scholarly journals Pichia pastoris Exhibits High Viability and a Low Maintenance Energy Requirement at Near-Zero Specific Growth Rates

2016 ◽  
Vol 82 (15) ◽  
pp. 4570-4583 ◽  
Author(s):  
Corinna Rebnegger ◽  
Tim Vos ◽  
Alexandra B. Graf ◽  
Minoska Valli ◽  
Jack T. Pronk ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Growth Rates ◽  
Specific Growth ◽  
Energy Requirement ◽  
Maintenance Energy ◽  
Content Type ◽  
Chemostat Cultures ◽  
Zero Growth ◽  
Maintenance Energy Requirement ◽  
Specific Growth Rates

ABSTRACTThe yeastPichia pastorisis a widely used host for recombinant protein production. Understanding its physiology at extremely low growth rates is a first step in the direction of decoupling product formation from cellular growth and therefore of biotechnological relevance. Retentostat cultivation is an excellent tool for studying microbes at extremely low specific growth rates but has so far not been implemented forP. pastoris. Retentostat feeding regimes were based on the maintenance energy requirement (mS) and maximum biomass yield on glucose (YX/Smax) estimated from steady-state glucose-limited chemostat cultures. Aerobic retentostat cultivation enabled reproducible, smooth transitions from a specific growth rate (μ) of 0.025 h−1to near-zero specific growth rates (μ < 0.001 h−1). At these near-zero specific growth rates, viability remained at least 97%. The value ofmSat near-zero growth rates was 3.1 ± 0.1 mg glucose per g biomass and h, which was 3-fold lower than themSestimated from faster-growing chemostat cultures. This difference indicated thatP. pastorisreduces its maintenance energy requirement at extremely low μ, a phenomenon not previously observed in eukaryotes. Intracellular levels of glycogen and trehalose increased, while μ progressively declined during retentostat cultivation. Transcriptional reprogramming toward zero growth included the upregulation of many transcription factors as well as stress-related genes and the downregulation of cell cycle genes. This study underlines the relevance of comparative analysis of maintenance energy metabolism, which has an important impact on large-scale industrial processes.IMPORTANCEThe yeastPichia pastorisnaturally lives on trees and can utilize different carbon sources, among them glucose, glycerol, and methanol. In biotechnology, it is widely used for the production of recombinant proteins. For both the understanding of life in its natural habitat and optimized production processes, a better understanding of cell physiology at an extremely low growth rate would be of extraordinary value. Therefore, we have grownP. pastorisin a retentostat, which allows the cultivation of metabolically active cells even at zero growth. Here we reached doubling times as long as 38 days and found thatP. pastorisdecreases its maintenance energy demand 3-fold during very slow growth, which enables it to survive with a much lower substrate supply than baker's yeast.

scholarly journals Isolated Microbial Single Cells and Resulting Micropopulations Grow Faster in Controlled Environments

2012 ◽  
Vol 78 (19) ◽  
pp. 7132-7136 ◽  
Author(s):  
Christian Dusny ◽  
Frederik Sven Ole Fritzsch ◽  
Oliver Frick ◽  
Andreas Schmid
Keyword(s):  
Pichia Pastoris ◽  
Corynebacterium Glutamicum ◽  
Growth Rates ◽  
Specific Growth ◽  
Single Cells ◽  
Hansenula Polymorpha ◽  
Content Type ◽  
Controlled Environments ◽  
Specific Growth Rates ◽  
Extracellular Environment

ABSTRACTSingularized cells ofPichia pastoris,Hansenula polymorpha, andCorynebacterium glutamicumdisplayed specific growth rates under chemically and physically constant conditions that were consistently higher than those obtained in populations. This highlights the importance of single-cell analyses by uncoupling physiology and the extracellular environment, which is now possible using the Envirostat 2.0 concept.

scholarly journals Quantitative Physiology of Saccharomyces cerevisiae at Near-Zero Specific Growth Rates

2009 ◽  
Vol 75 (17) ◽  
pp. 5607-5614 ◽  
Author(s):  
L�onie G. M. Boender ◽  
Erik A. F. de Hulster ◽  
Antonius J. A. van Maris ◽  
Pascale A. S. Daran-Lapujade ◽  
Jack T. Pronk
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Growth Rates ◽  
Specific Growth ◽  
Biomass Concentration ◽  
Product Formation ◽  
Energy Requirements ◽  
Maintenance Energy ◽  
Key Issues ◽  
Complete Cell ◽  
Specific Growth Rates

ABSTRACT Growth at near-zero specific growth rates is a largely unexplored area of yeast physiology. To investigate the physiology of Saccharomyces cerevisiae under these conditions, the effluent removal pipe of anaerobic, glucose-limited chemostat culture (dilution rate, 0.025 h−1) was fitted with a 0.22-μm-pore-size polypropylene filter unit. This setup enabled prolonged cultivation with complete cell retention. After 22 days of cultivation, specific growth rates had decreased below 0.001 h−1 (doubling time of >700 h). Over this period, viability of the retentostat cultures decreased to ca. 80%. The viable biomass concentration in the retentostats could be accurately predicted by a maintenance coefficient of 0.50 mmol of glucose g−1 of biomass h−1 calculated from anaerobic, glucose-limited chemostat cultures grown at dilution rates of 0.025 to 0.20 h−1. This indicated that, in contrast to the situation in several prokaryotes, maintenance energy requirements in S. cerevisiae do not substantially change at near-zero specific growth rates. After 22 days of retentostat cultivation, glucose metabolism was predominantly geared toward alcoholic fermentation to meet maintenance energy requirements. The strict correlation between glycerol production and biomass formation observed at higher specific growth rates was not maintained at the near-zero growth rates reached in the retentostat cultures. In addition to glycerol, the organic acids acetate, d-lactate, and succinate were produced at low rates during prolonged retentostat cultivation. This study identifies robustness and by-product formation as key issues in attempts to uncouple growth and product formation in S. cerevisiae.

scholarly journals Endoplasmic Reticulum-Associated rht-PA Processing in CHO Cells: Influence of Mild Hypothermia and Specific Growth Rates in Batch and Chemostat Cultures

PLoS ONE ◽  
2015 ◽  
Vol 10 (12) ◽  
pp. e0144224 ◽  
Author(s):  
Mauricio Vergara ◽  
Julio Berrios ◽  
Irene Martínez ◽  
Alvaro Díaz-Barrera ◽  
Cristian Acevedo ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Growth Rates ◽  
Cho Cells ◽  
Specific Growth ◽  
Mild Hypothermia ◽  
Chemostat Cultures ◽  
Specific Growth Rates

Morphogenetic expression of Arthrobacter globiformis 425 in continuous culture with carbon or biotin limitation

1978 ◽  
Vol 24 (1) ◽  
pp. 28-30 ◽  
Author(s):  
Adrian P. Wills ◽  
E. C. S. Chan
Keyword(s):  
Growth Rate ◽  
Growth Rates ◽  
Specific Growth ◽  
Normal Growth ◽  
Arthrobacter Globiformis ◽  
Glucose Limitation ◽  
Abnormal Morphology ◽  
Chemostat Cultures ◽  
Specific Growth Rates ◽  
Biotin Limitation

When deprived of biotin, Arthrobacter globiformis 425 exhibits abnormal morphology (large, branched forms of variable size) and a retardation of its normal growth rate. In chemostat cultures, when cells were grown under glucose limitation, the morphology was normal (coccoids or rods) at specific growth rates between 0.05 and 0.125 h−1 (doubling times between 14 and 5.5 h, respectively) at 25 °C. The coccoid-to-rod morphogenesis occurs at a specific growth rate of 0.11 h−1. At the same specific growth rates and temperature, but under biotin limitation, abnormal morphology was observed.

scholarly journals Molecular Analysis of theIn SituGrowth Rates of Subsurface Geobacter Species

2012 ◽  
Vol 79 (5) ◽  
pp. 1646-1653 ◽  
Author(s):  
Dawn E. Holmes ◽  
Ludovic Giloteaux ◽  
Melissa Barlett ◽  
Milind A. Chavan ◽  
Jessica A. Smith ◽  
...  
Keyword(s):  
Growth Rates ◽  
Ribosomal Proteins ◽  
Specific Growth ◽  
Expression Patterns ◽  
Transcript Abundance ◽  
Metal Reduction ◽  
Content Type ◽  
Specific Growth Rates ◽  
Dissimilatory Metal Reduction

ABSTRACTMolecular tools that can provide an estimate of thein situgrowth rate ofGeobacterspecies could improve understanding of dissimilatory metal reduction in a diversity of environments. Whole-genome microarray analyses of a subsurface isolate ofGeobacter uraniireducens, grown under a variety of conditions, identified a number of genes that are differentially expressed at different specific growth rates. Expression of two genes encoding ribosomal proteins,rpsCandrplL, was further evaluated with quantitative reverse transcription-PCR (qRT-PCR) in cells with doubling times ranging from 6.56 h to 89.28 h. Transcript abundance ofrpsCcorrelated best (r2= 0.90) with specific growth rates. Therefore, expression patterns ofrpsCwere used to estimate specific growth rates ofGeobacterspecies during anin situuranium bioremediation field experiment in which acetate was added to the groundwater to promote dissimilatory metal reduction. Initially, increased availability of acetate in the groundwater resulted in higher expression ofGeobacter rpsC, and the increase in the number ofGeobactercells estimated with fluorescentin situhybridization compared well with specific growth rates estimated from levels ofin situ rpsCexpression. However, in later phases, cell number increases were substantially lower than predicted fromrpsCtranscript abundance. This change coincided with a bloom of protozoa and increased attachment ofGeobacterspecies to solid phases. These results suggest that monitoringrpsCexpression may better reflect the actual rate thatGeobacterspecies are metabolizing and growing duringin situuranium bioremediation than changes in cell abundance.

Heat Production and Growth Kinetics of E. coli K12 from Flow Calorimetric Measurements on Chemostat Cultures

1989 ◽  
Vol 44 (11-12) ◽  
pp. 1036-1048 ◽  
Author(s):  
H. P. Leiseifer
Keyword(s):  
Heat Production ◽  
Growth Rates ◽  
Specific Growth ◽  
Batch Cultures ◽  
E Coli ◽  
Chemostat Cultures ◽  
Basic Parameters ◽  
Specific Growth Rates ◽  
The Relationship ◽  
Kinetics Of

The heat production of E. coli K12 growing aerobically in glucose limited chemostat cultures is determined in the range of specific growth rates μ ( = dilution rates D) from 0,058 h-1 to 0.852 h-1 for two different glucose concentrations Se in the instream of the chemostat. namely Se1=0.3182 g·1-1 and Se2 = 0.6364 g·1-1. Heat production Q and biomass production P per unit of culture volume show well correlated patterns for Se1 and Se2. For Se1 the highest value Q actually measured is 443-10-3 W·1-1 at D = 0.74 h-1 with P = 0.068 g·1-1·h-1 and for Se2 593·10-3 W·1-1 at D = 0.497 h-1 with P = 0.108 g·1-1·h-1. Heat production QB per unit of biomass appears to be independent of Se at least up to D - 0.5 h-1.At higher D there is strong indication that QB possesses a real maximum. The highest value of QB actually measured is 4.8 W·g-1 at D = 0.74 h-1. For Se1 and Se2 there were significantly higher specific growth rates verified in chemostat culture than μmaxBatch= 0.717 h-1 which is the maximum specific growth rate in comparable, unlimited batch cultures. The real maximum of QB is estimated to be in the vicinity of μmaxBatch. This suggests the hypothesis of a maximum principle for the growth in batch culture. For Se1 a closed analytical expression is derived for the relationship between μ and the substrate concentration S. μ[S] features a S-shaped characteristic with μmaxChemostat= 0.905 h-1; 1/2 μmaxChemostat is reached at S = 2.85·10-3 g·1-1. Three basic parameters which characterize the overall metabolism of the cells, namely the heat released per unit of substrate consumed, (Qs, the effective yield of biomass, Yeff, and μmaxChemostat are identified to depend on Se.

scholarly journals Contribution of Complex I NADH Dehydrogenase to Respiratory Energy Coupling in Glucose-Grown Cultures of Ogataea parapolymorpha

2020 ◽  
Vol 86 (15) ◽  
Author(s):  
Hannes Juergens ◽  
Xavier D. V. Hakkaart ◽  
Jildau E. Bras ◽  
André Vente ◽  
Liang Wu ◽  
...  
Keyword(s):  
Growth Rates ◽  
Complex I ◽  
Energy Coupling ◽  
Content Type ◽  
Batch Cultures ◽  
Cell Factories ◽  
Chemostat Cultures ◽  
Biomass Yields ◽  
Nadh Dehydrogenases ◽  
Specific Growth Rates

ABSTRACT The thermotolerant yeast Ogataea parapolymorpha (formerly Hansenula polymorpha) is an industrially relevant production host that exhibits a fully respiratory sugar metabolism in aerobic batch cultures. NADH-derived electrons can enter its mitochondrial respiratory chain either via a proton-translocating complex I NADH-dehydrogenase or via three putative alternative NADH dehydrogenases. This respiratory entry point affects the amount of ATP produced per NADH/O2 consumed and therefore impacts the maximum yield of biomass and/or cellular products from a given amount of substrate. To investigate the physiological importance of complex I, a wild-type O. parapolymorpha strain and a congenic complex I-deficient mutant were grown on glucose in aerobic batch, chemostat, and retentostat cultures in bioreactors. In batch cultures, the two strains exhibited a fully respiratory metabolism and showed the same growth rates and biomass yields, indicating that, under these conditions, the contribution of NADH oxidation via complex I was negligible. Both strains also exhibited a respiratory metabolism in glucose-limited chemostat cultures, but the complex I-deficient mutant showed considerably reduced biomass yields on substrate and oxygen, consistent with a lower efficiency of respiratory energy coupling. In glucose-limited retentostat cultures at specific growth rates down to ∼0.001 h−1, both O. parapolymorpha strains showed high viability. Maintenance energy requirements at these extremely low growth rates were approximately 3-fold lower than estimated from faster-growing chemostat cultures, indicating a stringent-response-like behavior. Quantitative transcriptome and proteome analyses indicated condition-dependent expression patterns of complex I subunits and of alternative NADH dehydrogenases that were consistent with physiological observations. IMPORTANCE Since popular microbial cell factories have typically not been selected for efficient respiratory energy coupling, their ATP yields from sugar catabolism are often suboptimal. In aerobic industrial processes, suboptimal energy coupling results in reduced product yields on sugar, increased process costs for oxygen transfer, and volumetric productivity limitations due to limitations in gas transfer and cooling. This study provides insights into the contribution of mechanisms of respiratory energy coupling in the yeast cell factory Ogataea parapolymorpha under different growth conditions and provides a basis for rational improvement of energy coupling in yeast cell factories. Analysis of energy metabolism of O. parapolymorpha at extremely low specific growth rates indicated that this yeast reduces its energy requirements for cellular maintenance under extreme energy limitation. Exploration of the mechanisms for this increased energetic efficiency may contribute to an optimization of the performance of industrial processes with slow-growing eukaryotic cell factories.

scholarly journals Maintenance-energy requirements and robustness of Saccharomyces cerevisiae at aerobic near-zero specific growth rates

2016 ◽  
Vol 15 (1) ◽  
Author(s):  
Tim Vos ◽  
Xavier D. V. Hakkaart ◽  
Erik A. F. de Hulster ◽  
Antonius J. A. van Maris ◽  
Jack T. Pronk ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Growth Rates ◽  
Specific Growth ◽  
Energy Requirements ◽  
Maintenance Energy ◽  
Specific Growth Rates

scholarly journals A Defined, Glucose-Limited Mineral Medium for the Cultivation of Listeria spp.

2013 ◽  
Vol 79 (8) ◽  
pp. 2503-2511 ◽  
Author(s):  
Rudolf Schneebeli ◽  
Thomas Egli
Keyword(s):  
Amino Acids ◽  
Growth Rates ◽  
Specific Growth ◽  
Synthetic Medium ◽  
Brain Heart Infusion ◽  
Mineral Medium ◽  
Content Type ◽  
Biomass Yields ◽  
Minimal Media ◽  
Specific Growth Rates

ABSTRACTMembers of the genusListeriaare fastidious bacteria with respect to their nutritional requirements, and several minimal media described in the literature fail to support growth of allListeriaspp. Furthermore, strict limitation by a single nutrient, e.g., the carbon source, has not been demonstrated for any of the published minimal media. This is an important prerequisite for defined studies of growth and physiology, including “omics.” Based on a theoretical analysis of previously published mineral media forListeria, an improved, well-balanced growth medium was designed. It supports the growth, not only of all testedListeria monocytogenesstrains, but of all otherListeriaspecies, with the exception ofL. ivanovii. The growth performance ofL. monocytogenesstrain Scott A was tested in the newly designed medium; glucose served as the only carbon and energy source for growth, whereas neither the supplied amino acids nor the buffering and complexing components (MOPS [morpholinepropanesulfonic acid] and EDTA) supported growth. Omission of amino acids, trace elements, or vitamins, alone or in combination, resulted in considerably reduced biomass yields. Furthermore, we monitored the specific growth rates of variousListeriastrains cultivated in the designed mineral medium and compared them to growth in complex medium (brain heart infusion broth [BHI]). The novel mineral medium was optimized for the commonly used strainL. monocytogenesScott A to achieve optimum cell yields and maximum specific growth rates. This mineral medium is the first published synthetic medium forListeriathat has been shown to be strictly carbon (glucose) limited.

scholarly journals Respiratory Physiology of Lactococcus lactis in Chemostat Cultures and Its Effect on Cellular Robustness in Frozen and Freeze-Dried Starter Cultures

2020 ◽  
Vol 86 (6) ◽  
Author(s):  
Anna Johanson ◽  
Anisha Goel ◽  
Lisbeth Olsson ◽  
Carl Johan Franzén
Keyword(s):  
Lactococcus Lactis ◽  
Specific Growth ◽  
Starter Cultures ◽  
Respiratory Metabolism ◽  
Cultivation Conditions ◽  
Content Type ◽  
Chemostat Cultures ◽  
Freeze Dried ◽  
Specific Growth Rates

ABSTRACT In this study, we used chemostat cultures to analyze the quantitative effects of the specific growth rate and respiration on the metabolism in Lactococcus lactis CHCC2862 and on the downstream robustness of cells after freezing or freeze-drying. Under anaerobic conditions, metabolism remained homofermentative, although biomass yields varied with the dilution rate (D). In contrast, metabolism shifted with the dilution rate under respiration-permissive conditions. At D = 0.1 h−1, no lactate was produced, while lactate formation increased with higher dilution rates. Thus, a clear metabolic shift was observed, from flavor-forming respiratory metabolism at low specific growth rates to mixed-acid respiro-fermentative metabolism at higher specific growth rates. Quantitative analysis of the respiratory activity, lactose uptake rate, and metabolite production rates showed that aerobic acetoin formation provided most of the NADH consumed in respiration. Moreover, the maintenance-associated lactose consumption under respiration-permissive conditions was only 10% of the anaerobic value, either due to higher respiratory yield of ATP on consumed lactose or due to lower maintenance-related ATP demand. The cultivation conditions also affected the quality of the starter cultures produced. Cells harvested under respiration-permissive conditions at D = 0.1 h−1 were less robust after freeze-drying and had lower acidification activity for subsequent milk acidification, whereas respiration-permissive conditions at the higher dilution rates led to robust cells that performed equally well or better than anaerobic cells. IMPORTANCE Lactococcus lactis is used in large quantities by the food and biotechnology industries. L. lactis can use oxygen for respiration if heme is supplied in the growth medium. This has been extensively studied in batch cultures using various mutants, but quantitative studies of how the cell growth affects respiratory metabolism, energetics, and cell quality are surprisingly scarce. Our results demonstrate that the respiratory metabolism of L. lactis is remarkably flexible and can be modulated by controlling the specific growth rate. We also link the physiological state of cells during cultivation to the quality of frozen or freeze-dried cells, which is relevant to the industry that may lack understanding of such relationships. This study extends our knowledge of respiratory metabolism in L. lactis and its impact on frozen and freeze-dried starter culture products, and it illustrates the influence of cultivation conditions and microbial physiology on the quality of starter cultures.

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