Segregostat: a novel concept to control phenotypic diversification dynamics on the example of Gram-negative bacteria.

Controlling and managing the degree of phenotypic diversification of microbial populations is a challenging task. This task not only requires detailed knowledge regarding diversification mechanisms but also advanced technical set-ups for the real-time analyses and control of population behaviour on single-cell level. In this work, set-up, design and operation of the so called segregostat are described which, in contrast to a traditional chemostat, allows the control of phenotypic diversification of microbial populations over time. Two exemplary case studies will be discussed, i.e. phenotypic diversification dynamics of Eschericia coli and Pseudomonas putida based on outer membrane permeabilization, emphasizing the applicability and versatility of the proposed approach. Upon nutrient limitation, cell population tends to diversify into several subpopulations exhibiting distinct phenotypic features (non-permeabilized and permeabilized cells). Online analysis leads to the determination of the ratio between cells in these two states, which in turn triggers the addition of glucose pulses in order to maintain a predefined diversification ratio. These results prove that phenotypic diversification can be controlled by means of defined pulse-frequency modulation within continuously running bioreactor set-ups. This lays the foundation for systematic studies, not only of phenotypic diversification but also for all processes where dynamics single-cell approaches are required, such as synthetic co-culture processes.

Growth-dependent recombinant product formation kinetics can be reproduced through engineering of glucose transport and is prone to phenotypic heterogeneity.

BACKGROUND: Escherichia coli W3110 and a group of six isogenic derivatives, each displaying distinct specific rates of glucose consumption were characterized to determine levels of GFP production and population heterogeneity. These strains have single or combinatory deletions in genes encoding phosphoenolpyruvate:sugar phosphotransferase system (PTS) permeases as PtsG and ManX, as well as common components EI, Hpr protein and EIIA, also the non-PTS Mgl galactose/glucose ABC transporter. They have been transformed for expressing GFP based on a lac-based expression vector, which is subject to bistability. RESULTS: These strains displayed specific glucose consumption and growth rates ranging from 1.75 to 0.45 g/g h and 0.54 to 0.16 h-1, respectively. The rate of acetate production was strongly reduced in all mutant strains when compared with W3110/pV21. In bioreactor cultures, wild type W3110/pV21 produced 50.51 mg/L GFP, whereas strains WG/pV21 with inactive PTS IICBGlc and WGM/pV21 with the additional inactivation of PTS IIABMan showed the highest titers of GFP, corresponding to 342 and 438 mg/L, respectively. Moreover, we showed experimentally that bistable expression systems, as lac-based ones, induce strong phenotypic segregation among microbial populations. CONCLUSIONS: We have demonstrated that reduction on glucose consumption rate in E. coli leads to an improvement of GFP production. Furthermore, from the perspective of phenotypic heterogeneity, we observed in this case that heterogeneous systems are also the ones leading to the highest performance. This observation suggests reconsidering the generally accepted proposition stating that phenotypic heterogeneity is generally unwanted in bioprocess applications.

pH level has a strong impact on population dynamics of the yeast Yarrowia lipolytica and oil micro-droplets in multiphasic bioreactor.

The oleaginous yeast Yarrowia lipolytica has the ability to use oils and fats as carbon source, making it a promising cell factory for the design of alternative bioprocesses based on renewable substrates. However, such a multiphasic bioreactor design is rather complex and leads to several constraints when considering emulsification of the oil-in-water mixture, foaming and cell growth/physiology on hydrophobic substrate. This study aims to shed light on the effect of pH changes on the physico-chemical properties of the cultivation medium and on cell physiology. It was indeed observed that at a pH value of 6, cell growth rate and intracellular lipid accumulation were optimized. Additionally, foaming was significantly reduced. In order to avoid over foaming in bioreactor, without impairing cell physiology, the use of alternative processes that can only act on the physical structure of culture medium, seems to be an effective alternative to usual chemical anti-foam agents.

Taking control over microbial populations: Current approaches for exploiting biological noise in bioprocesses.

Phenotypic plasticity of microbial cells has attracted much attention and several research efforts have been dedicated to the description of methods aiming at characterizing phenotypic heterogeneity and its impact on microbial populations. However, different approaches have also been suggested in order to take benefit from noise in a bioprocess perspective, e.g. by increasing the robustness or productivity of a microbial population. This review is dedicated to outline these controlling methods. A common issue, that has still to be addressed, is the experimental identification and the mathematical expression of noise. Indeed, the effective interfacing of microbial physiology with external parameters that can be used for controlling physiology depends on the acquisition of reliable signals. Latest technologies, like single cell microfluidics and advanced flow cytometric approaches, enable linking physiology, noise, heterogeneity in productive microbes with environmental cues and hence allow correctly mapping and predicting biological behavior via mathematical representations. However, like in the field of electronics, signals are perpetually subjected to noise. If appropriately interpreted, this noise can give an additional insight into the behavior of the individual cells within a microbial population of interest. This review focuses on recent progress made at describing, treating and exploiting biological noise in the context of microbial populations used in various bioprocess applications.

pH and Not Cell Morphology Modulate pLIP2 Induction in the Dimorphic Yeast Yarrowia lipolytica.

The dimorphic yeast Yarrowia lipolytica has become an emerging cell factory for recombinant proteins production. Expression vectors involving LIP2 promoter (pLIP2) have been developed and used successfully. However, the relationship between dimorphic transition (i.e., cell morphology) and pLIP2 regulation is still unclear and must be assessed to improve process robustness. This requests to discriminate the effect of cell morphology from that of effectors, such as pH, that trigger the dimorphic transition. This was performed using gene reporter system based on ß-galactosidase activity and DsRed fluorescence, single-cell analysis by flow cytometry, and quantification of gene expression. Our results clearly pointed out that cell morphology has not effect on the regulation of pLIP2. By contrast, pH modification yielded to phenotypic heterogeneity, potentially leading to a lack of robustness of the cell population. Taken altogether, our results demonstrated that, under appropriate environmental conditions (e.g., pH being an important factor), Y. lipolytica could be considered as a robust and reliable host for recombinant protein production.

Deciphering how LIP2 and POX2 promoters can optimally regulate recombinant protein production in the yeast Yarrowia lipolytica.

BACKGROUND: In recent years, the non-conventional model yeast species Yarrowia lipolytica has received much attention because it is a useful cell factory for producing recombinant proteins. In this species, expression vectors involving LIP2 and POX2 promoters have been developed and used successfully for protein production at yields similar to or even higher than those of other cell factories, such as Pichia pastoris. However, production processes involving these promoters can be difficult to manage, especially if carried out at large scales in fed-batch bioreactors, because they require hydrophobic inducers, such as oleic acid or methyl oleate. Thus, the challenge has become to reduce loads of hydrophobic substrates while simultaneously promoting recombinant protein production. One possible solution is to replace a portion of the inducer with a co-substrate that can serve as an alternative energy source. However, implementing such an approach would require detailed knowledge of how carbon sources impact promoter regulation, which is surprisingly still lacking for the LIP2 and POX2 promoters. This study's aim was thus to better characterize promoter regulation and cell metabolism in Y. lipolytica cultures grown in media supplemented with different carbon sources. RESULTS: pPOX2 induction could be detected when glucose or glycerol was used as sole carbon source, which meant these carbon source could not prevent promoter induction. In addition, when a mixture of glucose and oleic acid was used in complex medium, pPOX2 induction level was lower that that of pLIP2. In contrast, pLIP2 induction was absent when glucose was present in the culture medium, which meant that cell growth could occur without any recombinant gene expression. When a 40/60 mixture of glucose and oleic acid (w/w) was used, a tenfold increase in promoter induction, as compared to when an oleic-acid-only medium was observed. It was also clear that individual cells were adapting metabolically to use both glucose and oleic acid. Indeed, no distinct subpopulations that specialized on glucose versus oleic acid were observed; such an outcome would have led to producer and non-producer phenotypes. In medium containing both glucose and oleic acid, cells tended to directly metabolize oleic acid instead of storing it in lipid bodies. CONCLUSIONS: This study found that pLIP2 is a promoter of choice as compared to pPOX2 to drive gene expression for recombinant protein production by Y. lipolytica used as cell factory.

A quantitative study of methanol/sorbitol co-feeding process of a Pichia pastoris Mut⁺/pAOX1-lacZ strain.

BACKGROUND: One of the main challenges for heterologous protein production by the methylotrophic yeast Pichia pastoris at large-scale is related to its high oxygen demand. A promising solution is a co-feeding strategy based on a methanol/sorbitol mixture during the induction phase. Nonetheless, a deep understanding of the cellular physiology and the regulation of the AOX1 promoter, used to govern heterologous protein production, during this co-feeding strategy is still scarce. RESULTS: Transient continuous cultures with a dilution rate of 0.023 h(-1) at 25°C were performed to quantitatively assess the benefits of a methanol/sorbitol co-feeding process with a Mut+ strain in which the pAOX1-lacZ construct served as a reporter gene. Cell growth and metabolism, including O2 consumption together with CO2 and heat production were analyzed with regard to a linear change of methanol fraction in the mixed feeding media. In addition, the regulation of the promoter AOX1 was investigated by means of ß-galactosidase measurements. Our results demonstrated that the cell-specific oxygen consumption (qO2) could be reduced by decreasing the methanol fraction in the feeding media. More interestingly, maximal ß-galactosidase cell-specific activity (>7500 Miller unit) and thus, optimal pAOX1 induction, was achieved and maintained in the range of 0.45 ~ 0.75 C-mol/C-mol of methanol fraction. In addition, the qO2 was reduced by 30% at most in those conditions. Based on a simplified metabolic network, metabolic flux analysis (MFA) was performed to quantify intracellular metabolic flux distributions during the transient continuous cultures, which further shed light on the advantages of methanol/sorbitol co-feeding process. Finally, our observations were further validated in fed-batch cultures. CONCLUSION: This study brings quantitative insight into the co-feeding process, which provides valuable data for the control of methanol/sorbitol co-feeding, aiming at enhancing biomass and heterologous protein productivities under given oxygen supply. According to our results, ß-galactosidase productivity could be improved about 40% using the optimally mixed feed.