonas mobilis for uptake of unphosphorylated glucose and a NADP(+)-dependent glucose dehydrogenase from Bacillus megaterium that oxidizes glucose to gluconolactone. The engineered strain showed a nine times higher initial alpha-pinene oxide formation rate corresponding to a sixfold higher yield of 20 mg g(-1) cell dry weight after 1.5 h. The initial total product formation rate was 1,000 micromol h(-1) micromol(-1) P450 leading to a total of 32 mg oxidized products per gram cell of dry weight after 1.5 h. The physiological functioning of the heterologous cofactor regeneration system was illustrated by a sevenfold increased alpha-pinene oxide yield in the presence of glucose compared to glucose-free conditions.
A whole-cell biotransformation system for the conversion of d-fructose to d-mannitol was developed in Escherichia coli by constructing a recombinant oxidation/reduction cycle. First, the mdh gene, encoding mannitol dehydrogenase of Leuconostoc pseudomesenteroides ATCC 12291 (MDH), was expressed, effecting strong catalytic activity of an NADH-dependent reduction of D-fructose to D-mannitol in cell extracts of the recombinant E. coli strain. By contrast whole cells of the strain were unable to produce D-mannitol from D-fructose. To provide a source of reduction equivalents needed for d-fructose reduction, the fdh gene from Mycobacterium vaccae N10 (FDH), encoding formate dehydrogenase, was functionally co-expressed. FDH generates the NADH used for d-fructose reduction by dehydrogenation of formate to carbon dioxide. These recombinant E. coli cells were able to form D-mannitol from D-fructose in a low but significant quantity (15 mM). The introduction of a further gene, encoding the glucose facilitator protein of Zymomonas mobilis (GLF), allowed the cells to efficiently take up D-fructose, without simultaneous phosphorylation. Resting cells of this E. coli strain (3 g cell dry weight/l) produced 216 mM D-mannitol in 17 h. Due to equimolar formation of sodium hydroxide during NAD(+)-dependent oxidation of sodium formate to carbon dioxide, the pH value of the buffered biotransformation system increased by one pH unit within 2 h. Biotransformations conducted under pH control by formic-acid addition yielded d-mannitol at a concentration of 362 mM within 8 h. The yield Y(D-mannitol/D-fructose) was 84 mol%. These results show that the recombinant strain of E. coli can be utilized as an efficient biocatalyst for d-mannitol formation.
Recently, we reported on the construction of a whole-cell biotransformation system in Escherichia coli for the production of D: -mannitol from D: -fructose. Supplementation of this strain with extracellular glucose isomerase resulted in the formation of 800 mM D: -mannitol from 1,000 mM D: -glucose. Co-expression of the xylA gene of E. coli in the biotransformation strain resulted in a D: -mannitol concentration of 420 mM from 1,000 mM D: -glucose. This is the first example of conversion of D: -glucose to D: -mannitol with direct coupling of a glucose isomerase to the biotransformation system.
Chloroperoxidase (CPO) from Caldariomyces fumago was analysed for its ability to oxidize ten different monoterpenes with hydrogen peroxide as oxidant. In the absence of halide ions geraniol and, to a lesser extent, citronellol and nerol were converted into the corresponding aldehydes, whereas terpene hydrocarbons did not serve as substrates under these conditions. In the presence of chloride, bromide and iodide ions, every terpene tested was converted into one or more products. (1S)-(+)-3-carene was chosen as a model substrate for the CPO-catalysed conversion of terpenes in the presence of sodium halides. With chloride, bromide and iodide, the reaction products were the respective (1S,3R,4R,6R)-4-halo-3,7,7-trimethyl-bicyclo[4.1.0]-heptane-3-ols, as identified by 1H and 13C nuclear magnetic resonance. These product formations turned out to be strictly regio- and stereoselective and proceeded very rapidly and almost quantitatively. Initial specific activities of halohydrin formation increased from 4.22 U mg-1 with chloride to 12.22 U mg-1 with bromide and 37.11 U mg-1 with iodide as the respective halide ion. These results represent the first examples of the application of CPO as a highly efficient biocatalyst for monoterpene functionalization. This is a promising strategy for 'green' terpene chemistry overcoming drawbacks usually associated with cofactor-dependent oxygenases, whole-cell biocatalysts and conventional chemical methods used for terpene conversions.
Mannitol-2-dehydrogenase (EC 1.1.1.67) of Leuconostoc pseudomesenteroides ATCC 12291 catalyzing the NADH-dependent reduction of d-fructose to d-mannitol was purified to homogeneity. Native mannitol-2-dehydrogenase has a molecular mass of 155 kDa as determined by gel filtration chromatography. In SDS-PAGE, a single band appeared corresponding to a molecular mass of 43 kDa which indicated that the enzyme was composed of four identical subunits. Enzyme activity was completely inhibited by EDTA and could be restored by zinc ions, but not by Mn(2+) or Mg(2+) which demonstrated that zinc is a cofactor. Purified mannitol-2-dehydrogenase exhibited a maximal specific activity of 400 micromol fructose reduced min(-1) x (mg protein)(-1), using NADH as electron donor. The enzyme showed a high substrate specificity for d-fructose and d-mannitol, however it accepted NADPH as a cofactor with 32% activity ( V(max)) relative to NADPH (100%). The mdh gene, encoding mannitol-2-dehydrogenase, was identified by hybridization with a degenerate gene probe complementary to the nucleotide sequence encoding the first eight N-terminal amino acids of the enzyme. The mdh gene was cloned on a 4.2-kb DNA fragment, subcloned, and expressed in Escherichia coli. Sequencing of the gene revealed an open reading frame of 1017 bp, encoding a protein of 338 amino acids with a predicted molecular mass of 36.0 kDa. Plasmid-encoded mdh was functionally expressed, with 70 U/mg of cell-free protein in E. coli. Multiple sequence alignments showed that mannitol-2-dehydrogenase was affiliated with members of the Zn(2+)-containing medium-chain alcohol/polyol dehydrogenase/reductase protein family (MDR).
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