Guy Moulin

Inhibition of Clostridium butyricum by 1,3-propanediol and diols during glycerol fermentation

Abstract 1,3-Propanediol inhibition during glycerol fermentation to 1,3-propanediol by Clostridium butyricum CNCM 1211 has been studied. The initial concentration of the 1,3-propanediol affected the growth of the bacterium more than the glycerol fermentation. μmax was inversely proportional to the initial concentration of 1,3-propanediol (0–65 g l−1). For glycerol at 20 g l−1, the growth and fermentation were completely stopped at an initial 1,3-propanediol concentration of 65 g l−1. However, for an initial 1,3-propanediol concentration of 50 g l−1 and glycerol at 70 g l−1, the final concentration (initial and produced) of 1,3-propanediol reached 83.7 g l−1(1.1 M), with complete consumption of the glycerol. Therefore, during the fermentation, the strain tolerated a 1,3-propanediol concentration higher than the initial inhibitory concentration (65 g l−1). The addition of 1,2-propanediol or 2,3-butanediol (50 g l−1) in the presence of glycerol (50–100 g l−1), showed that 2-diols reduced the μmax in a similar way to 1,3-propanediol. The measurement of the osmotic pressure of glycerol solutions, diols and diol/glycerol mixtures did not indicate any differences between these compounds. The hypothesis of diol inhibition was discussed. Taking into account the strain tolerance of highly concentrated 1,3-propanediol during fermentation, the fermentation processes for optimising production were considered.

Effects of Acetate and Butyrate During Glycerol Fermentation by Clostridium butyricum

The effects of acetate and butyrate during glycerol fermentation to 1,3-propanediol at pH 7.0 by Clostridium butyricum CNCM 1211 were studied. At pH 7.0, the calculated quantities of undissociated acetic and butyric acids were insufficient to inhibit bacterial growth. The initial addition of acetate or butyrate at concentrations of 2.5 to 15 gL−1 had distinct effects on the metabolism and growth of Clostridium butyricum. Acetate increased the biomass and butyrate production, reducing the lag time and 1,3-propanediol production. In contrast, the addition of butyrate induced an increase in 1,3-propanediol production (yield: 0.75 mol/mol glycerol, versus 0.68 mol/mol in the butyrate-free culture), and reduced the biomass and butyrate production. It was calculated that reduction of butyrate production could provide sufficient NADH to increase 1,3-propanediol production. The effects of acetate and butyrate highlight the metabolic flexibility of Cl. butyricum CNCM 1211 during glycerol fermentation.

Identification of a triacylglycerol lipase gene family in Candida deformans: molecular cloning and functional expression

The yeast Candida deformans CBS 2071 produces an extracellular lipase which was shown to catalyse the production of various esters by the esterification of free fatty acids, even in the presence of a large molar excess of water. To clone the gene encoding this extracellular lipase, Saccharomyces cerevisiae was transformed with C. deformans genomic libraries and screened for lipolytic activity on a medium containing rapeseed oil emulsion and rhodamine B. Three members of a lipase gene family (CdLIP1, CdLIP2 and CdLIP3) were cloned and characterized. Each deduced lipase sequence has a Gly–His–Ser–Leu–Gly–(Gly/Ala)–Ala conserved motif, eight cysteine residues and encodes an N‐terminal signal sequence. MALDI–TOF mass spectrometry analysis of a proteolytic digest of the lipase produced was used to obtain experimental evidence that the CdLIP1 gene encoded the extracellular lipase. Recombinant expression studies confirmed that the cloned genes encoded functional lipases. The three lipases are very similar to lipases from the related species Yarrowia lipolytica. Significant homologies were also found with several yeast and fungal lipases. As C. deformans CBS 2071 was previously considered to be synonymous with Y. lipolytica, the strains were compared for the extent of nucleotide divergence in the variable regions (D1/D2) at the 5′‐end of the large‐subunit (26S) ribosomal DNA (rDNA) gene. This rDNA region has diverged sufficiently to suggest that C. deformans is a separate species. The nucleotide sequences of the CdLIP1, CdLIP2 and CdLIP3 genes will appear in the EMBL nucleotide sequence database under Accession Nos AJ428393, AJ428394 and AJ428395, respectively. Copyright

A comparative study of the inhibitory effect of ethanol and substrates on the fermentation rate of parent and a respiratory-deficient mutant

The inhibitory effect of alcohol and substrates on the fermentation rate of one strain of Candida pseudotropicalis and of a respiratory-deficient mutant of this strain is investigated. For the parent strain maximum fermentative activity is identical in the presence of glucose or lactose. For a respiratory-deficient mutant, the fermentation rate is always higher than that of the parent strain. The inhibitory effect of alcohol and substrate is always less with the respiratory-deficient mutant than with the parent strain.

Overexpression of ovine leptin in Pichia pastoris: physiological yeast response to leptin production and characterization of the recombinant hormone

Ovine leptin was cloned in the methylotrophic yeast Pichia pastoris using a pPIC9K vector. Leptin was produced and secreted into the culture medium using the Saccharomyces cerevisiae α‐mating factor prepro signal by five clones. Expression levels of leptin varied from clone to clone, depending on the copy number of the ob gene. Highest expression was observed with the single‐copy clone S27 (250 mg/l). The modifications of culture conditions in batch and fed‐batch culture increase the yield of protein. The use of higher cell concentration (63 g/l) before induction of oLept associate with a regulation of pH at 3.2, which decreases the effects of proteolysis, increases the expression level of the oLept to 402 mg/l. Moreover, compared with the non‐producer clone, we observed a drastic decrease in growth rate and biomass yield in the leptin‐producing clones. At the end of the fed‐batch phase at pH 3.2 with clone S27, mortality rate reached 17.3%. Results showed that recombinant leptin production induced metabolic stress, and a negative impact on biomass yield and growth rate. We characterized the recombinant leptin produced by clone S27. It exhibited a molecular mass of 16 kDa, an N‐terminal amino acid sequence identical to that of ovine leptin but with an additional tyrosine introduced by the cloning site. Moreover, it was found to be biologically active in vitro. The available production of a large quantity of oLept will strengthen the functional study for theorical and practical purposes. Copyright

Uptake of Galactose and Lactose by Kluyveromyces lactis: Biochemical Characteristics and Attempted Genetical Analysis

Study of the lactose and galactose transport systems in Kluyveromyces lactis has shown that lactose uptake is by active transport. The transport system is under monogenic control and is inducible. Galactose uptake is also by active transport but the system is controlled by two genes which, in the four strains we studied, are present only in K. lactis CBS 2359. Galactose uptake in the other K. lactis strains is by a simple diffusion process.

Influence of oxygen limitation on glucose metabolism in Hanseniaspora uvarum K5 grown in chemostat

SummaryGrowth and metabolite formation were studied as a function of oxygen feed rate, in glucose-limited chemostat cultures of Hanseniaspora uvarum K5 at a dilution rate of 0.26 h−1. Alcoholic fermentation occured at an oxygen feed rate of 80 mmol.l−1.h−1 . Below this value, pyruvate decarboxylase and alcohol dehydrogenase were present at high levels. In contrast, activities of oxidative metabolism enzymes, pyruvate dehydrogenase, aldehyde dehydrogenase and acetyl-CoA synthetase, decreased.

Strain selection in Kluyveromyces marxianus var. lactis for galactose production

After treatment with ethy1‐methane sulfonate (EMS) mutants of Kluyveromyces marxianus var, lactis altered in galactose metabolism were obtained which were further imporoved by genetic recombination. Six types of mutants were detected. In batch culture strains with double mutations — either in galactokinase and uridyltransferase, or in galactokinase and epimerase‐ and which also had a galactose permease transformed lactose to galactose and galactitol. Strains with the same double mutations but no galactose permease completely hydrolyzed lactose to galactose and galactitol under continuous culture.