Stress Tolerance in Doughs of Saccharomyces cerevisiae Trehalase Mutants Derived from Commercial Baker’s Yeast

Accumulation of trehalose is widely believed to be a critical determinant in improving the stress tolerance of the yeast Saccharomyces cerevisiae, which is commonly used in commercial bread dough. To retain the accumulation of trehalose in yeast cells, we constructed, for the first time, diploid homozygous neutral trehalase mutants (Δnth1), acid trehalase mutants (Δath1), and double mutants (Δnth1 ath1) by using commercial baker’s yeast strains as the parent strains and the gene disruption method. During fermentation in a liquid fermentation medium, degradation of intracellular trehalose was inhibited with all of the trehalase mutants. The gassing power of frozen doughs made with these mutants was greater than the gassing power of doughs made with the parent strains. The Δnth1 and Δath1 strains also exhibited higher levels of tolerance of dry conditions than the parent strains exhibited; however, the Δnth1 ath1 strain exhibited lower tolerance of dry conditions than the parent strain exhibited. The improved freeze tolerance exhibited by all of the trehalase mutants may make these strains useful in frozen dough.     

Model Predictive Temperature Control for Calorimetric Measurements in Baker’s Yeast Fermentations

We intended to establish a system for calorimetric measurements on a pilot scale. The on‐line calculation of model equations of the heat balance around the reactor is supposed to be simple at a constant fermentation temperature. To obtain higher accuracy in temperature control, a model predictive control (MPC) system is developed. MPC with the accuracy of 0.1 °C is much more precise than the classical single loop temperature control (SLC) with 1 °C. Calorimetry has a very high dynamic behavior compared with other on‐line determined parameters also representing metabolic activity. In a purely oxidative fed‐batch fermentation of baker’s yeast the accuracy of calorimetry is verified. The combustion enthalpy of glucose, calculated using on‐line data of microbial heat production and substrate consumption, corresponds very well with the data in the literature. It is now possible to determine the fraction of substrate uptake, utilized for energy (factor k) and biosynthesis (factor a) metabolisms on‐line.     

Characteristics and Efficiency of Glutamine Production by Coupling of a Bacterial Glutamine Synthetase Reaction with the Alcoholic Fermentation System of Baker’s Yeast

Glutamine production with bacterial glutamine synthetase (GS) and the sugar-fermenting system of baker’s yeast for ATP regeneration was investigated by determining the product yield obtained with the energy source for ATP regeneration (i.e., glucose) for yeast fermentation. Fructose 1,6-bisphosphate was accumulated temporarily prior to the formation of glutamine in mixtures which consisted of dried yeast cells, GS, their substrate (glucose and glutamate and ammonia), inorganic phosphate, and cofactors. By an increase in the amounts of GS and inorganic phosphate, the amounts of glutamine formed increased to 19 to 54 g/liter, with a yield increase of 69 to 72% based on the energy source (glucose) for ATP regeneration. The analyses of sugar fermentation of the yeast in the glutamine-producing mixtures suggested that the apparent hydrolysis of ATP by a futile cycle(s) at the early stage of glycolysis in the yeast cells reduces the efficiency of ATP utilization. Inorganic phosphate inhibits phosphatase(s) and thus improves glutamine yield. However, the analyses of GS activity in the glutamine-producing mixtures suggested that the higher concentration of inorganic phosphate as well as the limited amount of ATP-ADP caused the low reactivity of GS in the glutamine-producing mixtures. A result suggestive of improved glutamine yield under the conditions with lower concentrations of inorganic phosphate was obtained by using a yeast mutant strain that had low assimilating ability for glycerol and ethanol. In the mutant, the activity of the enzymes involved in gluconeogenesis, especially fructose 1,6-bisphosphatase, was lower than that in the wild-type strain.     

Multiple α-Glucoside Transporter Genes in Brewer’s Yeast

Maltose and maltotriose are the two most abundant fermentable sugars in brewer’s wort, and the rate of uptake of these sugars by brewer’s yeast can have a major impact on fermentation performance. In spite of this, no information is currently available on the genetics of maltose and maltotriose uptake in brewing strains of yeast. In this work, we studied 30 brewing strains of yeast (5 ale strains and 25 lager strains) with the aim of examining the alleles of maltose and maltotriose transporter genes contained by them. To do this, we hybridized gene probes to chromosome blots. Studies performed with laboratory strains have shown that maltose utilization is conferred by any one of five unlinked but highly homologous MAL loci (MAL1 to MAL4 and MAL6). Gene 1 at each locus encodes a maltose transporter. All of the strains of brewer’s yeast examined except two were found to contain MAL11 and MAL31 sequences, and only one of these strains lacked MAL41. MAL21 was not present in the five ale strains and 12 of the lager strains. MAL61 was not found in any of the yeast strains. In three of the lager strains, there was evidence that MAL transporter gene sequences occurred on chromosomes other than those known to carry MAL loci. Sequences corresponding to the AGT1 gene, which encodes a transporter of several α-glucosides, including maltose and maltotriose, were detected in all but one of the yeast strains. Homologues of AGT1 were identified in three of the lager strains, and two of these homologues were mapped, one to chromosome II and the other to chromosome XI. AGT1 appears to be a member of a family of closely related genes, which may have arisen in brewer’s yeast in response to selective pressure.     

Baker’s yeast mediated asymmetric reduction of cinnamaldehyde derivatives

The enantioselective reduction of cinnamaldehyde derivatives is an attractive strategy to prepare various optically active multifunctional molecules that can be used as chiral building blocks for the synthesis of some HIV-protease inhibitors. The asymmetric reduction with pH adjusted to 5.5 of α-substituted-cinnamaldehydes (Br, N₃) mediated by baker’s yeast (Saccharomyces cerevisiae) yielded α-substituted-3-phenyl-1-propanol in excellent enantiomeric excesses and yields.     

Protective Effect of Avian Myelomonocytic Growth Factor in Infection with Marek’s Disease Virus

Marek's disease virus (MDV) is a herpesvirus that induces T lymphomas in chickens. The aim of this study was to assess the role of the macrophage activator chicken myelomonocytic growth factor (cMGF) in controlling MDV infection. B13/B13 chickens, which are highly susceptible to MD, were either treated with cMGF delivered via a live fowlpox virus (fp/cMGF) or treated with the parent vector (fp/M3) or were left as untreated controls. Seven days later, when challenged with the very virulent RB-1B strain of MDV, the spleens of chickens treated with fp/cMGF showed increased expression of the inducible nitric oxide synthase (iNOS) gene compared to those of control chickens and fp/M3-treated chickens. Increased iNOS gene expression was also accompanied by greater induction of gamma interferon and macrophage inflammatory protein (K203) gene expression, both possible activators of iNOS. fp/cMGF treatment also increased the number of monocytes and systemic NO production in contrast to fp/M3 treatment. Even though cMGF treatment was unable to prevent death for the chickens, it did prolong their survival time, and viremia and tumor incidence were greatly reduced. In addition, cMGF treatment improved the partial protection induced by vaccination with HVT (herpesvirus isolated from turkeys) against RB-1B, preventing 100% mortality (versus 66% with vaccination alone) and greatly reducing tumor development. Treatment with fp/M3 did not have such effects. These results suggest that cMGF may play multiple roles in protection against MD. First, it may enhance the innate immune response by increasing the number and activity of monocytes and macrophages, resulting in increased NO production. Second, it may enhance the acquired immune response, indicated by its ability to enhance vaccine efficacy.     

Effect of Copper on Growth of Yeast

The effect of copper was tested on the growth of many strains of yeast. Plate culture on density gradient agar of copper was used for estimating the growth response to copper. Growth in many strains was more strongly inhibited by the copper-aquo complex than by the copper-amino acid complex. Debaryomyces hansenii IFO 023 was found a suitable strain for the present study, because it was not resistant, not producing H₂S, and copper absorption by this strain was similar to that of the resistant strain. Growth of yeast cells in medium containing copper was affected by pH and concentration of amino acid in medium. Absorption of copper into intact cells was almost saturated for the initial few minutes. It was also affected by the addition of amino acid to copper solution. Our results indicated that the growth response of yeast to copper was closely related to copper absorption into cells. About 60 percent of copper absorbed into cells was distributed in the soluble fraction of the cell homogenate which was obtained by centrifugation at 105,000 g for 60 min.     

几株啤酒酵母的筛选及发酵性能比较*

啤酒酵母是啤酒酿造的灵魂,可以直接影响啤酒品质。在啤酒酿造过程中,由于啤酒酵母被多次传代和保藏,造成优良菌种发酵性能衰退等问题,导致发酵不彻底,影响最后啤酒的风味质量。为此以8株Lager型啤酒酵母为出发菌株,通过平板分离纯化获得80株分离菌株,再经过三角瓶发酵初筛和复筛、发酵罐中试发酵实验最终获得了8株发酵性能优良的啤酒酵母。其中,6株酵母可应用于酿造双乙酰含量低于0.1 mg/L的啤酒;3株酵母发酵度高于70%,适合酿造干啤酒;1株酵母发酵度低于50%,适合酿造低醇啤酒。在风味方面:1株酵母酿造的啤酒醇酯比为3.3,啤酒酯香味较突出;另1株酵母酿造的啤酒醇酯比为4.5,啤酒高级醇含量较高。8株经过选育的啤酒酵母发酵特征明显,便于精酿啤酒厂实际应用。     

Effect of Photon Fluence Rate and Specific Growth Rate on Geosmin Production of the Cyanobacterium Oscillatoria brevis (Kütz.) Gom

The production of geosmin from the cyanobacterium Oscillatoria brevis was studied as a function of the photon fluence rate and appears to be related to the chlorophyll content.     

The Bloom’s and Werner’s syndrome proteins are DNA structure-specific helicases

BLM and WRN, the products of the Bloom’s and Werner’s syndrome genes, are members of the RecQ family of DNA helicases. Although both have been shown previously to unwind simple, partial duplex DNA substrates with 3′→5′ polarity, little is known about the structural features of DNA that determine the substrate specificities of these enzymes. We have compared the substrate specificities of the BLM and WRN proteins using a variety of partial duplex DNA molecules, which are based upon a common core nucleotide sequence. We show that neither BLM nor WRN is capable of unwinding duplex DNA from a blunt-ended terminus or from an internal nick. However, both enzymes efficiently unwind the same blunt-ended duplex containing a centrally located 12 nt single-stranded ‘bubble’, as well as a synthetic X-structure (a model for the Holliday junction recombination intermediate) in which each ‘arm’ of the 4-way junction is blunt-ended. Surprisingly, a 3′-tailed duplex, a standard substrate for 3′→5′ helicases, is unwound much less efficiently by BLM and WRN than are the bubble and X-structure substrates. These data show conclusively that a single-stranded 3′-tail is not a structural requirement for unwinding of standard B-form DNA by these helicases. BLM and WRN also both unwind a variety of different forms of G-quadruplex DNA, a structure that can form at guanine-rich sequences present at several genomic loci. Our data indicate that BLM and WRN are atypical helicases that are highly DNA structure specific and have similar substrate specificities. We interpret these data in the light of the genomic instability and hyper-recombination characteristics of cells from individuals with Bloom’s or Werner’s syndrome.     

Differential Effect of Culture Temperature and Specific Growth Rate on CHO Cell Behavior in Chemostat Culture

Mild hypothermia condition in mammalian cell culture technology has been one of the main focuses of research for the development of breeding strategies to maximize productivity of these production systems. Despite the large number of studies that show positive effects of mild hypothermia on specific productivity of r-proteins, no experimental approach has addressed the indirect effect of lower temperatures on specific cell growth rate, nor how this condition possibly affects less specific productivity of r-proteins. To separately analyze the effects of mild hypothermia and specific growth rate on CHO cell metabolism and recombinant human tissue plasminogen activator productivity as a model system, high dilution rate (0.017 h⁻¹) and low dilution rate (0.012 h⁻¹) at two cultivation temperatures (37 and 33°C) were evaluated using chemostat culture. The results showed a positive effect on the specific productivity of r-protein with decreasing specific growth rate at 33°C. Differential effect was achieved by mild hypothermia on the specific productivity of r-protein, contrary to the evidence reported in batch culture. Interestingly, reduction of metabolism could not be associated with a decrease in culture temperature, but rather with a decrease in specific growth rate.     

比生长速率和氮源对毕赤酵母生产重组鲈鱼生长激素的影响

对毕赤酵母胞内表达重组鲈鱼生长激素(rljGH)的发酵罐上生产进行了研究.建立了指数流加甲醇的策略并考察了不同比生长速率对rljGH生产的影响.结果表明,随着比生长速率的增加,平均比生产速率相应增加,但是胞内持续积累rljGH的时间减少.最大比rljGH产量(0.58 mg/g WCW)在比生长速率为0.029/h时获得.进一步考察了在诱导阶段添加硫酸铵、蛋白胨和酵母抽提物的影响.结果表明,添加硫酸铵和蛋白胨对于rljGH生产没有显著影响;添加2.5 g/L酵母抽提物有助于胞内rljGH的积累,并使胞内积累持续时间由17 h增加到23 h,提高了发酵稳定性.