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The oleaginous yeast Lipomyces starkeyi is a potential cost-effective source for the production of microbial lipids. Fatty acid elongases have vital roles in the syntheses of long-chain fatty acids. In this study, two genes encoding fatty acid elongases of L. starkeyi, LsELO1, and LsELO2 were identified and characterized. Heterologous expression of these genes in Saccharomyces cerevisiae revealed that LsElo1 is involved in the production of saturated long-chain fatty acids with 24 carbon atoms (C24:0) and that LsElo2 is involved in the conversion of C16 fatty acids to C18 fatty acids. In addition, both LsElo1 and LsElo2 were able to elongate polyunsaturated fatty acids. LsElo1 elongated linoleic acid (C18:2) to eicosadienoic acid (C20:2), and LsElo2 elongated α-linolenic acid (C18:3) to eicosatrienoic acid (C20:3). Overexpression of LsElo2 in L. starkeyi caused a reduction in C16 fatty acids, such as palmitic and palmitoleic acids, and an accumulation of C18 fatty acids such as oleic and linoleic acids. Our findings have the potential to contribute to the remodeling of fatty acid composition and the production of polyunsaturated long-chain fatty acids in oleaginous yeasts.

The oleaginous yeast Lipomyces starkeyi is an attractive organism for the industrial production of lipids; however, the amount of lipid produced by wild-type L. starkeyi is insufficient. The study aims to obtain L. starkeyi mutants that rapidly accumulate large amounts of triacylglycerol (TAG). Mutagenized yeast cells at the early stages of cultivation were subjected to Percoll density gradient centrifugation; cells with increased production of TAG were expected to be enriched in the resultant upper fraction because of their lower density. Among 120 candidates from the upper fractions, five mutants were isolated that accumulated higher amounts of TAG. Moreover, when omitting cells with mucoid colony morphology, 11 objective mutants from 11 candidates from the upper fraction were effectively (100%) isolated. Of total 16 mutants obtained, detailed characterization of five mutants was performed to reveal that five mutants achieved about 1.5-2.0 times TAG concentration (4.7-6.0 g/L) as compared with the wild-type strain (3.6 g/L) at day 5. Among these five mutants, strain E15 was the best for industrial use because only strain E15 showed significantly higher TAG concentration as well as significantly higher degree of lipid to glucose and biomass to glucose yields than the wild-type strain. Thus, Percoll density gradient centrifugation is an effective method to isolate mutant cells that rapidly accumulate large amounts of TAG. It is expected that by repeating this procedure as part of a yeast-breeding program, L. starkeyi mutants suitable for industrial lipid production can be easily and effectively obtained.

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.

Background Lipomyces starkeyi has been widely regarded as a promising oleaginous yeast with broad industrial application prospects because of its wide substrate spectrum, good adaption to fermentation inhibitors, excellent fatty acid composition for high-quality biodiesel, and negligible lipid remobilization. However, the currently low experimental lipid yield of L. starkeyi prohibits its commercial success. Metabolic model is extremely valuable to comprehend the complex biochemical processes and provide great guidance for strain modification to facilitate the lipid biosynthesis. Results A small-scale metabolic model of L. starkeyi NRRL Y-11557 was constructed based on the genome annotation information. The theoretical lipid yields of glucose, cellobiose, xylose, glycerol, and acetic acid were calculated according to the flux balance analysis (FBA). The optimal flux distribution of the lipid synthesis showed that pentose phosphate pathway (PPP) independently met the necessity of NADPH for lipid synthesis, resulting in the relatively low lipid yields. Several targets (NADP-dependent oxidoreductases) beneficial for oleaginicity of L. starkeyi with significantly higher theoretical lipid yields were compared and elucidated. The combined utilization of acetic acid and other carbon sources and a hypothetical reverse β-oxidation (RBO) pathway showed outstanding potential for improving the theoretical lipid yield. Conclusions The lipid biosynthesis potential of L. starkeyi can be significantly improved through appropriate modification of metabolic network, as well as combined utilization of carbon sources according to the metabolic model. The prediction and analysis provide valuable guidance to improve lipid production from various low-cost substrates.

The main objective of this study was to investigate the effects of a low cost minimal medium, developed by the UL Bioprocessing Lab, on the cultivation of Lipomyces starkeyi NRRL Y-11557 using repeated fed-batch and continuous fermentation strategies. The highest cell and lipid concentrations obtained were 22.7 g L–1 and 11.67 g L–1 under repeated fed-batch cultivation, respectively. Continuous cultivation with the dilution rate of 0.06 h–1 presented the highest cell (0.401 g g–1) and lipid yields (0.177g g–1). Exopolysaccharide production was observed when L. starkeyi was cultivated in the minimal media supplemented with 90 g L–1 glucose under repeated fed-batch fermentation. The produced exopolysaccharide is likely composed of 4–5 repeating sugar units, incorporating mannose and galactose and their respective uronic acids. This work is licensed under a Creative Commons Attribution 4.0 International License.

The aim of this study was to produce single cell oil (SCO) from Lipomyces starkeyi ATCC 56304 using a biphasic fed-batch fermentation strategy. Fed-batch fermentations were performed to compare oil production using glucose, xylose, mixed-sugars (1:1 ratio of glucose:xylose), and a biphasic system supplying glucose for cell growth and xylose for oil production. The fermentation using xylose as the sole carbon source generated an oil yield of 0.16 g g−1 and a productivity of 0.12 g L−1 h−1, two-times the oil yield (0.08 g g−1) and productivity (0.06 g L−1 h−1) achieved with glucose. At 120 h, the biphasic fermentation produced a greater oil productivity (0.13 g L−1 h−1) compared to the mixed-sugar fermentation (0.09 g L−1 h−1) but similar oil productivities (0.14 g L−1 h−1) were achieved for both at longer fermentation times. The biphasic fermentation generated a final oil yield of 0.17 g g−1 and an oil content of 60.1% (w w−1), but oil productivity showed minor improvement at 0.14 g L−1 h−1 after 255 h. Oleic acid accounted for at least 65% of the fatty acid content, suggesting that oil from L. starkeyi has potential for application in the biofuel and oleochemical industries.

The oleaginous yeast Lipomyces starkeyi is an excellent sustainable lipid producer, which can convert industrial wastes into lipids and accumulate triacylglycerols (TAG) by > 70% of its dry cell weight. Recent studies using omics technologies applied in L. starkeyi have aided in obtaining greater understanding of the important mechanisms of lipid metabolism in L. starkeyi. Therefore, the development of genetic engineering tools for L. starkeyi has led to accelerated efforts for a highly efficient production of lipids.This review focuses on the aspects of TAG and fatty acid synthesis pathways in L. starkeyi. We also present a quite effective strategy to obtain L. starkeyi mutants accumulating a larger amount of lipids and having a higher lipid production rate than the wild-type strain. The analysis of these mutants exhibiting high lipid production has led to the identification of important genes for achieving highly effective lipid production and thus advanced improvement in lipid production. Herein, our aim was to provide useful information to advance the development of L. starkeyi as a cost-effective TAG feedstock.Key Points•Oleaginous yeast Lipomyces starkeyi is an excellent sustainable lipid producer.•Efficient isolation of lipid-enriched L. starkeyi mutants depends on the low density of lipids.•Increased acyl-CoA synthesis pathway is important for improving lipid productivity.

Cocoa butter (CB) extracted from cocoa beans is the main raw material for chocolate production. However, growing chocolate demands and limited CB production has resulted in a shortage of CB supply. CB is mainly composed of three different kinds of triacylglycerols (TAGs), POP (C16:0–C18:1–C16:0), POS (C16:0–C18:1–C18:0), and SOS (C18:0–C18:1–C18:0). The storage lipids of yeasts, mainly TAGs, also contain relative high-level of C16 and C18 fatty acids and might be used as CB-like lipids (CBL). In this study, we cultivated six different yeasts, including one non-oleaginous yeast strain, Saccharomyces cerevisiae CEN.PK113-7D, and five oleaginous yeast strains, Trichosporon oleaginosus DSM11815, Rhodotorula graminis DSM 27356, Lipomyces starkeyi DSM 70296, Rhodosporidium toruloides DSM 70398, and Yarrowia lipolytica CBS 6124, in nitrogen-limited medium and compared their CBL production ability. Under the same growth conditions, we found that TAGs were the main lipids in all six yeasts and that T. oleaginosus can produce more TAGs than the other five yeasts. Less than 3% of the total TAGs were identified as potential SOS in the six yeasts. However, T. oleaginosus produced 27.8% potential POP and POS at levels of 378 mg TAGs/g dry cell weight, hinting that this yeast may have potential as a CBL production host after further metabolic engineering in future. Electronic supplementary material The online version of this article (doi:10.1007/s00253-017-8126-7) contains supplementary material, which is available to authorized users.

The goal of this project was to adapt the Yarrowia lipolytica plasmid based CRISPR/Cas9 system for usage in Lipomyces starkeyi. Lipomyces starkeyi is an oleaginous yeast, which synthesizes and stores high amounts of intracellular lipids. This specific yeast can store lipids at concentrations higher than 60% of its dry cell weight. Due to these high concentrations of lipids, L. starkeyi is a desired organism for the production of biofuels and other oleochemicals. However, there is a lack of knowledge and of genetic tools when trying to engineer the cells to produce these lipids for our use. The genome editing tool, CRISPR/Cas9 is efficient and simple, therefore desirable for the engineering of L. starkeyi. The goal was achieved by replacing the Y. lipolytica promoter with a L. starkeyi promoter, inserting guide RNA, as well as confirming cas9 protein expression.

Single cell oils (SCOs) are considered potential raw material for the production of biodiesel. Rhodosporidium sp. and Lipomyces sp. are good candidates for SCO production. Lipid extractability differs according to yeast species and literature on the most suitable method for each oleaginous yeast species is scarce. This work aimed to investigate the efficiency of the most cited strategies for extracting lipids from intact and pretreated cells of Rhodosporidium toruloides and Lipomyces starkeyi . Lipid extractions were conducted using hexane or combinations of chloroform and methanol. The Folch method resulted in the highest lipid yields for both yeasts (42% for R. toruloides and 48% for L. starkeyi ). Also, this method eliminates the cell pretreatment step. The Bligh and Dyer method underestimated the lipid content in the tested strains (25% for R. toruloides and 34% for L. starkeyi ). Lipid extractability increased after acid pretreatment for the Pedersen, hexane, and Bligh and Dyer methods. For R. toruloides unexpected fatty acid methyl esters (FAME) composition were found for some lipid extraction strategies tested. Therefore, this work provides useful information for analytical and process development aiming at biodiesel production from the SCO of these two yeast species.

Background Lipomyces starkeyi is one of the leading lipid-producing microorganisms reported to date; its genetic transformation was only recently reported. Our aim is to engineer L. starkeyi to serve in consolidated bioprocessing (CBP) to produce lipid or fatty acid-related biofuels directly from abundant and low-cost lignocellulosic substrates. Results To evaluate L. starkeyi in this role, we first conducted a genome analysis, which revealed the absence of key endo- and exocellulases in this yeast, prompting us to select and screen four signal peptides for their suitability for the overexpression and secretion of cellulase genes. To compensate for the cellulase deficiency, we chose two prominent cellulases, Trichoderma reesei endoglucanase II (EG II) and a chimeric cellobiohydrolase I (TeTrCBH I) formed by fusion of the catalytic domain from Talaromyces emersonii CBH I with the linker peptide and cellulose-binding domain from T. reesei CBH I. The systematically tested signal peptides included three peptides from native L. starkeyi and one from Yarrowia lipolytica. We found that all four signal peptides permitted secretion of active EG II. We also determined that three of these signal peptides worked for expression of the chimeric CBH I; suggesting that our design criteria for selecting these signal peptides was effective. Encouragingly, the Y. lipolytica signal peptide was able to efficiently guide secretion of the chimeric TeTrCBH I protein from L. starkeyi. The purified chimeric TeTrCBH I showed high activity against the cellulose in pretreated corn stover and the purified EG II showed high endocellulase activity measured by the CELLG3 (Megazyme) method. Conclusions Our results suggest that L. starkeyi is capable of expressing and secreting core fungal cellulases. Moreover, the purified EG II and chimeric TeTrCBH I displayed significant and potentially useful enzymatic activities, demonstrating that engineered L. starkeyi has the potential to function as an oleaginous CBP strain for biofuel production. The effectiveness of the tested secretion signals will also benefit future secretion of other heterologous proteins in L. starkeyi and, given the effectiveness of the cross-genus secretion signal, possibly other oleaginous yeasts as well. Electronic supplementary material The online version of this article (doi:10.1186/s12934-017-0742-5) contains supplementary material, which is available to authorized users.

The oleaginous yeast Lipomyces starkeyi (L. starkeyi) is an excellent intracellular lipid producer. Thus, extraction of protein from lipid-rich L. starkeyi samples following conventional methods can be difficult, leading to poor data in terms of proteomic analysis. The presence of lipophilic components in those samples may also interfere with the extraction process and the downstream analysis. In this work, we developed a sequential extraction method for preparation and analysis of L. starkeyi proteome combining to an online multidimensional nano reversed-phase liquid chromatography-tandem mass spectrometry (microRPLC-MS/MS) strategy. Protein hits of high confidence reached 227 with false positive rate less than 0.1, twice of those identified from the one-buffer extraction preparation. Moreover, the protein hits related to primary metabolism was increased, which may be important to establish the molecular mechanism of lipid accumulation. The method should be valuable for protein extraction from oleaginous species.

The oleaginous yeast, Lipomyces starkeyi, is an excellent lipid producer with great industrial potential. However, methods for molecular breeding have not been established for L. starkeyi. We describe the development of a system for targeted rDNA integration of multiple copies of a gene into L. starkeyi genome by spheroplast–polyethylene glycol transformation.

Producing microbial oils via oleaginous yeast fermentation has drawn broad attention in the biodiesel industry. The oleaginous yeast Lipomyces starkeyi utilizing diverse carbon sources including glucose, xylose, glycerol, and willow wood sawdust (WWS) hydrolysate for the biosynthesis of oils in its cell growth were investigated in this study. High carbon/nitrogen ratios within the glucose media significantly increased the lipid content of Lipomyces starkeyi cells and modified the fatty acid composition of lipids, promoting the accumulation of C16:0 fatty acids and saturated fatty acids (C16:0 and C18:0). The accumulation of C18 fatty acids (C18:0, C18:1, and C18:2) and unsaturated fatty acids (C16:1, C18:1, and C18:2) was restricted. When crude glycerol and WWS hydrolysate were used as the sole carbon sources for L. starkeyi fermentation, the dry cell weight, lipid content, and lipid productivity were 9.1 g/L, 46.2%, and 4.2 g/L, respectively, for glycerol, and 8.2 g/L, 42.7%, and 3.5 g/L, respectively, for the hydrolysate solution. This study provides useful information for producing oils with L. starkeyi fermentation using glycerol and WWS hydrolysate as the primary or secondary carbon substrates.

We report the development of an efficient genetic transformation system for Lipomyces starkeyi based on a modified lithium acetate transformation protocol. L. starkeyi is a highly lipogenic yeast that grows on a wide range of substrates. The initial transformation rate for this species was extremely low, and required very high concentrations of DNA. A systematic approach for optimizing the protocol resulted in an increase in the transformation efficiency by four orders of magnitude. Important parameters included cell density, the duration of incubation and recovery periods, the heat shock temperature, and the concentration of lithium acetate and carrier DNA within the transformation mixture. We have achieved efficiencies in excess of 8,000 transformants/µg DNA, which now make it possible to screen libraries in the metabolic engineering of this yeast. Metabolic engineering based on this transformation system could improve lipogenesis and enable formation of higher value products.

Due to the high organic compounds and high salinity of fishmeal wastewater (FW), it was firstly used as a novel medium to produce microbial lipid in this paper. Fermentation of FW without any additives adding showed that the broth was appropriate for the growth of strain Lipomyces starkeyi HL; however, production of 5.34 g l(-1) of biomass containing 20.8% of lipid was not satisfied. In order to enhance the accumulation of lipid and cell growth, FW was supplemented with various concentrations of glucose; meanwhile, the influence of initial pH was investigated. Biomass and lipid yield on FW were markedly affected by glucose concentration and initial pH. The addition of 20 g l(-1) glucose at initial pH 4.0 got the best results: 17.6 g l(-1) of biomass, 2.7 g l(-1) of lipid yield, 91.2% of protein removal and 43.4% of the chemical oxygen demand removal. The variation of fatty acid composition upon time course in the cellular lipid on FW or a mixture of glucose and FW was further studied.

In this work we performed assays for the genetic improvement of the oleaginous yeast Lipomyces starkeyi DSM 70296 focusing on its utilization for lipid biosynthesis from renewable sources. The genetic optimization was carried out by random mutagenesis by ultraviolet irradiation and mutant selection by cerulenin, a compound displaying inhibitory effects on lipid biosynthesis. Mutants demonstrating normal growth in presence of cerulenin were considered as good candidates for further studies. Using this strategy, we selected 6 mutants for further studies, in which their productivities were evaluated by fermentation in shaken flasks and bioreactor. The evaluation of the fermentative performance of mutants was carried out using xylose as sole carbon source; the fermentation of wild-type strain was used as reference. Using this strategy it was possible to identify one mutant (termed A1) presenting a significant increase in the productivity rates of both biomass and lipid in comparison to wild-type strain. A1 mutant was further studied in bioreactor using the same fermentation parameters optimized for L. starkeyi lipid production from a mixed carbon source (xylose:glucose), as previously determined by other studies in our laboratory. A1 presented a productivity increase of 15.1% in biomass and 30.7% in lipid productivity when compared to the wild-type strain with a similar fatty acid composition, despite a slight increase (approx. 7%) on the unsaturated fraction. Our work demonstrates the feasibility of the random mutagenesis strategy coupled with mutant selection based on cerulenin screening for the genetic improvement of the oleaginous yeast L. starkeyi.

SUMMARY: When eight strains of the oleaginous yeasts Candida curvata, Lipomyces starkeyi, Rhodosporidium toruloides and Trichosporon cutaneum were starved of carbon after having accumulated lipid up to 34% of their biomass, the lipid was readily converted to new biomass in all cases except the two strains of L. starkeyi. When C. curvata D was grown in a two-stage chemostat with the second stage as a carbon-starvation vessel (but containing NH+ 4) biosynthesis of new biomass reached 1.9 ± 0.2 g per g lipid utilized. Experiments in a single-stage chemostat undergoing transition from lipid accumulation (nitrogen-limited medium) to carbon-starvation conditions showed that the lipid in C. curvata was rapidly mobilized. When the lipid was pre-labelled with 14C, a transitory pool of rapidly metabolizable non-lipid material appeared within 1.5 h of the initiation of starvation. Rates of lipid loss indicated that initiation of lipid degradation occurred immediately carbon was lost from the external medium.

The optimal growth rate ofLipomyces starkeyi, with dextran as sole carbon source, was found within the pH range 2.5–4.0, and temperature between 25–30°C. This yeast was unable to grow above 33°C. Dextranase production optima paralleled growth optima, except at pH 2.5. Decrease in enzyme yield at this pH could not be attributed to poor yeast growth or enzyme stability.

ATP: citrate lyase from Lipomyces starkeyi was inhibited by oleoyl-CoA (50% inhibition at approx. 3 μM) and other long-chain fatty acyl-CoA esters but not by other fatty acyl compounds. The inhibition was readily reversed by adding bovine serum albumin. The molecular size of the protein was not altered even in the presence of 200 μM-oleoyl-CoA. It is concluded that this is a genuine feedback inhibition effect which may be significant in regulating the amount of triacylglycerol being produced by an oleaginous yeast.

A suitable medium was sought for controlling neutral lipid accumulation and cell proliferation of Lipomyces starkeyi. Deficiency of zinc sulfate and manganese chloride provided an increase in neutral lipid in cells (mg of neutral lipid/108 cells). The addition of large amounts of zinc sulfate, manganese chloride and potassium phosphate induced L. starkeyi to enhance cell proliferation (total cell number/ml) but limited neutral lipid accumulation. In medium consisting of low concentrations of zinc sulfate and manganese chloride, the neutral lipid in cells was approximately 22 times that of medium consisting of high concentrations of zinc sulfate, manganese chloride and potassium phosphate. The total cell number in medium for improving cell proliferation was approximately 20 times that of the medium for enhancing neutral lipid accumulation.

In this paper, we present a simple and sensitive method for the assay of intracellular neutral lipids of the yeast, Lipomyces starkeyi. In this method, we used a tower-shaped mixer for cell disruption and applied a diagnostic triglycerides assay kit as a sensitive color developer. This mixer could treat 14 samples at one time. No effect of pH on cell disruption was detected within the pH range of 1.6 to 6.8. Twenty min was chosen for cell disruption and for homogeneous dispersion of intracellular neutral lipids. The analytical results agreed well with those obtained by three conventional methods. The new method is simpler and of good reliability.

The effects of the concentration of the medium components and other cultural conditions on the total cell number and on the lipid content (mg of total lipid/108 cells) of the fat yeast Lipomyces starkeyi were examined. The no addition and deficiency of NH4+, K+, Mg2+, PO43-, SO42-, Zn2+, Fe3+, or Mn2+ decreased the total cell number. Mn2+ sufficiency increased the total cell number by a factor of 1.5 to 1.7, as compared with that of the standard concentration. The lipid content of the yeast was affected by six (NH4+, K+, Ca2+, Zn2+, Fe3+, and Mn2+) ion concentrations. The no addition and deficiency of Zn2+ increased the lipid content by a factor ranging from 2.4 to 2.8 in comparison with that of the standard concentration. The concentration of Zn2+ also altered the lipid yield (g of lipid/100g of glucose consumed) considerably. The concentration of Na+, Cl-, Cu2+, BO33-, I-, MoO42-, and biotin had almost no effect on the total cell number, lipid content, and lipid yield of L. starkeyi. The cultural temperature and the initial pH value of the medium affected the total cell number and lipid content; the optimum temperature ranged from 25.5 to 29.5°C, and the optimum pH value was 4.9. A low concentration of dissolved oxygen decreased both the total cell number and lipid content. D-Glucose, D-mannose, D-galactose, D-levulose, sucrose, D-xylose, and L-arabinose proved to be usable carbon sources for the growth and the lipid accumulation of L. starkeyi.

Different cell lipid accumulation and composition patterns were observed in the yeasts Lipomyces starkeyi and Candida steatolytica by varying the nutritive conditions. In Lipomyces starkeyi 303 which accumulates high levels of lipids as triglycerides, the composition of the different lipid fractions was relatively unaffected by the culture medium. In contrast, the lipid composition of Candida steatolytica 20C was strongly affected by the composition of the medium under conditions of nitrogen limitation and when starch was used as the carbon source, high levels of straight and branched hydrocarbons were observed. [Traduction de l'auteur]

The properties of the biotin uptake system of Lipomyces starkeyi IAM 4753 were investigated in proliferating and nonproliferating cells from the standpoint of explaining the characteristics of the biotin-requiring property of the organism in a medium with a pH from 5.5 to 6.5. The rate of biotin uptake was linear with time for at least the first 6hr. Uptake of 14C-biotin showed the system to be an aeration- and energy source-dependent process. This was confirmed by the almost complete inhibition of biotin uptake with 1mM of 2, 4-dinitrophenol, potassium cyanide, and sodium azide. Biotin uptake is pH-dependent, and the optimum condition for uptake is a pH of 4.2. Biotin uptake is operative between a pH of 5.5 and 6.5, and thus L. starkeyi IAM 4753 can grow in a medium with a pH from 5.5 to 6.5, when biotin is supplemented. The uptake of biotin by the cells is influenced by the biotin concentration in the medium employed. The greatest uptake was observed when the cells were grown in a minimal medium without any supplementation of biotin.Apparent activity for biotin uptake per mg dry cell was considerably influenced by culture age. The amount of 14C-biotin taken up by mg dry cell harvested during the logarithmic phase was the highest, decreasing gradually as the growth phase proceeded.

The biotin requirement of Lipomyces starkeyi IAM 4753 was tested in chemically defined media with variations in the initial pH. The strain exhibited normal growth in a glucose-mineral medium with a low initial pH (-5.5) and biphasic growth at a pH of 6.1. A supplement of biotin to the medium resulted in a rise of the pH to 6.5 at which normal growth occurred. Thus, it was considered that the yeast took up biotin from the medium with a high pH and supported their normal growth. When growing cells obtained from the glucose-mineral medium were analyzed for biotin, approximately 38 to 68 times as much biotin was detected as the amount of biotin which had been acquired from seed culture. Therefore, there was no obstruction in the biochemical sequence of the strain leading to the biosynthesis of biotin, but the strain has an enzyme system which is affected by culture pH.

The characteristics of the pH-dependent biotin requirement of Lipomyces starkeyi IAM 4753 were investigated in growing cells. Microbiological and radioisotope assays were employed to measure biotin content in the cells and in the medium. This yeast exhibited a strict biotin requirement in a medium with a pH ranging from 5.5 to 6.5. The minimum amount of biotin needed for normal growth in the bufferized medium with a pH of 6.0 was 0.1μg/liter. The maximum growth rate was observed when 0.5μg or more of biotin was added to the bufferized medium with a pH of 6.0; cellular radioactivity increased gradually accompanied by an increase of true biotin in the cells until the concentration of true biotin in the medium became undetectable. A part of the biotin added was degraded during the growth. Cellular biotin content remained constant for about 10hr followed by a gradual increase. The reincrease of the cellular true biotin content began at a point when the pH of the medium was below 5.5. From these results, it was concluded that the initiation of biotin biosynthesis in L. starkeyi IAM 4753 depends on the pH value of the medium.

Les analogues du glucose, le 2-deoxyglucose et le D-glucosamine, suppriment la formation de l'endodextranase extracellulaire de la levure L.b. Bien que ces deux analogues sont utilises avec succes dans la separation selective des mutants derepressifs et hyperproductifs, le 2-deoxyglucose s'avere etre superieur au D-glucosamine en ce qui concerne la concentration effective et la limitation de la croissance supplementaire

Cross-reactions of the extracellular polysaccharides of Lipomyces lipoferus and Lipomyces starkeyi are described, in some instances quantitatively. Non-reducing end-groups of D -glucuronic acid appear to account for most of the explicable reactions of the polysaccharide of L. lipoferus , whereas nonreducing end-groups of D -galactose account for many of those of L. starkeyi . Because of its strong cross-reaction in antipneumococcal type II serum, the polysaccharide of L. starkeyi is also presumed to have a portion of its D -glucuronic acid in the form of nonreducing end-groups.

Ascorbic acid was identified in cultures of the yeast Lipomyces starkeyi. The acid was identified by preparing the Roe and Kuether chromogen and comparing its infrared spectrum with the spectra of chromogens derived from ascorbic and araboascorbic acids. After 5 days of growth on glucose medium the ascorbic acid concentration in cellular extracts was 214 μg/g wet weight.