The increasing market demands of β‐carotene as colorant, antioxidant and vitamin precursor, requires novel biotechnological production platforms. Yarrowia lipolytica, is an industrial organism unable ...to naturally synthesize carotenoids but with the ability to produce high amounts of the precursor Acetyl‐CoA. We first found that a lipid overproducer strain was capable of producing more β‐carotene than a wild type after expressing the heterologous pathway. Thereafter, we developed a combinatorial synthetic biology approach base on Golden Gate DNA assembly to screen the optimum promoter‐gene pairs for each transcriptional unit expressed. The best strain reached a production titer of 1.5 g/L and a maximum yield of 0.048 g/g of glucose in flask. β‐carotene production was further increased in controlled conditions using a fed‐batch fermentation. A total production of β‐carotene of 6.5 g/L and 90 mg/g DCW with a concomitant production of 42.6 g/L of lipids was achieved. Such high titers suggest that engineered Y. lipolytica is a competitive producer organism of β‐carotene.
In this work the yeast Yarrowia lipolytica is engineered using synthetic biology techniques in order to become a producer of high amounts of the high value compound, beta‐carotene. The combination of multi‐copy integration and random selection of promoters, using Golden Gate based approach, further increased production, reaching production levels higher than 6 g/L.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
One of the most promising alternatives to petroleum for the production of fuels and chemicals is bio-oil based chemistry. Microbial oils are gaining importance because they can be engineered to ...accumulate lipids enriched in desired fatty acids. These specific lipids are closer to the commercialized product, therefore reducing pollutants and costly chemical steps. Yarrowia lipolytica is the most widely studied and engineered oleaginous yeast. Different molecular and bioinformatics tools permit systems metabolic engineering strategies in this yeast, which can produce usual and unusual fatty acids. Usual fatty acids, those usually found in triacylglycerol, accumulate through the action of several pathways, such as fatty acid/triacylglycerol synthesis, transport and degradation. Unusual fatty acids are enzymatic modifications of usual fatty acids to produce compounds that are not naturally synthetized in the host. Recently, the metabolic engineering of microorganisms has produced different unusual fatty acids, such as building block ricinoleic acid and nutraceuticals such as conjugated linoleic acid or polyunsaturated fatty acids. Additionally, microbial sources are preferred hosts for the production of fatty acid-derived compounds such as γ-decalactone, hexanal and dicarboxylic acids. The variety of lipids produced by oleaginous microorganisms is expected to rise in the coming years to cope with the increasing demand.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPUK, ZRSKP
Economically viable biotechnology processes must be characterized by a favorable ratio between the production costs and the product market price. In the bioproduction of bulk chemicals, costs must be ...minimized so that the process is competitive relative to petroleum-based production. The substrate costs must thus be reduced by employing inexpensive carbon sources, such as industrial wastes. Unfortunately, the most convenient microorganisms for a bioconversion are typically unable to degrade such substrates. Fortunately, the discovery of new enzymes together with advances in synthetic biology has moved metabolic engineering forward, expanding substrate ranges. Here we review the latest advances made using the industrial yeast Yarrowia lipolytica , which can exploit various carbon sources to produce biofuels and chemicals.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPUK, ZRSKP
Moving our society towards a bioeconomy requires efficient and sustainable microbial production of chemicals and fuels. Rhodotorula (Rhodosporidium) toruloides is a yeast that naturally synthesizes ...substantial amounts of specialty chemicals and has been recently engineered to (i) enhance its natural production of lipids and carotenoids, and (ii) produce novel industrially relevant compounds. The use of R. toruloides by companies and research groups has exponentially increased in recent years as a result of recent improvements in genetic engineering techniques and the availability of multiomics information on its genome and metabolism. This review focuses on recent engineering approaches in R. toruloides for bioproduction and explores its potential as a biotechnological chassis.
Finding the right microorganism is a key issue for a bioprocess. Ideally, it should be a good natural producer, use low-cost substrates, and perform well at large scale. An engineering process is often required to satisfy such requirements.
Lipids and carotenoids are two industrially relevant compounds. Lipids are a good source of fuels and chemicals which could facilitate the replacement of petroleum. Carotenoids are used as ingredients for the food, feed, pharma, and cosmetic industries. R. toruloides naturally synthesize high amounts of these compounds, which makes it a good candidate for their production.
R. toruloides has recently emerged as one of the most promising yeasts for bioproduction. In addition to its natural and convenient industrial features, it can now be metabolically engineered to boost production levels and to expand the range of compounds that are produced.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPUK, ZRSKP
Droplet-based microfluidics is becoming an increasingly attractive alternative to microtiter plate techniques for enzymatic high-throughput screening (HTS), especially for exploring large diversities ...with lower time and cost footprint. In this case, the assayed enzyme has to be accessible to the substrate within the water-in-oil droplet by being ideally extracellular or displayed at the cell surface. However, most of the enzymes screened to date are expressed within the cytoplasm of Escherichia coli cells, which means that a lysis step must take place inside the droplets for enzyme activity to be assayed. Here, we take advantage of the excellent secretion abilities of the yeast Yarrowia lipolytica to describe a highly efficient expression system particularly suitable for the droplet-based microfluidic HTS.
Five hydrolytic genes from Aspergillus niger genome were chosen and the corresponding five Yarrowia lipolytica producing strains were constructed. Each enzyme (endo-β-1,4-xylanase B and C; 1,4-β-cellobiohydrolase A; endoglucanase A; aspartic protease) was successfully overexpressed and secreted in an active form in the crude supernatant. A droplet-based microfluidic HTS system was developed to (a) encapsulate single yeast cells; (b) grow yeast in droplets; (c) inject the relevant enzymatic substrate; (d) incubate droplets on chip; (e) detect enzymatic activity; and (f) sort droplets based on enzymatic activity. Combining this integrated microfluidic platform with gene expression in Y. lipolytica results in remarkably low variability in the enzymatic activity at the single cell level within a given monoclonal population (<5%). Xylanase, cellobiohydrolase and protease activities were successfully assayed using this system. We then used the system to screen for thermostable variants of endo-β-1,4-xylanase C in error-prone PCR libraries. Variants displaying higher thermostable xylanase activities compared to the wild-type were isolated (up to 4.7-fold improvement).
Yarrowia lipolytica was used to express fungal genes encoding hydrolytic enzymes of interest. We developed a successful droplet-based microfluidic platform for the high-throughput screening (10
strains/h) of Y. lipolytica based on enzyme secretion and activity. This approach provides highly efficient tools for the HTS of recombinant enzymatic activities. This should be extremely useful for discovering new biocatalysts via directed evolution or protein engineering approaches and should lead to major advances in microbial cell factory development.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
The oleaginous yeast
Yarrowia lipolytica can accumulate up to 38% of its dry weight (DW) as lipids. Factors involved in lipid accumulation, particularly triglycerides, are not well identified. Using ...different mutations in the glycerol-3-phosphate (G3P) shuttle pathway (
Δgut2 affecting the anabolic dehydrogenase or overexpressing
GPD1 affecting the catabolic dehydrogenase), we were able to modulate G3P concentration. We show that in a Po1d genetic background,
GPD1 overexpression,
GUT2 inactivation or both mutations together result in 1.5, 2.9, and 5.6-fold respective increases in the level of G3P leading to an increase of triacylglyceride (TAG) accumulation. Moreover, our results indicate that each strain with an increased concentration of G3P, also presented a decreased concentration of glycerol. Analysis of the different genes involved in glycerol metabolism indicated that
Y. lipolytica does not possess a gene for glycerol-3-phosphatase. These findings suggest that
Y. lipolytica has a modified and unique metabolism of glycerol that is dedicated to G3P synthesis (and also to TAG synthesis) which may contribute to its oleaginous character. Furthermore, coupling the G3P shuttle disorders to a deficient β-oxidation pathway (by inactiving
POX1-6 or
MFE1 genes) increased TAG and free fatty acids content. Finally, we obtained strains that accumulated up to 65–75% of their DW as lipid. Transcriptional analysis in these strains, revealed that the high levels of lipids resulted from over-expression of genes involved in TAG synthesis (
SCT1, encoding a sn-1 acyltransferase; and
DGA1, encoding an acylCoA diacylglycerol acyltransferase) and the repression of genes involved in the degradation of TAG (
TGL3 and
TGL4, encoding triacylglycerol lipases). These findings indicate that TAG synthesis is limited by the availability of G3P and fatty acids, and that the expression of genes involved in TAG homeostasis is regulated by the G3P shuttle and the β-oxidation pathway. Finally, the synergistic contribution of acyltransferase gene expression to G3P synthesis is required for high levels of TAG synthesis and lipid accumulation in
Y. lipolytica.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPUK
Microbial oils are sustainable alternatives to petroleum for the production of chemicals and fuels. Oleaginous yeasts are promising source of oils and Yarrowia lipolytica is the most studied and ...engineered one. Nonetheless the commercial production of biolipids is so far limited to high value products due to the elevated production and extraction costs. In order to contribute to overcoming these limitations we exploited the possibility of secreting lipids to the culture broth, uncoupling production and biomass formation and facilitating the extraction. We therefore considered two synthetic approaches, Strategy I where fatty acids are produced by enhancing the flux through neutral lipid formation, as typically occurs in eukaryotic systems and Strategy II where the bacterial system to produce free fatty acids is mimicked. The engineered strains, in a coupled fermentation and extraction process using alkanes, secreted the highest titer of lipids described so far, with a content of 120% of DCW.
•Metabolic engineering strategies permit fatty acid (FA) secretion in Y. lipolytica.•Mimicking bacterial fatty acid production in Yarrowia allows secretion of 85% of FA.•Alkane layer stimulates secretion and facilitates extraction and recovery of FA.•Lipid synthesis was uncoupled to biomass formation allowing 120% of lipid content.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPUK, ZRSKP
Summary
In this study, we have adopted Golden Gate modular cloning strategy to develop a robust and versatile DNA assembly platform for the nonconventional yeast Yarrowia lipolytica. To this end, a ...broad set of destination vectors and interchangeable building blocks have been constructed. The DNA modules were assembled on a scaffold of predesigned 4 nt overhangs covering three transcription units (each bearing promoter, gene and terminator), selection marker gene and genomic integration targeting sequences, constituting altogether thirteen elements. Previously validated DNA modules (regulatory elements and selection markers) were adopted as the Golden Gate bricks. The system's operability was demonstrated based on synthetic pathway of carotenoid production. This technology greatly enriches a molecular biology toolbox dedicated to this industrially relevant microorganism enabling fast combinatorial cloning of complex synthetic pathways.
In this study we have developed a Golden Gate modular cloning strategy as a robust and versatile DNA assembly platform for the nonconventional yeast Yarrowia lipolytica.
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FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, UL, UM, UPUK
Microbial production of secretory proteins constitutes one of the key branches of current industrial biotechnology, earning billion dollar (USD) revenues each year. That industrial branch strongly ...relies on fluent operation of the secretory machinery within a microbial cell. The secretory machinery, directing the nascent polypeptide to its final destination, constitutes a highly complex system located across the eukaryotic cell. Numerous molecular identities of diverse structure and function not only build the advanced network assisting folding, maturation and secretion of polypeptides but also serve as sensors and effectors of quality control points. All these events must be harmoniously orchestrated to enable fluent processing of the protein traffic. Availability of these elements is considered to be the limiting factor determining capacity of protein traffic, which is of crucial importance upon biotechnological production of secretory proteins. The main purpose of this work is to review and discuss findings concerning secretory machinery operating in a non-conventional yeast species,
Yarrowia lipolytica
, and to highlight peculiarities of this system prompting its use as the production host. The reviewed literature supports the thesis that secretory machinery in
Y. lipolytica
is characterized by significantly higher complexity than a canonical yeast protein secretion pathway, making it more similar to filamentous fungi-like systems in this regard.
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CEKLJ, DOBA, EMUNI, FZAB, GEOZS, IJS, IMTLJ, IZUM, KILJ, KISLJ, MFDPS, NUK, OBVAL, OILJ, PILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UILJ, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
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