Yarrowia lipolytica as a model for bio-oil production

The yeast Yarrowia lipolytica has developed very efficient mechanisms for breaking down and using hydrophobic substrates. It is considered an oleaginous yeast, based on its ability to accumulate large amounts of lipids. Completion of the sequencing of the Y. lipolytica genome and the existence of suitable tools for genetic manipulation have made it possible to use the metabolic function of this species for biotechnological applications. In this review, we describe the coordinated pathways of lipid metabolism, storage and mobilization in this yeast, focusing in particular on the roles and regulation of the various enzymes and organelles involved in these processes. The physiological responses of Y. lipolytica to hydrophobic substrates include surface-mediated and direct interfacial transport processes, the production of biosurfactants, hydrophobization of the cytoplasmic membrane and the formation of protrusions. We also discuss culture conditions, including the mode of culture control and the culture medium, as these conditions can be modified to enhance the accumulation of lipids with a specific composition and to identify links between various biological processes occurring in the cells of this yeast. Examples are presented demonstrating the potential use of Y. lipolytica in fatty-acid bioconversion, substrate valorization and single-cell oil production. Finally, this review also discusses recent progress in our understanding of the metabolic fate of hydrophobic compounds within the cell: their terminal oxidation, further degradation or accumulation in the form of intracellular lipid bodies.     

Yarrowia lipolytica lipase production enhanced by increased air pressure

Aims: To study the cellular growth and morphology of Yarrowia lipolytica W29 and its lipase and protease production under increased air pressures. Methods and Results: Batch cultures of the yeast were conducted in a pressurized bioreactor at 4 and 8 bar of air pressure and the cellular behaviour was compared with cultures at atmospheric pressure. No inhibition of cellular growth was observed by the increase of pressure. Moreover, the improvement of the oxygen transfer rate (OTR) from the gas to the culture medium by pressurization enhanced the extracellular lipase activity from 96·6 U l^?1 at 1 bar to 533·5 U l^?1 at 8 bar. The extracellular protease activity was reduced by the air pressure increase, thereby eliciting further lipase productivity. Cell morphology was slightly affected by pressure, particularly at 8 bar, where cells kept the predominant oval form but decreased in size. Conclusions: OTR improvement by total air pressure rise up to 8 bar in a bioreactor can be applied to the enhancement of lipase production by Y. lipolytica. Significance and Impact of the Study: Hyperbaric bioreactors can be successfully applied for yeast cells cultivation, particularly in high‐density cultures used for enzymes production, preventing oxygen limitation and consequently increasing overall productivity.     

Beneficial effects of enhanced aeration using perfluorodecalin in <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> cultures for lipase production

The inadequate supply of oxygen to biomass is a critical factor to the productivity of most aerobic submerged fermentations. This happens because oxygen is sparingly soluble in the aqueous media. The use of a second liquid phase of perfluorocarbon (PFC), an oxygen-carrying compound, in the culture medium can increase the availability of oxygen to the microorganisms. The effect of perfluorodecalin on Yarrowia lipolytica cultures was investigated in shake-flask cultures. It was found that the specific growth rate of Y. lipolytica, a strictly aerobic yeast, increases with increasing PFC concentration. Extracellular lipase production was increased with 20% (v/v) of PFC and agitation of 250 rev/min. It was shown that the PFC presence benefitted lipase production and not just its secretion to the extracellular medium.     

Multiple gene integration to promote erythritol production on glycerol in Yarrowia lipolytica

Objective

Erythritol (1,2,3,4-butanetetrol) is a 4-carbon sugar alcohol that occurs in nature as a metabolite or storage compound. In this study, a multiple gene integration strategy was employed to enhance erythritol production in Y. lipolytica.

Results

The effects on the production of erythritol in Y. lipolytica of seven key genes involved in the erythritol synthesis pathway were evaluated individually, among which transketolase (TKL1) and transaldolase (TAL1) showed important roles in enhancing erythritol production. The combined overexpression of four genes (GUT1, TPI1, TKL1, TAL1) and disruption of the EYD1 gene (encoding erythritol dehydrogenase), resulted in produce approximately 40 g/L erythritol production from glycerol. Further enhanced erythritol synthesis was obtained by overexpressing the RKI1 gene (encoding ribose 5-phosphate isomerase) and the AMPD gene (encoding AMP deaminase), indicating for the first time that these two genes are also related to the enhancement of erythritol production in Y. lipolytica.

Conclusions

A combined gene overexpression strategy was developed to efficiently improve the production of erythritol in Y. lipolytica, suggesting a great capacity and promising potential of this non-conventional yeast in converting glycerol into erythritol.

     

Homology-independent genome integration enables rapid library construction for enzyme expression and pathway optimization in Yarrowia lipolytica

Yarrowia lipolytica is an important oleaginous industrial microorganism used to produce biofuels and other value-added compounds. Although several genetic engineering tools have been developed for Y. lipolytica, there is no efficient method for genomic integration of large DNA fragments. In addition, methods for constructing multigene expression libraries for biosynthetic pathway optimization are still lacking in Y. lipolytica. In this study, we demonstrate that multiple and large DNA fragments can be randomly and efficiently integrated into the genome of Y. lipolytica in a homology-independent manner. This homology-independent integration generates variation in the chromosomal locations of the inserted fragments and in gene copy numbers, resulting in the expression differences in the integrated genes or pathways. Because of these variations, gene expression libraries can be easily created through one-step integration. As a proof of concept, a LIP2 (producing lipase) expression library and a library of multiple genes in the β-carotene biosynthetic pathway were constructed, and high-production strains were obtained through library screening. Our work demonstrates the potential of homology-independent genome integration for library construction, especially for multivariate modular libraries for metabolic pathways in Y. lipolytica, and will facilitate pathway optimization in metabolic engineering applications.     

Dimorphism inYarrowia lipolytica: Filament formation is suppressed by nitrogen starvation and inhibition of respiration

In contrast toSaccharomyces cerevisiae, nitrogen starvation inhibited formation of hyphae in liquid cultures ofY. lipolytica, while carbon source did not seem to be important for filament formation. Inhibitors of mitochondrial respiration strongly suppressed the development of hyphae, indicating that energy conversion processes, and thus carbon metabolism, may be involved. pH of the medium also strongly affected the morphology, but only in the presence of a complex nitrogen source, implying that the cells respond to altered nutrition in media with different pH rather than to pH itself. The results suggest that theXPR2 gene encodingY. lipolytica alkaline extracellular proteinase is involved in the regulation of dimorphism in this species.     

Expression of a lipid-inducible,self-regulating form of <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> lipase <Emphasis Type="Italic">LIP2</Emphasis> in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Saccharomyces cerevisiae is frequently used as a bioreactor for conversion of exogenously acquired metabolites into value-added products, but has not been utilized for bioconversion of low-cost lipids such as triacylglycerols (TAGs) because the cells are typically unable to acquire these lipid substrates from the growth media. To help circumvent this limitation, the Yarrowia lipolytica lipase 2 (LIP2) gene was cloned into S. cerevisiae expression vectors and used to generate S. cerevisiae strains that secrete active Lip2 lipase (Lip2p) enzyme into the growth media. Specifically, LIP2 expression was driven by the S. cerevisiae PEX11 promoter, which maintains basal transgene expression levels in the presence of sugars in the culture medium but is rapidly upregulated by fatty acids. Northern blotting, lipase enzyme activity assays, and gas chromatographic measurements of cellular fatty acid composition after lipid feeding all confirmed that cells transformed with the PEX11 promoter–LIP2 construct were responsive to lipids in the media, i.e., cells expressing LIP2 responded rapidly to either free fatty acids or TAGs and accumulated high levels of the corresponding fatty acids in intracellular lipids. These data provided evidence of the creation of a self-regulating positive control feedback loop that allows the cells to upregulate Lip2p production only when lipids are present in the media. Regulated, autonomous production of extracellular lipase activity is a necessary step towards the generation of yeast strains that can serve as biocatalysts for conversion of low-value lipids to value-added TAGs and other novel lipid products.     

Engineering 4-coumaroyl-CoA derived polyketide production in Yarrowia lipolytica through a β-oxidation mediated strategy

Polyketides are a diverse class of molecules sought after for their valuable properties, including as potential pharmaceuticals. Previously, we demonstrated that the oleaginous yeast Yarrowia lipolytica is an optimal host for production of the simple polyketide, triacetic acid lactone (TAL). We here expand the capacities of this host by overcoming previous media challenges and enabling production of more complex polyketides. Specifically, we employ a β-oxidation related strategy to improve polyketide production directly from defined media. Beyond TAL production, we establish biosynthesis of the 4-coumaroyl-CoA derived polyketides: naringenin, resveratrol, and bisdemethoxycurcumin, as well as the diketide intermediate, (E)-5-(4-hydroxyphenyl)-3-oxopent-4-enoic acid. In this background, we enable high-level de novo production of naringenin through import of both a heterologous pathway and a mutant Y. lipolytica allele. In doing so, we generated an averaged maximum titer of 898 mg/L naringenin, the highest titer reported to date in any host. These results demonstrate that Y. lipolytica is an ideal polyketide production host for more complex 4-coumaroyl-CoA derived products.     

CO2 enrichment and supporting material impact the primary metabolism and 20-hydroxyecdysone levels in Brazilian ginseng grown under photoautotrophy

Plant cell and organ cultures via the implementation of effective elicitation strategies can offer attractive biotechnological platforms for the enhanced production of phytochemicals of pharmaceutical interest. For the first time, the elicitation of exogenous signal molecules was conducted to enhance the production of pharmacologically active alkaloids and flavonoids in Isatis tinctoria L. hairy root cultures (ITHRCs). ITHRCs III and V correspondingly possessing high alkaloid and flavonoid productivity were adopted for elicitation treatments. The maximum accumulation of alkaloids in ITHRCs III elicited by 142.61 µM salicylic acid for 28.18 h and flavonoids in ITHRCs V elicited by 179.54 µM methyl jasmonate for 41.87 h increased 5.89- and 11.21-folds as compared with controls, respectively. Moreover, expressions of 11 genes involved in alkaloid and flavonoid biosynthetic pathways were significantly up-regulated following elicitation, among which YUCCA, CHI and F3′H genes might play a crucial role in the target phytochemical augmentation. Overall, two effective elicitation protocols were provided here to improve the yields of bioactive alkaloids and flavonoids in ITHRCs, which was useful for the scale-up production of these valuable compounds to meet the demands for natural bioactive ingredients by pharmaceutical industries.

     

Effect of an exponential feeding regime on the production of Rhodotorula araucariae epoxide hydrolase in Yarrowia lipolytica

Aims: To evaluate the effect of and exponential feeding regime on the production of epoxide hydrolase (EH) enzyme in recombinant Yarrowia lipolytica in comparison to a constant feed strategy. Methods and Results: An exponential feed model was developed and fermentations were fed at six different exponential rates. A twofold increase in EH productivity and a 15% increase in volumetric EH activity was obtained by applying exponential glucose feed rates in fed‐batch cultivation. These responses were modelled to obtain a theoretical optimum feed rate that was validated in duplicate fermentations. The model optimum of 0·06 h^?1 resulted in a volumetric EH activity of c. 5500 U l^?1 h^?1 and a maximum activity of 206 000 U l^?1. This correlated well with model predictions, with a variance of <10%. Conclusions: The use of an exponential feed strategy at a rate of 0·06 h ^{? 1} yielded best results for all key responses which show a clear improvement over a constant feed strategy. Significance and Impact of the Study: The study was the first evaluation of an exponential feed strategy on recombinant Y. lipolytica for the production of EH enzyme. The results suggest a strategy for the commercial production of a valuable pharmaceutical enzyme.     

Tanshinone biosynthesis in Salvia miltiorrhiza and production in plant tissue cultures

Salvia miltiorrhiza Bunge (Lamiaceae) root, generally called Danshen, is an important herb in Chinese medicine widely used for treatment of cardiovascular diseases. Diterpenoid tanshinons are major bioactive constituents of Danshen with notable pharmacological activities and the potential as new drug candidates against some important human diseases. The importance of Danshen for traditional and modern medicines has motivated the research interest over two decades in the biosynthesis and biotechnological production of tanshinones. Although diterpenes in plants are presumably derived from the non-mevalonate (MVA) pathway, tanshinone biosynthesis in S. miltiorrhiza may also depend on the MVA pathway based on some key enzymes and genes detected in the early steps of these pathways. Plant tissue cultures are the major biotechnological processes for rapid production of tanshinones and other bioactive compounds in the herb. Various in vitro cultures of S. miltiorrhiza have been established, including cell suspension, adventitious root, and hairy root cultures, which can accumulate the major tanshinones as in the plant roots. Tanshinone production in cell and hairy root cultures has been dramatically enhanced with various strategies, including medium optimization, elicitor stimulation, and nutrient feeding operations. This review will summarize the above developments and also provide our views on future trends.     

Lignocellulose Biotechnology: Current and Future Prospects

Abstract

Lignocellulose is the most abundant biomass available on Earth. It has attracted considerable attention as an alternate feed stock and energy resource because of the large quantities available and its renewable nature. The potential uses of lignocelluloses are in pulp and paper industries, production of fuel alcohol and chemicals, protein for food, and feed using biotechnological means. The current industrial activity of lignocellulosic biomass fermentation is limited mainly because of the difficulty in economic bioconversion of these materials to value-added products. Considerable improvement in many processes related to lignocellulose biotechnology appeared during the last decade. Current uses of lignocellulosic biomass, process constraints, and areas of future research are discussed here.     

Fermentation of n-Paraffins by Yeast

α-Ketoglutarate productivity from n-paraffins of 141 strains of identified yeasts was studied. Among the strains tested, only strains of Candida lipolytica exclusively showed a high ability to produce α-ketoglutarale.

It was also observed that these strains of Candida lipolytica required thiamine for their growth and that exegenous thiamine stimulated the activity of α-ketoglutarate dehydrogenase of Candida lipolytica AJ 5004.

From these results, relationship between thiamine requirement and α-ketoglutarate productivity of Candida lipolytica was discussed.

α-Ketoglutarate fermentation by representative strains of Candida lipolytica was also carried out.     

Effect of culture conditions on antifouling compound production of a sponge-associated fungus

Microorganisms associated with invertebrate hosts have long been suggested to be a source for bioactive metabolites. In this study, we reported that a sponge-associated fungus, Letendraea helminthicola, produced two antifouling compounds: 3-methyl-N-(2-phenylethyl) butanamide and cyclo(D-Pro-D-Phe). To optimize the production of these antifouling compounds, we then examined the production of compounds under different culture conditions (temperature, salinity, pH, and carbon and nitrogen sources). This fungus grew well and produced more compounds at temperatures between 18 and 30°C; the fungus grew well at 75 parts per thousand (ppt) salinity but produced the highest amount of antifouling compounds at 30 and 45 ppt. The optimal initial pH value for mycelial growth was 5.5 to 6.5, whereas the production of the antifouling compounds was maximized at pH 3.5 and 4.5. Glucose and xylose (as carbon sources) increased the production of antifouling compounds. Yeast extract and peptone (as nitrogen sources) maximized the production of mycelial biomass and antifouling compounds. Our results indicate that culture conditions greatly affect the production of bioactive compounds from mycelial fungal cultures as exemplified by strain L. helminthicola and that the conditions favorable for fungal growth may not be the best conditions for bioactive compound production.     

Multi-copy expression and fed-batch production of Rhodotorula araucariae epoxide hydrolase in Yarrowia lipolytica

Epoxide hydrolases (EHs) of fungal origin have the ability to catalyze the enantioselective hydrolysis of epoxides to their corresponding diols. However, wild type fungal EHs are limited in substrate range and enantioselectivity. Additionally, the production of fungal epoxide hydrolase (EH) by wild-type strains is typically very low. In the present study, the EH-encoding gene from Rhodotorula araucariae was functionally expressed in Yarrowia lipolytica, under the control of a growth phase inducible hp4d promoter, in a multi-copy expression cassette. The transformation experiments yielded a positive transformant, with a final EH activity of 220 U/g dw in shake-flask cultures. Evaluation of this transformant in batch fermentations resulted in ~ 7-fold improvement in EH activity over the flask scale. Different constant specific feed rates were tested in fed-batch fermentations, resulting in an EH activity of 1,750 U/g dw at a specific feed rate of ~ 0.1 g/g/h, in comparison to enzyme production levels of 0.3 U/g dw for the wild type R. araucariae and 52 U/g dw for an Escherichia coli recombinant strain expressing the same gene. The expression of EH in Y. lipolytica using a multi-copy cassette demonstrates potential for commercial application.     

Evaluation of CV2025 ω-transaminase for the bioconversion of lignin breakdown products into value-added chemicals: synthesis of vanillylamine from vanillin

Abstract

Lignin is an essential component of the cell wall of various plants and represents an abundant and renewable natural resource. Both thermo-chemical and biological pre-treatment can be applied to break down the phenylpropanoid polymer subunits present in lignin. These liberate a range of phenolic compounds which represent potential substrates for bioconversion by ω-transaminases. In this work, the CV2025 ω-transaminase (ω-TAm) from Chromobacterium violaceum DSM30191, heterologously expressed in E. coli, was explored for selective amination of lignin breakdown intermediates into value-added products. Eight potential ω-TAm substrates were initially screened using (S)-α-methylbenzylamine (MBA) as the amino donor. Vanillin was identified as the best potential substrate which is converted into vanillylamine; an intermediate in the preparation of pelargonic acid vanillylamide used as a hyperemia inducing active substance in wound dressings. At low vanillin and MBA concentrations (< 10 mM) and with an excess of the amine donor (1:4 mol/mol) 100% w/w conversion of vanillin into vanillylamine was observed within 25 min. At vanillin concentrations above 10 mM, substrate inhibition was observed decreasing the rate and yield of the bioconversion. High concentrations of the reaction product (vanillylamine) and by-product (acetophenone) also limited the conversion due to increased backward reaction rate and inhibition. Vanillylamine synthesis could be carried out by both whole cell and clarified lysate forms of the CV2025 ω-TAm while fed-batch bioconversions (feeding low concentrations of both vanillin and MBA) could help overcome substrate inhibition and double the final product concentrations obtained. These results demonstrate the potential for bioconversion of lignin breakdown products into value-added chemicals but illustrate the need for enzymes with improved substrate range and implementation of techniques to overcome product inhibition and equilibrium constraints.     

Heavy metal tolerance in marine strains of Yarrowia lipolytica

Heavy metal tolerance of two marine strains of Yarrowia lipolytica was tested on solid yeast extract peptone dextrose agar plates. Based on minimum inhibitory concentration esteems, it is inferred that the two strains of Y. lipolytica were tolerant to heavy metals such as Pb(II), Cr(III), Zn(II), Cu(II), As(V), and Ni(II) ions. The impact of various heavy metal concentrations on the growth kinetics of Y. lipolytica was likewise assessed. With increased heavy metal concentration, the specific growth rate was reduced with delayed doubling time. Furthermore, biofilm development of both yeasts on the glass surfaces and in microtitre plates was assessed in presence of different heavy metals. In microtitre plates, a short lag phase of biofilm formation was noticed without the addition of heavy metals in yeast nitrogen base liquid media. A lag phase was extended over increasing metal concentrations of media. Heavy metals like Cr(VI), Cd(II), and As(V) are contrastingly influenced on biofilms’ formation of microtitre plates. Other heavy metals did not much influence on biofilms development. Thus, biofilm formation is a strategy of Y. lipolytica under stress of heavy metals has significance in bioremediation process for recovery of heavy metals from contaminated environment.

     

Pseudomonas: a promising biocatalyst for the bioconversion of terpenes

The Pseudomonas genus is one of the most diverse and ecologically significant groups of known bacteria, and it includes species that have been isolated worldwide in all types of environments. The bacteria from this genus are characterized by an elevated metabolic versatility, which is due to the presence of a complex enzymatic system. Investigations since the early 1960s have demonstrated their potential as biocatalysts for the production of industrially relevant and value-added flavor compounds from terpenes. Although terpenes are often removed from essential oils as undesirable components, its synthetic oxy-functionalized derivatives have broad applications in flavors/fragrances and pharmaceutical industries. Hence, biotransformation appears to be an effective tool for the structural modification of terpene hydrocarbons and terpenoids to synthesize novel and high-valued compounds. This review highlights the potential of Pseudomonas spp. as biocatalysts for the bioconversion of terpenes and summarizes the presently known bioflavors that are obtained from these processes.     

Bioactive compounds in seaweed: functional food applications and legislation

Seaweed is more than the wrap that keeps rice together in sushi. Seaweed biomass is already used for a wide range of other products in food, including stabilising agents. Biorefineries with seaweed as feedstock are attracting worldwide interest and include low-volume, high value-added products and vice versa. Scientific research on bioactive compounds in seaweed usually takes place on just a few species and compounds. This paper reviews worldwide research on bioactive compounds, mainly of nine genera or species of seaweed, which are also available in European temperate Atlantic waters, i.e. Laminaria sp., Fucus sp., Ascophyllum nodosum, Chondrus crispus, Porphyra sp., Ulva sp., Sargassum sp., Gracilaria sp. and Palmaria palmata. In addition, Undaria pinnatifida is included in this review as this is globally one of the most commonly produced, investigated and available species. Fewer examples of other species abundant worldwide have also been included. This review will supply fundamental information for biorefineries in Atlantic Europe using seaweed as feedstock. Preliminary selection of one or several candidate seaweed species will be possible based on the summary tables and previous research described in this review. This applies either to the choice of high value-added bioactive products to be exploited in an available species or to the choice of seaweed species when a bioactive compound is desired. Data are presented in tables with species, effect and test organism (if present) with examples of uses to enhance comparisons. In addition, scientific experiments performed on seaweed used as animal feed are presented, and EU, US and Japanese legislation on functional foods is reviewed.