Optimality of extracellular enzyme production and activity in dynamic flux balance modeling

In microbial communities, many vital metabolic functions, including the degradation of cellulose, proteins and other complex macromolecules, are carried out by costly, extracellularly secreted enzymes. While significant effort has been dedicated to analyzing genome-scale metabolic networks for individual microbes and communities, little is known about the interplay between global allocation of metabolic resources in the cell and extracellular enzyme secretion and activity. Here we introduce a method for modeling the secretion and catalytic functions of extracellular enzymes using dynamic flux balance analysis. This new addition, implemented within COMETS (Computation Of Microbial Ecosystems in Time and Space), simulates the costly production and secretion of enzymes and their diffusion and activity throughout the environment, independent of the producing organism. After tuning our model based on data for a Saccharomyces cerevisiae strain engineered to produce exogenous cellulases, we explored the dynamics of the system at different cellulose concentrations and enzyme production rates. We found that there are distinct rates of constitutive enzyme secretion which maximize either growth rate or biomass yield. These optimal rates are strongly dependent on enzyme kinetic properties and environmental conditions, including the amount of cellulose substrate available. Our framework will facilitate the development of more realistic simulations of microbial community dynamics within environments rich in complex macromolecules, with applications in the study of soil and plant-associated ecosystems, and other natural and engineered microbiomes.

Download Full-text

DYNAMIC FLUX BALANCE ANALYSIS FOR PREDICTING GENE OVEREXPRESSION EFFECTS IN BATCH CULTURES

Journal of Biological System ◽

10.1142/s0218339014500107 ◽

2014 ◽

Vol 22 (03) ◽

pp. 327-338 ◽

Cited By ~ 2

Author(s):

CAROL MILENA BARRETO-RODRIGUEZ ◽

JESSICA PAOLA RAMIREZ-ANGULO ◽

JORGE MARIO GOMEZ RAMIREZ ◽

LUKE ACHENIE ◽

HAROLD MOLINA-BULLA ◽

...

Keyword(s):

Flux Balance Analysis ◽

Metabolic Networks ◽

Ethanol Concentration ◽

Batch Cultures ◽

Dynamic Flux Balance Analysis ◽

Optimization Framework ◽

Flux Balance ◽

Balance Analysis ◽

Engineering Strategy ◽

Technological Platforms

The advent of numerous technological platforms for genome sequencing has led to increasing understanding and construction of metabolic networks. A popular system engineering strategy is used to analyze microbial metabolic networks is flux balance analysis (FBA). In recent times, there has been a lot of interest in the study of the metabolic network dynamics when genes are overexpressed in the system. Herein, an optimization framework, which employs dynamic flux balance analysis (DFBA) is proposed for predicting ethanol concentration profiles in glycerol fermentations using Escherichia coli. In silico results were experimentally validated by overexpressing alcohol/acetaldehyde dehydrogenase adhE, pyruvate kinase pykF, pyruvate formate-lyase pflB and isoleucine-valine enzymes ilvC and llvL.

Download Full-text

Unravelling the genetic basis for competence development of auxotrophic Bacillus licheniformis 9945A strains

Microbiology ◽

10.1099/mic.0.079236-0 ◽

2014 ◽

Vol 160 (10) ◽

pp. 2136-2147 ◽

Cited By ~ 6

Author(s):

Mareike Jakobs ◽

Kerstin Hoffmann ◽

Anja Grabke ◽

Stefania Neuber ◽

Heiko Liesegang ◽

...

Keyword(s):

Bacillus Licheniformis ◽

Extracellular Enzymes ◽

Random Mutagenesis ◽

Extracellular Enzyme ◽

Competence Development ◽

Colony Morphology ◽

Enzyme Secretion ◽

Dna Uptake ◽

Genetic Competence ◽

Regulator Genes

Bacterial natural genetic competence – well studied in Bacillus subtilis – enables cells to take up and integrate extracellularly supplied DNA into their own genome. However, little is known about competence development and its regulation in other members of the genus, although DNA uptake machineries are routinely encoded. Auxotrophic Bacillus licheniformis 9945A derivatives, obtained from repeated rounds of random mutagenesis, were long known to develop natural competence. Inspection of the colony morphology and extracellular enzyme secretion of two of these derivatives, M28 and M18, suggested that regulator genes are collaterally hit. M28 emerged as a 14 bp deletion mutant concomitantly displaying a shift in the reading frame of degS that encodes the sensor histidine kinase, which is part of the molecular switch that directs cells to genetic competence, the synthesis of extracellular enzymes or biofilm formation, while for M18, sequencing of the suspected gene revealed a 375 bp deletion in abrB, encoding the major transition state regulator. With respect to colony morphology, enzyme secretion and competence development, both of the mutations, when newly generated on the wild-type B. licheniformis 9945A genetic background, resulted in phenotypes resembling M28 and M18, respectively. All of the known naturally competent B. licheniformis representatives, hitherto thoroughly investigated in this regard, carry mutations in regulator genes, and hence genetic competence observed in domesticated strains supposedly results from deregulation.

Download Full-text

Effect of quorum sensing agents on the growth kinetics of Pseudomonas spp. of raw milk origin

Journal of Dairy Research ◽

10.1017/s0022029905000713 ◽

2005 ◽

Vol 72 (3) ◽

pp. 276-280 ◽

Cited By ~ 28

Author(s):

George Dunstall ◽

Michael T Rowe ◽

G Brian Wisdom ◽

David Kilpatrick

Keyword(s):

Quorum Sensing ◽

Pseudomonas Fluorescens ◽

Growth Kinetics ◽

Enzyme Production ◽

Extracellular Enzymes ◽

Extracellular Enzyme ◽

Raw Milk ◽

Homoserine Lactone ◽

Lag Phase ◽

Phase Duration

Psychrotrophs, particularly strains of Pseudomonas fluorescens, dominate the microflora of refrigerated raw milk and secrete heat-stable extracellular enzymes (proteases and lipases) which survive pasteurisation and even UHT heat treatments and degrade the casein and fat components of raw milk causing a reduction in cheese yield, gelation of UHT milk and off flavours in many dairy products. These enzymes are usually produced in the late log/early stationary growth phases when the cell density is high. This fact indicated that induction of these enzymes may be a candidate for quorum sensing, a phenomenon by which bacteria can sense and respond to cell population size by means of chemical signals based on the homoserine lactone molecule. It is assumed that the production of these enzymes contributes to the selective advantage of psychrotrophs and hence have a significant effect on their growth kinetics. In the work reported here nine proprietary homoserine lactone compounds were screened, using water as a control, for their effect on the lag phase duration (LPD) and exponential growth rate (EGR) of three strains of Pseudomonas fluorescens isolated from refrigerated raw milk after 1, 3 and 5 days storage. Two compounds viz. N-benzoyloxycarbonyl-L-homoserine lactone and N-3-oxyhexanoyl-DL-homoserine lactone were found to significantly (P<0·001) reduce the LPD and increase the EGR of the three strains. Further work with these compounds is warranted, monitoring growth in parallel with enzyme production, to determine the extent to which extracellular enzyme production is mediated by the quorum sensing phenomenon in this species.

Download Full-text