Key impact of an uncommon plasmid on bacillus amyloliquefaciens subsp. plantarum S499 developmental traits and lipopeptide production

The rhizobacterium Bacillus amyloliquefaciens subsp. plantarum S499 (S499) is particularly efficient in terms of the production of cyclic lipopeptides, which are responsible for the high level of plant disease protection provided by this strain. Sequencing of the S499 genome has highlighted genetic differences and similarities with the closely related rhizobacterium B. amyloliquefaciens subsp. plantarum FZB42 (FZB42). More specifically, a rare 8008 bp plasmid (pS499) harboring a rap-phr cassette constitutes a major distinctive element between S499 and FZB42. By curing this plasmid, we demonstrated that its presence is crucial for preserving the typical physiology of S499 cells. Indeed, the growth rate and extracellular proteolytic activity were significantly affected in the cured strain (S499 P-). Furthermore, pS499 made a significant contribution to the regulation of cyclic lipopeptide production. Surfactins and fengycins were produced in higher quantities by S499 P-, whereas lower amounts of iturins were detected. In line with the increase in surfactin release, bacterial motility improved after curing, whereas the ability to form biofilm was reduced in vitro. The antagonistic effect against phytopathogenic fungi was also limited for S499 P-, most probably due to the reduction of iturin production. With the exception of this last aspect, S499 P- behavior fell between that of S499 and FZB42, suggesting a role for the plasmid in shaping some of the phenotypic differences observed in the two strains. © 2017 Molinatto, Franzil, Steels, Puopolo, Pertot and Ongena

Coculture of Trichoderma harzianum and Bacillus velezensis Based on Metabolic Cross-Feeding Modulates Lipopeptide Production.

peer reviewedCocultures have been widely explored for their use in deciphering microbial interaction and its impact on the metabolisms of the interacting microorganisms. In this work, we investigate, in different liquid coculture conditions, the compatibility of two microorganisms with the potential for the biocontrol of plant diseases: the fungus Trichoderma harzianum IHEM5437 and the bacterium Bacillus velezensis GA1 (a strong antifungal lipopeptide producing strain). While the Bacillus overgrew the Trichoderma in a rich medium due to its antifungal lipopeptide production, a drastically different trend was observed in a medium in which a nitrogen nutritional dependency was imposed. Indeed, in this minimum medium containing nitrate as the sole nitrogen source, cooperation between the bacterium and the fungus was established. This is reflected by the growth of both species as well as the inhibition of the expression of Bacillus genes encoding lipopeptide synthetases. Interestingly, the growth of the bacterium in the minimum medium was enabled by the amendment of the culture by the fungal supernatant, which, in this case, ensures a high production yield of lipopeptides. These results highlight, for the first time, that Trichoderma harzianum and Bacillus velezensis are able, in specific environmental conditions, to adapt their metabolisms in order to grow together

Bacillus subtilis as a tool for screening soil metagenomic libraries for antimicrobial activities

peer reviewe

Stimulation of Fengycin-Type Antifungal Lipopeptides in Bacillus amyloliquefaciens in the Presence of the Maize Fungal Pathogen Rhizomucor variabilis.

Most isolates belonging to the Bacillus amyloliquefaciens subsp. plantarum clade retain the potential to produce a vast array of structurally diverse antimicrobial compounds that largely contribute to their efficacy as biocontrol agents against numerous plant fungal pathogens. In that context, the role of cyclic lipopeptides (CLPs) has been well-documented but still little is known about the impact of interactions with other soil-inhabiting microbes on the expression of these molecules. In this work, we wanted to investigate the antagonistic activity developed by this bacterium against Rhizomucor variabilis, a pathogen isolated from diseased maize cobs in Democratic Republic of Congo. Our data show that fengycins are the major compounds involved in the inhibitory activity but also that production of this type of CLP is significantly upregulated when co-cultured with the fungus compared to pure cultures. B. amyloliquefaciens is thus able to perceive fungal molecules that are emitted and, as a response, up-regulates the biosynthesis of some specific components of its antimicrobial arsenal

By-products show potential in glutathione production by engineered non-conventional yeast Yarrowia lipolytica

Strong constitutive promoter pTEF were used to overexpress genes encoding the two enzymes (L-cysteine gamma-ligase and glutathione synthase) which catalyze the synthesis of GSH in Yarrowia lipolytica. Several strains which can overexpress GSH1 or GSH2 or both of them have been successfully obtained. The newly constructed strains for glutathione overproduction show different compared to control

Impact of oxygen availability on heterologous geneexpression and polypeptide secretion dynamics in Yarrowia lipolytica-based protein production platforms

Industrially relevant traits of Yarrowia lipolytica, like high growth rate, capacity to grow at high cell density or to synthesize biomolecules with high productivities, strongly rely on sufficient oxygen provision. Although the impact of oxygen availability (OA) on the physiology of Y. lipolytica has been already studied, its influence on recombinant protein (rProt) synthesis and secretion has been largely neglected to date. With the aim to fill this gap, a fluorescent reporter protein (yellow fluorescent protein [YFP]) was used herein as a proxy to follow simultaneously rProt synthesis and secretion in Y. lipolytica under different OAs. This study covers the analysis of the reporter gene expression through reverse transcription quantitative polymerase chain reaction, polypeptide synthesis and its retention-to-secretion ratio using flow cytometry and fluorymetry during shake flasks and bioreactor cultivations under different OA. The results gathered demonstrate that OA has a dramatic impact on the kinetics of intracellular and extracellular YFP accumulation. Higher rProt production and secretion were favoured under high OA, and were largely related to OA and not to cell growth. Our observations also suggest the existence of some upper limit of secretory protein accumulation inside the cells above which massive secretion is initiated. Moreover, at low OA, the first bottleneck in rProt synthesis occurs as early as at transcription level, which could results from a lower availability of transcriptional machinery elements. Finally, using flow cytometry and bioreactor cultivations, we highlighted that ovoid cells are generally more efficient in terms of rProt synthesis. © 2020 John Wiley & Sons, Ltd

Impact of oxygen availability on heterologous geneexpression and polypeptide secretion dynamics in Yarrowia lipolytica

peer reviewedIndustrially relevant traits of Yarrowia lipolytica, like high growth rate, capacity to grow at high cell density or to synthesize biomolecules with high productivities, strongly rely on sufficient oxygen provision. Although the impact of oxygen availability (OA) on the physiology of Y. lipolytica has been already studied, its influence on recombinant protein (rProt) synthesis and secretion has been largely neglected to date. With the aim to fill this gap, a fluorescent reporter protein (yellow fluorescent protein [YFP]) was used herein as a proxy to follow simultaneously rProt synthesis and secretion in Y. lipolytica under different OAs. This study covers the analysis of the reporter gene expression through reverse transcription quantitative polymerase chain reaction, polypeptide synthesis and its retention-to-secretion ratio using flow cytometry and fluorymetry during shake flasks and bioreactor cultivations under different OA. The results gathered demonstrate that OA has a dramatic impact on the kinetics of intracellular and extracellular YFP accumulation. Higher rProt production and secretion were favoured under high OA, and were largely related to OA and not to cell growth. Our observations also suggest the existence of some upper limit of secretory protein accumulation inside the cells above which massive secretion is initiated. Moreover, at low OA, the first bottleneck in rProt synthesis occurs as early as at transcription level, which could results from a lower availability of transcriptional machinery elements. Finally, using flow cytometry and bioreactor cultivations, we highlighted that ovoid cells are generally more efficient in terms of rProt synthesis. © 2020 John Wiley & Sons, Ltd

Comprehensive comparison of Yarrowia lipolytica and Pichia pastoris for production of Candida antarctica lipase B

The large-scale production of recombinant proteins (rProt) is becoming increasingly economically important. Among the different hosts used for rProt production, yeasts are gaining popularity. The so-called non-conventional yeasts, such as the methylotrophic Pichia pastoris and the dimorphic Yarrowia lipolytica, are popular choices due to their favorable characteristics and well-established expression systems. Nevertheless, a direct comparison of the two systems for rProt production and secretion was lacking. This study therefore aimed to directly compare Y. lipolytica and P. pastoris for the production and secretion of lipase CalB in bioreactor. Y. lipolytica produced more than double the biomass and more than 5-fold higher extracellular lipase than P. pastoris. Furthermore, maximal CalB production levels were reached by Y. lipolytica in half the cultivation time required for maximal production by P. pastoris. Conversely, P. pastoris was found to express 7-fold higher levels of CalB mRNA. Secreted enhanced green fluorescent protein –in isolation and fused to CalB– and protease inhibitor MG-132 were used in P. pastoris to further investigate the reasons behind such discrepancy. The most likely explanation was ultimately found to be protein degradation by endoplasmic reticulum-associated protein degradation preceding successful secretion. This study highlighted the multifaceted nature of rProt production, prompting a global outlook for selection of rProt production systems. © 2020, The Author(s)

Insights into the impact of global regulators on the producon of bioacve secondary metabolites of Bacillus velezensis

Bacillus velezensis is one of the most promising biocontrol agents used for protecting crops against microbial diseases in sustainable agriculture. This biocontrol potential lies notably in its ability to coproduce a vast array of bioactive secondary metabolites (BSMs) retaining strong antimicrobial activity or other key ecological functions. It includes multiple non-ribosomally synthesized (NR-)BSMs such as cyclic lipopeptides, polyketides, small oligopeptides and siderophores but also some terpenes and diverse ribosomally synthesized and post-translationally modified peptides (RiPPs). Thereby, understanding the regulation of B. velezensis BSMs synthesis is crucial to understand the regulation of its biocontrol activities. In the model species B. subtilis, BSM synthesis is mainly under the control of some global regulators (GR) also referred as pleiotropic or master regulators. We assume that similar rules apply for other species but our knowledge about such BSMs regulation is still very limited in plant-beneficial bacilli such as B. velezensis which differs in its lifestyle and forms a larger and more diversified panoply of compounds. In this work we aimed to draw a global picture of BSM regulation in B. velezensis. Using a reverse genetics approach, we assessed the impact of 14 GRs known to control developmental traits, multicellular behavior or responses to environmental cues. Our data highlight a central role of some regulatory elements such as quorum-sensing related ComA and sporulation regulator Spo0A for the modulation of BSM production. Moreover, cyclic lipopeptides and polyketides also appears differentially fine-tuned by additional GRs. Finally, it appears that each BSM has its unique regulatory scheme. We also observed species-specific roles of GRs such as CodY, involved in central metabolism regulation, which appears to have little impact on NR-BSMs synthesis in opposition to its major role in B. subtilis. Using the same approach, we assessed the impact of those GRs on antimicrobial activities of B. velezensis against a range a phytopathogens. This later experiment further highlights how GRs impact antimicrobial potential of B. velezensis

Metabolic engineering of Yarrowia lipolytica for the synthesis of added value chemicals

Y. lipolytica is a non-conventional yeast, well-known for its unusual metabolic properties. Based on its ability to secrete high amounts of proteins and metabolites of biotechnological interest, Y. lipolytica has several industrial applications, including heterologous protein synthesis or citric acid production. We will report on strain development for the synthesis of two added value chemicals: erythritol and erythrulose. Erythritol is a four-carbon sugar alcohol with application as food additive due to its sweetening properties. Erythrulose is a derivative of erythritol and is an intermediate of its catabolism. It has application as sunless tanning agent and as precursor for the synthesis of different drugs. By overexpressing gene GUT1 and TKL1, which encode a glycerol kinase and a transketolase, respectively, strain-overproducing erythritol were obtained. Erythrulose producing strain was obtained by deleting gene YALI0F01606g than code for an erythrulose kinase