Lipomyces Starkeyi Research Articles

Strains and approaches for genetic crosses in the oleaginous yeast Lipomyces starkeyi.

The oleaginous yeast Lipomyces starkeyi is a powerful lipid producer with great industrial potential. Recent studies have reported the isolation of mutant L. starkeyi cells with higher lipid producing capacity. Although genetic engineering strategies have been applied to L. starkeyi, classical genetic approaches are lacking. The development of tools that facilitate genetic crosses in L. starkeyi would not only make it possible to build improved lipid‐producing strains but also facilitate molecular biological analysis of this species. In this study, I report a set of strains and approaches useful for performing genetic crosses with L. starkeyi. The homothallic L. starkeyi reportedly forms an ascus containing two to 20 spores. These spores were resistant to glusulase and could be dissected using a micromanipulator, suggesting that random spore and tetrad (spore dissection) analysis can be adapted for L. starkeyi. Additionally, to isolate a pair of heterothallic strains useful for genetic crosses, the homothallic strain was exposed to UV irradiation, and 10 self‐sterile strains were crossed with one another. One of these combinations, Ls75 and Ls100, sporulated stably. Moreover, to detect genetic recombination, I introduced a different drug resistance marker into each strain and crossed them. The resulting progeny exhibited Mendelian segregation of the resistance markers. Altogether, the work reported here provides a powerful resource for genetic analysis in L. starkeyi.

Identification and analysis of sugar transporters capable of co-transporting glucose and xylose simultaneously.

Simultaneous co-fermentation of glucose and xylose is a key desired trait of engineered Saccharomyces cerevisiae for efficient and rapid production of biofuels and chemicals. However, glucose strongly inhibits xylose transport by endogenous hexose transporters of S. cerevisiae. We identified structurally distant sugar transporters (Lipomyces starkeyi LST1_205437 and Arabidopsis thaliana AtSWEET7) capable of co-transporting glucose and xylose from previously unexplored oleaginous yeasts and plants. Kinetic analysis showed that LST1_205437 had lenient glucose inhibition on xylose transport and AtSWEET7 transported glucose and xylose simultaneously with no inhibition. Modelling studies of LST1_205437 revealed that Ala335 residue at sugar binding site can accommodates both glucose and xylose. Docking studies with AtSWEET7 revealed that Trp59, Trp183, Asn145, and Asn179 residues stabilized the interactions with sugars, allowing both xylose and glucose to be co-transported. In addition, we altered sugar preference of LST1_205437 by single amino acid mutation at Asn365. Our findings provide a new mechanistic insight on glucose and xylose transport mechanism of sugar transporters and the identified sugar transporters can be employed to develop engineered yeast strains for producing cellulosic biofuels and chemicals.

Two-Stage Fermentation of Lipomyces starkeyi for Production of Microbial Lipids and Biodiesel.

The high operating cost is currently a limitation to industrialize microbial lipids production by the yeast Lipomyces starkeyi. To explore economic fermentation technology, the two-stage fermentation of Lipomyces starkeyi using yeast extract peptone dextrose (YPD) medium, orange peel (OP) hydrolysate medium, and their mixed medium were investigated for seven days by monitoring OD600 values, pH values, cell growth status, C/N ratios, total carbon concentration, total nitrogen concentration, residual sugar concentration, lipid content, lipid titer, and fatty acids profiles of lipids. The results showed that two-stage fermentation with YPD and 50% YPD + 50% OP medium contributed to lipid accumulation, leading to larger internal lipid droplets in the yeast cells. However, the cells in pure OP hydrolysate grew abnormally, showing skinny and angular shapes. Compared to the one-stage fermentation, the two-stage fermentation enhanced lipid contents by 18.5%, 27.1%, and 21.4% in the flasks with YPD medium, OP medium, and 50%YPD + 50%OP medium, and enhanced the lipid titer by 77.8%, 13.6%, and 63.0%, respectively. The microbial lipids obtained from both one-stage and two-stage fermentation showed no significant difference in fatty acid compositions, which were mainly dominated by palmitic acid (33.36–38.43%) and oleic acid (46.6–48.12%). Hence, a mixture of commercial medium and lignocellulosic biomass hydrolysate could be a promising option to balance the operating cost and lipid production.

Citrate-Mediated Acyl-CoA Synthesis Is Required for the Promotion of Growth and Triacylglycerol Production in Oleaginous Yeast Lipomyces starkeyi.

The oleaginous yeast Lipomyces starkeyi is an excellent producer of triacylglycerol (TAG) as a feedstock for biodiesel production. To understand the regulation of TAG synthesis, we attempted to isolate mutants with decreased lipid productivity and analyze the expression of TAG synthesis-related genes in this study. A mutant with greatly decreased lipid productivity, sr22, was obtained by an effective screening method using Percoll density gradient centrifugation. The expression of citrate-mediated acyl-CoA synthesis-related genes (ACL1, ACL2, ACC1, FAS1, and FAS2) was decreased in the sr22 mutant compared with that of the wild-type strain. Together with a notion that L. starkeyi mutants with increased lipid productivities had increased gene expression, there was a correlation between the expression of these genes and TAG synthesis. To clarify the importance of citrate-mediated acyl-CoA synthesis pathway on TAG synthesis, we also constructed a strain with no ATP-citrate lyase responsible for the first reaction of citrate-mediated acyl-CoA synthesis and investigated the importance of ATP-citrate lyase on TAG synthesis. The ATP-citrate lyase was required for the promotion of cell growth and TAG synthesis in a glucose medium. This study may provide opportunities for the development of an efficient TAG synthesis for biodiesel production.

Mitochondrial dynamics and degradation in the oleaginous yeast Lipomyces starkeyi.

Emerging evidence implicates the vital role of mitochondria in lipid consumption and storage, highlighting the intimate link between energy production and saving. Although formation of giant lipid droplets, which is the key hallmark of the oleaginous yeast Lipomyces starkeyi, appears to be regulated in response to changes in mitochondrial shape and metabolism, technical limitations of genetic manipulation have become an obstacle to uncover the mitochondrial behavior in this nonconventional yeast. Here, we established an L. starkeyi strain stably expressing a fluorescent marker for monitoring mitochondrial morphology and degradation and found that mitochondria are mostly fragmented in L. starkeyi cells under fermentable, nonfermentable, and nitrogen depletion conditions. Notably, a fraction of mitochondria-specific fluorescent signals was localized to the vacuole, a lytic organelle in yeast, indicating degradation of mitochondria in those cells. This possible catabolic event was more predominant in cells under nutrient-poor conditions than that in cells under nutrient-rich conditions, concomitantly with lipid droplet formation. Collectively, our studies provide a new tool to investigate mitochondrial dynamics in L. starkeyi and decipher the potential role of mitochondrial degradation in lipid metabolism.

Microbial lipid accumulation through bioremediation of palm oil mill effluent using a yeast-bacteria co-culture

Co-cultures of different microorganisms are considered promising inocula for treating palm oil mill effluents (POME) and producing value-added bio-products (e.g., biofuels and fatty acid-derived materials). However, the efficiency of yeast-bacteria co-culture for microbial lipid production through bioremediation of wastewater remains a bottleneck. In this study, the performance of a co-culture for lipid accumulation through POME bioremediation was investigated using a yeast (Lipomyces starkeyi) and a bacterium (Bacillus cereus). A maximum biomass of 8.89 ± 0.33 g/L and lipid production of 2.27 ± 0.10 g/L were achieved by the co-culture inoculum, which were substantially higher than those of the monocultures. Besides, the co-culture inoculum attained a maximum chemical oxygen demand (COD) removal of 83.66 ± 1.9%, while the individual cultures of B. cereus and L. starkeyi obtained 74.35 ± 1.7% and 69.01 ± 2.3%, respectively. The bioremediation efficiency was confirmed by the seed germination index (GI) of Vigna radiata (Mung bean). It was observed that the co-culture inoculum had a higher GI compared to the untreated POME and even the monoculture-treated POME. We argue that the symbiotic association of a yeast-bacteria co-culture in POME could be an attractive approach for achieving maximum biomass as well as lipid production and simultaneous bioremediation of POME.

Microbial lipid production by the yeast Lipomyces starkeyi InaCC Y604 grown on various carbon sources

The use of non-renewable fuels in the transportation sector boosts the development of renewable biofuels, such as biodiesel. However, crops-based biodiesel production can cause the increment of deforestation and greenhouse gas emissions. This circumstance motivates the development of oleaginous microorganisms as alternative raw materials to overcome these problems. Lipomyces starkeyi is one of the potential oleaginous microorganisms due to its ability to produce higher lipid content. In addition, L. starkeyi is capable to grow in affordable carbon sources, such as lignocellulose and molases, which can reduce the production costs. The aim of this study is to determine the ability of L. starkeyi InaCC Y604 in producing lipids from various carbon sources and its effects on the composition of fatty acid methyl esters (FAME) and lipid contents. L. starkeyi InaCC Y604 were cultivated in nitrogen-limited mineral medium (-NMM) with various carbon sources, namely glucose, xylose, fructose, galactose, mannose, cellobiose and a mixture of glucose and xylose. The results showed that mixed glucose and xylose gave the best result with the lipid content achieved at 64.19% (w/w). The results obtained indicated that L. starkeyi InaCC Y604 could be a potential candidate for further optimizing biodiesel production.

Genetically Engineered Oleaginous Yeast Lipomyces starkeyi for Sesquiterpene α-Zingiberene Production.

Oleaginous yeast, such as Lipomyces starkeyi, are logical organisms for production of higher energy density molecules like lipids and terpenes. We demonstrate that transgenic L.starkeyi strains expressing an α-zingiberene synthase gene from lemon basil or Hall's panicgrass can produce up to 17 mg/L α-zingiberene in yeast extract peptone dextrose (YPD) medium containing 4% glucose. The transgenic strain was further examined in 8% glucose media with C/N ratios of 20 or 100, and YPD. YPD medium resulted in 59 mg/L α-zingiberene accumulation. Overexpression of selected genes from the mevalonate pathway achieved 145% improvement in α-zingiberene synthesis. Optimization of the growth medium for α-zingiberene production led to 15% higher titer than YPD medium. The final transgenic strain produced 700 mg/L α-zingiberene in fed-batch bioreactor culture. This study opens a new synthetic route to produce α-zingiberene or other terpenoids in L.starkeyi and establishes this yeast as a platform for jet fuel biosynthesis.

Screening and Growth Characterization of Non-conventional Yeasts in a Hemicellulosic Hydrolysate.

Lignocellulosic biomass is an attractive raw material for the sustainable production of chemicals and materials using microbial cell factories. Most of the existing bioprocesses focus on second-generation ethanol production using genetically modified Saccharomyces cerevisiae, however, this microorganism is naturally unable to consume xylose. Moreover, extensive metabolic engineering has to be carried out to achieve high production levels of industrially relevant building blocks. Hence, the use of non-Saccharomyces species, or non-conventional yeasts, bearing native metabolic routes, allows conversion of a wide range of substrates into different products, and higher tolerance to inhibitors improves the efficiency of biorefineries. In this study, nine non-conventional yeast strains were selected and screened on a diluted hemicellulosic hydrolysate from Birch. Kluyveromyces marxianus CBS 6556, Scheffersomyces stipitis CBS 5773, Lipomyces starkeyi DSM 70295, and Rhodotorula toruloides CCT 7815 were selected for further characterization, where their growth and substrate consumption patterns were analyzed under industrially relevant substrate concentrations and controlled environmental conditions in bioreactors. K. marxianus CBS 6556 performed poorly under higher hydrolysate concentrations, although this yeast was determined among the fastest-growing yeasts on diluted hydrolysate. S. stipitis CBS 5773 demonstrated a low growth and biomass production while consuming glucose, while during the xylose-phase, the specific growth and sugar co-consumption rates were among the highest of this study (0.17 h–1 and 0.37 g/gdw*h, respectively). L. starkeyi DSM 70295 and R. toruloides CCT 7815 were the fastest to consume the provided sugars at high hydrolysate conditions, finishing them within 54 and 30 h, respectively. R. toruloides CCT 7815 performed the best of all four studied strains and tested conditions, showing the highest specific growth (0.23 h–1), substrate co-consumption (0.73 ± 0.02 g/gdw*h), and xylose consumption (0.22 g/gdw*h) rates. Furthermore, R. toruloides CCT 7815 was able to produce 10.95 ± 1.37 gL–1 and 1.72 ± 0.04 mgL–1 of lipids and carotenoids, respectively, under non-optimized cultivation conditions. The study provides novel information on selecting suitable host strains for biorefinery processes, provides detailed information on substrate consumption patterns, and pinpoints to bottlenecks possible to address using metabolic engineering or adaptive evolution experiments.

Genome Sequence Analysis of the Oleaginous Yeast, Rhodotorula diobovata, and Comparison of the Carotenogenic and Oleaginous Pathway Genes and Gene Products with Other Oleaginous Yeasts.

Rhodotorula diobovata is an oleaginous and carotenogenic yeast, useful for diverse biotechnological applications. To understand the molecular basis of its potential applications, the genome was sequenced using the Illumina MiSeq and Ion Torrent platforms, assembled by AbySS, and annotated using the JGI annotation pipeline. The genome size, 21.1 MB, was similar to that of the biotechnological “workhorse”, R. toruloides. Comparative analyses of the R. diobovata genome sequence with those of other Rhodotorula species, Yarrowia lipolytica, Phaffia rhodozyma, Lipomyces starkeyi, and Sporidiobolus salmonicolor, were conducted, with emphasis on the carotenoid and neutral lipid biosynthesis pathways. Amino acid sequence alignments of key enzymes in the lipid biosynthesis pathway revealed why the activity of malic enzyme and ATP-citrate lyase may be ambiguous in Y. lipolytica and L. starkeyi. Phylogenetic analysis showed a close relationship between R. diobovata and R. graminis WP1. Dot-plot analysis of the coding sequences of the genes crtYB and ME1 corroborated sequence homologies between sequences from R. diobovata and R. graminis. There was, however, nonsequential alignment between crtYB CDS sequences from R. diobovata and those from X. dendrorhous. This research presents the first genome analysis of R. diobovata with a focus on its biotechnological potential as a lipid and carotenoid producer.

Biorefinery-Based Approach to Exploit Mixed Cultures of Lipomyces starkeyi and Chloroidium saccharophilum for Single Cell Oil Production

The mutualistic interactions between the oleaginous yeast Lipomyces starkeyi and the green microalga Chloroidium saccharophilum in mixed cultures were investigated to exploit possible synergistic effects. In fact, microalga could act as an oxygen generator for the yeast, while the yeast could provide carbon dioxide to microalga. The behavior of the two microorganisms alone and in mixed culture was studied in two synthetic media (YEG and BBM + G) before moving on to a real model represented by the hydrolysate of Arundo donax, used as low-cost feedstock, and previously subjected to steam explosion and enzymatic hydrolysis. The overall lipid content and lipid productivity obtained in the mixed culture of YEG, BBM + G and for the hydrolysate of Arundo donax were equal to 0.064, 0.064 and 0.081 glipid·gbiomass−1 and 30.14, 35.56 and 37.22 mglipid·L−1·day−1, respectively. The mixed cultures, in all cases, proved to be the most performing compared to the individual ones. In addition, this study provided new input for the integration of Single Cell Oil (SCO) production with agro-industrial feedstock, and the fatty acid distribution mainly consisting of stearic (C18:0) and oleic acid (C18:1) allows promising applications in biofuels, cosmetics, food additives and other products of industrial interest.

Isolation and characterization of Lipomyces starkeyi mutants with greatly increased lipid productivity following UV irradiation

The oleaginous yeast Lipomyces starkeyi is an intriguing lipid producer that can produce triacylglycerol (TAG), a feedstock for biodiesel production. We previously reported that the L.starkeyi mutant E15 with high levels of TAG production compared with the wild-type was efficiently obtained using Percoll density gradient centrifugation. However, considering its use for biodiesel production, it is necessary to further improve the lipid productivity of the mutant. In this study, we aimed to obtain mutants with better lipid productivity than E15, evaluate its lipid productivity, and analyze lipid synthesis-related gene expression in the wild-type and mutant strains. The mutants E15-11, E15-15, and E15-25 exhibiting higher lipid productivity than E15 were efficiently isolated from cells exposed to ultraviolet light using Percoll density gradient centrifugation. They exhibited approximately 4.5-fold higher lipid productivity than the wild-type on day 3. The obtained mutants did not exhibit significantly different fatty acid profiles than the wild-type and E15 mutant strains. E15-11, E15-15, and E15-25 exhibited higher expression of acyl-CoA synthesis- and Kennedy pathway-related genes than the wild-type and E15 mutant strains. Activation of the pentose phosphate pathway, which supplies NADPH, was also observed. These results suggested that the increased expression of acyl-CoA synthesis- and Kennedy pathway-related genes plays a vital role in lipid productivity in the oleaginous yeast L.starkeyi.

Yeast and bacteria co-culture-based lipid production through bioremediation of palm oil mill effluent: a statistical optimization

In the present study, a co-culture of yeast (Lipomyces starkeyi) and bacterium (Bacillus cereus) was used to optimize lipid accumulation capability and simultaneous treatment of wastewater using palm oil mill effluent (POME) as a carbon source. The influence of process parameters (i.e., inoculum composition, pH, temperature, and time) on the lipid accumulation and the chemical oxygen demand (COD) removal were optimized using design of experiments (DoE) as a statistical tool. The DoE results suggested that the maximum lipid accumulation of 2.95 g/L and COD removal efficiency of 86.54% could be obtained while the inoculum composition, pH, temperature, and incubation time were 50:50, 6.50, 32.5 ??C, and 90 h, respectively. The predicted results were very close to the experimental results (<???5% deviation); hence, the proposed model could be useful to predict the lipid accumulation and COD removal performance of a yeast and bacteria co-culture.

Chimeric cellobiohydrolase I expression, activity, and biochemical properties in three oleaginous yeast

Consolidated bioprocessing using oleaginous yeast is a promising modality for the economic conversion of plant biomass to fuels and chemicals. However, yeast are not known to produce effective biomass degrading enzymes naturally and this trait is essential for efficient consolidated bioprocessing. We expressed a chimeric cellobiohydrolase I gene in three different oleaginous, industrially relevant yeast: Yarrowia lipolytica, Lipomyces starkeyi, and Saccharomyces cerevisiae to study the biochemical and catalytic properties and biomass deconstruction potential of these recombinant enzymes. Our results showed differences in glycosylation, surface charge, thermal and proteolytic stability, and efficacy of biomass digestion. L. starkeyi was shown to be an inferior active cellulase producer compared to both the Y. lipolytica and S. cerevisiae enzymes, whereas the cellulase expressed in S. cerevisiae displayed the lowest activity against dilute-acid-pretreated corn stover. Comparatively, the chimeric cellobiohydrolase I enzyme expressed in Y. lipolytica was found to have a lower extent of glycosylation, better protease stability, and higher activity against dilute-acid-pretreated corn stover.

Carotenoid Production in Oleaginous Yeasts.

Oleaginous yeasts, Yarrowia lipolytica and Lipomyces starkeyi, can synthesize more than 20% of lipids per dry cell weight from a wide variety of substrates. This feature is attractive for cost-efficient production of industrial biodiesel fuel. These yeasts are also very promising hosts for the efficient production of more value-added lipophilic compound carotenoids, e.g., lycopene and astaxanthin, although they cannot naturally biosynthesize carotenoids. Here, we review recent progress in researches on carotenoid production by oleaginous yeasts, which include red yeasts that naturally produce carotenoids, e.g., Rhodotorula glutinis and Xanthophyllomyces dendrorhous. Our new results on pathway engineering of L. starkeyi for lycopene production are also revealed in the present review.

Optimizing lime pretreatment of rice straw for biolipid production using oleaginous microorganisms

This work focuses on the lime pretreatment of rice straw, agricultural waste in northeast Vietnam, for microbial lipid (bio-lipid) production. A response surface methodology approach was performed to optimize parameters for the pretreatment process. These parameters are the concentration of Ca(OH)2, hydrolysis temperature, and maintained time; the effect is considered bio-lipid production yield. The lipid yield was estimated through consolidated bioprocessing of the pretreated rice straw as a substrate and using the fungus strain (Aspergillus oryzae 32) and the yeast strain (Lipomyces starkeyi 22). As a result, the optimal pretreatment conditions for maximum lipid yield were obtained at Ca(OH)2 concentration of 12 g/L and hydrolysis temperature of 110 °C within 60 min. The accumulated lipid in fermentation using these oleaginous microorganisms was 8.5g/100g oven-dry weight of rice straw (10.9g/100g for pretreated rice straw). The biolipid consisted of 42.6% saturated, 21.1% monounsaturated (MUFA), and 35.1% polyunsaturated (PUFA) fatty acids.

Predictive capability evaluation and optimization of sustainable biodiesel production from oleaginous biomass grown on pulp and paper industrial wastewater

Biodiesel, as a green fuel, acts as a potential candidate to supplement conventional fossil fuels. This research study targets green environment (using biodiesel) and clean environment (reduce wastewater) by producing biodiesel through oleaginous biomasses (Yarrowia lipolytica, Metschnikowia pulcherrima and Lipomyces starkeyi) grown on pulp and paper industrial wastewater. Batch culture studies were explored for the potential feedstock of the oleaginous organism by the synthesis of single cell oil and fatty acid methyl ester (FAME) yield. Response surface methodology (RSM) was used to design the optimal experimental matrix and identify the optimal process conditions that enhance the FAME yield. To determine the inherent characteristics of the growth of oleaginous biomasses on the industrial wastewater, a data-driven adaptive neuro-fuzzy inference system (ANFIS) is implemented. Y. lipolytica strain cultured shown high biomass concentration of 32.36 g/l with biomass productivity of 5.39 g/l/d was considered for further scale-up for the transesterification process. Results indicated that the maximum yield of 0.48 (g-biodiesel/g-lipid) was obtained under the 2.5 g of lipid dosage with 0.02 g/ml of catalyst concentration by constant stirring at 70 °C. The optimum conditions to achieve maximum FAME yield of 1.154 g/g was obtained at 2.485 g, 70.87 °C and 0.021 g/ml for lipid dosage, temperature and catalyst concentrations, respectively.

Pretreatments for enhancing sewage sludge reduction and reuse in lipid production

BackgroundConverting wastewater sludge to lipid is considered as one of the best strategies of sludge management. The current problem of lipid production from wastewater sludge is the low yield (0.10–0.16 g lipid/g dry sludge) due to the low availability of easily uptaken materials (such as soluble monosaccharide and oligosaccharide) in sludge to oleaginous microorganism (Rhodotorula glutinis, Trichosporon oleaginosus, Lipomyces starkeyi). Pretreatments are efficient methods to improve sludge bioavailability. This study is aimed to achieve high lipid production from sludge and high sludge reduction.ResultsIn this study, it was observed that the soluble chemical oxygen demand (SCOD) had significantly increased after different pretreatment. The SCOD in the supernatant was increased from 32.64 to 180.25 mg/L, 924.16 mg/L, 1029.89 mg/L and 3708.31 mg/L after acidic (pH 2 for 2 h), alkaline (pH 12 for 2 h), microwave irradiation (15 min with 5 min interval), and ultrasonication (30 min at 450 W and 20 kHz frequency with 5 s on and 2 s off mode) pretreatment, respectively. Pretreatments have also increased the release of total nitrogen (TN) and total phosphorus (TP) from solids. The sludge after different pretreatments were used as a medium for lipid production, and the highest lipid content (36.67% g/g) was obtained in the fermentation with ultrasonication pretreatment sludge, and the sludge reduction was 63.10%. For other pretreatments, the lipid content and sludge reduction were 18.42% and 32.63% in acid pretreatment case, 21.08% and 36.44% in alkaline pretreatment case, and 26.31% and 43.03% in microwave pretreatment case, respectively.ConclusionIt was found that ultrasonication pretreatment was the most efficient way to increase the sludge biodegradability (SCOD) and to release TN and TP from solid phase to liquid phase. Pretreated sludge for lipid production achieved significant improvement in lipid yield and sludge reduction. Lipids produced from pretreated sludge were transesterified to biodiesel and the analysis showed that biodiesel had a similar composition as commercial biodiesel. The study reveals that pretreatment on sludge is a promising method for enhancing biological sludge management efficiency.

PRODUCTION AND PURIFICATION OF KILLER TOXIN FROM PROBIOTIC YEASTS AND ITS EFFECT ON FOODBORNE PATHOGENS

Probiotic yeast strains viz. Yarrowia lipolytica VIT-MN01, Kluyveromyces lactis VIT-MN02, Lipomyces starkeyi VIT-MN03, Saccharomycopsis fibuligera VIT-MN04 and Brettanomyces custersianus VIT-MN05 were investigated as producer of killer toxin. Among the five yeasts, three strains showed more killer toxin production. The maximum killer activity (12 aU/mg) against sensitive strain Saccharomyces cerevisiae was obtained at pH-3 with 0.5% NaCl at 25°C by using response surface methodology. The purified killer toxin K2 and K3 showed the highest killer activity against foodborne pathogens viz. Bacillus cereus, E. coli, Pseudomonas spp., Klebsiella spp. and Candida tropicalis. The purity of the killer toxin was confirmed by HPLC analysis. The molecular mass of killer toxin was 22 kDa (K2), 18 kDa (K3) and 14 kDa (K4). The results of the present study suggest that killer toxins produced by probiotic yeast strains can be used as an antimicrobial agent to control the microbial contamination in the food industry.

Inference of Regulatory System for TAG Biosynthesis in Lipomyces starkeyi.

Improving the bioproduction ability of efficient host microorganisms is a central aim in bioengineering. To control biosynthesis in living cells, the regulatory system of the whole biosynthetic pathway should be clearly understood. In this study, we applied our network modeling method to infer the regulatory system for triacylglyceride (TAG) biosynthesis in Lipomyces starkeyi, using factor analyses and structural equation modeling to construct a regulatory network model. By factor analysis, we classified 89 TAG biosynthesis-related genes into nine groups, which were considered different regulatory sub-systems. We constructed two different types of regulatory models. One is the regulatory model for oil productivity, and the other is the whole regulatory model for TAG biosynthesis. From the inferred oil productivity regulatory model, the well characterized genes DGA1 and ACL1 were detected as regulatory factors. Furthermore, we also found unknown feedback controls in oil productivity regulation. These regulation models suggest that the regulatory factor induction targets should be selected carefully. Within the whole regulatory model of TAG biosynthesis, some genes were detected as not related to TAG biosynthesis regulation. Using network modeling, we reveal that the regulatory system is helpful for the new era of bioengineering.