The Endophytic Fungus Cyanodermella asteris Influences Growth of the Nonnatural Host Plant Arabidopsis thaliana.

Cyanodermella asteris is a fungal endophyte from Aster tataricus, a perennial plant from the northern part of Asia. Here, we demonstrated an interaction of C. asteris with Arabidopsis thaliana, Chinese cabbage, rapeseed, tomato, maize, or sunflower resulting in different phenotypes such as shorter main roots, massive lateral root growth, higher leaf and root biomass, and increased anthocyanin levels. In a variety of cocultivation assays, it was shown that these altered phenotypes are caused by fungal CO2, volatile organic compounds, and soluble compounds, notably astins. Astins A, C, and G induced plant growth when they were individually included in the medium. In return, A. thaliana stimulates the fungal astin C production during cocultivation. Taken together, our results indicate a bilateral interaction between the fungus and the plant. A stress response in plants is induced by fungal metabolites while plant stress hormones induced astin C production of the fungus. Interestingly, our results not only show unidirectional influence of the fungus on the plant but also vice versa. The plant is able to influence growth and secondary metabolite production in the endophyte, even when both organisms do not live in close contact, suggesting the involvement of volatile compounds.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

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.

Yarrowia lipolytica morphological mutant enables lasting in situ immobilization in bioreactor.

In the present study, we have isolated and characterized a Yarrowia lipolytica morphological mutant growing exclusively in the pseudohyphal morphology. The gene responsible for this phenotype, YALI0E06519g, was identified as homologous to the mitosis regulation gene HSL1 from Saccharomyces cerevisiae. Taking advantage of its morphology, we achieved the immobilization of the Δhsl1 mutant on the metallic structured packing of immobilized-cell bioreactors. We obtained significant cell retention and growth on the support during shake flask and bioreactor experiments without an attachment step prior to the culture. The system of medium aspersion on the packing ensured oxygen availability in the absence of agitation and minimized the potential release of cells in the culture medium. Additionally, the metallic packing proved its facility of cleaning and sterilization after fermentation. This combined use of morphological mutation and bioreactor design is a promising strategy to develop continuous processes for the production of recombinant protein and metabolites using Y. lipolytica. Graphical Abstract.

Online flow cytometry, an interesting investigation process for monitoring lipid accumulation, dimorphism, and cells' growth in the oleaginous yeast Yarrowia lipolytica JMY 775.

This study aims to understand and better control the main biological mechanisms and parameters modulating the various phenomena affecting Yarrowia lipolytica JMY775 and its lipids accumulation. The results obtained in this study stress forward that the use of an original tool, consisting of coupling bioreactors to online flow cytometry, is highly efficient. Throughout 48 h of culturing, this emerging process allowed an online continuous observation of the effects of pH and/or aeration on the cell growth and dimorphism and lipid accumulation by Y. lipolytica. This present study showed clearly that online flow cytometry is an advantageous tool for the real-time monitoring of microbial culture at a single-cell level. Indeed, the present investigation showed for the first time that profiling of the various phenomena and their monitoring upon culture time is now possible by coupling online cytometry with culture bioreactors.

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.

Physical and physiological impacts of different foam control strategies during a process involving hydrophobic substrate for the lipase production by Yarrowia lipolytica.

The potentialities for the intensification of the process of lipase production by the yeast Yarrowia lipolytica on a renewable hydrophobic substrate (methyl oleate) have to be investigated. The key factor governing the lipase yield is the intensification of the oxygen transfer rate, considering the fact that Y. lipolytica is a strict aerobe. However, considering the nature of the substrate and the capacity for protein excretion and biosurfactant production of Y. lipolytica, intensification of oxygen transfer rate is accompanied by an excessive formation of foam. Two different foam control strategies have thus been implemented: a classical chemical foam control strategy and a mechanical foam control (MFM) based on the Stirring As Foam Disruption principle. The second strategy allows foam control without any modifications of the physico-chemical properties of the broth. However, the MFM system design induced the formation of a persistent foam layer in the bioreactor. This phenomenon has led to the segregation of microbial cells between the foam phase and the liquid phase in the case of the bioreactors operated with MFM control, and induced a reduction at the level of the lipase yield. More interestingly, flow cytometry experiments have shown that the residence time of microbial cells in the foam phase tends to induce a dimorphic transition which could potentially explain the reduction of lipase excretion.

Scale-down assessment of the sensitivity of Yarrowia lipolytica to oxygen transfer and foam management in bioreactors: investigation of the underlying physiological mechanisms.

A scale-down investigation of the impact of local dissolved oxygen limitation on lipase production by Y. lipolytica has been performed. One of the major issues encountered during this kind of process is foam formation, requiring a reduction of the overall oxygen transfer efficiency of the system in order to keep antifoam consumption to a reasonable level. A regulation strategy involving oxygen enrichment of the air flow through the reactor has allowed this issue to be partly overcome. For a second time, the scale dependency of the process operated with air enrichment has been investigated by a combination of scale-down and pilot-scale cultivation tests. The scale-down apparatus considered in this work comprised a well-mixed part connected to a plug-flow part subjected to dissolved oxygen limitation. Surprisingly, foaming intensity was greatly reduced in the case of the test performed in scale-down reactors (SDRs) while maintaining the same stirring and aeration intensities in the stirred part of the reactor. For mean residence time of 100 s in the recycle loop of the reactor, foam formation was significantly reduced while cell growth and lipase production were both unaltered. When the residence time in the recycle loop was raised to 200 s, the foam phenomena was also reduced, but the lipase yield was altered as well as lip2 gene transcription and translation as shown by real-time quantitative polymerase chain reaction (RT-qPCR) and reporter gene activity, respectively. Our results clearly show the importance of primarily taking into account cell physiology for the scaling-up procedure.