Abolishing storage lipids induces protein misfolding and stress responses in Yarrowia lipolytica.

Yarrowia lipolytica naturally saves excess carbon as storage lipids. Engineering efforts allow redirecting the high precursor flux required for lipid synthesis toward added-value chemicals such as polyketides, flavonoids, and terpenoids. To redirect precursor flux from storage lipids to other products, four genes involved in triacylglycerol and sterol ester synthesis (DGA1, DGA2, LRO1, and ARE1) can be deleted. To elucidate the effect of the deletions on cell physiology and regulation, we performed chemostat cultivations under carbon and nitrogen limitations, followed by transcriptome analysis. We found that storage lipid-free cells show an enrichment of the unfolded protein response, and several biological processes related to protein refolding and degradation are enriched. Additionally, storage lipid-free cells show an altered lipid class distribution with an abundance of potentially cytotoxic free fatty acids under nitrogen limitation. Our findings not only highlight the importance of lipid metabolism on cell physiology and proteostasis, but can also aid the development of improved chassy strains of Y. lipolytica for commodity chemical production.

Cinnamic acid and p-coumaric acid are metabolized to 4-hydroxybenzoic acid by Yarrowia lipolytica.

Yarrowia lipolytica has been explored as a potential production host for flavonoid synthesis due to its high tolerance to aromatic acids and ability to supply malonyl-CoA. However, little is known about its ability to consume the precursors cinnamic and p-coumaric acid. In this study, we demonstrate that Y. lipolytica can consume these precursors through multiple pathways that are partially dependent on the cultivation medium. By monitoring the aromatic acid concentrations over time, we found that cinnamic acid is converted to p-coumaric acid. We identified potential proteins with a trans-cinnamate 4-monooxygenase activity in Y. lipolytica and constructed a collection of 15 knock-out strains to identify the genes responsible for the reaction. We identified YALI1_B28430g as the gene encoding for a protein that converts cinnamic acid to p-coumaric acid (designated as TCM1). By comparing different media compositions we found that complex media components (casamino acids and yeast extract) induce this pathway. Additionally, we discover the conversion of p-coumaric acid to 4-hydroxybenzoic acid. Our findings provide new insight into the metabolic capabilities of Y. lipolytica and hold great potential for the future development of improved strains for flavonoid production.

Urea is a drop-in nitrogen source alternative to ammonium sulphate in Yarrowia lipolytica.

Media components, including the nitrogen source, are significant cost factors in cultivation processes. The nitrogen source also influences cell behavior and production performance. Ammonium sulfate is a widely used nitrogen source for microorganisms' cultivation. Urea is a sustainable and cheap alternative nitrogen source. We investigated the influence of urea as a nitrogen source compared to ammonium sulfate by cultivating phenotypically different Yarrowia lipolytica strains in chemostats under carbon or nitrogen limitation. We found no significant coherent changes in growth and lipid production. RNA sequencing revealed no significant concerted changes in the transcriptome. The genes involved in urea uptake and degradation are not upregulated on a transcriptional level. Our findings support urea usage, indicating that previous metabolic engineering efforts where ammonium sulfate was used are likely translatable to the usage of urea and can ease the way for urea as a cheap and sustainable nitrogen source in more applications.

Altering the fatty acid profile of Yarrowia lipolytica to mimic cocoa butter by genetic engineering of desaturases.

BACKGROUND: Demand for Cocoa butter is steadily increasing, but the supply of cocoa beans is naturally limited and under threat from global warming. One route to meeting the future demand for cocoa butter equivalent (CBE) could be to utilize microbial cell factories such as the oleaginous yeast Yarrowia lipolytica. RESULTS: The main goal was to achieve triacyl-glycerol (TAG) storage lipids in Y. lipolytica mimicking cocoa butter. This was accomplished by replacing the native Δ9 fatty acid desaturase (Ole1p) with homologs from other species and changing the expression of both Ole1p and the Δ12 fatty acid desaturase (Fad2p). We thereby abolished the palmitoleic acid and reduced the linoleic acid content in TAG, while the oleic acid content was reduced to approximately 40 percent of the total fatty acids. The proportion of fatty acids in TAG changed dramatically over time during growth, and the fatty acid composition of TAG, free fatty acids and phospholipids was found to be very different. CONCLUSIONS: We show that the fatty acid profile in the TAG of Y. lipolytica can be altered to mimic cocoa butter. We also demonstrate that a wide range of fatty acid profiles can be achieved while maintaining good growth and high lipid accumulation, which, together with the ability of Y. lipolytica to utilize a wide variety of carbon sources, opens up the path toward sustainable production of CBE and other food oils.

Strategies to increase tolerance and robustness of industrial microorganisms.

The development of a cost-competitive bioprocess requires that the cell factory converts the feedstock into the product of interest at high rates and yields. However, microbial cell factories are exposed to a variety of different stresses during the fermentation process. These stresses can be derived from feedstocks, metabolism, or industrial production processes, limiting production capacity and diminishing competitiveness. Improving stress tolerance and robustness allows for more efficient production and ultimately makes a process more economically viable. This review summarises general trends and updates the most recent developments in technologies to improve the stress tolerance of microorganisms. We first look at evolutionary, systems biology and computational methods as examples of non-rational approaches. Then we review the (semi-)rational approaches of membrane and transcription factor engineering for improving tolerance phenotypes. We further discuss challenges and perspectives associated with these different approaches.

Graphene coated magnetic nanoparticles facilitate the release of biofuels and oleochemicals from yeast cell factories.

Engineering of microbial cells to produce high value chemicals is rapidly advancing. Yeast, bacteria and microalgae are being used to produce high value chemicals by utilizing widely available carbon sources. However, current extraction processes of many high value products from these cells are time- and labor-consuming and require toxic chemicals. This makes the extraction processes detrimental to the environment and not economically feasible. Hence, there is a demand for the development of simple, effective, and environmentally friendly method for the extraction of high value chemicals from these cell factories. Herein, we hypothesized that atomically thin edges of graphene having ability to interact with hydrophobic materials, could be used to extract high value lipids from cell factories. To achieve this, array of axially oriented graphene was deposited on iron nanoparticles. These coated nanoparticles were used to facilitate the release of intracellular lipids from Yarrowia lipolytica cells. Our treatment process can be integrated with the growth procedure and achieved the release of 50% of total cellular lipids from Y. lipolytica cells. Based on this result, we propose that nanoparticles coated with axially oriented graphene could pave efficient, environmentally friendly, and cost-effective way to release intracellular lipids from yeast cell factories.

Tolerance of Yarrowia lipolytica to inhibitors commonly found in lignocellulosic hydrolysates.

BACKGROUND: Lignocellulosic material is a suitable renewable carbon and energy source for microbial cell factories, such as Yarrowia lipolytica. To be accessible for microorganisms, the constituent sugars need to be released in a hydrolysis step, which as a side effect leads to the formation of various inhibitory compounds. However, the effects of these inhibitory compounds on the growth of Y. lipolytica have not been thoroughly investigated. RESULTS: Here we show the individual and combined effect of six inhibitors from three major inhibitor groups on the growth of Y. lipolytica. We engineered a xylose consuming strain by overexpressing the three native genes XR, XDH, and XK and found that the inhibitor tolerance of Y. lipolytica is similar in glucose and in xylose. Aromatic compounds could be tolerated at high concentrations, while furfural linearly increased the lag phase of the cultivation, and hydroxymethylfurfural only inhibited growth partially. The furfural induced increase in lag phase can be overcome by an increased volume of inoculum. Formic acid only affected growth at concentrations above 25 mM. In a synthetic hydrolysate, formic acid, furfural, and coniferyl aldehyde were identified as the major growth inhibitors. CONCLUSION: We showed the individual and combined effect of inhibitors found in hydrolysate on the growth of Y. lipolytica. Our study improves understanding of the growth limiting inhibitors found in hydrolysate and enables a more targeted engineering approach to increase the inhibitor tolerance of Y. lipolytica. This will help to improve the usage of Y. lipolytica as a sustainable microbial cell factory.

Production of 10-methyl branched fatty acids in yeast.

BACKGROUND: Despite the environmental value of biobased lubricants, they account for less than 2% of global lubricant use due to poor thermo-oxidative stability arising from the presence of unsaturated double bonds. Methyl branched fatty acids (BFAs), particularly those with branching near the acyl-chain mid-point, are a high-performance alternative to existing vegetable oils because of their low melting temperature and full saturation. RESULTS: We cloned and characterized two pathways to produce 10-methyl BFAs isolated from actinomycetes and γ-proteobacteria. In the two-step bfa pathway of actinomycetes, BfaB methylates Δ9 unsaturated fatty acids to form 10-methylene BFAs, and subsequently, BfaA reduces the double bond to produce a fully saturated 10-methyl branched fatty acid. A BfaA-B fusion enzyme increased the conversion efficiency of 10-methyl BFAs. The ten-methyl palmitate production (tmp) pathway of γ-proteobacteria produces a 10-methylene intermediate, but the TmpA putative reductase was not active in E. coli or yeast. Comparison of BfaB and TmpB activities revealed a range of substrate specificities from C14-C20 fatty acids unsaturated at the Δ9, Δ10 or Δ11 position. We demonstrated efficient production of 10-methylene and 10-methyl BFAs in S. cerevisiae by secretion of free fatty acids and in Y. lipolytica as triacylglycerides, which accumulated to levels more than 35% of total cellular fatty acids. CONCLUSIONS: We report here the characterization of a set of enzymes that can produce position-specific methylene and methyl branched fatty acids. Yeast expression of bfa enzymes can provide a platform for the large-scale production of branched fatty acids suitable for industrial and consumer applications.

Deletion of MHY1 abolishes hyphae formation in Yarrowia lipolytica without negative effects on stress tolerance.

There is a need for development of sustainable production processes for production of fats/oils and lipid derived chemicals. The dimorphic oleaginous yeast Yarrowia lipolytica is a promising organism for conversion of biomass hydrolysate to lipids, but in many such processes hyphae formation will be problematic. We have therefore constructed and compared the performance of strains carrying deletions in several published gene targets suggested to abolish hyphae formation (MHY1, HOY1 and CLA4). The MHY1-deletion was the only of the tested strains which did not exhibit hyphae formation under any of the conditions tested. The MHY1-deletion also had a weak positive effect on lipid accumulation without affecting the total fatty acid composition, irrespective of the nitrogen source used. MHY1 has been suggested to constitute a functional homolog of the stress responsive transcription factors MSN2/4 in Saccharomyces cerevisiae, the deletion of which are highly stress sensitive. However, the deletion of MHY1 displayed only minor difference on survival of a range of acute or long term stress and starvation conditions. We conclude that the deletion of MHY1 in Y.lipolytica is a reliable way of abolishing hyphae formation with few detectable negative side effects regarding growth, stress tolerance and lipid accumulation and composition.

Engineering high-level production of fatty alcohols by Saccharomyces cerevisiae from lignocellulosic feedstocks.

Fatty alcohols in the C12-C18 range are used in personal care products, lubricants, and potentially biofuels. These compounds can be produced from the fatty acid pathway by a fatty acid reductase (FAR), yet yields from the preferred industrial host Saccharomyces cerevisiae remain under 2% of the theoretical maximum from glucose. Here we improved titer and yield of fatty alcohols using an approach involving quantitative analysis of protein levels and metabolic flux, engineering enzyme level and localization, pull-push-block engineering of carbon flux, and cofactor balancing. We compared four heterologous FARs, finding highest activity and endoplasmic reticulum localization from a Mus musculus FAR. After screening an additional twenty-one single-gene edits, we identified increasing FAR expression; deleting competing reactions encoded by DGA1, HFD1, and ADH6; overexpressing a mutant acetyl-CoA carboxylase; limiting NADPH and carbon usage by the glutamate dehydrogenase encoded by GDH1; and overexpressing the Δ9-desaturase encoded by OLE1 as successful strategies to improve titer. Our final strain produced 1.2g/L fatty alcohols in shake flasks, and 6.0g/L in fed-batch fermentation, corresponding to ~ 20% of the maximum theoretical yield from glucose, the highest titers and yields reported to date in S. cerevisiae. We further demonstrate high-level production from lignocellulosic feedstocks derived from ionic-liquid treated switchgrass and sorghum, reaching 0.7g/L in shake flasks. Altogether, our work represents progress towards efficient and renewable microbial production of fatty acid-derived products.