Enhanced lactic acid bacteria viability with yeast coincubation under acidic conditions

ABSTRACT

The enhancing effects of yeasts on the viability of lactic acid bacteria (LAB) under acidic conditions were investigated. Meyerozyma guilliermondii, coaggregative with both LAB strains under acidic conditions, significantly enhanced the viability of Lactobacillus pentosus and L. paracasei in pH 3.0 lactic acid (LA) buffer at 10°C (p < 0.05). Non-coaggregative yeasts (Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Cyberlindnera saturnus) also significantly enhanced the LAB viability (p < 0.05), and physical contact between LAB and yeasts was not essential for the viability-enhancing effect, indicating that the coaggregation had no relation to the enhancing mechanism. Although yeast metabolites and LA assimilation had no enhancing effect, hydrogen peroxide (H₂O₂) decreased after yeast coincubation, and H₂O₂ elimination improved L. pentosus viability. H₂O₂ elimination alone did not sufficiently improve L. paracasei viability, but the addition of antioxidants was effective. These results suggest that the antioxidant activity of yeast increased the LAB viability under acidic conditions.     

Succinate production from sucrose by metabolic engineered Escherichia coli strains under aerobic conditions

Two metabolically engineered E. coli strains HL2765k and HL27659k, while capable of producing succinate from glucose with high yields, are not able to grow and produce succinate on sucrose. Consequently, the pUR400 plasmid containing scrK, Y, A, B, and R genes was introduced into HL2765k and HL27659k, respectively. Shake flask culture studies showed that the resulting strains can utilize sucrose; the strain HL2765k pUR400 and HL27659k pUR400 can produce succinate aerobically with a molar yield of 0.78 ± 0.02 mol/mol and 1.35 ± 0.13 mol/mol, respectively. On introduction of the plasmid pHL413, which encodes the heterologous pyruvate carboxylase (PYC) from Lactococcus lactis, the molar succinate yield increased to 1.60 ± 0.01 mol of succinate per mole of sucrose by the HL2765k pUR400 pHL413 strain and to 1.84 ± 0.10 by the HL27659k pUR400 pHL413 strain. In aerobic batch bioreactor studies, the succinate production rate was faster, and succinate production reached 101.83 mM with a yield of 1.90 when dissolved oxygen (DO) was controlled at 40 ± 7%. In addition, the results showed that DO had an important effect on succinate production by influencing PYC activity. This work demonstrates the possibility of producing succinate aerobically using sucrose as the carbon source.     

Fluorescent differential display analysis of Lactobacillus sakei strains under stress conditions

Lactobacillus (Lb.) sakei is widely used as starter in the production process of Italian fermented sausages and its growth and survival are affected by various factors such as temperature, pH and salt concentration. We studied the behaviour of Lb. sakei strains under various growth conditions relative to acid, osmotic and heat stress treatments by a novel fluorescent differential display (FDD) technique. This study obtained the development and the optimization of a technique that allows the identification of genome expression changes, associated with differential microbial behaviour under different stress conditions with a better stress response definition and a better discrimination of starter cultures. DNA sequence information from the FDD products provided an important tool to assess and observe the response to a variety of environmental stimuli and the adaptation to bacterial stress.Our work provided an innovative FDD method, with a high level of reproducibility and quality for studying and probing the knowledge of the relation between differential genome expression and different stresses tolerance.     

Succinate production from different carbon sources under anaerobic conditions by metabolic engineered Escherichia coli strains

Succinic acid has drawn much interest as a precursor of many industrially important chemicals. Using a variety of feedstocks for the bio-production of succinic acid would be economically beneficial to future industrial processes. Escherichia coli SBS550MG is able to grow on both glucose and fructose, but not on sucrose. Therefore, we derived a SBS550MG strain bearing both the pHL413 plasmid, which contains Lactococcus lactis pycA gene, and the pUR400 plasmid, which contains the scrK, Y, A, B, and R genes for sucrose uptake and catalyzation. Succinic acid production by this modified strain and the SBS550pHL413 strain was tested on fructose, sucrose, a mixture of glucose and fructose, a mixture of glucose, fructose and sucrose, and sucrose hydrolysis solution. The modified strain can produce succinic acid efficiently from all combinations of different carbon sources tested with minimal byproduct formation and with high molar succinate yields close to that of the maximum theoretic values. The molar succinic acid yield from fructose was the highest among the carbon sources tested. Using the mixture of glucose and fructose as the carbon source resulted in slightly lower yields and much higher productivity than using fructose alone. Fermenting sucrose mixed with fructose and glucose gave a 1.76-fold higher productivity than that when sucrose was used as the sole carbon source. Using sucrose pretreated with sulfuric acid as carbon source resulted in a similar succinic acid yield and productivity as that when using the mixture of sucrose, fructose, and glucose. The results of the effect of agitation rate in aerobic phase on succinate production showed that supplying large amount of oxygen in aerobic phase resulted in higher productions of formate and acetate, and therefore lower succinate yield. This study suggests that fructose, sucrose, mixture of glucose and fructose, mixture of glucose, fructose and sucrose, or sucrose hydrolysis solution could be used for the economical and efficient production of succinic acid by our metabolic engineered E. coli strain.     

Astrocytic amino acid metabolism under control conditions and during oxygen and/or glucose deprivation

Amino acid contents were measured in 1- and 3-week-old primary cultures of astrocytes and in their incubation media, an amino acid-free salt solution with or without glucose, during 3-h incubation under normoxic or anoxic conditions. Most essential amino acids were rapidly released to the medium during the beginning of the incubation. A subsequent slow medium increase reflected proteolysis. Glutamate and aspartate were absent from the media during all conditions, indicating fueling of their uptake by either glycolytically or oxidatively derived energy. The total content of glutamine increased, except during incubation in glucose-deprived media, when it declined or remained constant. Changes in aspartate were negligible, suggesting oxidative degradation of aspartate-derived oxaloacetate during normoxia and its reduction to succinate during anoxia, driving regeneration of NAD⁺ from NADH. An increase of alanine was reduced in glucose-free media, whereas serine showed especially large increase during isolated glucose deprivation, suggesting its production from glutamine via 3-phosphoglycerate.     

Comparison of glycogen store in two strains of rat and guinea-pig under fed and fasted conditions

Glycogen content in the liver, skeletal muscle and heart has been determined in Sprague-Dawley (SD) and Wistar (W) rats and in tricoloured (T) and albino Dunkin Hartley (DH) guinea-pigs. The 12-week-old animals were studied under non-fasted or control conditions (N) and after 48 hr of fast (F48). Hepatic glycogen was higher in DH guinea-pigs (95.6 +/- 3.8 mg g-1) than in W (77.2 +/- 5.3 mg g-1) and SD (80.2 +/- 2.3 mg g-1) rats under N conditions. Mean values for the two strains were slightly higher in guinea-pigs than in rats. After fasting, hepatic glycogen was almost exhausted in the two species but was higher in W (1.5 +/- 0.08 mg g-1) and T (1.5 +/- 0.2 mg g-1) than in SD and DH (0.6 +/- 0.1 mg g-1). The content of glycogen in the anterior muscles of the thigh was comparable in the two strains of rat and guinea-pig, but was twice as high in the guinea-pigs (DH:15.1 +/- 0.6; T: 16.4 +/- 0.7 mg g-1) as in the rats (SD: 8.1 +/- 0.2; W: 7.1 +/- 0.5 mg g-1) under N conditions. In F48 animals, muscular glycogen decreased by 41-46% (rats) and 38-39% (guinea-pigs). Hepatic and extra-liver glycogen stores were calculated and found higher in the guinea-pigs than in the rats. The total utilization during fasting was larger in the guinea-pigs (6140 mg/kg body wt) than in the rats (4500 mg/kg body wt).(ABSTRACT TRUNCATED AT 250 WORDS)     

Genetically engineered Pichia pastoris yeast for conversion of glucose to xylitol by a single-fermentation process

Xylitol is industrially synthesized by chemical reduction of d-xylose, which is more expensive than glucose. Thus, there is a growing interest in the production of xylitol from a readily available and much cheaper substrate, such as glucose. The commonly used yeast Pichia pastoris strain GS115 was shown to produce d-arabitol from glucose, and the derivative strain GS225 was obtained to produce twice amount of d-arabitol than GS115 by adaptive evolution during repetitive growth in hyperosmotic medium. We cloned the d-xylulose-forming d-arabitol dehydrogenase (DalD) gene from Klebsiella pneumoniae and the xylitol dehydrogenase (XDH) gene from Gluconobacter oxydans. Recombinant P. pastoris GS225 strains with the DalD gene only or with both DalD and XDH genes could produce xylitol from glucose in a single-fermentation process. Three-liter jar fermentation results showed that recombinant P. pastoris cells with both DalD and XDH converted glucose to xylitol with the highest yield of 0.078 g xylitol/g glucose and productivity of 0.29 g xylitol/L h. This was the first report to convert xylitol from glucose by the pathway of glucose–d-arabitol–d-xylulose–xylitol in a single process. The recombinant yeast could be used as a yeast cell factory and has the potential to produce xylitol from glucose.     

Optimization of glucose feeding approaches for enhanced glucosamine and N-acetylglucosamine production by an engineered Escherichia coli

In this work, a recombinant Escherichia coli was constructed by overexpressing glucosamine (GlcN) synthase and GlcN-6-P N-acetyltransferase for highly efficient production of GlcN and N-acetylglucosamine (GlcNAc). For further enhancement of GlcN and GlcNAc production, the effects of different glucose feeding strategies including constant-rate feeding, interval feeding, and exponential feeding on GlcN and GlcNAc production were investigated. The results indicated that exponential feeding resulted in relatively high cell growth rate and low acetate formation rate, while constant feeding contributed to the highest specific GlcN and GlcNAc production rate. Based on this, a multistage glucose supply approach was proposed to enhance GlcN and GlcNAc production. In the first stage (0–2 h), batch culture with initial glucose concentration of 27 g/l was conducted, whereas the second culture stage (2–10 h) was performed with exponential feeding at μ _set = 0.20 h⁻¹, followed by feeding concentrated glucose (300 g/l) at constant rate of 32 ml/h in the third stage (10–16 h). With this time-variant glucose feeding strategy, the total GlcN and GlcNAc yield reached 69.66 g/l, which was enhanced by 1.59-fold in comparison with that of batch culture with the same total glucose concentration. The time-dependent glucose feeding approach developed here may be useful for production of other fine chemicals by recombinant E. coli.     

Continuous ethanol production and evaluation of yeast cell lysis and viability loss under very high gravity medium conditions

A combined bioreactor system, composed of a stirred tank and a three-stage tubular bioreactor in series and with a total working volume of 3260 ml, was established. Continuous ethanol production was carried out using Saccharomyces cerevisiae and a very high gravity (VHG) medium containing 280 g l⁻¹ glucose. An average ethanol concentration of 124.6 g l⁻¹ or 15.8% (v) was produced when the bioreactor system was operated at a dilution rate of 0.012 h⁻¹. The yield of ethanol to glucose consumed was calculated to be 0.484 or 94.7% of its theoretical value of 0.511 when ethanol entrapped in the exhaust gas was incorporated. Meanwhile, quasi-steady states and non-steady oscillations were observed for residual glucose, ethanol and biomass concentrations for all of these bioreactors during their operations. Models that can be used to predict yeast cell lysis and viability loss were developed.     

Direct measurement of glucose profiles in immobilized yeast gels with a pH-insensitive micro-electrode under anaerobic conditions

Summary a 10 μm glucose sensor was developed based on a glucose oxidase coated Pt-electrode inserted in a capillary shaft. The internal buffer medium effected in a glucose that was insensitive for the external pH. The sensor was sucessfully utilized at pH 4 under anaerobic conditions in gel particles containing homogeneously dispersed immobilized yeast cells.     

Overexpression of genes encoding glycolytic enzymes in Corynebacterium glutamicum enhances glucose metabolism and alanine production under oxygen deprivation conditions

We previously reported that Corynebacterium glutamicum strain ΔldhAΔppc+alaD+gapA, overexpressing glyceraldehyde-3-phosphate dehydrogenase-encoding gapA, shows significantly improved glucose consumption and alanine formation under oxygen deprivation conditions (T. Jojima, M. Fujii, E. Mori, M. Inui, and H. Yukawa, Appl. Microbiol. Biotechnol. 87:159-165, 2010). In this study, we employ stepwise overexpression and chromosomal integration of a total of four genes encoding glycolytic enzymes (herein referred to as glycolytic genes) to demonstrate further successive improvements in C. glutamicum glucose metabolism under oxygen deprivation. In addition to gapA, overexpressing pyruvate kinase-encoding pyk and phosphofructokinase-encoding pfk enabled strain GLY2/pCRD500 to realize respective 13% and 20% improved rates of glucose consumption and alanine formation compared to GLY1/pCRD500. Subsequent overexpression of glucose-6-phosphate isomerase-encoding gpi in strain GLY3/pCRD500 further improved its glucose metabolism. Notably, both alanine productivity and yield increased after each overexpression step. After 48 h of incubation, GLY3/pCRD500 produced 2,430 mM alanine at a yield of 91.8%. This was 6.4-fold higher productivity than that of the wild-type strain. Intracellular metabolite analysis showed that gapA overexpression led to a decreased concentration of metabolites upstream of glyceraldehyde-3-phosphate dehydrogenase, suggesting that the overexpression resolved a bottleneck in glycolysis. Changing ratios of the extracellular metabolites by overexpression of glycolytic genes resulted in reduction of the intracellular NADH/NAD(+) ratio, which also plays an important role on the improvement of glucose consumption. Enhanced alanine dehydrogenase activity using a high-copy-number plasmid further accelerated the overall alanine productivity. Increase in glycolytic enzyme activities is a promising approach to make drastic progress in growth-arrested bioprocesses.     

Construction of engineered fructosyl peptidyl oxidase for enzyme sensor applications under normal atmospheric conditions

Current enzymatic methods for the analysis of glycated proteins use flavoenzymes that catalyze the oxidative deglycation of fructosyl peptides, designated as fructosyl peptidyl oxidases (FPOXs). However, as FPOXs are oxidases, the signals derived from electron mediator-type electrochemical monitoring based on them are affected by dissolved O₂. Improvement of dye-mediated dehydrogenase activity of FPOXs and its application to enzyme electrode construction were therefore undertaken. Saturation mutagenesis study on Asn56 of FPOX from Phaeosphaeria nodorum, produced mutants with marked decreases in the catalytic ability to employ O₂ as the electron acceptor, while showing higher dye-mediated dehydrogenase activity employing artificial electron acceptors than the parental enzyme. Thus constructed virtually fructosyl peptide dehydrogenase, Asn56Ala, was then applied to produce an enzyme electrode for the measurement of fructosyl-^α N-valyl-histidine (f-^αVal-His), the protease-digested product of HbA1c. The enzyme electrode could measure f-^αVal-His in the physiological target range in air.     

Azospirillum strains use phenolic compounds as intermediates for electron transfer under oxygen-limiting conditions

The effects of catechol, vanillic, caffeic (CAF), 2-hydroxyphenylacetic, 4-hydroxy- and 3,4-dihydroxybenzoic (3,4-DHBA) acids on the growth of a common rice rhizosphere inhabitant, Azospirillum lipoferum were studied. Two strains of this nonfermenting nitrogen-fixing bacterium were used: a motile strain (4B), and a nonmotile strain (4T). Under atmospheric conditions (pO₂ = 21 kPa), the growth of strain 4T was inhibited by catechol (0.1 mm) only. None of these compounds affected the growth of strain 413. Under 5 kPa O₂, no effect was observed on strain 413, whereas three of the six tested phenolics stimulated the growth of strain 4T; maximum effects were observed for 3,4-DHBA and CAF. As revealed by TLC and HPLC, under low oxygen, more new lipophilic compounds were formed from CAF by strain 4T, differing from CAF autooxydation products and from the products obtained under 21 kPa O₂. It was hypothesized that strain 4T had the ability to use an oxidized derivative of CAF as a terminal electron acceptor. This hypothesis was tested in experiments under nitrogen-fixing conditions, in the absence of oxygen, and in the presence of N₂O as a reoxidizing agent for CAF. Acetylene was used both as a substrate to measure nitrogenase activity (ARA) and to inhibit the biological transfer of electrons to N₂O. The addition of CAF in the presence of N₂O had the same effect on ARA rates as an addition of oxygen. It is concluded that the strain 4T of Azospirillum lipoferum is able to sustain some of its activities (e.g., N₂ fixation) using phenolics as alternative electron acceptors under low oxygen conditions.     

DNA-PK is responsible for enhanced phosphorylation of histone H2AX under hypertonic conditions

Exposure of cells to hypertonic medium after X-irradiation results in a 3-4-fold increase in the phosphorylation of histone H2AX (gammaH2AX) at sites of radiation-induced DNA double-strand breaks. This increase was previously associated with salt-induced radiosensitization and inhibition of repair of DNA double-strand breaks. To examine possible mechanisms for the increase in foci size, chemical inhibitors of kinase and phosphatase activity and cell lines deficient in ATM and DNA-PK, two kinases known to phosphorylate H2AX, were examined. H2AX kinase and phosphatase activity were maintained in the presence of high salt. ATM mutant HT144 melanoma cells showed the expected 3-4-fold increase in H2AX phosphorylation in the presence of 0.5M Na(+). However, DNA-PKcs deficient M059J cells failed to respond to hypertonic treatment and M059J Fus1 cells corrected for this deficiency showed the expected increase in foci size. Although the active phosphoform of ATM, phosphoserine-1981, increased after irradiation, the level was unaffected by the addition of 0.5M Na(+). Instead, 0.5M Na(+) caused a partial redistribution of serine-1981-ATM to perinuclear regions. Hypertonic medium added after irradiation was effective in inhibiting rejoining of the radiation-induced double-strand breaks even in DNA-PK deficient M059J cells. We suggest that hypertonic treatment following irradiation inhibits double-strand break rejoining that in turn maintains DNA-PK activity at the site of the break, enhancing the size of the gammaH2AX foci.     

Metallothionein is part of a zinc-scavenging mechanism for cell survival under conditions of extreme zinc deprivation

Metallothionein (MT) is a small cysteine-rich protein thought to play a critical role in cellular detoxification of inorganic species by sequestering metal ions that are present in elevated concentrations. We demonstrate here that metallothionein can play an important role at the other end of the homeostatic spectrum by scavenging an essential metal in a mouse fibroblast cell line that has been cultured under conditions of extreme zinc deprivation (LZA-LTK-). These cells unexpectedly produce constitutively high levels of metallothionein mRNA; however, the MT protein accumulates only when high concentrations of zinc are provided in the media. Until this MT pool is saturated, no measurable zinc remains in the external media. In this case, zinc deprivation leads to amplification of the MT gene locus in the LZA-LTK- cell line. Furthermore, the intracellular zinc levels in the fully adapted cells remain at the normal level of 0.4 fmol zinc/cell, even when extracellular zinc concentration is decreased by 2 orders of magnitude relative to normal media.     

Survival of genetically-engineered and wild-type strains of the yeast Saccharomyces cerevisiae under simulated environmental conditions: a contribution on risk assessment

H. FUJIMURA, Y. SAKUMA AND E. AMANN. 1994. A genetically-engineered strain of Saccharomyces cerevisiae employed for the industrial production of the human coagulation Factor XIIIa (rhFXIIIa) was used for a survival study under simulated environmental conditions. The homologous strain devoid of the recombinant plasmid and the homologous strain bearing the 2 μm-based vector plasmid without the rhFXIIIa-encoding DNA insert were compared. The strains were introduced into natural soil/water suspension, into soil/medium suspension and into waste water. After intervals, samples of cell suspensions were taken and viable cell numbers were determined by plating on antibiotic-containing medium. In addition, a non-radioactive technique involving enhanced chemiluminescence was employed to detect plasmid-bearing yeast cells. The rhFXIIIa expression plasmid showed a high stability during the simulated environmental condition. No differences in survival rates, however, could be detected for the plasmid-bearing and plasmid-less strains under the three conditions tested, suggesting that the presence of plasmid does not confer selective advantages on the survival of the yeast cells. It is concluded that, even after accidental release of the engineered yeast cells into the environment, elimination rates would be comparable to those for non-recombinant yeast strains.