Metabolic footprint analysis of recombinant Escherichia coli strains during fed-batch fermentations

Metabolic footprinting has become a valuable analytical approach for the characterization of phenotypes and the distinction of specific metabolic states resulting from environmental and/or genetic alterations. The metabolic impact of heterologous protein production in Escherichia coli cells is of particular interest, since there are numerous cellular stresses triggered during this process that limit the overall productivity, e.g. the stringent response. Because the knowledge on the metabolic responses in recombinant bioprocesses is still scarce, metabolic footprinting can provide relevant information on the intrinsic metabolic adjustments. Thus, the metabolic footprints generated by E. coli W3110 and the ΔrelA mutant strain during recombinant fed-batch fermentations at different experimental conditions were measured and interpreted. The IPTG-induction of the heterologous protein expression resulted in the rapid accumulation of inhibitors of the glyoxylate shunt in the culture broth, suggesting the clearance of this anaplerotic route to replenish the TCA intermediaries withdrawn for the additional formation of the heterologous protein. Nutritional shifts were also critical in the recombinant cellular metabolism, indicating that cells employ diverse strategies to counteract imbalances in the cellular metabolism, including the secretion of certain metabolites that are, most likely, used as a metabolic relief to survival processes.     

Analysis of yeast diversity during spontaneous and induced alcoholic fermentations

The diversity of yeast species and strains was monitored by physiological tests and a simplified method of karyotyping of yeast chromosomes. During the first phase of investigated alcoholic fermentations, the yeast species Metschnikowia pulcherrima and Hanseniaspora uvarum were predominant, irrespective of the origin of the grape must. At the beginning of fermentation H. uvarum was even present in the case of induced fermentations with dried yeast. Middle and end phase of the alcoholic fermentation were clearly dominated by the yeast species Saccharomyces cerevisiae . In the case of spontaneous fermentations, several different strains of S. cerevisiae were present and competed with each other, whereas in induced fermentations only the inoculated strain of S. cerevisiae was observed. A competition of strains of S. cerevisiae also occurred during the fermentation with dried yeast product consisting of two different strains. An effect of H. uvarum on taste and flavour of wines can be postulated according to the frequency of its appearance during the first phase of fermentation. With the method of rapid karyotyping and supplementary physiological tests it was possible to make reliable assertions about the yeast diversity during alcoholic fermentation.     

Analytical differentiation of wine fermentations using pure and mixed yeast cultures

Summary Thirty-three fermentations of Pedro Ximénez grapes, collected in three degrees of ripeness, were carried out by inoculation with three types of inoculum: pure cultures ofSaccharomyces cerevisiae races and ofTorulaspora delbrueckii, indigenous yeasts, and mixed cultures of indigenous yeasts enriched with the pure cultures. By means of variance analysis 21 compounds were determined whose final concentrations in the wines significantly depended on the musts, the inocula or both. Eleven products that depended significantly on the inocula were subjected to a discriminant analysis in which most of the pure cultures gathered in a discriminant space area different from that occupied by the indigenous yeasts. The centroids corresponding to most of the mixed cultures were shifted to the central area of the discriminant space, moved away from their corresponding pure cultures and approached the indigenous yeasts. The results show a high similarity between the fermentations carried out with mixed cultures with the addedS. cerevisiae races and those fermentations carried out with the indigenous yeasts, with regard to those compounds which were significantly dependent on the inocula.     

Metabolic flux analysis of the two astaxanthin-producing microorganisms Haematococcus pluvialis and Phaffia rhodozyma in the pure and mixed cultures

The two major astaxanthin-producing microorganisms Phaffia rhodozyma and Haematococcus pluvialis exhibited elevated astaxanthin yields under the mixed culture regime, and the changes in flux distribution were investigated by means of metabolic flux analysis (MFA). In the mixed culture of the two strains, the carbon flux towards astaxanthin formation in P. rhodozyma increased by 20%, which may be due to the enriched oxygen evolved through the photosynthesis of H. pluvialis. On the other hand, the uptake of pyruvate and CO(2) excreted by P. rhodozyma also facilitated astaxanthin synthesis in H. pluvialis, which reduced 33% of the carbon flux exported from Calvin cycle to the catabolic pathway, and in turn raised the carbon flux to glyceraldehyde-3-phosphate by 25%. As a result, the carbon flux diverted to astaxanthin synthesis increased 2.8-fold in comparison with that in the pure culture.     

Cell cycle analysis of Taxus suspension cultures at the single cell level as an indicator of culture heterogeneity

Single cell growth and division was measured via flow cytometry in order to characterize the metabolic variability of Taxus cuspidata suspension cultures, which produce the valuable secondary metabolite Taxol. Good agreement was observed between the cell cycle distribution and biomass accumulation over the batch culture period. Specific growth rates of 0.13 days(-1) by fresh weight and 0.15 days(-1) by dry weight were measured. Elicitation with methyl jasmonate (MJ) significantly decreased both cell cycle progression and biomass accumulation, as the specific growth rate decreased to 0.027 days(-1) by fresh and dry weight. Despite the decrease in biomass accumulation for MJ elicited cultures, sucrose utilization was not significantly different from control cultures. MJ elicitation also increased the accumulation of paclitaxel and other taxanes. The accumulation of upstream taxanes (baccatin III and 10-deactylbaccatin III) increased during exponential growth, reached a maximum around day 12, and then declined throughout the stationary phase. The paclitaxel concentration increased during both exponential growth and stationary phase, reaching a maximum around days 20-25. Throughout the culture period, greater than 70% of the cells were in G(0)/G(1) phase of the cell cycle. Studies using bromodeoxyuridine (BrdU) incorporation showed that approximately 65% of the Taxus cells are noncycling, even during exponential growth. Although the role of these cells is currently unknown, the presence of a large, noncycling subpopulation can have a significant impact on the utilization of plant cell culture technology for the large-scale production of paclitaxel. These results demonstrate that there is a high degree of metabolic heterogeneity in Taxus cuspidata suspension cultures. Understanding this heterogeneity is important for the optimization of plant cell cultures, particularly the reduction of production variability.     

Evaluation of yeast diversity during wine fermentations with direct inoculation and pied de cuve method at an industrial scale

The diversity and composition of yeast populations may greatly impact wine quality. This study investigated the yeast microbiota in two different types of wine fermentations: direct inoculation of a commercial starter versus pied de cuve method at an industrial scale. The pied de cuve fermentation entailed growth of the commercial inoculum used in the direct inoculation fermentation for further inoculation of additional fermentations. Yeast isolates were collected from different stages of wine fermentation and identified to the species level using Wallersterin Laboratory nutrient (WLN) agar followed by analysis of the 26S rDNA D1/D2 domain. Genetic characteristics of the Saccharomyces cerevisiae strains were assessed by a rapid PCR-based method, relying on the amplification of interdelta sequences. A total of 412 yeast colonies were obtained from all fermentations and eight different WL morphotypes were observed. Non-Saccharomyces yeast mainly appeared in the grape must and at the early stages of wine fermentation. S. cerevisiae was the dominant yeast species using both fermentation techniques. Seven distinguishing interdelta sequence patterns were found among S. cerevisiae strains, and the inoculated commercial starter, AWRI 796, dominated all stages in both direct inoculation and pied de cuve fermentations. This study revealed that S. cerevisiae was the dominant species and an inoculated starter could dominate fermentations with the pied de cuve method under controlled conditions.     

Theoretical and experimental recovery of oscillations during batch growth of a mixed culture of yeast in water

Theoretical and experimental evidence of substantial damped oscillations during batch interaction of two strains of Saccharomyces cerevisiae grown as a mixed culture in pure (distilled) water is provided. The experimental results show that in the limited nutrient conditions of growth in pure water both strains coexist if the initial concentration ratio between the two strains is 1:1. A new theoretical model that was originally developed for recovering the growth of single species in isolation is extended and applied to the two strains of yeast competing over a common ecological niche. The model solutions are shown to recover all the qualitative features captured in the experiments.     

Non-invasive nuclear magnetic resonance analysis of male and female embryo metabolites during in vitro embryo culture

Introduction

In the past 20+ years, several studies of bovine embryo production showed how the ratio of male to female embryos changes if embryos are made in vivo or in vitro. It is known that in in vitro systems, the sex ratio is in favor of males when there are high levels of glucose, and favors females when the principal energetic substrate is one other than glucose, like citrate.

Objectives

The aim of this study was to evaluate the embryo metabolism during three important periods of in vitro development: the early development (from day 1 until day 3), the middle of culture (day 3 until day 5), and later development (day 5 until day 7).

Methods

To obtain this information we evaluated the spent medium from each time period by ¹H NMR.

Results

Our results confirm that embryo metabolism is different between sexes. The new information obtained by identifies markers that we can use to predict the embryo sex.

Conclusion

These results open a new, non-invasive method to evaluate sex of the embryos before the transfer. In the first period of embryo culture, valine concentration is good indicator (66.7% accurate), while in the last phase of culture, pyruvate depletion is the best marker (64% accurate) to evaluate the sex of the embryo.

     

Substrate interactions of benzene, toluene, and para-xylene during microbial degradation by pure cultures and mixed culture aquifer slurries.

Benzene, toluene, and p-xylene (BTX) were degraded by indigenous mixed cultures in sandy aquifer material and by two pure cultures isolated from the same site. Although BTX compounds have a similar chemical structure, the fate of individual BTX compounds differed when the compounds were fed to each pure culture and mixed culture aquifer slurries. The identification of substrate interactions aided the understanding of this behavior. Beneficial substrate interactions included enhanced degradation of benzene and p-xylene by the presence of toluene in Pseudomonas sp. strain CFS-215 incubations, as well as benzene-dependent degradation of toluene and p-xylene by Arthrobacter sp. strain HCB. Detrimental substrate interactions included retardation in benzene and toluene degradation by the presence of p-xylene in both aquifer slurries and Pseudomonas incubations. The catabolic diversity of microbes in the environment precludes generalizations about the capacity of individual BTX compounds to enhance or inhibit the degradation of other BTX compounds.     

甲醇营养型酵母高密度培养过程中甲醇和乙醇的GC快速检测

采用气相色谱法（GC）快速检测甲醇营养型酵母发酵液中的甲醇、乙醇含量,具有样品处理简便,测定时间较短,结果重视性好的特点。在1－10mg/mL范围内具有很好的线性关系。在毕赤酵母高密度表达发酵过程中应用此法对甲醇和乙醇进行实时监空,细胞终密度超过300g/L（干重）。本方法为甲醇营养型酵母工程菌的发酵中试工艺研究提供了重要 的发酵生化参数。     

Substrate interactions of benzene, toluene, and para-xylene during microbial degradation by pure cultures and mixed culture aquifer slurries.

Benzene, toluene, and p-xylene (BTX) were degraded by indigenous mixed cultures in sandy aquifer material and by two pure cultures isolated from the same site. Although BTX compounds have a similar chemical structure, the fate of individual BTX compounds differed when the compounds were fed to each pure culture and mixed culture aquifer slurries. The identification of substrate interactions aided the understanding of this behavior. Beneficial substrate interactions included enhanced degradation of benzene and p-xylene by the presence of toluene in Pseudomonas sp. strain CFS-215 incubations, as well as benzene-dependent degradation of toluene and p-xylene by Arthrobacter sp. strain HCB. Detrimental substrate interactions included retardation in benzene and toluene degradation by the presence of p-xylene in both aquifer slurries and Pseudomonas incubations. The catabolic diversity of microbes in the environment precludes generalizations about the capacity of individual BTX compounds to enhance or inhibit the degradation of other BTX compounds.     

Expression of cell growth and bone specific genes at single cell resolution during development of bone tissue-like organization in primary osteoblast cultures.

Primary cultures of calvarial derived normal diploid osteoblasts undergo a developmental expression of genes reflecting growth, extracellular matrix maturation, and mineralization during development of multilayered nodules having a bone tissue-like organization. Scanning electron microscopy of the developing cultures indicates the transition from the uniform distribution of cuboidal osteoblasts to multilayered nodules of smaller cells with a pronounced orientation of perinodular cells towards the apex of the nodule. Ultrastructural analysis of the nodule by transmission electron microscopy indicates that the deposition of mineral is confined to the extracellular matrix where cells appear more osteocytic. The cell body contains rough endoplasmic reticulum and golgi, while these intracellular organelles are not present in the developing cellular processes. To understand the regulation of temporally expressed genes requires an understanding of which genes are selectively expressed on a single cell basis as the bone tissue-like organization develops. In situ hybridization analysis using 35S labelled histone gene probes, together with 3H-thymidine labelling and autoradiography, indicate that greater than 98% of the pre-confluent osteoblasts are proliferating. By two weeks, both the foci of multilayered cells and internodular cell regions have down-regulated cell growth associated genes. Post-proliferatively, but not earlier, initial expression of both osteocalcin and osteopontin are restricted to the multilayered nodules where all cells exhibit expression. While total mRNA levels for osteopontin and osteocalcin are coordinately upregulated with an increase in mineral deposition, in situ hybridization has revealed that expression of osteocalcin and osteopontin occurs predominantly in cells associated with the developing nodules. In contrast, proliferating rat osteosarcoma cells (ROS 17/2.8) concomitantly express histone H4, along with osteopontin and osteocalcin. These in situ analyses of gene expression during osteoblast growth and differentiation at the single cell level establish that a population of proliferating calvarial-derived cells subsequently expresses osteopontin and osteocalcin in cells developing into multilayered nodules with a tissue-like organization.     

A quantitative real‐time PCR assay for identification and enumeration of the occasionally co‐occurring ichthyotoxic Pseudochattonella farcimen and P. verruculosa (Dictyochophyceae) and analysis of variation in gene copy numbers during the growth phase of single and mixed cultures

The ichthyotoxic genus Pseudochattonella forms recurrent extensive blooms in coastal waters in Japan, New Zealand and Northern Europe. It comprises of two morphologically similar species, P. verruculosa and P. farcimen, which complicates visual species identification and enumeration of live and fixed material. Primers designed previously could not quantitatively distinguish species in mixed assemblages. To address this issue we developed two primer sets: one revealed itself to be genus specific for Pseudochattonella and the other species‐specific for P. verruculosa. By subtracting cell estimates for P. verruculosa from combined results we could calculate cell numbers for P. farcimen. This approach has overcome the challenges posed by the very limited sequence availability and low gene variability between the two species. The qPCR assay was extensively tested for specificity, efficiency and sensitivity over an entire growth cycle in both single and mixed assemblages. Comparison of cell abundance estimates obtained by qPCR assay and microscopy showed no statistically significant difference until stationary and death phases. The assay was also tested on environmental samples collected during a small Pseudochattonella bloom in Denmark in March–April 2015. It was impossible to distinguish P. farcimen and P. verruculosa by light microscopy but qPCR showed both species were present. The two methods provided nearly identical cell numbers but the assay provided discrimination and enumeration of both species.     

Nuclear genes coding for four subunits of the yeast ubiquinol-cytochrome c reductase complex are present in single copies in the haploid genome and at least two of these are located on different chromosomes

Summary Genes coding for the 40 kilodaltons (kDa), 17-kDa, 14-kDa and 11-kDa subunits of the ubiquinol-cytochrome c reductase in yeast are present in single copies in the haploid genome. We have mapped each gene to a unique genomic environment and demonstrate that integration of cloned segments into nuclear DNA by homologous crossing-over with the endogenous gene results in the replacement of the corresponding chromosomal restriction fragment by fragments of predicted sizes. Chromosomal mapping, carried out by the procedure of Falco and Botstein 1983, indicates that the gene for the 17-kDa subunit lies on chromosome VI and that for the 11-kDa subunit on chromosome XII.     

Effects of feeding dried grass pasture and a grain ration differing in rumen undegradable protein at two feeding frequencies on nutrient digestibility and microbial protein synthesis during continuous culture

Four continuous culture fermenters were used in a randomized block design to evaluate the effects of grain feeding frequency and rumen undegradable protein (RUP) on nutrient utilization and microbial protein synthesis with high quality grass. Two grain rations fed two or four times per day were arranged in a 2×2 factorial with four replications. The grain rations contained 60% and 45% of CP as RUP, and similar total nonstructural carbohydrate (TNC) content (64% of DM). Grain was fed separately from freeze dried, pelleted pasture (56% of DM). Fermenters were maintained at a liquid dilution rate of 12%/h and a solids retention time of 20 h. Regardless of RUP level or grain feeding frequency, true DM, OM, and CP digestibilities averaged 72.1%, 73.3%, and 61.8%, respectively with no differences due to treatments. Apparent digestibility of ADF, NDF, and TNC were also not affected by treatments, and averaged 55.9%, 46.2%, and 80.9%, respectively. The production of the major VFA (acetic, propionic, and butyric) were not affected by treatment and averaged 56.4, 21.2 and 16.3 Mmol/ml, respectively. The interaction of grain feeding frequency and dietary RUP content was significant for isovalerate production. Increasing the grain feeding frequency significantly increased valerate production. Ammonia N (NH₃–N) concentration and flows were decreased with higher RUP supplementation, and by feeding grain 4X/day (P<0.05). Non NH₃–N flows were also higher with 4X/day grain feeding. Feeding a high RUP grain reduced the CP percentage in bacteria and tended to cause a reduction in rumen pH (P>0.10). The interaction of time during the day when pH was measured and feeding frequency was significant (P<0.001) reduced the diurnal variation in pH and when grain feeding frequency was measured to 4X/day.