A computational tool for the simulation and optimization of microbial strains accounting integrated metabolic/regulatory information

Background and scope

Recently, a number of methods and tools have been proposed to allow the use of genome-scale metabolic models for the phenotype simulation and optimization of microbial strains, within the field of Metabolic Engineering (ME). One of the limitations of most of these algorithms and tools is the fact that only metabolic information is taken into account, disregarding knowledge on regulatory events.

Implementation and performances

This work proposes a novel software tool that implements methods for the phenotype simulation and optimization of microbial strains using integrated models, encompassing both metabolic and regulatory information. This tool is developed as a plug-in that runs over OptFlux, a computational platform that aims to be a reference tool for the ME community.

Availability

The plug-in is made available in the OptFlux web site (www.optflux.org) together with examples and documentation.     

Metabolic engineering in dark fermentative hydrogen production; theory and practice

Dark fermentation is an attractive option for hydrogen production since it could use already existing reactor technology and readily available substrates without requiring a direct input of solar energy. However, a number of improvements are required before the rates and yields of such a process approach those required for a practical process. Among the options for achieving the required advances, metabolic engineering offers some powerful tools for remodeling microbes to increase product production rates and molar yields. Here we review the current metabolic engineering tool box that is available, discuss the current status of engineering efforts as applied to dark hydrogen production, and suggest areas for future improvements.     

核磁共振技术在微生物代谢研究中的应用

介绍了核磁共振的种类,并结合具体实例阐述了核磁共振技术在微生物代谢研究领域的应用。主要包括在代谢工程、环境保护及生态学研究、以及人与动物健康几个方面的应用。     

Clostridium cellulolyticum: model organism of mesophilic cellulolytic clostridia

Clostridium cellulolyticum ATCC 35319 is a non-ruminal mesophilic cellulolytic bacterium originally isolated from decayed grass. As with most truly cellulolytic clostridia, C. cellulolyticum possesses an extracellular multi-enzymatic complex, the cellulosome. The catalytic components of the cellulosome release soluble cello-oligosaccharides from cellulose providing the primary carbon substrates to support bacterial growth. As most cellulolytic bacteria, C. cellulolyticum was initially characterised by limited carbon consumption and subsequent limited growth in comparison to other saccharolytic clostridia. The first metabolic studies performed in batch cultures suggested nutrient(s) limitation and/or by-product(s) inhibition as the reasons for this limited growth. In most recent investigations using chemostat cultures, metabolic flux analysis suggests a self-intoxication of bacterial metabolism resulting from an inefficiently regulated carbon flow. The investigation of C. cellulolyticum physiology with cellobiose, as a model of soluble cellodextrin, and with pure cellulose, as a carbon source more closely related to lignocellulosic compounds, strengthen the idea of a bacterium particularly well adapted, and even restricted, to a cellulolytic lifestyle. The metabolic flux analysis from continuous cultures revealed that (i) in comparison to cellobiose, the cellulose hydrolysis by the cellulosome introduces an extra regulation of entering carbon flow resulting in globally lower metabolic fluxes on cellulose than on cellobiose, (ii) the glucose 1-phosphate/glucose 6-phosphate branch point controls the carbon flow directed towards glycolysis and dissipates carbon excess towards the formation of cellodextrins, glycogen and exopolysaccharides, (iii) the pyruvate/acetyl-CoA metabolic node is essential to the regulation of electronic and energetic fluxes. This in-depth analysis of C. cellulolyticum metabolism has permitted the first attempt to engineer metabolically a cellulolytic microorganism.     

ATP in current biotechnology: Regulation,applications and perspectives

Adenosine tri-phosphate (ATP), the most important energy source for metabolic reactions and pathways, plays a vital role in the growth of industrial strain and the production of target metabolites. In this review, current advances in manipulating ATP in industrial strains, including altering NADH availability, and regulating NADH oxidation pathway, oxygen supply, proton gradient, the electron transfer chain activity and the F₀F₁-ATPase activity, are summarized and discussed. By applying these strategies, optimal product concentrations, yields and productivity in industrial biotechnology have been achieved. Furthermore, the mechanisms by which ATP extends the substrate utilization spectra and enhances the ability to challenge harsh environmental stress have been elucidated. Finally, three critical issues related to ATP manipulation have been addressed.     

固碳产油微藻的基因工程改造

藻种的选育和基因工程改造是微藻生物柴油研究的核心。为此,简要综述了微藻从光合作用到甘油三酯(TAG)合成过程中的关键基因及其代谢调控等方面的研究进展。从光合作用固碳、中心碳代谢、脂肪酸合成、TAG的组装、抑制TAG合成的竞争途径及脂类的分解途径等几个方面入手,浅析各个代谢途径中关键基因的作用及其表达调控。在此基础上,探讨微藻基因工程改造的可行性并指出微藻生物柴油在生物质能源领域中的前景及其综合利用的发展优势。     

Engineering glycolysis branch pathways of Escherichia coli to improve heterologous protein expression

Escherichia coli expression systems are still preferred to other bacterial expression systems. However, by-product formation via glycolytic pathways inhibits protein production efficiency. In this paper, by-product-forming pathways were engineered to evaluate their effect on foreign protein production. Elimination of d-lactate dehydrogenase (encoded by ldhA) resulted in enhanced cell performance and 17.8% increase in recombinant β-mannanase production. Single deletions of pflB (encoding pyruvate formate lyase), pps (encoding phoenolpyruvate synthase) or poxB (encoding pyruvate oxidase) also had an affirmative impact on recombinant protein production. Furthermore, simultaneous deletions of ldhA, pflB, pps and poxB increased cell mass by 29% and β-mannanase production by 56% under shake-flasks cultivation. Meanwhile, overall acetate concentration showed a decrease of 33%. This quadruple mutant showed the best performance under bioreactor process, in which volume and specific activity of β-mannanase increased by 1.9 and 1.46 fold compared to the control strain respectively. The approach shown here indicated that rational engineering of glycolytic pathways can efficiently improve foreign protein production in E. coli.     

Development of a kinetic metabolic model: application to Catharanthus roseus hairy root

A kinetic metabolic model describing Catharanthus roseus hairy root growth and nutrition was developed. The metabolic network includes glycolysis, pentose-phosphate pathway, TCA cycle and the catabolic reactions leading to cell building blocks such as amino acids, organic acids, organic phosphates, lipids and structural hexoses. The central primary metabolic network was taken at pseudo-steady state and metabolic flux analysis technique allowed reducing from 31 metabolic fluxes to 20 independent pathways. Hairy root specific growth rate was described as a function of intracellular concentration in cell building blocks. Intracellular transport and accumulation kinetics for major nutrients were included. The model uses intracellular nutrients as well as energy shuttles to describe metabolic regulation. Model calibration was performed using experimental data obtained from batch and medium exchange liquid cultures of C. roseus hairy root using a minimal medium in Petri dish. The model is efficient in estimating the growth rate.     

Optimal re-design of primary metabolism in Escherichia coli using linlog kinetics

This paper examines the validity of the linlog approach, which was recently developed in our laboratory, by comparison of two different kinetic models for the metabolic network of Escherichia coli. The first model is a complete mechanistic model; the second is an approximative model in which linlog kinetics are applied. The parameters of the linlog model (elasticities) are derived from the mechanistic model. Three different optimization cases are examined. In all cases, the objective is to calculate the enzyme levels that maximize a certain flux while keeping the total amount of enzyme constant and preventing large changes of metabolite concentrations. For an average variation of metabolite levels of 10% and individual changes of a factor 2, the predicted enzyme levels, metabolite concentrations and fluxes of both models are highly similar. This similarity holds for changes in enzyme level of a factor 4-6 and for changes in fluxes up to a factor 6. In all three cases, the predicted optimal enzyme levels could neither have been found by intuition-based approaches, nor on basis of flux control coefficients. This demonstrates that kinetic models are essential tools in Metabolic Engineering. In this respect, the linlog approach is a valuable extension of MCA, since it allows construction of kinetic models, based on MCA parameters, that can be used for constrained optimization problems and are valid for large changes of metabolite and enzyme levels.