Yves Waché

Extracellular Oxidoreduction Potential Modifies Carbon and Electron Flow in Escherichia coli

Wild-type Escherichia coli K-12 ferments glucose to a mixture of ethanol and acetic, lactic, formic, and succinic acids. In anoxic chemostat culture at four dilution rates and two different oxidoreduction potentials (ORP), this strain generated a spectrum of products which depended on ORP. Whatever the dilution rate tested, in low reducing conditions (-100 mV), the production of formate, acetate, ethanol, and lactate was in molar proportions of approximately 2.5:1:1:0.3, and in high reducing conditions (-320 mV), the production was in molar proportions of 2:0.6:1:2. The modification of metabolic fluxes was due to an ORP effect on the synthesis or stability of some fermentation enzymes; thus, in high reducing conditions, lactate dehydrogenase-specific activity increased by a factor of 3 to 6. Those modifications were concomitant with a threefold decrease in acetyl-coenzyme A (CoA) needed for biomass synthesis and a 0.5- to 5-fold decrease in formate flux. Calculations of carbon and cofactor balances have shown that fermentation was balanced and that extracellular ORP did not modify the oxidoreduction state of cofactors. From this, it was concluded that extracellular ORP could regulate both some specific enzyme activities and the acetyl-CoA needed for biomass synthesis, which modifies metabolic fluxes and ATP yield, leading to variation in biomass synthesis.

Role of β-Oxidation Enzymes in γ-Decalactone Production by the Yeast Yarrowia lipolytica

ABSTRACT Some microorganisms can transform methyl ricinoleate into γ-decalactone, a valuable aroma compound, but yields of the bioconversion are low due to (i) incomplete conversion of ricinoleate (C18) to the C10 precursor of γ-decalactone, (ii) accumulation of other lactones (3-hydroxy-γ-decalactone and 2- and 3-decen-4-olide), and (iii) γ-decalactone reconsumption. We evaluated acyl coenzyme A (acyl-CoA) oxidase activity (encoded by the POX1 throughPOX5 genes) in Yarrowia lipolytica in lactone accumulation and γ-decalactone reconsumption inPOX mutants. Mutants with no acyl-CoA oxidase activity could not reconsume γ-decalactone, and mutants with a disruption ofpox3, which encodes the short-chain acyl-CoA oxidase, reconsumed it more slowly. 3-Hydroxy-γ-decalactone accumulation during transformation of methyl ricinoleate suggests that, in wild-type strains, β-oxidation is controlled by 3-hydroxyacyl-CoA dehydrogenase. In mutants with low acyl-CoA oxidase activity, however, the acyl-CoA oxidase controls the β-oxidation flux. We also identified mutant strains that produced 26 times more γ-decalactone than the wild-type parents.

Measurement of the intracellular pH in Escherichia coli with the internally conjugated fluorescent probe 5- (and 6-)carboxyfluorescein succinimidyl ester

The 5- (and 6-)carboxyfluorescein succinimidyl ester (cFDASE) is a pH-dependent fluorescent probe which conjugates with the aliphatic amine functions of the cells. Because of this property, the fluorescence efflux rate is low, 10 to 40% after 30 min of fermentable carbon source incubation. Thus, this technique allows a continuous high-time measurement (>30 min) without correction of the signal, but also a dynamic study phenomenon of few seconds. Due to the advantages presented by this technique, the method was adapted for Escherichia coli. The internal pH measurement obtained under various conditions are in accuracy with those from radiolabelled technique and from other fluorescence probe, with the advantages mentioned previously.

Biochemistry of lactone formation in yeast and fungi and its utilisation for the production of flavour and fragrance compounds

The consumers’ demand for natural flavour and fragrances rises. To be natural, compounds have to result from the extraction of natural materials and/or to be transformed by natural means such as the use of enzymes or whole cells. Fungi are able to transform some fatty acids into lactones that can thus be natural. Although some parts of this subject have been reviewed several times, the present article proposes to review the different pathways utilised, the metabolic engineering strategies and some current concerns on the reactor application of the transformation including scaling up data. The main enzymatic steps are hydroxylation and β-oxidation in the traditional way, and lactone desaturation or Baeyer–Villiger oxidation. Although the pathway to produce γ-decalactone is rather well known, metabolic engineering strategies may result in significant improvements in the productivity. For the production of other lactones, a key step is the hydroxylation of fatty acids. Beside the biotransformation, increasing the production of the various lactones requires from biotechnologists to solve two main problems which are the toxicity of lactones toward the producing cell and the aeration of the emulsified reactor as the biochemical pathway is very sensitive to the level of available oxygen. The strategies employed to resolve these problems will be presented.

Potential of nisin-incorporated sodium caseinate films to control Listeria in artificially contaminated cheese.

A sodium caseinate film containing nisin (1000 IU/cm(2)) was produced and used to control Listeria innocua in an artificially contaminated cheese. Mini red Babybel cheese was chosen as a model semi-soft cheese. L. innocua was both surface- and in-depth inoculated to investigate the effectiveness of the antimicrobial film as a function of the distance from the surface in contact with the film. The presence of the active film resulted in a 1.1 log CFU/g reduction in L. innocua counts in surface-inoculated cheese samples after one week of storage at 4 degrees C as compared to control samples. With regard to in-depth inoculated cheese samples, antimicrobial efficiency was found to be dependent on the distance from the surface in contact with the active films to the cheese matrix. The inactivation rates obtained were 1.1, 0.9 and 0.25 log CFU/g for distances from the contact surface of 1 mm, 2 mm and 3 mm, respectively. Our study demonstrates the potential application of sodium caseinate films containing nisin as a promising method to overcome problems associated with post-process contamination, thereby extending the shelf life and possibly enhancing the microbial safety of cheeses.

Involvement of Acyl Coenzyme A Oxidase Isozymes in Biotransformation of Methyl Ricinoleate into γ-Decalactone by Yarrowia lipolytica

ABSTRACT We reported previously on the function of acyl coenzyme A (acyl-CoA) oxidase isozymes in the yeast Yarrowia lipolytica by investigating strains disrupted in one or several acyl-CoA oxidase-encoding genes (POX1 throughPOX5) (H. Wang et al., J. Bacteriol. 181:5140–5148, 1999). Here, these mutants were studied for lactone production. Monodisrupted strains produced similar levels of lactone as the wild-type strain (50 mg/liter) except for Δpox3, which produced 220 mg of γ-decalactone per liter after 24 h. The Δpox2 Δpox3 double-disrupted strain, although slightly affected in growth, produced about 150 mg of lactone per liter, indicating that Aox2p was not essential for the biotransformation. The Δpox2 Δpox3 Δpox5 triple-disrupted strain produced and consumed lactone very slowly. On the contrary, the Δpox2 Δpox3 Δpox4 Δpox5 multidisrupted strain did not grow or biotransform methyl ricinoleate into γ-decalactone, demonstrating that Aox4p is essential for the biotransformation.

Cloning and Characterization of the Peroxisomal Acyl CoA Oxidase ACO3 Gene from the Alkane-utilizing Yeast Yarrowia lipolytica

The ACO3 gene, which encodes one of the acyl‐CoA oxidase isoenzymes, was isolated from the alkane‐utilizing yeast Yarrowia lipolytica as a 10 kb genomic fragment. It was sequenced and found to encode a 701‐amino acid protein very similar to other ACOs, 67·5% identical to Y. lipolytica Aco1p and about 40% identical to S. cerevisiae Pox1p. Haploid strains with a disrupted allele were able to grow on fatty acids. The levels of acyl‐CoA oxidase activity in the ACO3 deleted strain, in an ACO1 deleted strain and in the wild‐type strain, suggested that ACO3 encodes a short chain acyl‐CoA oxidase isoenzyme. This narrow substrate spectrum was confirmed by expression of Aco3p in E. coli.

Use of a Doehlert factorial design to investigate the effects of pH and aeration on the accumulation of lactones by Yarrowia lipolytica

Aims:  To detect rate‐limiting steps in the production of lactones by studying the combined effect of pH and aeration on their accumulation.

Optimization of Yarrowia lipolytica’s β-oxidation pathway for γ-decalactone production

The yeast Yarrowia lipolytica growing on methyl ricinoleate produces various lactones, γ-decalactone, the worthy aroma compound, 3-hydroxy-γ-decalactone without sensorial properties and two decenolides of various interest. Unfortunately, these three latter lactones are produced at high levels by this yeast, decreasing yields and complicating the extraction of γ-decalactone. In this study, the production of γ-decalactone was increased through a genetic engineering of the strain and the accumulation of the three other lactones was lowered. Theses results show that it is possible to improve the mastering of the complex β-oxidation pathway (the metabolic pathway involved in these bioconversions) by playing on genetic factors.

Acyl-CoA oxidase, a key step for lactone production by Yarrowia lipolytica

γ-Decalactone is a peachy aroma compound resulting from the peroxisomal β-oxidation of ricinoleic acid by yeasts. During this oxidation, the action of acyl-CoA oxidase is fundamental. In Yarrowia lipolytica, it was shown, using conserved blocks, that five acyl-CoA-oxidase genes (ACO1 to ACO5) were present. In order to investigate the role of each ACO isozymes, mono-disrupted strains were constructed (Δaco1 to Δaco5). The acyl-CoA activity was measured for each strain showing that a long-chain oxidase was encoded by ACO2 and a short one by ACO3. Lactone production, for its part, was increased for Δaco3 and, to a lesser extend, for Δaco5 whereas lactone consumption was higher for Δaco4.