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Interest in using renewable sources of carbon, especially lignocellulosic biomass, for the production of hydrocarbon fuels and chemicals has fueled interest in exploring various organisms capable of producing hydrocarbon biofuels and chemicals or their precursors. The oleaginous (oil-producing) yeast Lipomyces starkeyi is the subject of active research regarding the production of triacylglycerides as hydrocarbon fuel precursors using a variety of carbohydrate and nutrient sources. The genome of L. starkeyi has been published, which opens the door to production strain improvements through the development and use of the tools of synthetic biology for this oleaginous species. The first step in establishment of synthetic biology tools for an organism is the development of effective and reliable transformation methods with suitable selectable marker genes and demonstration of the utility of the genetic elements needed for expression of introduced genes or deletion of endogenous genes. Chemical-based methods of transformation have been published but suffer from low efficiency. To address these problems, Agrobacterium -mediated transformation was investigated as an alternative method for L. starkeyi and other Lipomyces species. In this study, Agrobacterium -mediated transformation was demonstrated to be effective in the transformation of both L. starkeyi and other Lipomyces species. The deletion of the peroxisomal biogenesis factor 10 gene was also demonstrated in L. starkeyi . In addition to the bacterial antibiotic selection marker gene hygromycin B phosphotransferase, the bacterial β-glucuronidase reporter gene under the control of L. starkeyi translation elongation factor 1α promoter was also stably expressed in six different Lipomyces species. The results from this study demonstrate that Agrobacterium -mediated transformation is a reliable and effective genetic tool for homologous recombination and expression of heterologous genes in L. starkeyi and other Lipomyces species.

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.

Non-invasive methods for observing the morphology of living oleaginous yeast are ideal for optimizing the production of various oils, such as food oils, oleochemicals, and biodiesel, from oleaginous yeast. However, existing methods have been developed to target budding yeast without high oil production ability and extract regions of entire cells. This study is the first to target oleaginous yeast, namely, Lipomyces starkeyi , demonstrating a method for extracting regions of L. starkeyi directly influencing oil production through the unique optical properties of L. starkeyi . Specifically, we exploited changes in the brightness along the z-stack depth of multiple z-stack images obtained using confocal microscopy. Because this brightness change was unique to lipid droplets, pixels corresponding to lipid droplets were easily identified, allowing calculations of the parameters of visual features. The obtained parameters of visual features were then used as input for a semantic segmentation algorithm to accurately distinguish lipid droplets from other organelles, including organelles similar to lipid droplets in shape. Experimental results showed that our method successfully estimated the growth status of L. starkeyi , which, to date, is obtainable through invasive biochemical methods only. Moreover, our method non-invasively determined the shape of each yeast cell over the cultivation period to enable single-cell analysis, which has not been achieved with conventional biochemical methods. Key points ∙ We propose a method to extract regions of L. starkeyi influencing oil production. ∙ Our method requires only confocal microscopy images and is completely non-invasive. ∙ Our method estimated the growth status of L. starkeyi to enable single-cell analysis.

Fatty acid desaturases play vital roles in the synthesis of unsaturated fatty acids. In this study, Δ12 and Δ12/Δ15 fatty acid desaturases of the oleaginous yeast Lipomyces starkeyi, termed LsFad2 and LsFad3, respectively, were identified and characterized. Saccharomyces cerevisiae expressing LsFAD2 converted oleic acid (C18:1) to linoleic acid (C18:2), while a strain of LsFAD3-expressing S. cerevisiae converted oleic acid to linoleic acid, and linoleic acid to α-linolenic acid (C18:3), indicating that LsFad2 and LsFad3 were Δ12 and bifunctional Δ12/Δ15 fatty acid desaturases, respectively. The overexpression of LsFAD2 in L. starkeyi caused an accumulation of linoleic acid and a reduction in oleic acid levels. In contrast, overexpression of LsFAD3 induced the production of α-linolenic acid. Deletion of LsFAD2 and LsFAD3 induced the accumulation of oleic acid and linoleic acid, respectively. Our findings are significant for the commercial production of polyunsaturated fatty acids, such as ω-3 polyunsaturated fatty acids, in L. starkeyi.

Lipomyces starkeyi is an oleaginous yeast, and has been classified in four distinct groups, i.e., sensu stricto and custers α, β, and γ. Recently, L. starkeyi clusters α, β, and γ were recognized independent species, Lipomyces mesembrius, Lipomyces doorenjongii, and Lipomyces kockii, respectively. In this study, we investigated phylogenetic relationships within L. starkeyi, including 18 Japanese wild strains, and its related species, based on internal transcribed spacer sequences and evaluated biochemical characters which reflected the phylogenetic tree. Phylogenetic analysis showed that most of Japanese wild strains formed one clade and this clade is more closely related to L. starkeyi s.s. clade including one Japanese wild strain than other clades. Only three Japanese wild strains were genetically distinct from L. starkeyi. Lipomyces mesembrius and L. doorenjongii shared one clade, while L. kockii was genetically distinct from the other three species. Strains in L. starkeyi s.s. clade converted six sugars, D: -glucose, D: -xylose, L: -arabinose, D: -galactose, D: -mannose, and D: -cellobiose to produce high total lipid yields. The Japanese wild strains in subclades B, C, and D converted D: -glucose, D: -galactose, and D: -mannose to produce high total lipid yields. Lipomyces mesembrius was divided into two subclades. Lipomyces mesembrius CBS 7737 converted D: -xylose, L: -arabinose, D: -galactose, and D: -cellobiose, while the other L. mesembrius strains did not. Lipomyces doorenjongii converted all the sugars except D: -cellobiose. In comparison to L. starkeyi, L. mesembrius, and L. doorenjongii, L. kockii produced higher total lipid yields from D: -glucose, D: -galactose, and D: -mannose. The type of sugar converted depended on the subclade classification elucidated in this study.

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.

Microbial lipids are sustainable feedstock for the production of oleochemicals and biodiesel. Oleaginous yeasts have recently been proposed as alternative lipid producers to plants and animals to promote sustainability in the chemical and fuel industries. The oleaginous yeast Lipomyces starkeyi has great industrial potential as an excellent lipid producer. However, improvement of its lipid productivity is essential for the cost-effective production of oleochemicals and fuels. Genetic and metabolic engineering of L. starkeyi via gene manipulation techniques may result in improvements in lipid production and our understanding of the mechanisms behind lipid biosynthesis pathways. We previously described an integrative transformation system using a drug-resistant marker for L. starkeyi . However, gene-targeting frequencies were very low because non-homologous recombination is probably predominant in L. starkeyi . Genetic engineering tools for L. starkeyi have not been sufficiently developed. In this study, we describe a new genetic tool and its application in L. starkeyi . To develop a highly efficient gene-targeting system for L. starkeyi , we constructed a series of mutants by disrupting genes for LsKu70p, LsKu80p, and/or LsLig4p, which share homology with other yeasts Ku70p, Ku80p, and Lig4p, respectively, being involved in non-homologous end-joining pathway. Deletion of the LsLIG4 gene dramatically improved the homologous recombination efficiency (80.0%) at the LsURA3 locus compared with that in the wild-type strain (1.4%), when 2000-bp homologous flanking regions were used. The homologous recombination efficiencies of the double mutant ∆ l sku70 ∆ lslig4 and the triple mutant ∆ lsku70 ∆ lsku80 ∆ lslig4 were also markedly enhanced. Therefore, the L. starkeyi ∆ lslig4 background strains have promise as efficient recipient strains for genetic and metabolic engineering approaches in this yeast.

Background Lipomyces starkeyi is an oleaginous yeast with a native metabolism well-suited for production of lipids and biofuels from complex lignocellulosic and waste feedstocks. Recent advances in genetic engineering tools have facilitated the development of L. starkeyi into a microbial chassis for biofuel and chemical production. However, the feasibility of redirecting L. starkeyi lipid flux away from lipids and towards other products remains relatively unexplored. Here, we engineer the native metabolism to produce malic acid by introducing the reductive TCA pathway and a C4-dicarboxylic acid transporter to the yeast. Results Heterogeneous expression of two genes, the Aspergillus oryzae malate transporter and malate dehydrogenase, enabled L. starkeyi malic acid production. Overexpression of a third gene, the native pyruvate carboxylase, allowed titers to reach approximately 10 g/L during shaking flasks cultivations, with production of malic acid inhibited at pH values less than 4. Corn-stover hydrolysates were found to be well-tolerated, and controlled bioreactor fermentations on the real hydrolysate produced 26.5 g/L of malic acid. Proteomic, transcriptomic and metabolomic data from real and mock hydrolysate fermentations indicated increased levels of a S. cerevisiae hsp9/hsp12 homolog (proteinID: 101453), glutathione dependent formaldehyde dehydrogenases (proteinIDs: 2047, 278215), oxidoreductases, and expression of efflux pumps and permeases during growth on the real hydrolysate. Simultaneously, machine learning based medium optimization improved production dynamics by 18% on mock hydrolysate and revealed lower tolerance to boron (a trace element included in the standard cultivation medium) than other yeasts. Conclusions Together, this work demonstrated the ability to produce organic acids in L. starkeyi with minimal byproducts. The fermentation characterization and omic analyses provide a rich dataset for understanding L. starkeyi physiology and metabolic response to growth in hydrolysates. Identified upregulated genes and proteins provide potential targets for overexpression for improving growth and tolerance to concentrated hydrolysates, as well as valuable information for future L. starkeyi engineering work.

Oily yeasts have been described to be able to accumulate lipids up to 20% of their cellular dry weight. These yeasts represent a minor proportion of the total yeast population, and only 5% of them have been reported as able to accumulate more than 25% of lipids. The oily yeast genera include Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon, and Lipomyces. More specifically, examples of oleaginous yeasts include the species: Lipomyces starkeyi, Rhodosporidium toruloides, Rhodotorula glutinis, and Yarrowia lipolytica. Yeast do exhibit advantages for lipid production over other microbial sources, namely, their duplication times are usually lower than 1 h, are much less affected than plants by season or climate conditions, and their cultures are more easily scaled up than those of microalgae. Additionally, some oily yeasts have been reported to accumulate oil up to 80% of their dry weight and can indeed generate different lipids from different carbon sources or from lipids present in the culture media. Thus, they can vary their lipid composition by replacing the fatty acids present in their triglycerides. Due to the diversity of microorganisms and growth conditions, oily yeasts can be useful for the production of triglycerides, surfactants, or polyunsaturated fatty acids.

The objective of this study was to disrupt the non-homologous end-joining (NHEJ) pathway gene (Ls ku70 Δ) and evaluate the effects of selected gene deletions related to glycogen synthesis (Ls GSY1 ) and lipid degradation (Ls MFE1 , Ls PEX10 , and Ls TGL4 ) on lipid production in the oleaginous yeast Lipomyces starkeyi . Disruption of the NHEJ pathway to reduce the rate of non-homologous recombination is a common approach used to overcome low-efficiency targeted deletion or insertion in various organisms. Here, the homologue of the Ls KU70 gene was identified and disrupted in L. starkeyi NRRL Y-11558. The Ls GSY1 , Ls MFE1 , Ls PEX10 , Ls TGL4 , and Ls URA3 genes were then replaced with a resistance marker in the Ls ku70 Δ strain and several site-specific insertions were assessed for targeted over-expression of selected genes. The targeted disruption efficiency of five selected genes (Ls GSY1 , Ls MFE1 , Ls PEX10 , Ls TGL4 , and Ls URA3 ) was increased from 0 to 10% in the parent to 50–100% of transformants screened in the Ls ku70 Δ strain with 0.8–1.4 kb homologous flanking sequences, while the efficiency of site-specific gene insertion with the β-glucuronidase reporter gene was 100% in the locus near the 3′-end coding (Ls KU70 ) and non-coding (Ls GSY1 , Ls MFE1 , and Ls PEX10 ) regions. Disruption of Ls KU70 in isolation and in conjunction with Ls GSY1 , Ls MFE1 , Ls PEX10 , or Ls TGL4 did not affect lipid production in L. starkeyi . Furthermore, β-glucuronidase reporter gene activity was similar in strains containing site-specific targeted insertions. Therefore, over-expression of genes related to lipid synthesis at targeted loci can be further examined for improvement of total lipid production in L. starkeyi .