Effect of gene amplification on threonine production by yeast

In this work, we have studied the effect of amplifying different alleles involved in the threonine biosynthesis on the amino acid production by Saccharomyces cerevisiae. The genes used were wild-type HOM3, HOM2, HOM6, THR1, and THR4, and two mutant alleles of HOM3 (namely HOM3-R2 and HOM3-R6), that code for feedback-insensitive aspartate kinases. The results show that only the amplification of the HOM3 alleles leads to threonine and, in some instances, to homoserine overproduction. In terms of the regulation of the pathway, the data indicate that the main control is exerted by inhibition of the aspartate kinase and that, probably, a second and less important regulation takes place at the level of the homoserine kinase, the THR1 gene product. However, amplification of THR1 in two related Hom3-R2 strains does not increase the amount of threonine but, in one of them, it does induce accumulation of more homoserine. This result probably reflects differences between these strains in some undetermined genetic factor/s related with threonine metabolism. In general, the data indicate that the common laboratory yeast strains are genetically rather heterogeneous and, thus, extrapolation of conclusions must be done carefully. (c) 1996 John Wiley & Sons, Inc.     

Genetic analysis of the metabolic pathways responsible for aroma metabolite production by Saccharomyces cerevisiae

During alcoholic fermentation, higher alcohols, esters, and acids are formed from amino acids via the Ehrlich pathway by yeast, but many of the genes encoding the enzymes have not yet been identified. When the BAT1/2 genes, encoding transaminases that deaminate amino acids in the first step of the Ehrlich pathway are deleted, higher metabolite formation is significantly decreased. Screening yeast strains with deletions of genes encoding decarboxylases, dehydrogenases, and reductases revealed nine genes whose absence had the most significant impact on higher alcohol production. The seven most promising genes (AAD6, BAT2, HOM2, PAD1, PRO2, SPE1, and THI3) were further investigated by constructing double- and triple-deletion mutants. All double-deletion strains showed a greater decrease in isobutanol, isoamyl alcohol, isobutyric, and isovaleric acid production than the corresponding single deletion strains with the double-deletion strains in combination with ?bat2 and the ?hom2-?aad6 strain revealing the greatest impact. BAT2 is the dominant gene in these deletion strains and this suggests the initial transaminase step of the Ehrlich pathway is rate-limiting. The triple-deletion strains in combination with BAT2 (?bat2-?thi3-?aad6 and ?bat2-?thi3-?hom2) had the greatest impact on the end metabolite production with the exception of isoamyl alcohol and isovaleric acid. The strain deleted for two dehydrogenases and a reductase (?hom2-?pro2-?aad6) had a greater effect on the levels of these two compounds. This study contributes to the elucidation of the Ehrlich pathway and its significance for aroma production by fermenting yeast cells.     

Structure of the yeast HOM3 gene which encodes aspartokinase

The yeast HOM3 gene has been cloned molecularly by complementation of a HOM3 mutant. The gene is located about 8 kilobase pairs from HIS1 and is present as a single copy in the yeast genome. Mutations in HOM3 result in a requirement for threonine and methionine (or homoserine) for growth and a lack of detectable aspartokinase activity. The nucleotide sequence of HOM3 predicts an enzyme 414 amino acids long that shows homology to the three Escherichia coli aspartokinases, indicating that it is the structural gene for yeast aspartokinase. An approximately 1800-base pair mRNA is transcribed from the HOM3 gene, initiating at several start sites, 80 and 70 base pairs downstream, respectively, from two TATA boxes. Upstream of the TATA boxes is a single TGACTC sequence. This sequence has been shown to be essential for regulation of several genes that encode amino acid biosynthetic enzymes by the general control system. However, no increase in aspartokinase mRNA is observed under general control derepressing conditions.     

Metabolic and bioprocess engineering of the yeast Candida famata for FAD production

Flavins in the form of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) play an important role in metabolism as cofactors for oxidoreductases and other enzymes. Flavin nucleotides have applications in the food industry and medicine; FAD supplements have been efficiently used for treatment of some inheritable diseases. FAD is produced biotechnologically; however, this compound is much more expensive than riboflavin. Flavinogenic yeast Candida famata synthesizes FAD from FMN and ATP in the reaction catalyzed by FAD synthetase, a product of the FAD1 gene. Expression of FAD1 from the strong constitutive promoter TEF1 resulted in 7- to 15-fold increase in FAD synthetase activity, FAD overproduction, and secretion to the culture medium. The effectiveness of FAD production under different growth conditions by one of these recombinant strains, C. famata T-FD-FM 27, was evaluated. First, the two-level Plackett–Burman design was performed to screen medium components that significantly influence FAD production. Second, central composite design was adopted to investigate the optimum value of the selected factors for achieving maximum FAD yield. FAD production varied most significantly in response to concentrations of adenine, KH₂PO₄, glycine, and (NH₄)₂SO₄. Implementation of these optimization strategies resulted in 65-fold increase in FAD production when compared to the non-optimized control conditions. Recombinant strain that has been cultivated for 40 h under optimized conditions achieved a FAD accumulation of 451 mg/l. So, for the first time yeast strains overproducing FAD were obtained, and the growth media composition for maximum production of this nucleotide was designed.     

A comparison and optimization of methods and factors affecting the transformation of Escherichia coli

DNA manipulation routinely requires competent bacteria that can be made using one of numerous methods. To determine the best methods, we compared four commonly used chemical methods (DMSO, MgCl₂–CaCl₂, CaCl₂ and Hanahan''s methods) on frequently used Escherichia coli (E. coli) strains: DH5α, XL-1 Blue, SCS110, JM109, TOP10 and BL21-(DE3)-PLysS. Hanahan''s method was found to be most effective for DH5α, XL-1 Blue and JM109 strains (P<0.05), whilst the CaCl₂ method was best for SCS110, TOP10 and BL21 strains (P<0.05). The use of SOB (super optimal broth) over LB [Luria–Bertani (broth)] growth media was found to enhance the competency of XL-1 Blue (P<0.05), dampened JM109′s competency (P<0.05), and had no effect on the other strains (P>0.05). We found no significant differences between using 45 or 90 s heat shock across all the six strains (P>0.05). Through further optimization by means of concentrating the aliquots, we were able to get further increases in transformation efficiencies. Based on the optimized parameters and methods, these common laboratory E. coli strains attained high levels of TrE (transformation efficiency), thus facilitating the production of highly efficient and cost-effective competent bacteria.     

Molecular Analysis of Maltotriose Active Transport and Fermentation by Saccharomyces cerevisiae Reveals a Determinant Role for the AGT1 Permease

Incomplete and/or sluggish maltotriose fermentation causes both quality and economic problems in the ale-brewing industry. Although it has been proposed previously that the sugar uptake must be responsible for these undesirable phenotypes, there have been conflicting reports on whether all the known α-glucoside transporters in Saccharomyces cerevisiae (MALx1, AGT1, and MPH2 and MPH3 transporters) allow efficient maltotriose utilization by yeast cells. We characterized the kinetics of yeast cell growth, sugar consumption, and ethanol production during maltose or maltotriose utilization by several S. cerevisiae yeast strains (both MAL constitutive and MAL inducible) and by their isogenic counterparts with specific deletions of the AGT1 gene. Our results clearly showed that yeast strains carrying functional permeases encoded by the MAL21, MAL31, and/or MAL41 gene in their plasma membranes were unable to utilize maltotriose. While both high- and low-affinity transport activities were responsible for maltose uptake from the medium, in the case of maltotriose, the only low-affinity (K_m, 36 ± 2 mM) transport activity was mediated by the AGT1 permease. In conclusion, the AGT1 transporter is required for efficient maltotriose fermentation by S. cerevisiae yeasts, highlighting the importance of this permease for breeding and/or selection programs aimed at improving sluggish maltotriose fermentations.     

Rock phosphate solubilization by four yeast strains

The solubilization of rock phosphate (RP) by four yeast strains, Rhodotorula sp., Candida rugosa, Saccharomyces cerevisiae and Saccharomyces rouxii, which were isolated from wheat rhizospheric soils, was investigated in this study. The yeast isolates demonstrated diverse levels of soluble phosphate releasing abilities in modified Pikovskaya liquid medium containing RP as sole phosphate source. C. rugosa was the most effective solubilizer under different conditions, followed by Rhodotorula sp., S. rouxii and S. cerevisiae. Acidification of the broth seemed to be the major mechanism for RP solubilization by the yeast isolates, and the increase in soluble phosphate released was correlated significantly with an increase in titratable acidity and a drop in pH. The optimal composition for the solubilization of RP by the yeast isolates in the broth was 20 g L^?1 glucose, 1 g L^?1 yeast extract, 0.5 g L^?1 (NH₄)₂SO₄, and 5 g L^?1 RP, respectively. The yeast isolates were able to solubilize RP at wide range of temperature and initial pH, with the maximum percentage of soluble phosphate released being recorded at 30–35 °C and pH 5–6, respectively.     

Respiratory capacity of the Kluyveromyces marxianus yeast isolated from the mezcal process during oxidative stress

During the mezcal fermentation process, yeasts are affected by several stresses that can affect their fermentation capability. These stresses, such as thermal shock, ethanol, osmotic and growth inhibitors are common during fermentation. Cells have improved metabolic systems and they express stress response genes in order to decrease the damage caused during the stress, but to the best of our knowledge, there are no published works exploring the effect of oxidants and prooxidants, such as H₂O₂ and menadione, during growth. In this article, we describe the behavior of Kluyveromyces marxianus isolated from spontaneous mezcal fermentation during oxidative stress, and compared it with that of Saccharomyces cerevisiae strains that were also obtained from mezcal, using the W303-1A strain as a reference. S. cerevisiae strains showed greater viability after oxidative stress compared with K. marxianus strains. However, when the yeast strains were grown in the presence of oxidants in the media, K. marxianus exhibited a greater ability to grow in menadione than it did in H₂O₂. Moreover, when K. marxianus SLP1 was grown in a minibioreactor, its behavior when exposed to menadione was different from its behavior with H₂O₂. The yeast maintained the ability to consume dissolved oxygen during the 4 h subsequent to the addition of menadione, and then stopped respiration. When exposed to H₂O₂, the yeast stopped consuming oxygen for the following 8 h, but began to consume oxygen when stressors were no longer applied. In conclusion, yeast isolated from spontaneous mezcal fermentation was able to resist oxidative stress for a long period of time.     

PVA-gel (Lentikats®) as an effective matrix for yeast strain immobilization aimed at heterologous protein production

Three different yeast strains, namely Saccharomyces cerevisiae mnn1mnn9, Kluyveromyces lactis JA6/GAA and Zygosaccharomyces bailii [pZ₃klIL-1β], were entrapped in polyvinyl alcohol (PVA) gel particles, obtained following the commercially available immobilization kit named Lentikat^®. After immobilization in the PVA-gel particles, yeast cells remained viable: colonization of the gel matrix reached up 100 mg d.w. of cells cm⁻³ gel for all the strains examined.Lentikats^® of K. lactis JA6/GAA and Z. bailii [pZ₃klIL-1β] showed to be suitable for the continuous production of glucoamylase and interleukin 1β, respectively, when employed under non-selective conditions. They were of easy handiness and showed excellent mechanical properties during prolonged operation in stirred tank reactors.     

Microbial Production of Lysine and Threonine from Whey Permeate

Extracellular accumulation of lysine and threonine was investigated in modified whey permeate by using Brevibacterium lactofermentum ATCC 21086 and Escherichia coli ATCC 21151. Whey permeate was prepared from whey by membrane ultrafiltration, and lactose was hydrolyzed by treating permeate with HCl or β-galactosidase. The highest amount of lysine (3.3 g/liter) was produced from a mixture of acid-hydrolyzed whey permeate and yeast extract (0.2%). The highest amount of threonine (3.6 g/liter) was produced from a mixture of whey permeate, (NH₄)₂SO₄ (1.4%), yeast extract (0.1%), and Na₂CO₃ (0.3%).     

Stability of recombinant plasmids containing the ars sequence of yeast extrachromosomal rDNA in several strains of Saccharomyces cerevisiae

The mitotic stabilities of hybrid plasmid Rcp21/11, which contains the replicator of yeast rDNA, have been compared for four yeast host strains of different origins. In two related strains, Saccharomyces cerevisiae A62-1G-P188 and 1A-P3812 from the Peterhof genetic stocks, the plasmid was much more stable than in strains DC5 and GRF18 from the USA stocks.The enhanced mitotic stability of Rcp21/11 in these two yeast strains is obviously attributable to a higher rate of integration of the plasmid into the chromosomal rDNA repeats of the hosts.The centromeric locus CEN3 was inserted into Rcp21/11 because it provides high mitotic and meiotic stability of plasmids with yeast replicators, due to an ordered distribution of plasmids throughout cell division. Using the new centromeric plasmid RcpCEN3, transformation of the four above-described yeast strains was carried out. It was found that, similarly to centromeric plasmids with other chromosomal replicators, RcpCEN3 remains in the cell as a single copy. In strains DC5, GRF18 and A62-1G-P188 the mitotic stability of RcpCEN3 was 20–50%, i.e., less than half that of plasmids containing locus CEN3 and other yeast repliiators, ars1, ars2 and the 2μ DNA replicator. The mitotic stability of RcpCEN3 in strains 1A-P3812 (from the Peterhof genetic stocks) for individual clones reached 85%, i.e. close to that of the other plasmids. Genetic analysis showed that the capacity of strain 1A-P3812 to stably retain RcpCEN3 has a recessive polygenic character. We suggest that the observed differences in mitotic stability of centromeric plasmid RcpCEN3 between various yeast strains reflects the differences in activity of rDNA replicator in these strains. The nature of extrachromosomal rDNA circles, found in some strains of S. cerevisiae, is discussed from the point of view of the data.     

Catabolic Activities of Neisseria meningitidis: Utilization of Succinate

Two strains of Saccharomycopsis guttulata, JB-1 and JB-3, isolated from stomach contents of domestic rabbits, were grown under different gas phases, and their growth rates were compared. Strain JB-1 grew exponentially at a maximal growth rate under a continuous gas phase of 15% CO₂, 2% O₂ in nitrogen. High cell yields with low cell granulation were obtained. The growth rates were almost the same between oxygen concentrations of 0.25 and 20% at 15% CO₂. Poor growth and early cell granulation occurred in the absence of oxygen at 15% CO₂. Growth increased at 2% O₂ in direct proportion to the carbon dioxide concentration up to 10 to 15% CO₂. A very high carbon dioxide content (e.g. 98%) was somewhat inhibitory. Cell granulation always occurred during the maximal stationary phase in media at pH 4, but was relatively slight at pH 5.6 or higher. Strain JB-3 responded to various gas phases in a similar manner except that it grew slowly in the absence of oxygen at 15% CO₂ (pH 4). The effect of an optimal gas phase on the growth of strain JB-1 was examined in relation to other environmental conditions. In the presence of 15% CO₂, 2% O₂, this strain grew exponentially in yeast autolysate-Proteose Peptone-glucose medium at 37 C at pH 2, 4, and 5.6 at approximately the same rate; the growth rate was somewhat lower at pH 6.2. Under similar conditions, strain JB-1 grew at 30 C and pH 4 at one-sixth its maximal growth rate. Cell granulation was greatly reduced at this temperature. With adequate CO₂ strain JB-1 also grew at a reduced rate in a yeast autolysate medium previously reported not to support growth. Results indicate that continuous gassing with an optimal gas phase increases the growth rate to the extent that the growth rate surpasses the death rate by a significant margin; as a result, granulated cells can be avoided almost entirely in the log phase.     

Exploring Three PIPs and Three TIPs of Grapevine for Transport of Water and Atypical Substrates through Heterologous Expression in aqy-null Yeast

Aquaporins are membrane channels that facilitate the transport of water and other small molecules across the cellular membranes. We examined the role of six aquaporins of Vitis vinifera (cv. Touriga nacional) in the transport of water and atypical substrates (other than water) in an aqy-null strain of Saccharomyces cerevisiae. Their functional characterization for water transport was performed by stopped-flow fluorescence spectroscopy. The evaluation of permeability coefficients (P_f) and activation energies (Ea) revealed that three aquaporins (VvTnPIP2;1, VvTnTIP1;1 and VvTnTIP2;2) are functional for water transport, while the other three (VvTnPIP1;4, VvTnPIP2;3 and VvTnTIP4;1) are non-functional. TIPs (VvTnTIP1;1 and VvTnTIP2;2) exhibited higher water permeability than VvTnPIP2;1. All functional aquaporins were found to be sensitive to HgCl₂, since their water conductivity was reduced (24–38%) by the addition of 0.5 mM HgCl₂. Expression of Vitis aquaporins caused different sensitive phenotypes to yeast strains when grown under hyperosmotic stress generated by KCl or sorbitol. Our results also indicate that Vitis aquaporins are putative transporters of other small molecules of physiological importance. Their sequence analyses revealed the presence of signature sequences for transport of ammonia, boron, CO₂, H₂O₂ and urea. The phenotypic growth variations of yeast cells showed that heterologous expression of Vitis aquaporins increased susceptibility to externally applied boron and H₂O₂, suggesting the contribution of Vitis aquaporins in the transport of these species.     

Extracellular phosphomannan as a phosphate reserve in the yeast Kuraishia capsulata

We have found that extracellular phosphomannan is the main phosphate reserve in the yeast Kuraishia capsulata, in contrast to other yeast species effectively absorbing P_i. Under nitrogen starvation, K. capsulata absorbed essentially all P_i from the medium containing 240 mM glucose, 2.5 mM MgSO₄, and 11 mM KH₂PO₄. Inorganic polyphosphate level in the cells was about 14% of the P_i absorbed. Most of the P_i (～60%) was found in the fraction of extracellular phosphomannan that can be used as a carbon and phosphorus source by this yeast in deficient media.     

The patterns of utilization and accumulation of polyphosphates in the cytosol of the yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> under inactivation of exopolyphosphatase genes <Emphasis Type="Italic">PPX1</Emphasis> and <Emphasis Type="Italic">PPN1</Emphasis>

The effect of inactivation of the PPX1 and PPN1 genes encoding the yeast exopolyphosphatases on the activities of these enzymes and polyphosphate content in the cytosol of Saccharomyces cerevisiae was studied under P_i deficit and P_i excess in the cultivation medium. Under P_i deficit, exopolyphosphatase activity in strain CRN (with inactivated PPN1 gene) and in the parent strain CRY increased 3- and 1.5-fold, respectively. In the strain CRX (with inactivated PPX1 gene), exopolyphosphatase activity did not change under P_i deficit. Transfer from P_i-deficient to P_i-rich medium was accompanied by an ～1.7-fold increase of exopolyphosphatase activities in the cytosol preparations of strains CRY, CRX, and CRN. In the cytosol of the double mutant, exopolyphosphatase activity was practically absent under all of the above cultivation conditions. The content of polyphosphates in the cytosol preparations of all strains under study substantially decreased under P_i deficit. Transfer from P_i-deficient to P_i-rich medium was accompanied by polyphosphate over-accumulation only in the cytosol preparations of stains CRX and CNX, where their levels increased ～1.3 and 3.5-fold, respectively. No over-accumulation was observed in the parent strain CRY and in the PPN1-deficient strain CRN. These data suggest that the exopolyphosphatases encoded by the PPX1 and PPN1 genes are not involved in polyphosphate synthesis.