Application of New Yarrowia lipolytica Transformants in Production of Citrates and Erythritol from Glycerol.

Citric acid and erythritol are obtained on an industrial scale using biotechnological methods. Due to the growing market demand for these products, research is underway to improve the process economics by introducing new microorganisms, in particular of the species Yarrowia lipolytica. The aim of this study was to evaluate transformants of Y. lipolytica for growth and ability to overproduce citric acids and erythritol from glycerol. The transformants were constructed by overexpressing glycerol kinase, methylcitrate synthase and mitochondrial succinate-fumarate transporter in the mutant Wratislavia 1.31. Next, strains were assessed for biosynthesis of citrate (pH 5.5; nitrogen limitation) and erythritol (pH 3.0; high osmotic pressure) from glycerol. Regardless of culture conditions strains, 1.31.GUT1/6 and 1.31.GUT1/6.CIT1/3 exhibited high rates of substrate utilization. Under conditions favoring citrate biosynthesis, both strains produced several percent more citrates, accompanied by higher erythritol production compared to the parental strain. During erythritol biosynthesis, the strain 1.31.GUT1/6.CIT1/3.E34672g obtained as a result of co-expression of all three genes stood out, producing 84.0 g/L of erythritol with yield and productivity of 0.54 g/g and 0.72 g/Lh, respectively, which places it in the group of the highest-ranked producers of erythritol among Y. lipolytica species.

Bioconversion of waste glycerol into viscosinamide by Pseudomonas fluorescens DR54 and its activity evaluation.

Lipopeptides, derived from microorganisms, are promising surface-active compounds known as biosurfactants. However, the high production costs of biosurfactants, associated with expensive culture media and purification processes, limit widespread industrial application. To enhance the sustainability of biosurfactant production, researchers have explored cost-effective substrates. In this study, crude glycerol was evaluated as a promising and economical carbon source in viscosinamide production by Pseudomonas fluorescens DR54. Optimization studies using the Box - Behnken design and response surface methodology were performed. Optimal conditions for viscosinamide production including glycerol 70.8 g/L, leucine 2.7 g/L, phosphate 3.7 g/L, and urea 9.3 g/L were identified. Yield of viscosinamide production, performed under optimal conditions, reached 7.18 ± 0.17 g/L. Preliminary characterization of viscosinamide involved the measurement of surface tension. The critical micelle concentration of lipopeptide was determined to be 5 mg/L. Furthermore, the interactions between the viscosinamide and lipase from Candida rugosa (CRL) were investigated by evaluating the impact of viscosinamide on lipase activity and measuring circular dichroism. It was observed that the α-helicity of CRL increases with increasing viscosinamide concentration, while the random coil structure decreases.

Honey's Yeast-New Source of Valuable Species for Industrial Applications.

Honey is a rich source of compounds with biological activity; moreover, it is a valuable source of various microorganisms. The aim of this study was to isolate and identify yeast from a sample of lime honey from Poland as well as to assess its ability to biosynthesize value-added chemicals such as kynurenic acid, erythritol, mannitol, and citric acid on common carbon sources. Fifteen yeast strains belonging to the species Yarrowia lipolytica, Candida magnolia, and Starmerella magnoliae were isolated. In shake-flask screening, the best value-added compound producers were chosen. In the last step, scaling up of the culture in the bioreactor was performed. A newly isolated strain of Y. lipolytica No. 12 produced 3.9 mg/L of kynurenic acid growing on fructose. Strain Y. lipolytica No. 9 synthesized 32.6 g/L of erythritol on technical glycerol with a low concentration of byproducts. Strain Y. lipolytica No. 5 produced 15.1 g/L of mannitol on technical glycerol, and strain No. 3 produced a very high amount of citric acid (76.6 g/L) on technical glycerol. In conclusion, to the best of our knowledge this is the first study to report the use of yeast isolates from honey to produce valuable chemicals. This study proves that natural products such as lime honey can be an excellent source of wild-type yeasts with valuable production properties.

Comparative Analysis of the Alkaline Proteolytic Enzymes of Yarrowia Clade Species and Their Putative Applications.

Proteolytic enzymes are commercially valuable and have multiple applications in various industrial sectors. The most studied proteolytic enzymes produced by Yarrowia lipolytica, extracellular alkaline protease (Aep) and extracellular acid protease (Axp), were shown to be good candidates for different biotechnological applications. In this study, we performed a comprehensive analysis of the alkaline proteolytic enzymes of Yarrowia clade species, including phylogenetic studies, synteny analysis, and protease production and application. Using a combination of comparative genomics approaches based on sequence similarity, synteny conservation, and phylogeny, we reconstructed the evolutionary scenario of the XPR2 gene for species of the Yarrowia clade. Furthermore, except for the proteolytic activity of the analyzed Yarrowia clade strains, the brewers' spent grain (BSG) was used as a substrate to obtain protein hydrolysates with antioxidant activity. For each culture, the degree of hydrolysis was calculated. The most efficient protein hydrolysis was observed in the cultures of Y. lipolytica, Y. galli, and Y. alimentaria. In contrast, the best results obtained using the 2,2-azinobis (3-ethyl-benzothiazoline-6-sulfonic acid (ABTS) method were observed for the culture medium after the growth of Y. divulgata, Y. galli, and Y. lipolytica on BSG.

Studies on the Anticancer and Antioxidant Activities of Resveratrol and Long-Chain Fatty Acid Esters.

Resveratrol (RES) is gaining recognition as a natural bioactive compound. To expand the possible applications of RES with its enhanced bioactivity as well as to increase the health benefits of long-chain fatty acids, a lipophilization process of RES was performed using three fatty acids: palmitic acid (PA), oleic acid (OA), and conjugated linoleic acid (CLA). The obtained mono-, di-, and tri-esters of RES were evaluated for their anticancer and antioxidant properties against lung carcinoma (A549), colorectal adenocarcinoma (HT29), and pancreatic ductal adenocarcinoma (BxPC3) cell lines. Human fibroblast (BJ) cells were used as a control. Several parameters were investigated: cell viability and apoptosis, including the expression of major pro- and anti-apoptotic markers, as well as the expression of superoxide dismutase, a key enzyme of the body's antioxidant barrier. Three of the obtained esters: mono-RES-OA, mono-RES-CLA, and tri-RES-PA, which significantly reduced the tumor cell viability up to 23%, at concentrations 25, 10, 50 µg/mL, respectively, turned out to be particularly interesting. The above-mentioned resveratrol derivatives similarly increased the tumor cells' apoptosis by modifying their caspase activity of pro-apoptotic pathways (p21, p53, and Bax). Moreover, among the mentioned esters, mono-RES-OA induced apoptosis of the analyzed cell lines most strongly, reducing the number of viable cells up to 48% for HT29 cells versus 36% for pure RES. Furthermore, the selected esters exhibited antioxidant properties towards the normal BJ cell line by regulating the expression of major pro-antioxidant genes (superoxide dismutases-SOD1 and SOD2) without the effect on their expression in the tumor, and therefore reducing the defense of cancer cells against increased oxidative stress induced by high ROS accumulation. The obtained results indicate that the esters of RES and long-chain fatty acids allow enhancement of their biological activity. The RES derivatives have the potential for being applied in cancer prevention and treatment, as well as for oxidative stress suppression.

Cost-effective rhamnolipid production by Burkholderia thailandensis E264 using agro-industrial residues.

The agro-industrial by-products corn steep liquor (CSL) and olive mill wastewater (OMW) were evaluated as low-cost substrates for rhamnolipid production by Burkholderia thailandensis E264. In a culture medium containing CSL (7.5% (v/v)) as sole substrate, B. thailandensis E264 produced 175 mg rhamnolipid/L, which is about 1.3 times the amount produced in the standard medium, which contains glycerol, peptone, and meat extract. When the CSL medium was supplemented with OMW (10% (v/v)), rhamnolipid production further increased up to 253 mg/L in flasks and 269 mg/L in a bioreactor. Rhamnolipids produced in CSL + OMW medium reduced the surface tension up to 27.1 mN/m, with a critical micelle concentration of 51 mg/L, better than the values obtained with the standard medium (28.9 mN/m and 58 mg/L, respectively). However, rhamnolipids produced in CSL + OMW medium displayed a weak emulsifying activity when compared to those produced in the other media. Whereas di-rhamnolipid congeners represented between 90 and 95% of rhamnolipids produced by B. thailandensis E264 in CSL and the standard medium, the relative abundance of mono-rhamnolipids increased up to 55% in the culture medium containing OMW. The difference in the rhamnolipid congeners produced in each medium explains their different surface-active properties. To the best of our knowledge, this is the first report of rhamnolipid production by B. thailandensis using a culture medium containing agro-industrial by-products as sole ingredients. Furthermore, rhamnolipids produced in the different media recovered around 60% of crude oil from contaminated sand, demonstrating its potential application in the petroleum industry and bioremediation. KEY POINTS: • B. thailandensis produced RL using agro-industrial by-products as sole substrates • Purified RL displayed excellent surface activity (minimum surface tension 27mN/m) • Crude RL (cell-free supernatant) recovered 60% of crude oil from contaminated sand.

The microbial production of kynurenic acid using Yarrowia lipolytica yeast growing on crude glycerol and soybean molasses.

Yarrowia lipolytica yeast are able to produce kynurenic acid-a very valuable compound acting as a neuroprotective and antioxidant agent in humans. The recent data proved the existence of the kynurenine biosynthesis pathway in this yeast cells. Due to this fact, the aim of this work was to enhance kynurenic acid production using crude glycerol and soybean molasses as cheap and renewable carbon and nitrogen sources. The obtained results showed that Y. lipolytica GUT1 mutants are able to produce kynurenic acid in higher concentrations (from 4.5 mg dm-3 to 14.1 mg dm-3) than the parental strain (3.6 mg dm-3) in the supernatant in a medium with crude glycerol. Moreover, the addition of soybean molasses increased kynurenic acid production by using wild type and transformant strains. The A-101.1.31 GUT1/1 mutant strain produced 17.7 mg dm-3 of kynurenic acid in the supernatant during 150 h of the process and 576.7 mg kg-1 of kynurenic acid in dry yeast biomass. The presented work proves the great potential of microbial kynurenic acid production using waste feedstock. Yeast biomass obtained in this work is rich in protein, with a low content of lipid, and can be a healthy ingredient of animal and human diet.

Elevating Phospholipids Production Yarrowia lipolytica from Crude Glycerol.

Phospholipids (PLs) are a class of lipids with many proven biological functions. They are commonly used in lipid replacement therapy to enrich cell membranes damaged in chronic neurodegenerative diseases, cancer, or aging processes. Due to their amphipathic nature, PLs have been widely used in food, cosmetic, and pharmaceutical products as natural emulsifiers and components of liposomes. In Yarrowia lipolytica, PLs are synthesized through a similar pathway like in higher eukaryotes. However, PL biosynthesis in this yeast is still poorly understood. The key intermediate in this pathway is phosphatidic acid, which in Y. lipolytica is mostly directed to the production of triacylglycerols and, in a lower amount, to PL. This study aimed to deliver a strain with improved PL production, with a particular emphasis on increased biosynthesis of phosphatidylcholine (PC). Several genetic modifications were performed: overexpression of genes from PL biosynthesis pathways as well as the deletion of genes responsible for PL degradation. The best performing strain (overexpressing CDP-diacylglycerol synthase (CDS) and phospholipid methyltransferase (OPI3)) reached 360% of PL improvement compared to the wild-type strain in glucose-based medium. With the substitution of glucose by glycerol, a preferred carbon source by Y. lipolytica, an almost 280% improvement of PL was obtained by transformant overexpressing CDS, OPI3, diacylglycerol kinase (DGK1), and glycerol kinase (GUT1) in comparison to the wild-type strain. To further increase the amount of PL, the optimization of culture conditions, followed by the upscaling to a 2 L bioreactor, were performed. Crude glycerol, being a cheap and renewable substrate, was used to reduce the costs of PL production. In this process 653.7 mg/L of PL, including 352.6 mg/L of PC, was obtained. This study proved that Y. lipolytica is an excellent potential producer of phospholipids, especially from waste substrates.

Chokeberry Pomace as a Component Shaping the Content of Bioactive Compounds and Nutritional, Health-Promoting (Anti-Diabetic and Antioxidant) and Sensory Properties of Shortcrust Pastries Sweetened with Sucrose and Erythritol.

In this study, an attempt was made to develop shortcrust pastries containing different amounts of chokeberry pomace (0%, 10%, 30%, 50%), modulating their degree of sweetness via the application of sucrose or erythritol. The obtained products were assessed for their nutritional value (energy value, protein, fats, dietary fibre, sugars, minerals). Bioactive compounds, as well as antioxidant and anti-diabetic properties in an in vitro model and sensory attributes, were also analysed. Increasing the proportion of chokeberry pomace in shortcrust pastries improved their nutritional value, especially their energy value (reduction of nearly 30% for shortcrust pastries with 50% pomace sweetened with erythritol), nutritional fibre content (10-fold higher in shortcrust pastries with the highest proportion of pomace) and potassium, calcium, magnesium, and iron content. Chokeberry pomace was also a carrier of 14 bioactive compounds. The most beneficial antioxidant and anti-diabetic effect was shown for shortcrust pastries containing 50% chokeberry pomace. In addition, it was shown that the use of erythritol as a sweetener has a beneficial effect on the perception of sensory attributes. Finally, it was shown that the developed products could be excellent alternatives to traditional shortcrust pastries and, at the same time, be a good way to utilize waste from the fruit industry.

Advances in production of high-value lipids by oleaginous yeasts.

The global market for high-value fatty acids production, mainly omega-3/6, hydroxy fatty-acids, waxes and their derivatives, has seen strong development in the last decade. The reason for this growth was the increasing utilization of these lipids as significant ingredients for cosmetics, food and the oleochemical industries. The large demand for these compounds resulted in a greater scientific interest in research focused on alternative sources of oil production - among which microorganisms attracted the most attention. Microbial oil production offers the possibility to engineer the pathways and store lipids enriched with the desired fatty acids. Moreover, costly chemical steps are avoided and direct commercial use of these fatty acids is available. Among all microorganisms, the oleaginous yeasts have become the most promising hosts for lipid production - their efficient lipogenesis, ability to use various (often highly affordable) carbon sources, feasible large-scale cultivations and wide range of available genetic engineering tools turns them into powerful micro-factories. This review is an in-depth description of the recent developments in the engineering of the lipid biosynthetic pathway with oleaginous yeasts. The different classes of valuable lipid compounds with their derivatives are described and their importance for human health and industry is presented. The emphasis is also placed on the optimization of culture conditions in order to improve the yield and titer of these valuable compounds. Furthermore, the important economic aspects of the current microbial oil production are discussed.

High value-added products derived from crude glycerol via microbial fermentation using Yarrowia clade yeast.

BACKGROUND: Contemporary biotechnology focuses on many problems related to the functioning of developed societies. Many of these problems are related to health, especially with the rapidly rising numbers of people suffering from civilization diseases, such as obesity or diabetes. One factor contributing to the development of these diseases is the high consumption of sucrose. A very promising substitute for this sugar has emerged: the polyhydroxy alcohols, characterized by low caloric value and sufficient sweetness to replace table sugar in food production. RESULTS: In the current study, yeast belonging to the Yarrowia clade were tested for erythritol, mannitol and arabitol production using crude glycerol from the biodiesel and soap industries as carbon sources. Out of the 13 tested species, Yarrowia divulgata and Candida oslonensis turned out to be particularly efficient polyol producers. Both species produced large amounts of these compounds from both soap-derived glycerol (59.8-62.7 g dm-3) and biodiesel-derived glycerol (76.8-79.5 g dm-3). However, it is equally important that the protein and lipid content of the biomass (around 30% protein and 12% lipid) obtained after the processes is high enough to use this yeast in the production of animal feed. CONCLUSIONS: The use of waste glycerol for the production of polyols as well as utilization of the biomass obtained after the process for the production of feed are part of the development of modern waste-free technologies.

New Cytoplasmic Virus-Like Elements (VLEs) in the Yeast Debaryomyces hansenii.

Yeasts can have additional genetic information in the form of cytoplasmic linear dsDNA molecules called virus-like elements (VLEs). Some of them encode killer toxins. The aim of this work was to investigate the prevalence of such elements in D. hansenii killer yeast deposited in culture collections as well as in strains freshly isolated from blue cheeses. Possible benefits to the host from harboring such VLEs were analyzed. VLEs occurred frequently among fresh D. hansenii isolates (15/60 strains), as opposed to strains obtained from culture collections (0/75 strains). Eight new different systems were identified: four composed of two elements and four of three elements. Full sequences of three new VLE systems obtained by NGS revealed extremely high conservation among the largest molecules in these systems except for one ORF, probably encoding a protein resembling immunity determinant to killer toxins of VLE origin in other yeast species. ORFs that could be potentially involved in killer activity due to similarity to genes encoding proteins with domains of chitin-binding/digesting and deoxyribonuclease NucA/NucB activity, could be distinguished in smaller molecules. However, the discovered VLEs were not involved in the biocontrol of Yarrowia lipolytica and Penicillium roqueforti present in blue cheeses.

The Role of Hexokinase and Hexose Transporters in Preferential Use of Glucose over Fructose and Downstream Metabolic Pathways in the Yeast Yarrowia lipolytica.

The development of efficient bioprocesses requires inexpensive and renewable substrates. Molasses, a by-product of the sugar industry, contains mostly sucrose, a disaccharide composed of glucose and fructose, both easily absorbed by microorganisms. Yarrowia lipolytica, a platform for the production of various chemicals, can be engineered for sucrose utilization by heterologous invertase expression, yet the problem of preferential use of glucose over fructose remains, as fructose consumption begins only after glucose depletion what significantly extends the bioprocesses. We investigated the role of hexose transporters and hexokinase (native and fructophilic) in this preference. Analysis of growth profiles and kinetics of monosaccharide utilization has proven that the glucose preference in Y. lipolytica depends primarily on the affinity of native hexokinase for glucose. Interestingly, combined overexpression of either hexokinase with hexose transporters significantly accelerated citric acid biosynthesis and enhanced pentose phosphate pathway leading to secretion of polyols (31.5 g/L vs. no polyols in the control strain). So far, polyol biosynthesis was efficient in glycerol-containing media. Moreover, overexpression of fructophilic hexokinase in combination with hexose transporters not only shortened this process to 48 h (84 h for the medium with glycerol) but also allowed to obtain 23% more polyols (40 g/L) compared to the glycerol medium (32.5 g/L).

Application of a New Engineered Strain of Yarrowia lipolytica for Effective Production of Calcium Ketoglutarate Dietary Supplements.

The present study aimed to develop a technology for the production of dietary supplements based on yeast biomass and α-ketoglutaric acid (KGA), produced by a new transformant of Yarrowia lipolytica with improved KGA biosynthesis ability, as well to verify the usefulness of the obtained products for food and feed purposes. Transformants of Y. lipolytica were constructed to overexpress genes encoding glycerol kinase, methylcitrate synthase and mitochondrial organic acid transporter. The strains were compared in terms of growth ability in glycerol- and oil-based media as well as their suitability for KGA biosynthesis in mixed glycerol-oil medium. The impact of different C:N:P ratios on KGA production by selected strain was also evaluated. Application of the strain that overexpressed all three genes in the culture with a C:N:P ratio of 87:5:1 allowed us to obtain 53.1 g/L of KGA with productivity of 0.35 g/Lh and yield of 0.53 g/g. Finally, the possibility of obtaining three different products with desired nutritional and health-beneficial characteristics was demonstrated: (1) calcium α-ketoglutarate (CaKGA) with purity of 89.9% obtained by precipitation of KGA with CaCO3, (2) yeast biomass with very good nutritional properties, (3) fixed biomass-CaKGA preparation containing 87.2 µg/g of kynurenic acid, which increases the health-promoting value of the product.

Sustainable Surfactin Production by Bacillus subtilis Using Crude Glycerol from Different Wastes.

Most biosurfactants are obtained using costly culture media and purification processes, which limits their wider industrial use. Sustainability of their production processes can be achieved, in part, by using cheap substrates found among agricultural and food wastes or byproducts. In the present study, crude glycerol, a raw material obtained from several industrial processes, was evaluated as a potential low-cost carbon source to reduce the costs of surfactin production by Bacillus subtilis #309. The culture medium containing soap-derived waste glycerol led to the best surfactin production, reaching about 2.8 g/L. To the best of our knowledge, this is the first report describing surfactin production by B. subtilis using stearin and soap wastes as carbon sources. A complete chemical characterization of surfactin analogs produced from the different waste glycerol samples was performed by liquid chromatography-mass spectrometry (LC-MS) and Fourier transform infrared spectroscopy (FTIR). Furthermore, the surfactin produced in the study exhibited good stability in a wide range of pH, salinity and temperatures, suggesting its potential for several applications in biotechnology.

A 37-amino acid loop in the Yarrowia lipolytica hexokinase impacts its activity and affinity and modulates gene expression.

The oleaginous yeast Yarrowia lipolytica is a potent cell factory as it is able to use a wide variety of carbon sources to convert waste materials into value-added products. Nonetheless, there are still gaps in our understanding of its central carbon metabolism. Here we present an in-depth study of Y. lipolytica hexokinase (YlHxk1), a structurally unique protein. The greatest peculiarity of YlHxk1 is a 37-amino acid loop region, a structure not found in any other known hexokinases. By combining bioinformatic and experimental methods we showed that the loop in YlHxk1 is essential for activity of this protein and through that on growth of Y. lipolytica on glucose and fructose. We further proved that the loop in YlHxk1 hinders binding with trehalose 6-phosphate (T6P), a glycolysis inhibitor, as hexokinase with partial deletion of this region is 4.7-fold less sensitive to this molecule. We also found that YlHxk1 devoid of the loop causes strong repressive effect on lipase-encoding genes LIP2 and LIP8 and that the hexokinase overexpression in Y. lipolytica changes glycerol over glucose preference when cultivated in media containing both substrates.

De novo production of resveratrol from glycerol by engineering different metabolic pathways in Yarrowia lipolytica.

Resveratrol is a polyphenol with multiple applications in pharma, cosmetics and food. The aim of this study was to construct Yarrowia lipolytica strains able to produce resveratrol. For this purpose, resveratrol-biosynthesis genes from bacteria and plants were expressed in this host. Since resveratrol can be produced either via tyrosine or phenylaniline, both pathways were tested, first with a single copy and then with two copies. The phenylalanine pathway resulted in slightly higher production in glucose media, although when the media was supplemented with amino acids, the best production was found in the strain with two copies of the tyrosine pathway, which reached 0.085 â€‹g/L. When glucose was replaced by glycerol, a preferred substrate for bioproduction, the best results, 0.104 â€‹g/L, were obtained in a strain combining the expression of the two synthesis pathways. Finally, the best producer strain was tested in bioreactor conditions where a production of 0.43 â€‹g/L was reached. This study suggests that Y. lipolytica is a promising host for resveratrol production from glycerol.

Nitrogen as the major factor influencing gene expression in Yarrowia lipolytica.

Yarrowia lipolytica is an important industrial microorganism used for the production of oleochemicals. The design of effective biotechnological processes with this cell factory requires an in-depth knowledge of its metabolism. Here we present a transcriptomic study of Y. lipolytica grown in the presence of glycerol and glucose, and mixture of both at different carbon to nitrogen ratios. It emerged that the transcriptomic landscape of Y. lipolytica is more sensitive to the nitrogen availability than to the utilized carbon source, as evidenced by more genes being differentially expressed in lower carbon to nitrogen ratio. Specifically, expression of hexokinase (HXK1) is significantly susceptible to changes in nitrogen concentrations. High HXK1 expression in low nitrogen seems to impact other genes which are implicated in tricarboxylic acid cycle and erythritol biosynthesis. We further show that expression of HXK1 and two genes belonging to the sugar porter family might be controlled by GATA-like zinc-finger proteins.

Enhancing isoprenoid synthesis in Yarrowia lipolytica by expressing the isopentenol utilization pathway and modulating intracellular hydrophobicity.

The abundant supply of biosynthetic precursors and product compatibility with the intracellular environment play important roles for microbial isoprenoid production. In this study, we tailor to both of these requirements by introducing the two-step isopentenol utilization pathway (IUP) to augment the native pathway in the oleaginous yeast Yarrowia lipolytica. With shortcut access to the common isoprenoid precursor, isopentenyl pyrophosphate (IPP) and its isomer dimethylallyl pyrophosphate (DMAPP), IUP is capable of elevating IPP + DMAPP levels by 15.7-fold compared to the mevalonate pathway alone. The increase in IPP + DMAPP levels can directly lead to better isoprenoid synthesis, which is illustrated using lycopene as a model compound. Moreover, we also demonstrate that higher lipid contents in the cells correlate with improved intracellular lycopene production, suggesting the importance of having a substantial hydrophobic environment to sequester isoprenoids. Combining these strategies with further genetic and fermentation optimizations, we achieved a final lycopene titer of 4.2 g/L. Overall, these strategies hold great potential for strengthening the synthesis of long-chain isoprenoids and fat-soluble natural products in microbes.

Production of high titer of citric acid from inulin.

BACKGROUND: Citric acid is considered as the most economically feasible product of microbiological production, therefore studies on cheap and renewable raw materials for its production are highly desirable. In this study citric acid was synthesized by genetically engineered strains of Yarrowia lipolytica from widely available, renewable polysaccharide - inulin. Hydrolysis of inulin by the Y. lipolytica strains was established by expressing the inulinase gene (INU1 gene; GenBank: X57202.1) with its native secretion signal sequence was amplified from genomic DNA from Kluyveromyces marxianus CBS6432. To ensure the maximum citric acid titer, the optimal cultivation strategy-repeated-batch culture was applied. RESULTS: The strain Y. lipolytica AWG7 INU 8 secreted more than 200 g dm- 3 of citric acid during repeated-batch culture on inulin, with a productivity of 0.51 g dm- 3 h- 1 and a yield of 0.85 g g- 1. CONCLUSIONS: The citric acid titer obtained in the proposed process is the highest value reported in the literature for Yarrowia yeast. The obtained results suggest that citric acid production from inulin by engineered Y. lipolytica may be a very promising technology for industrial citric acid production.