Carbon and nitrogen sources modulate lipase production in the yeast Yarrowia lipolytica

AIMS: To analyse the influence of nitrogen and carbon sources on extracellular lipase production by Yarrowia lipolytica-overproducing mutant in order to optimize its production in large-scale bioreactors. METHODS AND RESULTS: The level of lipase production and LIP2 induction, measured using an LIP2-LacZ reporter gene, were compared for different carbon and nitrogen sources and for different concentrations. The localization of the enzyme during growth was also determined by Western blotting analysis using a six-histidine-tagged lipase. SIGNIFICANCE AND IMPACT OF THE STUDY: Tryptone N1 and oleic acid are the most suitable nitrogen and carbon sources for the production of the extracellular lipase by the Y. lipolytica mutant. Higher levels of lipase production were obtained as the tryptone concentration increased in the culture medium. Such a positive correlation was not observed with oleic acid media where the highest lipolytic productivities were obtained in the presence of low concentration. We also demonstrate that in the presence of oleic acid, lipase is cell-bound during the growth phase before being released in the media. CONCLUSIONS: This work provides a better understanding of the mechanism controlling LIP2 expression and, thus, extracellular lipase production in the yeast Y. lipolytica.     

Effect of tank mixed adjuvants on the drift potential of phenmedipham formulations

The aim of this paper is to analyse the effect of adjuvants and formulations on drift. The spray liquids consisted of four adjuvants (Actirob 0.4 %, Tensiofix 0.2%, Breakthru 0.2%, Silwet L-77 0.1%) with water and with two formulations of Phenmediphame (C16H16N2O4, 4.45%): an emulsion-forming (EC) and a suspension concentrate (SC). A standard flat fan nozzle at a pressure of 3 bar was used. The droplet size spectrum of each combination was determined using a Malvern granulometer. The droplet size was characterized by the volume median diameter (VMD) and the percentage of spray volume contained in droplets <100 microm (%<100). The relative drift potential was measured for each combination of formulation and adjuvant in a wind-tunnel. This latter has a working section 2.0 m wide by 2.0 m high by 6.0 m long. The air-stream is drawn by a 1.2 m diameter axial flow fan, powered by a 22 kW electric motor. Wind speed was 5 m/s. Its uniformity was controlled by a three-dimensional sonic anemometer able to move on a linear translation beam placed in the tunnel cross section. The wind-tunnel was operated under ambient conditions and three repetitions were performed randomized in order to eliminate variations in temperature and humidity for each combination. The ground spray deposits were measured on glass fibber collectors using a fluorescent tracer dye (sodium fluorescein), at a concentration of 2.5 mg/l. The statistical analysis of the droplet spectrum showed that the Phenmediphame SC formulation generated droplets of higher size than the EC. The mean VMD values were respectively equal to 228+/-11 microm and 185+/-11 microm for these formulations. For SC formulation, Break-thru decreased the VMD while Tensiofix increased the %<100. This confirmed that the degree to which an adjuvant influences spray characteristics is very variable. The drift profiles produced by the different combinations were similar, but the relative drift potential was significantly different comparing SC and EC formulations: it respectively reached 0.8+/-0,08% and 1.2+/-0,08%, whatever the adjuvant used in the liquid. Clearly, when using a flat fan nozzle to spray Phenmediphame, the droplet size and the drift potential are mainly governed by the kind of formulation, even if an interaction between the formulation and the adjuvant exists.     

Selection of new over-producing derivatives for the improvement of extracellular lipase production by the non-conventional yeast Yarrowia lipolytica

The non-conventional yeast Yarrowia lipolytica produces an extracellular lipase encoded by the LIP2 gene. Mutant strains with enhanced productivity were previously obtained either by chemical mutagenesis or genetic engineering. In this work, we used one of these mutants, named LgX64.81 to select new overproducing strains following by amplification of the LIP2 gene. We also developed a process for lipase production in bioreactors and compared lipase production levels in batch and fed-batch cultures. Batch culture led to a lipase production of 26450 U ml(-1) in a media containing olive oil and tryptone as carbon and nitrogen sources. Feeding of a combination of tryptone and olive oil at the end of the exponential growth phase yielded to lipase activity of 158246 U ml(-1) after 80 h of cultivation. In addition this production system developed for the extracellular lipase could also be applied for other heterologous protein production since we have demonstrated that LgX64.81 is an interesting alternative host strain.     

Involvement of Hexokinase Hxk1 in Glucose Catabolite Repression of LIP2 Encoding Extracellular Lipase in the Yeast Yarrowia lipolytica

The yeast Yarrowia lipolytica produces an extracellular lipase encoded by the LIP2 gene. However, very little is known about the mechanisms controlling its expression, especially on glucose media. In this work, the involvement of hexokinase Hxk1 in the glucose catabolite repression of LIP2 was investigated in a lipase overproducing mutant less sensitive to glucose repression. This mutant has a reduced capacity to phosphorylate hexose compared with the wild-type strain, but no differences could be observed between the HXK1 sequences in the two isolates. This suggested that the reduced phosphorylating activity of the mutant strain probably resulted from a modification in the level of HXK1 expression. However, overexpression of the HXK1 gene in this mutant led to a decrease of both LIP2 induction and extracellular lipase activity, suggesting that the hexokinase is involved in the glucose catabolite repression of LIP2 in Y lipolytica.     

Fertilizer nitrogen budget of Na15NO3 and (15NH4)2SO4 split-applied to winter wheat in microplots on a loam soil

Labelled fertilizer N applied to winter wheat as Na¹⁵NO₃ and (¹⁵NH₄)₂SO₄ at a total N dressing of 100kg ha⁻¹ was used in a microplot balance study to investigate the fate of each split fraction at three growth stages: end of tillering, heading and beginning of flowering. Results indicated that while the percentage utilization of the applied N by the grain and total crop increased considerably from the first to the third split application, these values diminished steadily in the straw. Grain recovery values for the first, second and third split applications were 34.2%, 51.5% and 55.7% for the NO₃ and 32.3%, 48.4% and 52.5% for the NH₄ carrier, respectively. The corresponding recovery values for the whole plant were 54.6%, 67.8% and 69.9% for the NO₃ and 51.7%, 63.5% and 66.1% for the NH₄ carrier. A greater proportion of the fertilizer N applied at the end of tillering stage was found in the vegetative plant components as compared with the grain. The reverse occurred for the N applied at the heading and at the beginning of the flowering stages. The residual fertilizer N found in the soil amounted to 18.0%, 10.4% and 11.6% of the applied NO₃−N and to 22.5%, 12.7% and 15.2% of the applied NH₄−N for the respective split applications. No differences were found for each split application between the two carriers as far as the unaccounted fertilizer N was concerned. The losses were 26.6%, 22.3% and 18.6% of the applied N for the three split applications, respectively. The application of fertilizer N did not lead to any increase in soil N uptake by the crop.     

New glucosidase activities identified by functional screening of a genomic DNA library from the gut microbiota of the termite Reticulitermes santonensis

β-Glucosidases are widely distributed in living organisms and play a major role in the degradation of wood, hydrolysing cellobiose or cello-oligosaccharides to glucose. Termites are among the rare animals capable of digesting wood, thanks to enzyme activities of their own and to enzymes produced by their gut microbiota. Many bacteria have been identified in the guts of lower termites, some of which possess cellulolytic or/and hemicellulolytic activity, required for digesting wood. Here, having isolated bacterial colonies from the gut of Reticulitermes santonensis, we constructed in Escherichia coli a genomic DNA library corresponding to all of the colonies obtained and screened the library for clones displaying β-glucosidase activity. This screen revealed 8 positive clones. Sequence analysis with the BLASTX program revealed putative enzymes belonging to three glycoside hydrolase families (GH1, GH3 and GH4). Agar-plate tests and enzymatic assays revealed differences between the GH1- and GH3-type enzymes (as regards substrate specificity and regulation) and a difference in substrate specificity within the GH3 group. The substrate specificities and characteristic activities of these enzymes suggest that they may intervene in the depolymerisation of cellulose and hemicellulose.     

Fate of nitrogen fertilizer applied on two main arables crops,winter wheat (Triticum aestivum) and sugar beet (Beta vulgaris) in the loam region of Belgium

Since 1986, the fate of fertilizer N (NH₄NO₃ or NaNO₃) applied in field conditions on two main arable crops, winter wheat (Triticum aestivum) and sugar beet (Beta vulgaris), has been studied using ¹⁵N. Up to a rate of 200 kg ha^-1 of N, mean recovery of fertilizer by winter wheat was 70%, provided it had been split applied. Single application (with or without dicyandiamid) was less effective. For sugar beet, in 1990, 1991 and 1992, 40% of fertilizer N was found in the crop at harvest when NH₄NO₃ had been broadcast at 100 to 160 kg N ha^-1 at sowing time. For the same N rate, recovery was 50% when row applied near the seeds and 60% for 80 kg N ha^-1. For the two experimental crops, residual fertilizer N in soil was exclusively organic. It ranged from 15 to 30% of applied N and was located in the 30 cm upper layer. Losses were generally lower with winter wheat (12%) than with sugar beet (20–40%) and could be ascribed to volatilization and denitrification. Soil derived N taken up by the plant was site and year dependent.     

Investigation of the effect of different extracellular factors on the lipase production by <Emphasis Type="Italic">Yarrowia lipolityca</Emphasis> on the basis of a scale-down approach

The influence of three extracellular factors (namely, the methyl oleate dispersion in the broth, the dissolved oxygen variations, and the pH fluctuation) on the lipase production by Y. lipolytica in batch bioreactor has been investigated in different scale-down apparatus. These systems allow to reproduce the hydrodynamic phenomena encountered in large-scale equipments for the three specified factors. The effects of the extracellular factors have been observed at three distinct levels: the microbial growth, the extracellular lipase production, and the induction of the gene LIP2 encoding for the main lipase of Y. lipolytica. Among the set of environmental factors investigated, the dissolved oxygen fluctuations generated in a controlled scale-down reactor (C-SDR) have led to the more pronounced physiological effect by decreasing the LIP2 gene expression level. The other environmental factors observed in a partitioned scale-down reactor, i.e., the methyl oleate dispersion and the pH fluctuations, have led to a less severe stress traduced only by a decrease of the microbial yield and thus of the extracellular lipase specific production rate.     

Overproduction of lipase by<Emphasis Type="Italic"> Yarrowia lipolytica</Emphasis> mutants

Non-genetically modified mutants with increased capacities of extracellular lipase production were obtained from Yarrowia lipolytica strain CBS6303 by chemical mutagenesis. Of the 400 mutants isolated, LgX64.81 had the highest potential for the development of an industrial lipase production process. This mutant exhibits lipase production uncoupled from catabolite repression by glucose, and a 10-fold increased productivity upon addition of oleic acid. Using a LIP2- LacZ reporter gene, we demonstrate that the mutant phenotype originates from a trans-acting mutation. The glucose uptake capacity of LgX64.81 is reduced 2.5-fold compared to the wild-type-strain, and it exhibits high lipase production on glucose medium. A trans-acting mutation in a gene involved in glucose transport could thus explain this mutant phenotype.     

Physical and physiological impacts of different foam control strategies during a process involving hydrophobic substrate for the lipase production by Yarrowia lipolytica

The potentialities for the intensification of the process of lipase production by the yeast Yarrowia lipolytica on a renewable hydrophobic substrate (methyl oleate) have to be investigated. The key factor governing the lipase yield is the intensification of the oxygen transfer rate, considering the fact that Y. lipolytica is a strict aerobe. However, considering the nature of the substrate and the capacity for protein excretion and biosurfactant production of Y. lipolytica, intensification of oxygen transfer rate is accompanied by an excessive formation of foam. Two different foam control strategies have thus been implemented: a classical chemical foam control strategy and a mechanical foam control (MFM) based on the Stirring As Foam Disruption principle. The second strategy allows foam control without any modifications of the physico-chemical properties of the broth. However, the MFM system design induced the formation of a persistent foam layer in the bioreactor. This phenomenon has led to the segregation of microbial cells between the foam phase and the liquid phase in the case of the bioreactors operated with MFM control, and induced a reduction at the level of the lipase yield. More interestingly, flow cytometry experiments have shown that the residence time of microbial cells in the foam phase tends to induce a dimorphic transition which could potentially explain the reduction of lipase excretion.