Christopher T. Evans

A comparison of the oleaginous yeast,Candida curvata, grown on different carbon sources in continuous and batch culture

The oleaginous yeast,Candida curvata D, was grown in both batch and continuous culture on 5 different carbon sources to compare the efficiency of fat production from the various substrates. Maximum lipid accumulation occurred in batch culture with xylose as the carbon source on nitrogenlimited medium reaching a level of 49% (w/w) of the biomass, but this was reduced to 37% at the optimum dilution rate (D=0.05/hr) in a chemostat. Both the highest biomass and lipid yields were attained in continuous culture with lactose as the sole carbon source at a dilution rate of D=0.04/hr, giving an efficiency of substrate conversion of 60 g of biomass and 18.6 g lipid per 100 g lactose utilized. The relative proportions of the major fatty acids (16∶0, 18∶0, 18∶1, 18∶2) in the lipid were found to vary considerably in batch culture and in continuous culture under carbon-limited conditions. However, on nitrogen-limited media in the chemostat, the fatty acid composition remained relatively constant over the whole range of dilution rates employed. Lipid from xylose-grown cells contained the greatest percentage of stearic acid (18∶0) 15% and the lowest linoleic acid (18∶2) 4%, whereas lipid from ethanol-grown cells contained elevated levels of oleic acid (18∶1) 51% and decreased palmitic acid (16∶0) 25%.

Effect of Nitrogen Source on Lipid Accumulation in Oleaginous Yeasts

The effect of various nitrogen sources on lipid accumulation by 17 species and strains of yeast was examined. Organic nitrogen sources resulted in considerably increased lipid contents only in strains of Rhodosporidium toruloides. Lipid accumulation in Rs. toruloides CBS 14 increased from 18% (w/w), with NH4Cl as nitrogen source, to above 50% (w/w) when glutamate, urea or arginine was used. Stimulation of lipid production by glutamate was not observed when the yeast was grown in continuous culture with nitrogen-limiting medium. The increase in lipid content of glutamate-grown cells in batch culture was accompanied by a marked increase in the intracellular citrate concentration and its excretion from the cells. The pattern of citrate accumulation in glutamate-grown cells was mirrored by the accumulation of other metabolites, especially 2-oxoglutarate and NH+ 4 ions, which were produced as a result of the increased catabolism of glutamate. It is proposed that the products of glutamate metabolism in Rs. toruloides play a major role in regulating the flux of carbon to precursors of lipid biosynthesis, such as citrate.

Induction of xylulose-5-phosphate phosphoketolase in a variety of yeasts grown ond-xylose: the key to efficient xylose metabolism

Activity of a pentulose (xylulose 5-phosphate) phosphoketolase was detected in 20 out of 25 yeasts examined. No significant activity was detected in any yeast grown with glucose, and the enzyme was induced by up to 70-fold when the yeasts were grown on xylose as sole carbon source. Biomass yields from xylose were greater than, or approximately equal to, those from glucose in 15 of the 19 yeasts which possessed phosphoketolase activity. The molar yield of C2 units from xylose, by metabolism via the pentose phosphate pathway, can be calculated to be insufficient to account for the high yields of biomass and ethanol obtained from xylose. We have shown that the presence of a phosphoketolase system can account for such yields by producing 2 mol C2 from 1 mol C5. This pathway must therefore be regarded as a major route of pentose dissimilation in such yeasts.

Influence of Nitrogen Metabolism on Lipid Accumulation by Rhodosporidium toruloides CBS 14

Rhodosporidium toruloides CBS 14 was grown in batch culture with urea as principal nitrogen source. The lipid content increased from 18% (w/w) with NH+ 4-grown cells to 52% (w/w) after 90 h growth. Urea was rapidly taken up and catabolized to release intracellular NH+ 4, which accumulated between 10 and 30 h growth. The increase in pool NH+ 4 content was mirrored by an increase in citric acid accumulation and excretion from the cells. The production of intracellular NH+ 4, sufficient to permit lipid accumulation, could be attributed to the increase in activity of urease over this period. Similarly, other catabolic enzymes, such as arginase, threonine dehydratase and NAD+: glutamate dehydrogenase, were also induced (or derepressed) when the respective amino acids were used as medium nitrogen source. Growth with mixed organic and inorganic nitrogen compounds considerably decreased the lipid content and was accompanied by a reduction in activity of the various catabolic enzymes concerned. The significance of nitrogen catabolism during lipid accumulation in this yeast is discussed.

Biochemical activities during lipid accumulation inCandida curvata

Intracellular and extracellular concentrations of citrate and the specific activities of ten different enzymes inCandida curvata D were examined in relation to lipid biosynthesis in batch and continuous culture. Citrate was found to accumulate prior to lipid production and declined markedly as lipid accumulated in batch culture. The cells excreted citrate as the culture became nitrogen-limiting after 30 hr of growth, but little more was expelled after 40 hr when lipid accumulation was more marked. In continuous culture, only low levels of citrate were detected at the lower dilution rates and citrate was completely absent from both the cells and medium above a dilution rate of 0.1/hr. The activity of malic enzyme, malate dehydrogenase and ATP:citrate lyase increased in batch culture on lipid accumulated, and, in continuous culture, both malic enzyme and ATP: citrate lyase varied in parallel with the specific rate of lipid synthesis which increased with increasing dilution rate. Activity of malate dehydrogenase, citrate synthase and glucose-6-phosphate dehydrogenase decrease with increasing dilution rate. The regulatory significance of these enzymes in lipid accumulation byC. curvata is discussed.

Phosphofructokinase and the Regulation of the Flux of Carbon from Glucose to Lipid in the Oleaginous Yeast Rhodosporidium toruloides

Summary: Changes in cell composition of Rhodosporidium toruloides CBS 14 were monitored during growth of batch cultures with NH4Cl and glutamate as nitrogen sources. Carbohydrate was synthesized at the expense of lipid in NH4 +-grown cells, whereas in glutamate-grown cells lipid accumulation was predominant. Total biomass and protein concentration were similar in both cultures. Uptake of [U-14C], [1-14C] and [6-14C]glucose, and evolution of 14CO2 from these sources, by washed suspensions of cells grown on glutamate revealed the flux of carbon during glucose dissimilation was principally via the Embden-Meyerhof pathway (72%), with 28% being metabolized by the pentose phosphate pathway. Both urea and glutamate, when added to the cell suspensions, significantly stimulated glucose catabolism, with the flux of carbon via the former pathway increasing to about 89% of the total. Phosphofructokinase (PFK) was implicated as the likely controlling enzyme to explain these events. PFK was only detected in extracts prepared from the yeast grown in a carbon-limited (nitrogen-excess) medium; no activity was detected in extracts of cells grown in nitrogen-limited medium. The presence of a protease in these latter extracts was revealed. PFK was purified 92-fold to a final specific activity (in the presence of 10 mM-NH4 +) of 4.2 units (mg protein)−1 and exhibited one broad band on polyacrylamide gel electrophoresis. The apparent mol. wt (Mt ) of the enzyme was approx. 700000. The major properties of the enzyme were examined to determine its regulatory role during lipid biosynthesis. Unlike the enzyme from Saccharomyces cerevisiae, no inhibition was found with 10 mM-ATP. ADP was not inhibitory either. NH4 + ions increased activity 11-fold by increasing the affinity of the enzyme for both fructose 6-phosphate and ATP. K+ ions also stimulated activity but to a lesser extent. Activity was severely inhibited by citrate, isocitrate and cis-aconitate but this inhibition was dramatically alleviated by NH4 +. Inhibition by citrate was competitive with fructose 6-phosphate in the absence of NH4 + ions. The K i values for citrate were 1.0 mM (with no NH4 +) and 7.2 mM (with 10 mM-NH4 +). Long-chain fatty acyl-CoA esters had no significant inhibitory effect. It is concluded that the interplay between the prevailing intracellular concentrations of NH4 + and citrate is the major determinant of the activity of PFK in vivo and thus governs the extent to which glucose is converted either to lipid or carbohydrate.

Novel stabilization of phenylalanine ammonia-lyase catalyst during bioconversion of trans-cinnamic acid to L-phenylalanine

SummaryProduction of l-phenylalanine from trans-cinnamic acid using isolate SPA10 cells was reduced to 26% of that observed initially when cells were reacted a second time with fresh substrate mixture. The stability (reuseability) of Phenylalanine Ammonia-Lyase (PAL) containing cells was significantly influenced by both the trans-cinnamate concentration and initial reaction pH. Using 2% t-cinnamate, l-phenylalanine production was 7-fold greater after 3 successive runs at pH 9.0 than at the optimum of pH 10.2. Cells reacted in the presence of 5% t-cinnamate were relatively unstable. Permeabilising agents, such as toluene and xylene, stimulated l-phenylalanine production but also enhanced instability of the catalyst. Several effectors were shown to stimulate the initial rate of the PAL bioconversion, but only sorbitol, alginate, glutaraldehyde, polyethylene glycol and glycerol conferred any significant degree of stability. Sparging of cultures and bioreactors with various gases revealed that oxygen enhanced PAL inactivation, CO2 had little effect and nitrogen conferred remarkable stability on PAL activity for several weeks in culture medium. The presence of chloride ions (from HCl) and aeration of substrate mixtures resulted in poor reuseability of catalyst. A combination of H2SO4 substitution for HCl and N2-sparging resulted in excellent initial conversions and good catalyst stability at 26°C but less at 30°C. The inclusion of 1.5 M sorbitol in reaction mixtures maintained PAL stability over several successive incubations.

Production of phenylalanine ammonia-lyase (PAL): isolation and evaluation of yeast strains suitable for commercial production of l-phenylalanine

SummaryPhenylalanine Ammonia-Lyase (PAL) containing microorganisms were isolated from a wide variety of natural habitats. The best 21 strains to emerge from the primary screen were screened for PAL activities in both directions using l-phenylalanine and t-cinnamate substrates. Twelve of the latter strains were compared for total cell production and PAL activity and 7 isolates were chosen for examination of the extent of PAL induction in various media. On the basis of these screens, isolate SPA 10 (identified as Rhodotorula rubra) was selected for further optimization. Growth was optimal at 28° C and pH 5.0, although cellular PAL activity was shown to be higher at sub-optimal temperatures (36° C) and pH (8.0) for growth. Synthesis of PAL was repressed when grown in the presence of various sugars and NH4+ions. Manipulation of fermentation conditions enabled PAL synthesis to occur at maximum biomass levels, upon glucose exhaustion. PAL was rapidly inactivated within cells shortly after maximum synthesis was attained: feeding of d,l-isoleucine and low concentrations of d,l-phenylalanine, and shifting of fermentation temperature conferred catalyst stability for fermentations over 100 h. These results demonstrate the suitability and superiority of isolate SPA 10 for the commercial production of l-phenylalanine from trans-cinnamic acid.

A rapid screening method for lipid-accumulating yeast using a replica-printing technique

Abstract A replica-printing technique has been developed which enables the tentative identification of a high lipid-containing yeast colony on an agar plate. The method involves a simple permeabilization and staining procedure, followed by destaining, and is able to detect colonies that accumulate lipid (that is those from Candida curvata, Lipomyces starkeyi and Trichosporon cutaneum ) whilst easily distinguishing the latter from a low-lipid containing colony ( Saccharomyces cerevisiae ). The technique could not be successfully applied to the red yeast, Rhodosporidium toruloides . The method is simple and rapid and so provides for efficient screening and identification of high lipid-producing hybrids (protoplast fusion, recombinant DNA), mutants and natural isolates.

Biotransformation of phenylpyruvic acid to L-phenylalanine using a strain of Pseudomonas fluorescens ATCC 11250 with high transaminase activity

The rate of l-phenylalanine production from phenylpyruvic acid by whole cells of Pseudomonas fluorescens strain ATCC 11250 was greater than 3 g·l-1 h-1. Synthesis of transaminase was constitutive but activity was greatest in medium containing d- or l- phenylalanine as sole nitrogen source. Maximum conversion was observed at 34–40° C and at alkaline pH, with over six times initial rate of conversion at pH 12 than at pH 5. The optimum catalyst (cell) concentration was between 10–20 mg ml-1 dry weight. The initial rate of conversion was directly proportional to phenylpyruvate concentration, up to 4%, but the conversion yield steadily decreased between 2% and 4% substrate concentration. The rate of conversion, as expected, increased as the concentration of glutamate increased. Whole cells were still capable of over 63% conversion after 40 days providing reactions were supplemented with pyridoxal phosphate. Immobilisation of cells in calcium alginate and operation of a packed bed bioreactor enabled the continuous production of l-phenylalanine in concentrations greater than 15 g·l-1 after 60 days operation.