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1.
Appl Environ Microbiol ; 87(6)2021 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-33452022

RESUMO

Yarrowia lipolytica has been extensively used to produce essential chemicals and enzymes. As in most other eukaryotes, nonhomologous end joining (NHEJ) is the major repair pathway for DNA double-strand breaks in Y. lipolytica Although numerous studies have attempted to achieve targeted genome integration through homologous recombination (HR), this process requires the construction of homologous arms, which is time-consuming. This study aimed to develop a homology-independent and CRISPR/Cas9-mediated targeted genome integration tool in Y. lipolytica Through optimization of the cleavage efficiency of Cas9, targeted integration of a hyg fragment was achieved with 12.9% efficiency, which was further improved by manipulation of the fidelity of NHEJ repair, the cell cycle, and the integration sites. Thus, the targeted integration rate reached 55% through G1 phase synchronization. This tool was successfully applied for the rapid verification of intronic promoters and iterative integration of four genes in the pathway for canthaxanthin biosynthesis. This homology-independent integration tool does not require homologous templates and selection markers and achieves one-step targeted genome integration of the 8,417-bp DNA fragment, potentially replacing current HR-dependent genome-editing methods for Y. lipolytica IMPORTANCE This study describes the development and optimization of a homology-independent targeted genome integration tool mediated by CRISPR/Cas9 in Yarrowia lipolytica This tool does not require the construction of homologous templates and can be used to rapidly verify genetic elements and to iteratively integrate multiple-gene pathways in Y. lipolytica This tool may serve as a potential supplement to current HR-dependent genome-editing methods for eukaryotes.


Assuntos
Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Yarrowia/genética , Cantaxantina/metabolismo , Reparo do DNA por Junção de Extremidades , Edição de Genes , Genoma Fúngico , Yarrowia/metabolismo , beta Caroteno/metabolismo
2.
J Agric Food Chem ; 69(1): 275-285, 2021 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-33356235

RESUMO

α-Pinene, an important biologically active natural monoterpene, has been widely used in fragrances, medicines, and fine chemicals, especially, in high-density renewable fuels such as jet fuel. The development of an α-pinene production platform in a highly modifiable microbe from renewable substitute feedstocks could lead to a green, economical avenue, and sustainable biotechnological process for the biosynthesis of α-pinene. Here, we report engineering of an orthogonal biosynthetic pathway for efficient production of α-pinene in oleaginous yeast Yarrowia lipolytica that resulted in an α-pinene titer of 19.6 mg/L when using glucose as the sole carbon source, a significant 218-fold improvement than the initial titer. In addition, the potential of using waste cooking oil and lignocellulosic hydrolysate as carbon sources for α-pinene production from the engineered Y. lipolytica strains was analyzed. The results indicated that α-pinene titers of 33.8 and 36.1 mg/L were successfully obtained in waste cooking oil and lignocellulosic hydrolysate medium, thereby representing the highest titer reported to date in yeast. To our knowledge, this is also the first report related to microbial production of α-pinene from waste cooking oil and lignocellulosic hydrolysate.


Assuntos
Monoterpenos Bicíclicos/metabolismo , Yarrowia/genética , Yarrowia/metabolismo , Biocombustíveis/análise , Vias Biossintéticas , Fermentação , Glucose/metabolismo , Lignina/metabolismo , Engenharia Metabólica
3.
PLoS One ; 15(12): e0239882, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33332385

RESUMO

Alkane-based biofuels are desirable to produce at a commercial scale as these have properties similar to current petroleum-derived transportation fuels. Rationally engineering microorganisms to produce a desirable compound, such as alkanes, is, however, challenging. Metabolic engineers are therefore increasingly implementing evolutionary engineering approaches combined with high-throughput screening tools, including metabolite biosensors, to identify productive cells. Engineering Saccharomyces cerevisiae to produce alkanes could be facilitated by using an alkane-responsive biosensor, which can potentially be developed from the native alkane-sensing system in Yarrowia lipolytica, a well-known alkane-assimilating yeast. This putative alkane-sensing system is, at least, based on three different transcription factors (TFs) named Yas1p, Yas2p and Yas3p. Although this system is not fully elucidated in Y. lipolytica, we were interested in evaluating the possibility of translating this system into an alkane-responsive biosensor in S. cerevisiae. We evaluated the alkane-sensing system in S. cerevisiae by developing one sensor based on the native Y. lipolytica ALK1 promoter and one sensor based on the native S. cerevisiae CYC1 promoter. In both systems, we found that the TFs Yas1p, Yas2p and Yas3p do not seem to act in the same way as these have been reported to do in their native host. Additional analysis of the TFs suggests that more knowledge regarding their mechanism is needed before a potential alkane-responsive sensor based on the Y. lipolytica system can be established in S. cerevisiae.


Assuntos
Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica/genética , Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Yarrowia/genética , Alcanos/metabolismo , Regiões Promotoras Genéticas/genética , Saccharomyces cerevisiae/metabolismo , Transcrição Genética/genética , Yarrowia/metabolismo
4.
Nat Commun ; 11(1): 3803, 2020 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-32732991

RESUMO

Microbial communities comprised of phototrophs and heterotrophs hold great promise for sustainable biotechnology. Successful application of these communities relies on the selection of appropriate partners. Here we construct four community metabolic models to guide strain selection, pairing phototrophic, sucrose-secreting Synechococcus elongatus with heterotrophic Escherichia coli K-12, Escherichia coli W, Yarrowia lipolytica, or Bacillus subtilis. Model simulations reveae metabolic exchanges that sustain the heterotrophs in minimal media devoid of any organic carbon source, pointing to S. elongatus-E. coli K-12 as the most active community. Experimental validation of flux predictions for this pair confirms metabolic interactions and potential production capabilities. Synthetic communities bypass member-specific metabolic bottlenecks (e.g. histidine- and transport-related reactions) and compensate for lethal genetic traits, achieving up to 27% recovery from lethal knockouts. The study provides a robust modelling framework for the rational design of synthetic communities with optimized growth sustainability using phototrophic partners.


Assuntos
Bacillus subtilis/metabolismo , Escherichia coli/metabolismo , Processos Heterotróficos/fisiologia , Processos Fototróficos/fisiologia , Synechococcus/metabolismo , Yarrowia/metabolismo , Aldeídos/metabolismo , Bacillus subtilis/genética , Reatores Biológicos/microbiologia , Escherichia coli/genética , Etanol/metabolismo , Formaldeído/metabolismo , Metanol/metabolismo , Microbiota/fisiologia , Modelos Biológicos , Ácido Succínico/metabolismo , Synechococcus/genética , Yarrowia/genética
5.
Arch Biochem Biophys ; 689: 108475, 2020 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-32585312

RESUMO

The unconventional yeast Yarrowia lipolytica is known as a producer of extracellular lipases. Here we overexpressed extracellular lipase (YlLip2) in yeast strain Y. lipolytica AJD ΔXΔA-Lip2 harboring the overexpression cassette of the YALI0A20350 gene under the strong hybrid promoter UAS1B16-TEF. To maintain a high level of YlLip2 production, two extracellular proteases of Y. lipolytica, AEPp and AXPp, were deleted. The purified recombinant YlLip2 presented optimal catalytic activities at 37 °C and pH 8.0. The effect of two lipopeptide biosurfactants, i.e., amphisin produced by Pseudomonas fluorescens DSS73 and viscosinamide secreted by P. fluorescens DR54, on the conformation and activity of YlLip2 was evaluated using spectral methods, surface tension, and the enzyme activity assay. YlLip2 demonstrated high tolerance of the tested biosurfactants and had greater activity retention after incubation with both biosurfactants. Finally, we observed that intrinsic fluorescence intensity of YlLip2 decreased significantly with increasing lipopeptides concentration ranging from 2.5 to 60 µM. Our results showed that both biosurfactants improve enzymatic activity of YlLip2 and might suggest better interaction of the substrate with the active site. These favorable characteristics make YlLip2 a prospective additive in the pharmaceutical, food, cosmetic, and detergent industries.


Assuntos
Lipase/metabolismo , Lipopeptídeos/metabolismo , Yarrowia/enzimologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Lipase/genética , Pseudomonas fluorescens/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Regulação para Cima , Yarrowia/genética , Yarrowia/metabolismo
6.
J Ind Microbiol Biotechnol ; 47(4-5): 403-412, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32372295

RESUMO

Past research has sought to improve the production of cyclopropane fatty acids by the oleaginous yeast Yarrowia lipolytica by heterologously expressing the E. coli fatty acid synthase gene and improving cultivation processes. Cyclopropane fatty acids display properties that hold promise for biofuel applications. The E. coli fatty acid synthase gene was introduced into several genetic backgrounds of the yeast Y. lipolytica to optimize lipid synthesis; the mean cyclopropane fatty acid productivity was 43 mg L-1 h-1 on glucose, and the production rate reached its maximum (3.06 g L-1) after 72 h of cultivation in a bioreactor. The best strain (JMY6851) overexpressed simultaneously the E. coli cyclopropane fatty acid synthase gene under a hybrid promoter (hp8d) and Y. lipolytica LRO1 gene. In fed-batch process using crude glycerol as carbon source, JMY6851 strain displayed high lipid accumulation (78% of dry cell weight) and high biomass production (56 g L-1). After 165 h of cultivation, cyclopropane fatty acids represented 22% of the lipids produced; cyclopropane fatty acid productivity (103.3 mg L-1 h-1) was maximal at 72.5 h of cultivation.


Assuntos
Ácidos Graxos/biossíntese , Fermentação , Yarrowia/metabolismo , Biomassa , Reatores Biológicos , Ciclopropanos , Escherichia coli/genética , Escherichia coli/metabolismo , Glucose , Yarrowia/genética
7.
Microb Cell Fact ; 19(1): 100, 2020 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-32393258

RESUMO

BACKGROUND: The secretory production of recombinant proteins in yeast simplifies isolation and purification but also faces possible complications due to the complexity of the secretory pathway. Therefore, correct folding, maturation and intracellular transport of the recombinant proteins are important processing steps with a higher effort needed for complex and large proteins. The aim of this study was to elucidate the secretion potential of Yarrowia lipolytica for low and high molecular weight ß-glycosidases in a comparative cultivation approach. RESULTS: A low sized ß-glucosidase from Pyrococcus furiosus (CelB; 55 kDa) and a large sized ß-galactosidase isolated from the metagenome (M1; 120 kDa) were integrated into the acid extracellular protease locus using the CRISPR-Cas9 system to investigate the size dependent secretion of heterologous proteins in Y. lipolytica PO1f. The recombinant strains were cultivated in the bioreactor for 78 h and the extra- and intracellular enzyme activities were determined. The secretion of CelB resulted in an extracellular volumetric activity of 187.5 µkatoNPGal/Lmedium, while a volumetric activity of 2.98 µkatoNPGal/Lmedium was measured during the M1 production. However, when the amount of functional intra- and extracellular enzyme was investigated, the high molecular weight M1 (85%) was secreted more efficiently than CelB (27%). Real-time PCR experiments showed a linear correlation between the transcript level and extracellular activity for CelB, while a disproportional high mRNA level was observed regarding M1. Interestingly, mass spectrometry data revealed the unexpected secretion of two endogenous intracellular glycolytic enzymes, which is reported for the first time for Y. lipolytica. CONCLUSION: The results of this study provide deeper insights into the secretion potential of Y. lipolytica. A secretion limitation for the low-size CelB was observed, while the large size M1 enzyme was produced in lower amounts but was secreted efficiently. It was shown for the first time that Y. lipolytica is a promising host for the secretion of heterologous high molecular weight proteins (> 100 kDa), although the total secreted amount has to be increased further.


Assuntos
Proteínas Arqueais/biossíntese , Glucosidases/biossíntese , Yarrowia/metabolismo , Proteínas Arqueais/classificação , Reatores Biológicos , Glucosidases/classificação , Peso Molecular , Pyrococcus furiosus/enzimologia , Pyrococcus furiosus/genética , Proteínas Recombinantes/biossíntese
8.
NPJ Syst Biol Appl ; 6(1): 14, 2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32415097

RESUMO

Cells can sense changes in their extracellular environment and subsequently adapt their biomass composition. Nutrient abundance defines the capability of the cell to produce biomass components. Under nutrient-limited conditions, resource allocation dramatically shifts to carbon-rich molecules. Here, we used dynamic biomass composition data to predict changes in growth and reaction flux distributions using the available genome-scale metabolic models of five eukaryotic organisms (three heterotrophs and two phototrophs). We identified temporal profiles of metabolic fluxes that indicate long-term trends in pathway and organelle function in response to nitrogen depletion. Surprisingly, our calculations of model sensitivity and biosynthetic cost showed that free energy of biomass metabolites is the main driver of biosynthetic cost and not molecular weight, thus explaining the high costs of arginine and histidine. We demonstrated how metabolic models can accurately predict the complexity of interwoven mechanisms in response to stress over the course of growth.


Assuntos
Eucariotos/crescimento & desenvolvimento , Eucariotos/metabolismo , Nitrogênio/metabolismo , Animais , Bacteroidetes/metabolismo , Biomassa , Células CHO/metabolismo , Carbono/metabolismo , Isótopos de Carbono , Chlorella vulgaris/metabolismo , Cricetulus , Genoma , Saccharomyces cerevisiae/metabolismo , Inanição , Yarrowia/metabolismo
9.
Enzyme Microb Technol ; 135: 109498, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32146931

RESUMO

Whole-cell biocatalysts could be used in wide-ranging applications. In this study, a new kind of whole-cell biocatalyst was successfully constructed by genetically immobilizing soybean seed coat peroxidase (SBP) on the cell surface of Yarrowia lipolytica Po1h, using a new integrative surface display expression vector (pMIZY05). The coding sequence of SBP was optimized and chemically synthesized, then inserted into pMIZY05 to generate expression plasmid pMIZY05-oEp. A DNA fragment corresponding to SBP and selection marker expression cassettes, without bacterial sequences, was released from pMIZY05-oEp by enzyme digestion and used to transform host yeast cells. A transformant (CM11) with a high recombinant SBP activity of 1571.9 U/mL was obtained, and recombinant SBP was proved to be successfully anchored on cell surface by testing the activities of different cellular fractions. After optimization of culture conditions, the recombinant SBP activity of CM11 was increased to 4187.8 U/mL. Afterwards, biochemical properties of the recombinant SBP were determined: optimum catalytic conditions were 37.5℃ at pH 3.5, and recombinant SBP exhibited high stability during thermal or acidic treatment. Recombinant activity of cell-displayed SBP was re-examined at optimum temperature and pH, which promoted an increase up to 4432.5 U/mL. To our knowledge, this represents the highest activity ever reported for heterologous expression of SBP. This study also provides a useful strategy for heterologous expression of proteins which could be toxic to intracellular content of host cells.


Assuntos
Peroxidases/genética , Proteínas de Soja/genética , Yarrowia/genética , Biocatálise , Clonagem Molecular , Estabilidade Enzimática , Expressão Gênica , Peroxidases/química , Peroxidases/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo , Proteínas de Soja/química , Proteínas de Soja/metabolismo , Yarrowia/metabolismo
10.
Electron. j. biotechnol ; 44: 19-24, Mar. 2020. ilus, graf
Artigo em Inglês | LILACS | ID: biblio-1087631

RESUMO

BACKGROUND: Pyruvic acid (PA), a vital α-oxocarboxylic acid, plays an important role in energy and carbon metabolism. The oleaginous yeast Yarrowia lipolytica (Y. lipolytica) has considerable potential for the production of PA. An increased NaCl concentration reportedly increases the biomass and PA yield of Y. lipolytica. RESULTS: To increase the yield of PA, the NaCl-tolerant Y. lipolytica A4 mutant was produced using the atmospheric and room temperature plasma method of mutation. The A4 mutant showed growth on medium containing 160 g/L NaCl. The PA yield of the A4 mutant reached 97.2 g/L at 120 h (0.795 g/g glycerol) in a 20-L fermenter with glycerol as the sole carbon source, which was 28.9% higher than that of the parental strain. CONCLUSION: The PA yield from Y. lipolytica can be improved by increasing its NaCl tolerance.


Assuntos
Ácido Pirúvico/metabolismo , Yarrowia/genética , Yarrowia/metabolismo , Pressão Osmótica , Leveduras , Carbono/metabolismo , Cloreto de Sódio , Reatores Biológicos , Tolerância ao Sal/genética , Fermentação , Glicerol/metabolismo , Mutação
11.
Molecules ; 25(4)2020 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-32093025

RESUMO

The demand for natural lactone gamma-decalactone (GDL) has increased in the fields of food and cosmetic products. However, low productivity during bioprocessing limits its industrial production. In this study, a novel composite porous cell carrier, bacterial cellulose-alginate (BC-ALG), was used for long-term biotransformation and production of GDL. The effects of this carrier on biotransformation and related mechanisms were investigated. BC-ALG carriers showed improved mechanical strength over ALG carriers, with their internal embedded cell pattern changed to an interconnected porous structure. In five repeated-batch biotransformation experiments, the maximum concentration of GDL obtained in culture with BC-ALG carriers was 8.37 g/L, approximately 3.7 times higher than that from the medium with an ALG carrier alone. The result indicated that multiple hydrogen bonding interactions at the interface between BC and ALG contributed to the compatibility and stability of BC-ALG carriers. On the basis of the above results, the BC-ALG composite carrier can be considered ideal for immobilisation of cells for the production of GDL on an industrial scale, and has the potential to be utilised in other biological processes.


Assuntos
Alginatos/química , Células Imobilizadas/metabolismo , Celulose/química , Lactonas/metabolismo , Yarrowia/metabolismo
12.
Microb Cell Fact ; 19(1): 38, 2020 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-32070349

RESUMO

BACKGROUND: Synthetic biology requires toolbox of promoters to finely tune gene expression levels for building up efficient cell factories. Yeast promoters owned variable core promoter regions between the TATA-box and transcriptional starting site (TSS) at the length mostly around 20-80 bases. This region allowed flexible design of artificial promoter but potentially demand special base motifs to maintain or enhance the promoter's strength. RESULTS: Here, we designed and screened the base motifs and tested the activities of yeast artificial core promoters. Different 30 bases of artificial sequences led to variable expression levels of CrtY enzyme which determined the lycopene-carotene compositions, represented in the colony-color spectrum of red-orange-yellow. The upstream sequences of two strong promoter PEXP1 and PGPD and two starting strains with distinguishable lycopene production levels were utilized to characterize the promoter sequences. Different partition designs of T-rich or G/C-rich base motifs led to distinguishable colony-color distributions. Finally, we screened a champion promoter with a highest 5.5-fold enhancement of lycopene-carotene transformation. Another selected promoter generated a highest beta-carotene production as 7.4 mg/g DCW. CONCLUSIONS: This work offered an approach to redesign promoter with artificial sequences. We concluded that the core promoter region could be designated as 30 bases and different base motifs would enhance or weaken the promoter's strength. Generally, more T-rich elements, higher %T and lower G/C percentage were beneficial to enhance the strength of artificial core promoter.


Assuntos
Proteínas Fúngicas/metabolismo , Genes Fúngicos , Liases Intramoleculares/metabolismo , Regiões Promotoras Genéticas , Yarrowia/genética , Proteínas Fúngicas/genética , Engenharia Genética , Liases Intramoleculares/genética , Licopeno/metabolismo , Biologia Sintética , Transcrição Genética , Yarrowia/metabolismo , beta Caroteno/biossíntese
13.
FEMS Microbiol Lett ; 367(5)2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-32053204

RESUMO

The discovery of non-fish sources of polyunsaturated fatty acids (PUFAs) is of great biotechnological importance. Although various oleaginous microalgae and fungi are able of accumulating storage lipids (single cell oils - SCOs) containing PUFAs, the industrial applications utilizing these organisms are rather limited due to the high-fermentation cost. However, combining SCO production with other biotechnological applications, including waste and by-product valorization, can overcome this difficulty. In the current review, we present the major sources of fungi (i.e. members of Mucoromycota, fungoid-like Thraustochytrids and genetically modified strains of Yarrowia lipolytica) and microalgae (e.g. Isochrysis, NannochloropsisandTetraselmis) that have come recently to the forefront due to their ability to produce PUFAs. Approaches adopted in order to increase PUFA productivity and the potential of using various residues, such as agro-industrial, food and aquaculture wastes as fermentation substrates for SCO production have been considered and discussed. We concluded that several organic residues can be utilized as feedstock in the SCO production increasing the competitiveness of oleaginous organisms against conventional PUFA producers.


Assuntos
Meios de Cultura , Ácidos Graxos Insaturados/biossíntese , Fungos/metabolismo , Engenharia Metabólica , Microalgas/metabolismo , Biocombustíveis , Biotecnologia , Fermentação , Fungos/genética , Microalgas/genética , Óleos/metabolismo , Yarrowia/genética , Yarrowia/metabolismo
14.
Microb Cell Fact ; 19(1): 22, 2020 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-32024536

RESUMO

BACKGROUND: Yarrowia lipolytica is an oleaginous yeast that can be genetically engineered to produce lipid and non-lipid biochemicals from a variety of feedstocks. Metabolic engineering of this organism usually requires genetic markers in order to select for modified cells. The potential to combine multiple genetic manipulations depends on the availability of multiple or recyclable selectable markers. RESULTS: We found that Y. lipolytica has the ability to utilize acetamide as the sole nitrogen source suggesting that the genome contains an acetamidase gene. Two potential Y. lipolytica acetamidase gene candidates were identified by homology to the A. nidulans acetamidase amdS. These genes were deleted in the wild-type Y. lipolytica strain YB-392, and deletion strains were evaluated for acetamide utilization. One deletion strain was unable to grow on acetamide and a putative acetamidase gene YlAMD1 was identified. Transformation of YlAMD1 followed by selection on acetamide media and counterselection on fluoroacetamide media showed that YlAMD1 can be used as a recyclable genetic marker in Saccharomyces cerevisiae and Ylamd1Δ Y. lipolytica. CONCLUSIONS: These findings add to our understanding of Y. lipolytica nitrogen utilization and expand the set of genetic tools available for engineering this organism, as well as S. cerevisiae.


Assuntos
Acetamidas/metabolismo , Amidoidrolases/genética , Engenharia Metabólica , Yarrowia/genética , Yarrowia/metabolismo , Marcadores Genéticos/genética , Saccharomyces cerevisiae/genética , Transformação Genética
15.
Sci Rep ; 10(1): 1741, 2020 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-32015397

RESUMO

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.


Assuntos
Clonagem Molecular , Proteínas Fúngicas/biossíntese , Lipase/biossíntese , Pichia/metabolismo , Proteínas Recombinantes/biossíntese , Yarrowia/metabolismo , Biomassa
16.
Microb Cell Fact ; 19(1): 49, 2020 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-32103761

RESUMO

BACKGROUND: The GRAS and oleaginous yeast Yarrowia lipolytica (Y. lipolytica) is an attractive cell factory for the production of chemicals and biofuels. The production of many natural products of commercial interest have been investigated in this cell factory by introducing heterologous biosynthetic pathways and by modifying the endogenous pathways. However, since natural products anabolism involves long pathways and complex regulation, re-channelling carbon into the product of target compounds is still a cumbersome work, and often resulting in low production performance. RESULTS: In this work, the carotenogenic genes contained carB and bi-functional carRP from Mucor circinelloides and carotenoid cleavage dioxygenase 1 (CCD1) from Petunia hybrida were introduced to Y. lipolytica and led to the low production of ß-ionone of 3.5 mg/L. To further improve the ß-ionone synthesis, we implemented a modular engineering strategy for the construction and optimization of a biosynthetic pathway for the overproduction of ß-ionone in Y. lipolytica. The strategy involved the enhancement of the cytosolic acetyl-CoA supply and the increase of MVA pathway flux, yielding a ß-ionone titer of 358 mg/L in shake-flask fermentation and approximately 1 g/L (~ 280-fold higher than the baseline strain) in fed-batch fermentation. CONCLUSIONS: An efficient ß-ionone producing GRAS Y. lipolytica platform was constructed by combining integrated overexpressed of heterologous and native genes. A modular engineering strategy involved the optimization pathway and fermentation condition was investigated in the engineered strain and the highest ß-ionone titer reported to date by a cell factory was achieved. This effective strategy can be adapted to enhance the biosynthesis of other terpenoids in Y. lipolytica.


Assuntos
Engenharia Metabólica , Norisoprenoides/metabolismo , Yarrowia/metabolismo , Acetilcoenzima A/metabolismo , Fermentação , Microbiologia Industrial , Microrganismos Geneticamente Modificados/genética , Microrganismos Geneticamente Modificados/metabolismo , Oxigênio/metabolismo , Yarrowia/genética
17.
J Agric Food Chem ; 68(5): 1364-1372, 2020 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-31903751

RESUMO

Arbutin, a glycoside, is derived from the leaves of several plants, including wheat, pear, and bearberry plants, and has a significant role in the treatment of melanoma, cystitis, and cough. Here, we aimed to modify Yarrowia lipolytica to produce arbutin. To construct the arbutin synthetic pathway in Y. lipolytica, three genes (chorismate pyruvate-lyase (UbiC), 4-hydroxybenzoate 1-hydroxylase (MNX1), and hydroquinone glucosyltransferase (AS)) were codon-optimized and heterologously expressed. To maximize arbutin production, seven arbutin-biosynthesis molecular targets were overexpressed, and we found that the individual strengthening of DHS1 and DHS2 led to an 8.9- and 7.8-fold improvement in arbutin yield, respectively. Through optimization, a maximum arbutin titer of 8.6 ± 0.7 g/L was achieved using the finally engineered strain, po1f-At09. Overall, this is the first report of heterologous arbutin synthesis in Y. lipolytica at a high titer. Furthermore, this work opens a possibility for the overproduction of shikimate pathway derivatives in Y. lipolytica.


Assuntos
Arbutina/biossíntese , Yarrowia/genética , Yarrowia/metabolismo , Arbutina/química , Engenharia Metabólica , Ácido Chiquímico/química , Ácido Chiquímico/metabolismo , Yarrowia/química
18.
N Biotechnol ; 56: 123-129, 2020 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-31953202

RESUMO

Microbial oil biosynthesis is envisaged as a promising technology for sustainable production of chemicals and fuels. Sugar-based substrates are the most typical carbon sources used for this purpose where metabolic pathways and stoichiometry are well known. However, the use of low-cost substrates is crucial for the economic viability of the process. Volatile fatty acids (VFAs) are considered to be a novel low-cost carbon source for microbial lipid production. They can be utilized by oleaginous yeasts to produce and store fatty acids in form of intracellular lipid bodies. In this work, Yarrowia lipolytica growth and substrate consumption were evaluated using the major VFAs present in anaerobic effluents. Individual VFAs as well as synthetic mixtures were tested at different concentrations to determine uptake rates and potential toxicity. Increasing VFA chain length resulted in greater biomass yield although, when added individually, 4 g Carbon/L VFA (e.g. 6.45 g/L of caproic and 10 g/L of acetic acid) caused inhibitory effects. Remarkably, biomass growth increased by 2.5-fold on real anaerobic fermentation effluent compared with synthetic mixtures. When real digestate was supplemented with synthetic VFAs up to 26.5 g/L, the inhibitory effect of the acids was counterbalanced. The results provided evidence of robustness of Y. lipolytica towards low-cost fermentation effluents and present this yeast as a promising candidate for the sustainable production of microbial oil using real digestates.


Assuntos
Carbono/metabolismo , Ácidos Graxos Voláteis/metabolismo , Eliminação de Resíduos Líquidos , Yarrowia/metabolismo , Carbono/química , Carbono/economia , Ácidos Graxos Voláteis/química , Ácidos Graxos Voláteis/economia , Yarrowia/crescimento & desenvolvimento
19.
Appl Environ Microbiol ; 86(3)2020 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-31704686

RESUMO

Thiamine is a vitamin that functions as a cofactor for key enzymes in carbon and energy metabolism in all living cells. While most plants, fungi, and bacteria can synthesize thiamine de novo, the oleaginous yeast Yarrowia lipolytica cannot. In this study, we used proteomics together with physiological characterization to elucidate key metabolic processes influenced and regulated by thiamine availability and to identify the genetic basis of thiamine auxotrophy in Y. lipolytica Specifically, we found that thiamine depletion results in decreased protein abundance for the lipid biosynthesis pathway and energy metabolism (i.e., ATP synthase), leading to the negligible growth and poor sugar assimilation observed in our study. Using comparative genomics, we identified the missing 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate synthase (THI13) gene for the de novo thiamine biosynthesis in Y. lipolytica and discovered an exceptional promoter, P3, that exhibits strong activation and tight repression by low and high thiamine concentrations, respectively. Capitalizing on the strength of our thiamine-regulated promoter (P3) to express the missing gene from Saccharomyces cerevisiae (scTHI13), we engineered a thiamine-prototrophic Y. lipolytica strain. By comparing this engineered strain to the wild-type strain, we revealed the tight relationship between thiamine availability and lipid biosynthesis and demonstrated enhanced lipid production with thiamine supplementation in the engineered thiamine-prototrophic Y. lipolytica strain.IMPORTANCE Thiamine plays a crucial role as an essential cofactor for enzymes involved in carbon and energy metabolism in all living cells. Thiamine deficiency has detrimental consequences for cellular health. Yarrowia lipolytica, a nonconventional oleaginous yeast with broad biotechnological applications, is a native thiamine auxotroph whose affected cellular metabolism is not well understood. Therefore, Y. lipolytica is an ideal eukaryotic host for the study of thiamine metabolism, especially because mammalian cells are also thiamine auxotrophic and thiamine deficiency is implicated in several human diseases. This study elucidates the fundamental effects of thiamine deficiency on cellular metabolism in Y. lipolytica and identifies genes and novel thiamine-regulated elements that eliminate thiamine auxotrophy in Y. lipolytica Furthermore, the discovery of thiamine-regulated elements enables the development of thiamine biosensors with useful applications in synthetic biology and metabolic engineering.


Assuntos
Proteínas Fúngicas/metabolismo , Proteoma , Deficiência de Tiamina/metabolismo , Tiamina/metabolismo , Yarrowia/metabolismo
20.
Cell Biol Int ; 44(2): 651-660, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31750586

RESUMO

In response to osmotic stress, the yeast Yarrowia lipolytica produces erythritol, a four-carbon sugar alcohol, from erythrose-P, an intermediate of the pentose phosphate pathway. Under non-stressing conditions (isotonic environment), the produced erythritol is subsequently recycled into erythrose-P that can feed the pentose phosphate pathway. Herein, gene YALI0F01584g was characterized as involved in the erythritol catabolic pathway. Several experimental evidences suggested that it encodes an erythrulose-1P isomerase that converts erythrulose-1P into erythrulose-4P. On the basis of our previous reports and results gathered in this study with genetically modified strains, including ΔYALI0F01584g and ΔYALI0F01628g disrupted mutants, the entire erythritol catabolic pathway has been characterized.


Assuntos
Eritritol/metabolismo , Proteínas Fúngicas/metabolismo , Fosfatos/metabolismo , Tetroses/metabolismo , Yarrowia/metabolismo , Sequência de Aminoácidos , Proteínas Fúngicas/genética , Homologia de Sequência , Yarrowia/genética , Yarrowia/crescimento & desenvolvimento
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