Sophorolipid Biosynthesis Research Articles

Handling the inhibitory role of product accumulation during sophorolipid fermentation in a bubble column reactor with an in situ foam recovery.

Sophorolipid (SL) production by Candida catenulata from sunflower fatty acids was studied in a bubble column reactor (BCR). The specific oxygen uptake rate was 0.021mggcell-1 min-1 which indicates the importance of aeration in SL biosynthesis. The measurement of oxygen transfer rate (OTR) in the BCR showed a satisfactory OTR value of about 0.093min-1 in the system. However, further SL production was stopped after 30h in the BCR mainly due to the product accumulation in the culture and its inhibitory effects on cell growth and SL synthesis. Since an extensive foam was generated in the BCR under the absence of an antifoam agent, the development of an in situ foam recovery system provided the integration of production and separation of SL to handle the problem. The application of the foam recovery system enhanced biomass and titer SL concentration by 38.5 and 28.2% in comparison with the conventional BCR, respectively. Further studies in the system were performed by monitoring the size of bubbles and their effects on the biomass and SL enrichment in the foam stream at different aeration rates where the SL enrichment varied from 900 to 100% at 12 and 50h of the fermentation.

Oleic acid‐rich waste fleshing oil as a secondary carbon source for the synthesis of sophorolipids

AbstractBiosurfactants are nontoxic, renewable, and environmentally benign; and have excellent emulsification and antibacterial properties. Oleic acid‐rich oils have a crucial role as a hydrophobic substrate in the biological production of sophorolipids (SL). The major purpose of this study is to identify a low‐cost oleic acid‐rich hydrophobic substrate that may be used as a secondary carbon source in the synthesis of SL by Starmerella bombicola (S. bombicola). Thus, the biosynthesis of SL was investigated utilizing a variety of edible and nonedible oils rich in oleic acid, which acts as a precursor. Characterization of SL generated from different oils using FT‐IR and NMR indicated the presence of both lactonic and acidic SL. SL production was greatest with secondary carbon sources, with 38.9, 27.57, and 19 g/L, respectively, in the order olive oil > sunflower oil > fleshing oil. Surprisingly, the cost analysis for fleshing oil synthesis indicated that it was the lowest at 0.43 INR/g, followed by sunflower oil at 0.52 INR/g and olive oil at 1.71 INR/g. Our findings indicate that fleshing oil is a potential secondary carbon source with significant SL yield as well as environmental and economic benefits.

Comprehensive metabolomics reveals correlation between sophorolipid biosynthesis and autophagy

Sophorolipids are biobased and biodegradable glycolipid surface-active agents contributing to the shift from petroleum to biobased surfactants, associated with clear environmental benefits. However, their production cost is currently too high to allow commercialisation. Therefore, a continuous sophorolipid production process was evaluated, i.e., a retentostat with an external filtration unit. Despite an initial increase in volumetric productivity, productivity eventually declined to almost 0 g L−1 h−1. Following comprehensive metabolomics on supernatant obtained from a standardised retentostat, we hypothesised exhaustion of the N-starvation-induced autophagy as the main mechanism responsible for the decline in bolaform sophorolipid productivity. Thirty-six metabolites that correlate with RNA/protein autophagy and high sophorolipid productivity were putatively identified. In conclusion, our results unveil a plausible cause of this bola sophorolipid productivity decline in an industrially relevant bioreactor set-up, which may thus impact majorly on future yeast biosurfactant regulation studies and the finetuning of bola sophorolipid production processes.

Vitreoscilla Hemoglobin Improves Sophorolipid Production in Starmerella Bombicola O-13-1 Under Oxygen Limited Conditions.

Sophorolipids (SLs) are homologous microbial secondary metabolites produced by Starmerella bombicola and have been widely applied in many industrial fields. The biosynthesis of SLs is a highly aerobic process and is often limited by low dissolved oxygen (DO) levels. In this study, the Vitreoscilla hemoglobin (VHb) gene was transformed into S. bombicola O-13–1 by homologous recombination to alleviate oxygen limitation. VHb expression improved the intracellular oxygen utilization efficiency under either oxygen-rich or oxygen-limited conditions. In shake flask culture, the production of SLs was higher in the recombinant (VHb+) strain than in the wild-type (VHb−) strain, while the oxygen uptake rate of the recombinant (VHb+) strain was significantly lower than that of the wild-type (VHb−) strain. In a 5 L bioreactor, the production of SLs did not increase significantly, but the DO level in the fermentation broth of the VHb+ strain was 21.8% higher than that of VHb− strain under oxygen-rich conditions. Compared to wide-type strains (VHb−), VHb expression enhanced SLs production by 25.1% in the recombinants (VHb+) under oxygen-limited conditions. In addition, VHb expression raised the transcription levels of key genes involved in the electron transfer chain (NDH, SDH, COX), TCA cycle (CS, ICD, KDG1) and SL synthesis (CYP52M1 and UGTA1) in the recombinant (VHb+) strains. VHb expression in S. bombicola could enhance SLs biosynthesis and intracellular oxygen utilization efficiency by increasing ATP production and cellular respiration. Our findings highlight the potential use of VHb to improve the oxygen utilization efficiency of S. bombicola in the industrial-scale production of SLs using industrial and agricultural by-products like molasses and waste oil as fermentation feedstock.

Hydrophobic cryogels prepared via cryo-polymerization as oil carriers for biosynthesis of sophorolipids

Microbial synthesis of high-value biochemicals like biosurfactants using renewable substrates has attracted an intensive interest in the past decade. In this work, the bioproduction of sophorolipids was achieved by fermentation of different vegetable oils with the strain of Candida bombicola A0803 using novel hydrophobic poly(butyl methacrylate)(pBMA) cryogels as the oil substrate delivery carriers. The hydrophobic cryogels were prepared successfully via the cryo-polymerization of the reactive emulsion containing hydrophobic monomers under freezing conditions, and the characterization of cryogels was investigated experimentally. The results showed that the polymerization yields of 70 %–89 % were achieved and the cryogels had supermacropores with the maximum porosities of 92 %–96 % and good elasticity in organic solvents but rigid in water, which could provide preferable release properties for vegetable oils. Theie Young’s elasticity modulus varied from 0.096 to 0.951 kPa in ethanol and acetone and the related aqueous solutions. With the pBMA cryogels as the delivery carriers of oil substrates, the maximum concentrations of sophorolipids produced for rapeseed, perilla seed and linseed oils were improved 14.5 %, 54.5 % and 53.7 %, while the maximum productivities were improved 14.5 %, 72.1 % and 42.7 % compared with those under the same fermentation condition of free carriers after 204 h–252 h of cultivation, indicating that the hydrophobic cryogels could be an interesting material for the potential applications in biosynthesis areas.

Identification and importance of mitochondrial citrate carriers and ATP citrate lyase for glycolipid production in Starmerella bombicola.

Starmerella bombicola is a non-conventional yeast commercially used as a microbial cell factory for sophorolipid production. Sophorolipids are glycolipid biosurfactants composed of a glucose disaccharide sophorose and a fatty acid. In de novo sophorolipid synthesis, the fatty acid moiety is derived from the fatty acid synthesis (FAS) complex; therefore, the yeast's lipid metabolism plays a crucial role in sophorolipid biosynthesis. As a fatty acid precursor, citric acid is a key primary metabolite that connects carbohydrate and lipid metabolism, and in S. bombicola, it also has a regulatory effect on sophorolipid composition and productivity. We aimed to identify the mitochondrial transporters involved in citrate shuttling and the ATP citrate lyase (Acl), the enzyme that converts citric acid into acetyl-CoA. Subsequently, we studied their role in the citric acid shuttle and glycolipid synthesis and the potential of citrate metabolism as a genetic manipulation target for increased glycolipid synthesis. Bioinformatics analyses predicted 32 mitochondrial carriers of which two were identified as citrate transporters, named SbCtp1 and SbYhm2. Deletion of these mitochondrial carriers led to a lesser sophorolipid yield and a shift in the lactonic/acidic sophorolipid ratio. However, only the knockout of SbYhm2 caused a decrease of citric and an increase of malic acid extracellular concentrations. Additionally, deletion of SbAcl1 had a negative effect on S. bombicola's specific growth rate and sophorolipid synthesis and contributed to extra- and intracellular citric acid accumulation. Unexpectedly, SbAcl1 overexpression also decreased glycolipid production.Key Points• Starmerella bombicola is an industrially relevant microbial cell factory for biosurfactant production.• There are 32 predicted mitochondrial carriers in S. bombicola.• Citrate mitochondrial carriers SbYhm2 and SbCtp1 are essential for glycolipid synthesis in S. bombicola.• Deletion of SbAcl1 negatively affects growth and sophorolipid production in S. bombicola. Graphical abstract.

Unraveling the regulation of sophorolipid biosynthesis in Starmerella bombicola.

Starmerella bombicola very efficiently produces the secondary metabolites sophorolipids (SLs). Their biosynthesis is not-growth associated and highly upregulated in the stationary phase. Despite high industrial and academic interest, the underlying regulation of SL biosynthesis remains unknown. In this paper, potential regulation of SL biosynthesis through the telomere positioning effect (TPE) was investigated, as the SL gene cluster is located adjacent to a telomere. An additional copy of this gene cluster was introduced elsewhere in the genome to investigate if this results in a decoy of regulation. Indeed, for the new strain, the onset of SL production was shifted to the exponential phase. This result was confirmed by RT-qPCR analysis. The TPE effect was further investigated by developing and applying a suitable reporter system for this non-conventional yeast, enabling non-biased comparison of gene expression between the subtelomeric CYP52M1- and the URA3 locus. This was done with a constitutive endogenous promotor (pGAPD) and one of the endogenous promotors of the SL biosynthetic gene cluster (pCYP52M1). A clear positioning effect was observed for both promotors with significantly higher GFP expression levels at the URA3 locus. No clear GFP upregulation was observed in the stationary phase for any of the new strains.

Redirecting the lipid metabolism of the yeast Starmerella bombicola from glycolipid to fatty acid production.

Free fatty acids are basic oleochemicals implemented in a range of applications including surfactants, lubricants, paints, plastics, and cosmetics. Microbial fatty acid biosynthesis has gained much attention as it provides a sustainable alternative for petrol- and plant oil-derived chemicals. The yeast Starmerella bombicola is a microbial cell factory that naturally employs its powerful lipid metabolism for the production of the biodetergents sophorolipids (> 300g/L). However, in this study we exploit the lipidic potential of S. bombicola and convert it from the glycolipid production platform into a free fatty acid cell factory. We used several metabolic engineering strategies to promote extracellular fatty acid accumulation which include blocking competing pathways (sophorolipid biosynthesis and β-oxidation) and preventing free fatty acid activation. The best producing mutant (Δcyp52m1Δfaa1Δmfe2) secreted 0.933g/L (± 0.04) free fatty acids with a majority of C18:1 (43.8%) followed by C18:0 and C16:0 (40.0 and 13.2%, respectively). Interestingly, deletion of SbFaa1 in a strain still producing sophorolipids also resulted in 25% increased de novo sophorolipid synthesis (P = 0.0089) and when oil was supplemented to the same strain, a 50% increase in sophorolipid production was observed compared to the wild type (P = 0.03). We believe that our work is pivotal for the further development and exploration of S. bombicola as a platform for synthesis of environmentally friendly oleochemicals.

Sophorolipid biosynthesis and production from diverse hydrophilic and hydrophobic carbon substrates.

Sophorolipids (SLs), mainly synthesized by yeasts, were a sort of biosurfactant with the highest fermentation level at present. In recent years, SLs have drawn extensive attention for their excellent physiochemical properties and physiological activities. Besides, issues such as economics, sustainability, and use of renewable resources also stimulate the shift from chemical surfactants towards green or microbial-derived biosurfactants. SLs' large-scale production and application were restricted by the relatively high production costs. Currently, waste streams from agriculture, food and oil refining industries, etc., have been exploited as low-cost renewable substrates for SL production. Advanced cultivation method, uncommonly used substrates, and new genetically modified SL-producing mutants were also designed and applied to improve the productivity or the special properties of SLs. In this review, a systematic and detailed description of primary and secondary metabolism pathways involved in SL biosynthesis was summarized firstly. Furthermore, based on the pathways of SL biosynthesis from different carbon substrates, we reviewed the current knowledge and advances in the exploration of cost-effective and infrequently used hydrophilic and hydrophobic substrates for large or specialized SL production.

Production of long-chain hydroxy fatty acids by Starmerella bombicola.

To decrease our dependency for the diminishing source of fossils resources, bio-based alternatives are being explored for the synthesis of commodity and high-value molecules. One example in this ecological initiative is the microbial production of the biosurfactant sophorolipids by the yeast Starmerella bombicola. Sophorolipids are surface-active molecules mainly used as household and laundry detergents. Because S. bombicola is able to produce high titers of sophorolipids, the yeast is also used to increase the portfolio of lipophilic compounds through strain engineering. Here, the one-step microbial production of hydroxy fatty acids by S. bombicola was accomplished by the selective blockage of three catabolic pathways through metabolic engineering. Successful production of 17.39 g/l (ω-1) linked hydroxy fatty acids was obtained by the successive blockage of the sophorolipid biosynthesis, the β-oxidation and the ω-oxidation pathways. Minor contamination of dicarboxylic acids and fatty aldehydes were successfully removed using flash chromatography. This way, S. bombicola was further expanded into a flexible production platform of economical relevant compounds in the chemical, food and cosmetic industries.

Metabolic profiling and flux distributions reveal a key role of acetyl-CoA in sophorolipid synthesis by Candida bombicola

Sophorolipids are important biosurfactants produced by the wild-type strain of Candida bombicola ATCC 22214, with antimicrobial, antiviral, and anticancer activities. Our previous study showed that a mutant strain lacking multifunctional enzyme type 2 displayed extremely low sophorolipid production capacity compared to the wild-type strain. In the present study, we aimed to reveal the possible reasons for this deficiency through a series of analytical methods. First, comparative metabolic profiling analysis between the wild-type and mutant strains was performed. Multivariate statistical analysis, including principal component analysis and orthogonal partial least squares discriminant analysis, showed that nine potential metabolites were closely associated with the metabolic differences between strains. Second, metabolic flux analysis revealed that an inadequate supply of intracellular acetyl-CoA in the mutant strain could significantly affect the biosynthesis of sophorolipids. In addition, supplementation of citric acid recovered the sophorolipid production in the mutant strain by approximately 83%, suggesting that citrate metabolism plays an important role in sophorolipid biosynthesis. These findings provide novel theoretical insights for further improving the production of sophorolipids in C. bombicola.

Increasing Uniformity of Biosurfactant Production in Starmerella bombicola via the Expression of Chimeric Cytochrome P450s

Sophorolipids are one of the best known microbial biosurfactants and are produced by several yeast species. The best studied producer is Starmerella bombicola, a non-pathogenic yeast associated in nature with bumblebees. Sophorolipids are built up of the rare disaccharide sophorose, which is attached to a fatty acid through a glyosidic bound. Sophorolipids produced by S. bombicola mainly contain oleic acid as the incorporated hydrophobic group. Other chain lengths can, to a certain content, be incorporated by feeding the yeast with substrates of alternative chain lengths. However, the efficiency for such substrates is low as compared to the preferred C18 chain length and defined by the substrate specificity of the first enzymatic step in sophorolipid biosynthesis, i.e., the cytochrome P450 enzyme CYP52M1. To increase product uniformity and diversity at the same time, a new strain of S. bombicola was developed that produces sophorolipids with a palmitic acid acyl chain. This was achieved by heterologous expression of the cytochrome P450 cyp1 gene of Ustilago maydis and feeding with palmitic acid. Optimization of the production was done by protein and process engineering.

Progress of Biosynthesis of Sophorolipids and Its Derivatives Production in Starmerella bombicola

Progress of Biosynthesis of Sophorolipids and Its Derivatives Production in Starmerella bombicola

SILAC-Based Proteome Analysis of Starmerella bombicola Sophorolipid Production

Starmerella (Candida) bombicola is the biosurfactant-producing species that caught the greatest deal of attention in the academic and industrial world due to its ability of producing large amounts of sophorolipids. Despite its high economic potential, the biochemistry behind the sophorolipid biosynthesis is still poorly understood. Here we present the first proteomic characterization of S. bombicola for which we created a lys1Δ mutant to allow the use of SILAC for quantitative analysis. To characterize the processes behind the production of these biosurfactants, we compared the proteome of sophorolipid producing (early stationary phase) and nonproducing cells (exponential phase). We report the simultaneous production of all known enzymes involved in sophorolipid biosynthesis including a predicted sophorolipid transporter. In addition, we identified the heme binding protein Dap1 as a possible regulator for Cyp52M1. Our results further indicate that ammonium and phosphate limitation are not the sole limiting factors inducing sophorolipid biosynthesis.

Identification of the UDP-glucosyltransferase gene UGTA1, responsible for the first glucosylation step in the sophorolipid biosynthetic pathway of Candida bombicola ATCC 22214

Candida bombicola ATCC 22214 is applied commercially for the production of sophorolipids from renewable resources such as vegetable oils or waste streams. Although much research has been performed on optimization of fermentation conditions and on the influence of feed source and process parameters on sophorolipid structures and yields, the metabolic pathway of these important bioproducts remains unclear. Here, we identify a glucosyltransferase gene UGTA1 and show that the gene product is responsible for the first glucosylation step in the biosynthetic pathway of sophorolipids. Moreover, we provide evidence that the second glucosylation step is catalysed by a different glucosyltransferase that acts independently from the first. Therefore, the biosynthesis of sophorolipids by C. bombicola involves two glucosyltransferases that act in a stepwise manner. The UGTA1 gene described here is the first identified gene with a clear function in sophorolipid production by this economically important yeast.

The influence of increasing media methanol concentration on sophorolipid biosynthesis from glycerol-based feedstocks

Candida bombicola, a known producer of sophorolipids (SLs; glycolipid surfactants), was grown on glycerol and oleic acid with up to 1.5% (v/v) methanol in the fermentation growth media to assess the effects of methanol presence on SL synthesis and structural distribution. Increasing methanol concentrations had little effect on the growth of the organism resulting in average cell dry weights (CDW; after SL separation) of 20.8 ± 0.7 g/l between 0 and 1.5% methanol. However, increasing methanol concentrations decreased SL production by 56% (from 12.7 to 5.6 g/l at 1.5% methanol) which translated to SL yields on a cellular basis of between 0.60 g SL/g cells (in the absence of methanol) to 0.27 g SL/g cells (in the presence of 1.5% methanol). LC/MS revealed that increased methanol concentrations also resulted in larger concentrations (up to 20 mol%) of free acid SLs but had little effect on the ratios of diacetylated SL lactones synthesized with palmitic acid (4 mol%), linoleic acid (3 mol%), oleic acid (80 mol%), and stearic acid (13 mol%) as the hydrophobic moieties.

Microbial production and application of sophorolipids

Sophorolipids are surface-active compounds synthesized by a selected number of yeast species. They have been known for over 40 years, but because of growing environmental awareness, they recently regained attention as biosurfactants due to their biodegradability, low ecotoxicity, and production based on renewable resources. In this paper, an overview is given of the producing yeast strains and various aspects of fermentative sophorolipid production. Also, the biochemical pathways and regulatory mechanisms involved in sophorolipid biosynthesis are outlined. To conclude, a summary is given on possible applications of sophorolipids, either as native or modified molecules.

Sophorolipid biosynthesis by Candida bombicola from industrial fatty acid residues

Yeast Candida bombicola is known to produce sophorolipids from a variety of saccharic and lipidic feed-stocks. In the current study, the influence of fatty acid carbon chain length, unsaturation, source of low-cost industrial lipid feed-stocks, and nickel content in lipid feed-stocks on sophorolipid production by C. bombicola were investigated. Saturated and monounsaturated C18 fatty acid gave the highest levels of sophorolipid production while fatty acids with more than one site of unsaturation gave much lower yields. Sophorolipid production was reduced when shorter (≤C16) and longer (≥C20) fatty acids were fed. The possibility of exploiting low-cost industrial wastes or by-products for sophorolipid production by batch or fed-batch processes was investigated. Tallow fatty acid residue was the best lipid feed-stock for sophorolipid production (120 g/L, fed-batch cultures), while coconut fatty acid residue resulted in the lowest production (40 g/L, fed-batch cultures). Co-feeding coconut fatty acid residue with other feed-stocks improved its substrate value in batch cultures. C. bombicola was tolerant to nickel contamination of up to 112.5 mg/L during sophorolipid production. Nickel contaminated stearic fatty acid residue was transformed to value added sophorolipid product while nickel was sequestered primarily in biomass for safe removal and recovery. Sophorolipid product obtained from nickel contaminated lipid wastes had low nickel levels (<5 mg/L) that could allow its use in low-end consumer products and household applications.

Sophorolipid biosynthesis from a biodiesel co‐product stream

AbstractWe applied a biodiesel co‐product stream (BCS) as a fermentation feedstock for the microbial synthesis of sophorolipids (SL). The BCS was composed of 40% glycerol, 34% hexane‐solubles (made up of 92% FA soaps/FAME and 6% MAG/DAG), and 26% water. Batch culture fermentations of the yeast Candida bombicola on pure glycerol resulted in low‐level synthesis of SL (∼ g/L). HPLC associated with atmospheric pressure CI‐MS (LC/APCI‐MS) revealed that the SL derived from pure glycerol had 99% of the FA side chains linked to the 4″ hydroxyl group of the sophorose sugar, resulting in a lactonic structure. In contrast, the use of the BCS as feedstock increased the SL yield to 60 g/L and the open‐chain form to 75% including both oleic acid and linoleic acid (along with their methyl esters) as the dominant species comprising the side chains. By favoring the open‐chain structure, the SL molecules (particularly the FA side chain) can be chemically modified without the need to open a lactone ring first. The ability to use the BCS as a feedstock for SL synthesis will provide an outlet for this residual material, thus helping to stimulate growth in the biodiesel market and the use of agricultural fats and oils from which the biodiesel was synthesized.