Academic literature on the topic 'L. starkeyi'

The goal of this project was to adapt the Yarrowia lipolytica plasmid based CRISPR/Cas9 system for usage in Lipomyces starkeyi. Lipomyces starkeyi is an oleaginous yeast, which synthesizes and stores high amounts of intracellular lipids. This specific yeast can store lipids at concentrations higher than 60% of its dry cell weight. Due to these high concentrations of lipids, L. starkeyi is a desired organism for the production of biofuels and other oleochemicals. However, there is a lack of knowledge and of genetic tools when trying to engineer the cells to produce these lipids for our use. The genome editing tool, CRISPR/Cas9 is efficient and simple, therefore desirable for the engineering of L. starkeyi. The goal was achieved by replacing the Y. lipolytica promoter with a L. starkeyi promoter, inserting guide RNA, as well as confirming cas9 protein expression.

The oleaginous yeast Lipomyces starkeyi can accumulate intracellular lipids to over 60% of its cell dry mass under a nitrogen-limited condition. We showed that extracellular and intracellular citrate concentrations of L. starkeyi AS 2.1560 increased and the nicotinamide adenine dinucleotide – isocitrate dehydrogenase (NAD+–IDH) activity decreased at the beginning of the lipid accumulation, suggesting that the attenuation of the NAD+–IDH activity might initiate lipid storage. We next cloned the IDH gene by the methods of degenerate PCR and rapid amplification of cDNA ends. Phylogenetic analyses of the evolutionary relationships among LsIDH1, LsIDH2, and other yeast NAD+–IDHs revealed that the L. starkeyi IDH had a closer relationship with the IDHs of Yarrowia lipolytica . Further real-time PCR analysis showed that the expression levels of both LsIDH1 and LsIDH2 decreased concurrently with the evolution of cellular lipids. Our data should be valuable for understanding the biology of oleaginous yeasts and for further strain engineering of L. starkeyi.

Six yeast strains belonging to Rhodosporidium toruloides, Lipomyces starkeyi, Rhodotorula glutinis and Cryptococcus curvatus were shake-flask cultured on xylose (initial sugar—S0 = 70 ± 10 g/L) under nitrogen-limited conditions. C. curvatus ATCC 20509 and L. starkeyi DSM 70296 were further cultured in media where process waters were partially replaced by the phenol-containing olive mill wastewaters (OMWs). In flasks with S0 ≈ 100 g/L and OMWs added yielding to initial phenolic compounds concentration (PCC0) between 0.0 g/L (blank experiment) and 2.0 g/L, C. curvatus presented maximum total dry cell weight—TDCWmax ≈ 27 g/L, in all cases. The more the PCC0 increased, the fewer lipids were produced. In OMW-enriched media with PCC0 ≈ 1.2 g/L, TDCW = 20.9 g/L containing ≈ 40% w/w of lipids was recorded. In L. starkeyi cultures, when PCC0 ≈ 2.0 g/L, TDCW ≈ 25 g/L was synthesized, whereas lipids in TDCW = 24–28% w/w, similar to the experiments without OMWs, were recorded. Non-negligible dephenolization and species-dependent decolorization of the wastewater occurred. A batch-bioreactor trial by C. curvatus only with xylose (S0 ≈ 110 g/L) was performed and TDCW = 35.1 g/L (lipids in TDCW = 44.3% w/w) was produced. Yeast total lipids were composed of oleic and palmitic and to lesser extent linoleic and stearic acids. C. curvatus lipids were mainly composed of nonpolar fractions (i.e., triacylglycerols).

Centromeres function as a platform for the assembly of multiple kinetochore proteins and are essential for chromosome segregation. An active centromere is characterized by the presence of a centromere-specific histone H3 variant, CENP-A. Faithful centromeric localization of CENP-A is supported by heterochromatin in almost all eukaryotes; however, heterochromatin proteins have been lost in most Saccharomycotina. Here, identification of CENP-A (CENP-AL.s.) and heterochromatin protein 1 (Lsw1) in a Saccharomycotina species, the oleaginous yeast Lipomyces starkeyi, is reported. To determine if these proteins are functional, the proteins in S. pombe, a species widely used to study centromeres, were ectopically expressed. CENP-AL.s. localizes to centromeres and can be replaced with S. pombe CENP-A, indicating that CENP-AL.s. is a functional centromere-specific protein. Lsw1 binds at heterochromatin regions, and chromatin binding is dependent on methylation of histone H3 at lysine 9. In other species, self-interaction of heterochromatin protein 1 is thought to cause folding of chromatin, triggering transcription repression and heterochromatin formation. Consistent with this, it was found that Lsw1 can self-interact. L. starkeyi chromatin contains the methylation of histone H3 at lysine 9. These results indicated that L. starkeyi has a primitive heterochromatin structure and is an attractive model for analysis of centromere heterochromatin evolution.

Genomic clustering of functionally related genes is rare in yeasts and other eukaryotes with only few examples available. Here, we summarize our data on a nontelomeric MAL cluster of a non-conventional methylotrophic yeast Ogataea (Hansenula) polymorpha containing genes for α-glucosidase MAL1, α-glucoside permease MAL2 and two hypothetical transcriptional activators. Using genome mining, we detected MAL clusters of varied number, position and composition in many other maltose-assimilating non-conventional yeasts from different phylogenetic groups. The highest number of MAL clusters was detected in Lipomyces starkeyi while no MAL clusters were found in Schizosaccharomyces pombe and Blastobotrys adeninivorans. Phylograms of α-glucosidases and α-glucoside transporters of yeasts agreed with phylogenesis of the respective yeast species. Substrate specificity of unstudied α-glucosidases was predicted from protein sequence analysis. Specific activities of Scheffersomycesstipitis α-glucosidases MAL7, MAL8, and MAL9 heterologously expressed in Escherichia coli confirmed the correctness of the prediction—these proteins were verified promiscuous maltase-isomaltases. α-Glucosidases of earlier diverged yeasts L. starkeyi, B. adeninivorans and S. pombe showed sequence relatedness with α-glucosidases of filamentous fungi and bacilli.

In this paper, potato starch wastewater as culture medium was treated by the oleaginous yeast Lipomyces starkeyi to biosynthesize microbial lipid. The result indicated that carbon source types, carbon source concentration, nitrogen source types, nitrogen source concentration, inoculum size, and cultivation time all had a significant effect on cell growth and microbial lipid accumulation in batch cultures. A measure of 120 g/L of glucose concentration, 3.0 g/L of (NH4)2SO4 concentration, 10% inoculum size, and incubation time 96 h cultivated in a shaking flask at 30 °C were found to be the optimal conditions not only for cell growth but also for lipid synthesis. Under this condition, the cellular biomass and lipid content could reach 2.59 g/L and 8.88%, respectively. This work provides a new method for effective utilization of potato starch wastewater, which has particular social and economic benefits for yeast treatment technology.

The current research aimed to solve the environmental pollution of mature coconut water by Lipomyces starkeyi and provide a study of its high value utilization. The innovation firstly investigated the rheological properties and interface behavior of a crude exopolysaccharide and provided a technical support for its application in food. A response surface methodology was performed to ameliorate the fermentation factors of the new exopolysaccharide with mature coconut water as a substrate, and the consequences suggested that the maximum yield was 7.76 g/L under optimal conditions. Rotary shear measurements were used to study the influence of four variables on the viscosity of the exopolysaccharide solution. The results illustrated that the exopolysaccharide solution demonstrated a shear-thinning behavior and satisfactory thermal stability within the test range. The viscosity of the exopolysaccharide solution was significantly affected by ionic strength and pH; it reached the peak viscosity when the concentration of NaCl was 0.1 mol/L and the pH was neutral. The adsorption behavior of the exopolysaccharide at the medium chain triglyceride–water interface was investigated by a quartz crystal microbalance with a dissipation detector. The results demonstrated that the exopolysaccharide might form a multilayer adsorption layer, and the thickness of the adsorption layer was at its maximum at a concentration of 1.0%, while the interfacial film was the most rigid at a concentration of 0.4%. Overall, these results suggest that the exopolysaccharide produced by Lipomyces starkeyi is an excellent biomaterial for usage in drink, makeup and drug fabrications as a thickening and stabilizing agent.

Rhodotorula diobovata is an oleaginous and carotenogenic yeast, useful for diverse biotechnological applications. To understand the molecular basis of its potential applications, the genome was sequenced using the Illumina MiSeq and Ion Torrent platforms, assembled by AbySS, and annotated using the JGI annotation pipeline. The genome size, 21.1 MB, was similar to that of the biotechnological “workhorse”, R. toruloides. Comparative analyses of the R. diobovata genome sequence with those of other Rhodotorula species, Yarrowia lipolytica, Phaffia rhodozyma, Lipomyces starkeyi, and Sporidiobolus salmonicolor, were conducted, with emphasis on the carotenoid and neutral lipid biosynthesis pathways. Amino acid sequence alignments of key enzymes in the lipid biosynthesis pathway revealed why the activity of malic enzyme and ATP-citrate lyase may be ambiguous in Y. lipolytica and L. starkeyi. Phylogenetic analysis showed a close relationship between R. diobovata and R. graminis WP1. Dot-plot analysis of the coding sequences of the genes crtYB and ME1 corroborated sequence homologies between sequences from R. diobovata and R. graminis. There was, however, nonsequential alignment between crtYB CDS sequences from R. diobovata and those from X. dendrorhous. This research presents the first genome analysis of R. diobovata with a focus on its biotechnological potential as a lipid and carotenoid producer.

The oleaginous yeast Lipomyces starkeyi is an excellent producer of triacylglycerol (TAG) as a feedstock for biodiesel production. To understand the regulation of TAG synthesis, we attempted to isolate mutants with decreased lipid productivity and analyze the expression of TAG synthesis-related genes in this study. A mutant with greatly decreased lipid productivity, sr22, was obtained by an effective screening method using Percoll density gradient centrifugation. The expression of citrate-mediated acyl-CoA synthesis-related genes (ACL1, ACL2, ACC1, FAS1, and FAS2) was decreased in the sr22 mutant compared with that of the wild-type strain. Together with a notion that L. starkeyi mutants with increased lipid productivities had increased gene expression, there was a correlation between the expression of these genes and TAG synthesis. To clarify the importance of citrate-mediated acyl-CoA synthesis pathway on TAG synthesis, we also constructed a strain with no ATP-citrate lyase responsible for the first reaction of citrate-mediated acyl-CoA synthesis and investigated the importance of ATP-citrate lyase on TAG synthesis. The ATP-citrate lyase was required for the promotion of cell growth and TAG synthesis in a glucose medium. This study may provide opportunities for the development of an efficient TAG synthesis for biodiesel production.

Abstract A mixed culture of oleaginous yeast Lipomyces starkeyi and wastewater native microalgae (mostly Scenedesmus sp. and Chlorella sp.) was performed to enhance lipid and biomass production from urban wastewaters. A 400 L raceway pond, operating outdoors, was designed and used for biomass cultivation. Microalgae and yeast were inoculated into the cultivation pond with a 2:1 inoculum ratio. Their concentrations were monitored for 14 continuous days of batch cultivation. Microalgal growth presented a 3-day initial lag-phase, while yeast growth occurred in the first few days. Yeast activity during the microalgal lag-phase enhanced microalgal biomass productivity, corresponding to 31.4 mgTSS m−2 d−1. Yeast growth was limited by low concentrations in wastewater of easily assimilated organic substrates. Organic carbon was absorbed in the first 3 days with a 3.7 mgC L−1 d−1 removal rate. Complete nutrient removal occurred during microalgal linear growth with 2.9 mgN L−1 d−1 and 0.96 mgP L−1 d−1 removal rates. Microalgal photosynthetic activity induced high pH and dissolved oxygen values resulted in natural bactericidal and antifungal activity. A 15% lipid/dry weight was measured at the end of the cultivation time. Fatty acid methyl ester (FAME) analysis indicated that the lipids were mainly composed of arachidic acid.

Il ruolo principale delle biotecnologie industriali è quello di fornire soluzioni innovative per alcune delle più grandi sfide del mondo. Nonostante il potenziale e le tecniche innovative applicate, i processi microbiologici bio-based necessitano ancora di ulteriori studi per diventare pervasivi e quindi sostituire i processi di produzione tradizionali. Per rendere i processi microbici economicamente fattibili e rispettosi dell'ambiente, uno dei fattori chiave risiede nella scelta della biomassa di partenza. In una logica di bioeconomia circolare, i sottoprodotti e le biomasse residue devono essere considerati come materie prime di partenza del processo. L'uso di queste biomasse non solleva questioni etiche e allo stesso tempo è economicamente vantaggioso e orientato all'ambiente. La maggior parte di queste biomasse residue sono residui agricoli e forestali, una famiglia di biomasse caratterizzate da una struttura lignocellulosica. Il problema legato al loro utilizzo nelle bioraffinerie a base microbica è quello di trovare un pretrattamento efficiente per convertirli in zuccheri fermentabili e altri nutrienti, riducendo al minimo il rilascio di inibitori della crescita microbica. Parlando di bioraffinerie microbiche, ci sono due aspetti principali da tenere a mente durante la progettazione del processo: la biomassa di partenza e l'ospite microbico. L’host finale può essere scelto seguendo due approcci complementari: i) sfruttare la biodiversità microbica già presente in natura, scegliendo l'ospite finale in base alle sue caratteristiche innate, particolarmente vantaggiose in uno specifico processo produttivo; ii) lavorare su una cell factory già nota, customizzandola secondo le necessità. Nel Capitolo 2 è stata valutata una specifica classe di lieviti non convenzionali, denominata lieviti oleaginosi, per ottenere oli microbici (SCOs) per la produzione di biodiesel a partire da scarti dell'industria della barbabietola da zucchero. Lipomyces starkeyi è stato selezionato come cell factory per la conversione della polpa di barbabietola da zucchero e della melassa di barbabietola da zucchero per massimizzare l'accumulo di SCOs. Con questo esempio applicativo abbiamo dimostrato la possibilità di sfruttare microrganismi non convenzionali per ottenere bio-carburanti più sostenibili. D'altra parte, la scelta di Saccharomyces cerevisiae come ospite finale ha il grande vantaggio di sfruttare l'ampia conoscenza che lo circonda, compreso l’enorme numero di approcci di biologia sintetica per disegnarlo nella forma finale necessaria. Nel Capitolo 3 presento una nuova combinazione di approcci di biologia sintetica per accelerare le procedure di ingegnerizzazione, consentendo l’over-espressione e lo studio di vie biosintetiche eterologhe sempre più complesse. Inoltre, mostro l'applicazione di questo nuovo kit di strumenti alla produzione di un metabolita secondario di pianta. Nel capitolo 4 descrivo la progettazione di un nuovo vettore per migliorare le procedure di editing del genoma in S. cerevisiae. Anche in questo secondo progetto l'obiettivo finale è stato quello di velocizzare le fasi di progettazione e costruzione e le procedure di laboratorio, standardizzandole il più possibile per semplificare una parte del lavoro e lasciare più spazio alle fasi successive di test & learn. Nel Capitolo 5 propongo il concetto di co-localizzazione spaziale degli enzimi come campo d'avanguardia nella biologia sintetica per massimizzare il flusso di carbonio verso il prodotto di interesse, sfruttando l'uso di scaffold proteici sintetici e domini di interazione sintetici. La tesi qui presentata vuole porsi come esempio pratico di come le biotecnologie industriali possano essere utilizzate come potente strumento nella difficile transizione da una società basata sul petrolio e una più sostenibile.
The role of industrial biotechnology is to provide game-changing solutions for some of the world’s greatest challenges. From climate change to alternative energy sources and to sustainable productions, industrial biotechnology is fighting to find new sustainable solutions. Despite the promising potential and the innovative techniques applied, bio-based biological processes still need further studies for becoming pervasive and therefore substituting the traditional processes of production. To make microbial processes economically feasible and environmentally friendly, one of the key factors resides in the choice of the starting biomass. In a logic of circular bioeconomy, by-products and residual biomasses have to be considered as starting feedstocks of the process. The use of these biomasses does not raise ethical issues and at the same time is economically advantageous and environment oriented. Indeed, they do not compete with the food industry, as they are usually production waste. Most of these residual biomasses are agricultural and forest residues, a family of biomasses characterised by a lignocellulosic structure. The problem related to their use in microbial-based biorefineries is to find an efficient pretreatment to convert them into fermentable sugars and other nutrients, while reducing to a minimum the release of inhibitors of microbial growth. Talking about microbial-based biorefinery as a substitute to petrol-based refinery, there are two main topics to keep in mind during the process design: the starting biomass and the microbial host. The chassis which will be involved in the final production process can be chosen following two complementary approaches: i) exploiting microbial biodiversity already present in nature by picking the final host depending on its innate characteristics, particularly advantageous in a specific production process; ii) working on a well-known cell factory by customising it as needed. In this thesis both principles were followed. In Chapter 2 a specific class of non-conventional yeasts, named oleaginous yeasts, was evaluated to obtain single cell oils (SCOs) for biodiesel production starting from wastes of the sugar beet industry. Lipomyces starkeyi was selected as cell factory for the conversion of sugar beet pulp and sugar beet molasses to maximise SCOs accumulation. With this applicative example we showed the possibility to take advantage of non-conventional microorganisms to achieve a more sustainable way to produce fuels. On the other hand, choosing Saccharomyces cerevisiae as final host has the major advantage of exploiting the wide knowledge around it, starting from its genome and physiology, and arriving at the tremendous number of synthetic biology approaches to engineer it and manipulate it in the desired final form. In Chapter 3 I introduce a novel toolkit: a new combination of synthetic biology approaches to accelerate the engineering procedures allowing the overexpression and the study of more and more complex biosynthetic heterologous pathways. Moreover, I show the application of this novel toolkit to the production of a selected plant secondary metabolite. In Chapter 4 I describe the design of a new vector to improve genome editing procedures in S. cerevisiae. Even in this second project the final goal was to speed up the design and build stages and laboratory procedures, standardising them as much as possible to simplify one part of scientists' work, to leave more space to the subsequent phases of testing and learning. In Chapter 5 I propose the concept of enzyme spatial co-localisation as a forefront field in synthetic biology to maximise the carbon flux toward the product of interest, exploiting the use of protein synthetic scaffolds and synthetic interaction domains. The presented thesis wants to pose itself as a practical example on how industrial biotechnology can be used as a powerful tool in the difficult transition to a more sustainable society.