Ezequiel Franco-Lara

Flux Design: In silico design of cell factories based on correlation of pathway fluxes to desired properties

BackgroundThe identification of genetic target genes is a key step for rational engineering of production strains towards bio-based chemicals, fuels or therapeutics. This is often a difficult task, because superior production performance typically requires a combination of multiple targets, whereby the complex metabolic networks complicate straightforward identification. Recent attempts towards target prediction mainly focus on the prediction of gene deletion targets and therefore can cover only a part of genetic modifications proven valuable in metabolic engineering. Efficient in silico methods for simultaneous genome-scale identification of targets to be amplified or deleted are still lacking.ResultsHere we propose the identification of targets via flux correlation to a chosen objective flux as approach towards improved biotechnological production strains with optimally designed fluxes. The approach, we name Flux Design, computes elementary modes and, by search through the modes, identifies targets to be amplified (positive correlation) or down-regulated (negative correlation). Supported by statistical evaluation, a target potential is attributed to the identified reactions in a quantitative manner. Based on systems-wide models of the industrial microorganisms Corynebacterium glutamicum and Aspergillus niger, up to more than 20,000 modes were obtained for each case, differing strongly in production performance and intracellular fluxes. For lysine production in C. glutamicum the identified targets nicely matched with reported successful metabolic engineering strategies. In addition, simulations revealed insights, e.g. into the flexibility of energy metabolism. For enzyme production in A.niger flux correlation analysis suggested a number of targets, including non-obvious ones. Hereby, the relevance of most targets depended on the metabolic state of the cell and also on the carbon source.ConclusionsObjective flux correlation analysis provided a detailed insight into the metabolic networks of industrially relevant prokaryotic and eukaryotic microorganisms. It was shown that capacity, pathway usage, and relevant genetic targets for optimal production partly depend on the network structure and the metabolic state of the cell which should be considered in future metabolic engineering strategies. The presented strategy can be generally used to identify priority sorted amplification and deletion targets for metabolic engineering purposes under various conditions and thus displays a useful strategy to be incorporated into efficient strain and bioprocess optimization.

High-yield intra- and extracellular protein production using Bacillus megaterium.

ABSTRACT The Bacillus megaterium protein production system based on the inducible promoter of the xyl operon (PxylA) was systematically optimized. Multiple changes in basic promoter elements, such as the −10 and −35 region and the ribosome-binding site, resulted in an 18-fold increase of protein production compared to the production of the previously established system. The production in shaking-flask culture of green fluorescent protein (Gfp) as a model product led to 82.5 mg per g cell dry weight (gCDW) or 124 mg liter−1. In fed-batch cultivation, the volumetric protein yield was increased 10-fold to 1.25 g liter−1, corresponding to 36.8 mg protein per gCDW. Furthermore, novel signal peptides for Sec-dependent protein secretion were predicted in silico using the B. megaterium genome. Subsequently, leader peptides of Vpr, NprM, YngK, YocH, and a computationally designed artificial peptide were analyzed experimentally for their potential to facilitate the secretion of the heterologous model protein Thermobifida fusca hydrolase (Tfh). The best extracellular protein production, 5,000 to 6,200 U liter−1 (5.3 to 6.6 mg liter−1), was observed for strains where the Tfh export was facilitated by a codon-optimized leader peptide of YngK and by the signal peptide of YocH. Further increases in extracellular protein production were achieved when leader peptides were used in combination with the optimized expression system. In this case, the greatest extracellular enzyme amount of 7,200 U liter−1, 7.7 mg liter−1, was achieved by YocH leader peptide-mediated protein export. Nevertheless, the observed principal limitations in protein export might be related to components of the Sec-dependent protein transport system.

Microfluidic reactor for continuous cultivation of Saccharomyces cerevisiae

A diffusion‐based microreactor system operated with a reaction volume of 8 μL is presented and characterized to intensify the process understanding in microscale cultivations. Its potential as screening tool for biological processes is evaluated. The advantage of the designed microbioreactor is the use for the continuous cultivation mode by integrating online measurement technique for dissolved oxygen (DO) and optical density (OD). A further advantage is the broaden application for biological systems. The bioreactor geometry was chosen to achieve homogeneous flow during continuous process operation. The device consisted of a microstructured top layer made of poly(dimethylsiloxane) (PDMS), which was designed and fabricated using UV‐depth and soft lithography assembled with a glass bottom. CFD simulation data used for geometry design were verified via microparticle‐image‐velocimetry (μPIV). In the used microreactor geometry no concentration gradients occurred along the entire reaction volume because of rapid diffusive mixing, the homogeneous medium flow inside the growth chamber of the microreactor could be realized. Undesirable bubble formation before and during operation was reduced by using degassed medium as well as moistened and moderate incident air flow above the gas permeable PDMS membrane. Because of this a passive oxygen supply of the culture medium in the device is ensured by diffusion through the PDMS membrane. The oxygen supply itself was monitored online via integrated DO sensors based on a fluorescent dye complex. An adequate overall volumetric oxygen transfer coefficient KLa as well as mechanical stability of the device were accomplished for a membrane thickness of 300 μm. Experimental investigations considering measurements of OD (online) and several metabolite concentrations (offline) in a modified Verduyn medium. The used model organism Saccharomyces cerevisiae DSM 2155 tended to strong reactor wall growth resembling a biofilm.

Process Optimization of the Integrated Synthesis and Secretion of Ectoine and Hydroxyectoine Under Hyper/Hypo-Osmotic Stress

The synthesis and secretion of the industrial relevant compatible solutes ectoine and hydroxyectoine using the halophile bacterium Chromohalobacter salexigens were studied and optimized. For this purpose, a cascade of two continuously operated bioreactors was used. In the first bioreactor, cells were grown under constant hyperosmotic conditions and thermal stress driving the cells to accumulate large amounts of ectoines. To enhance the overall productivity, high cell densities up to 61 g L−1 were achieved using a cross‐flow ultrafiltration connected to the first bioreactor. In the coupled second bioreactor the concentrated cell broth was subjected to an osmotic and thermal down‐shock by addition of fresh distilled water. Under these conditions, the cells are forced to secrete the accumulated intracellular ectoines into the medium to avoid bursting. The cultivation conditions in the first bioreactor were optimized with respect to growth temperature and medium salinity to reach the highest synthesis (productivity); the second bioreactor was optimized using a multi‐objective approach to attain maximal ectoine secretion with simultaneous minimization of cell death and product dilution caused by the osmotic and thermal down‐shock. Depending on the cultivation conditions, intracellular ectoine and hydroxyectoine contents up to 540 and 400 mg per g cell dry weight, respectively, were attained. With a maximum specific growth rate of 0.3 h−1 in defined medium, productivities of approximately 2.1 g L−1 h−1 secreted ectoines in continuous operation were reached. Biotechnol. Bioeng. 2010;107: 124–133.

Proteome analysis of the Escherichia coli heat shock response under steady-state conditions

In this study a proteomic approach was used to investigate the steady-state response of Escherichia coli to temperature up-shifts in a cascade of two continuously operated bioreactors. The first reactor served as cell source with optimal settings for microbial growth, while in the second chemostat the cells were exposed to elevated temperatures. By using this reactor configuration, which has not been reported to be used for the study of bacterial stress responses so far, it is possible to study temperature stress under well-defined, steady-state conditions. Specifically the effect on the cellular adaption to temperature stress using two-dimensional gel electrophoresis was examined and compared at the cultivation temperatures of 37°C and 47.5°C. As expected, the steady-state study with the double bioreactor configuration delivered a different protein spectrum compared to that obtained with standard batch experiments in shaking flasks and bioreactors. Setting a high cut-out spot-to-spot size ratio of 5, proteins involved in defence against oxygen stress, functional cell envelope proteins, chaperones and proteins involved in protein biosynthesis, the energy metabolism and the amino acid biosynthesis were found to be differently expressed at high cultivation temperatures. The results demonstrate the complexity of the stress response in a steady-state culture not reported elsewhere to date.

Fast sampling and quenching procedures for microbial metabolic profiling

A reliable quantification of intracellular concentrations of intermediates in microorganisms depends on a proper sampling procedure and the subsequent fast inactivation of metabolism via quenching. A single device integrating both operations was developed and simultaneously the quenching procedure on cells was assessed too, without finding negative effects on viability or metabolite leakage. Moreover, supported by an experimental design, the influences of process parameters in its dynamic operation were characterized and optimized. The novel in-situ rapid sampling and quenching apparatus can be employed on any laboratory glass fermenters accessible from the top of the bioreactor.

Rapid sampling devices for metabolic engineering applications

A number of rapid sampling devices for metabolic engineering applications have been developed over the last years with the purpose of the estimation of in vivo metabolic concentrations and dynamics. This review outlines the designs and characteristics as well as the developments and changes in diverse approaches over the years. Primary performance parameters for these constructions are sampling time and rate and, for an accurate representation of the in vivo condition in cells, the reproducibility of results and easy handling throughout the sampling operation.

Debottlenecking recombinant protein production in Bacillus megaterium under large-scale conditions—targeted precursor feeding designed from metabolomics

In the present work the impact of large production scale was investigated for Bacillus megaterium expressing green fluorescent protein (GFP). Specifically designed scale‐down studies, mimicking the intermittent and continuous nutrient supply of large‐ and small‐scale processes, were carried out for this purpose. The recombinant strain revealed a 40% reduced GFP yield for the large‐scale conditions. In line with extended carbon loss via formation of acetate and carbon dioxide, this indicated obvious limitations in the underlying metabolism of B. megaterium under the large‐scale conditions. Quantitative analysis of intracellular amino acids via validated fast filtration protocols revealed that their level strongly differed between the two scenarios. During cultivation in large‐scale set‐up, the availability of most amino acids, serving as key building blocks of the recombinant protein, was substantially reduced. This was most pronounced for tryptophan, aspartate, histidine, glutamine, and lysine. In contrast alanine was increased, probably related to a bottleneck at the level of pyruvate which also triggered acetate overflow metabolism. The pre‐cursor quantifications could then be exploited to verify the presumed bottlenecks and improve recombinant protein production under large‐scale conditions. Addition of only 5 mM tryptophan, aspartate, histidine, glutamine, and lysine to the feed solution increased the GFP yield by 100%. This rational concept of driving the lab scale productivity of recombinant microorganisms under suboptimal feeding conditions emulating large scale can easily be extended to other processes and production hosts. Biotechnol. Bioeng. 2012; 109:1538–1550.

Polyelectrolyte multilayer surface functionalization of poly(dimethylsiloxane) (PDMS) for reduction of yeast cell adhesion in microfluidic devices

Polyelectrolyte multilayers (PEMs) based on the combinations poly(diallyldimethylammonium chloride)∕poly(acrylic acid) (PDADMAC∕PAA) and poly(allylamine hydrochloride)∕PAA (PAH∕PAA) were adsorbed on poly(dimethylsiloxane) (PDMS) and tested for nonspecific surface attachment of hydrophobic yeast cells using a parallel plate flow chamber. A custom-made graft copolymer containing poly(ethylene glycol) (PEG) side chains (PAA-g-PEG) was additionally adsorbed on the PEMs as a terminal layer. A suitable PEM modification effectively decreased the adhesion strength of Saccharomyces cerevisiae DSM 2155 to the channel walls. However, a further decrease in initial cell attachment and adhesion strength was observed after adsorption of PAA-g-PEG copolymer onto PEMs from aqueous solution. The results demonstrate that a facile layer-by-layer surface functionalization from aqueous solutions can be successfully applied to reduce cell adhesion strength of S. cerevisiae by at least two orders of magnitude compared to bare PDMS. Therefore, this method is potentially suitable to promote planktonic growth inside capped PDMS-based microfluidic devices if the PEM deposition is completed by a dynamic flow-through process.

Monitoring and control of microbioreactors: An expert opinion on development needs

This perspective article is based on an expert panel review on microbioreactor applications in biochemical and biomedical engineering that was organized by the M³C (measurement, monitoring, modelling and control) Working Group of the European Section of Biochemical Engineering Science (ESBES) in the European Federation of Biotechnology (EFB). The aim of the panel was to provide an updated view on the present status of the subject and to identify critical needs and issues for furthering the successful development of microbioreactor monitoring and control. This will benefit future bioprocess development and in vitro toxicity testing. The article concludes with a set of recommendations for extended use and further development of microbioreactors.