Metabolic engineering of the oleaginous yeast Yarrowia lipolytica PO1f for production of erythritol from glycerol

Abstract Background Sugar alcohols are widely used as low-calorie sweeteners in the food and pharmaceutical industries. They can also be transformed into platform chemicals. Yarrowia lipolytica, an oleaginous yeast, is a promising host for producing many sugar alcohols. In this work, we tested whether heterologous expression of a recently identified sugar alcohol phosphatase (PYP) from Saccharomyces cerevisiae would increase sugar alcohol production in Y. lipolytica. Results Y. lipolytica was found natively to produce erythritol, mannitol, and arabitol during growth on glucose, fructose, mannose, and glycerol. Osmotic stress is known to increase sugar alcohol production, and was found to significantly increase erythritol production during growth on glycerol. To better understand erythritol production from glycerol, since it was the most promising sugar alcohol, we measured the expression of key genes and intracellular metabolites. Osmotic stress increased the expression of several key genes in the glycerol catabolic pathway and the pentose phosphate pathway. Analysis of intracellular metabolites revealed that amino acids, sugar alcohols, and polyamines are produced at higher levels in response to osmotic stress. Heterologous overexpression of the sugar alcohol phosphatase increased erythritol production and glycerol utilization in Y. lipolytica. We further increased erythritol production by increasing the expression of native glycerol kinase (GK), and transketolase (TKL). This strain was able to produce 27.5 ± 0.7 g/L erythritol from glycerol during batch growth and 58.8 ± 1.68 g/L erythritol during fed-batch growth in shake-flasks experiments. In addition, the glycerol utilization was increased by 2.5-fold. We were also able to demonstrate that this strain efficiently produces erythritol from crude glycerol, a major byproduct of the biodiesel production. Conclusions We demonstrated the application of a promising enzyme for increasing erythritol production in Y. lipolytica. We were further able to boost production by combining the expression of this enzyme with other approaches known to increase erythritol production in Y. lipolytica. This suggest that this new enzyme provides an orthogonal route for boosting production and can be stacked with existing designs known to increase sugar alcohol production in yeast such as Y. lipolytica. Collectively, this work establishes a new route for increasing sugar alcohol production and further develops Y. lipolytica as a promising host for erythritol production from cheap substrates such as glycerol.

Download Full-text

Production of galactitol from galactose by the oleaginous yeast Rhodosporidium toruloides IFO0880

Biotechnology for Biofuels ◽

10.1186/s13068-019-1586-5 ◽

2019 ◽

Vol 12 (1) ◽

Cited By ~ 5

Author(s):

Sujit Sadashiv Jagtap ◽

Ashwini Ashok Bedekar ◽

Jing-Jing Liu ◽

Yong-Su Jin ◽

Christopher V. Rao

Keyword(s):

Oleaginous Yeast ◽

Lipid Production ◽

Value Added ◽

Building Blocks ◽

Rhodosporidium Toruloides ◽

Sugar Alcohols ◽

Galactose Metabolism ◽

Alcohol Production ◽

Wide Range ◽

Multiple Value

Abstract Background Sugar alcohols are commonly used as low-calorie sweeteners and can serve as potential building blocks for bio-based chemicals. Previous work has shown that the oleaginous yeast Rhodosporidium toruloides IFO0880 can natively produce arabitol from xylose at relatively high titers, suggesting that it may be a useful host for sugar alcohol production. In this work, we explored whether R. toruloides can produce additional sugar alcohols. Results Rhodosporidium toruloides is able to produce galactitol from galactose. During growth in nitrogen-rich medium, R. toruloides produced 3.2 ± 0.6 g/L, and 8.4 ± 0.8 g/L galactitol from 20 to 40 g/L galactose, respectively. In addition, R. toruloides was able to produce galactitol from galactose at reduced titers during growth in nitrogen-poor medium, which also induces lipid production. These results suggest that R. toruloides can potentially be used for the co-production of lipids and galactitol from galactose. We further characterized the mechanism for galactitol production, including identifying and biochemically characterizing the critical aldose reductase. Intracellular metabolite analysis was also performed to further understand galactose metabolism. Conclusions Rhodosporidium toruloides has traditionally been used for the production of lipids and lipid-based chemicals. Our work demonstrates that R. toruloides can also produce galactitol, which can be used to produce polymers with applications in medicine and as a precursor for anti-cancer drugs. Collectively, our results further establish that R. toruloides can produce multiple value-added chemicals from a wide range of sugars.

Download Full-text

Effects of a high-cultivation temperature on the physiology of three different Yarrowia lipolytica strains

FEMS Yeast Research ◽

10.1093/femsyr/foz068 ◽

2019 ◽

Vol 19 (7) ◽

Cited By ~ 2

Author(s):

S Hackenschmidt ◽

F Bracharz ◽

R Daniel ◽

A Thürmer ◽

S Bruder ◽

...

Keyword(s):

Yarrowia Lipolytica ◽

Academic Research ◽

Genomic Analysis ◽

Industrial Applications ◽

Sugar Alcohol ◽

Alcohol Production ◽

Physiological Properties ◽

Sequence Variations ◽

Distinct Strain ◽

Substrate Concentrations

ABSTRACT Despite the increasing relevance, ranging from academic research to industrial applications, only a limited number of non-conventional, oleaginous Yarrowia lipolytica strains are characterized in detail. Therefore, we analyzed three strains in regard to their metabolic and physiological properties, especially with respect to important characteristics of a production strain. By investigating different cultivation conditions and media compositions, similarities and differences between the distinct strain backgrounds could be derived. Especially sugar alcohol production, as well as an agglomeration of cells were found to be connected with growth at high temperatures. In addition, sugar alcohol production was independent of high substrate concentrations under these conditions. To investigate the genotypic basis of particular traits, including growth characteristics and metabolite concentrations, genomic analysis were performed. We found sequence variations for one third of the annotated proteins but no obvious link to all phenotypic features.

Download Full-text

Recent Advances in Producing Sugar Alcohols and Functional Sugars by Engineering Yarrowia lipolytica

Frontiers in Bioengineering and Biotechnology ◽

10.3389/fbioe.2021.648382 ◽

2021 ◽

Vol 9 ◽

Author(s):

Abdul Rahman Abbasi ◽

Jinle Liu ◽

Zhi Wang ◽

Anqi Zhao ◽

Hanjie Ying ◽

...

Keyword(s):

Yarrowia Lipolytica ◽

Pathway Engineering ◽

Sugar Alcohol ◽

Breeding Methods ◽

Biological Functions ◽

Sugar Alcohols ◽

Bioprocess Engineering ◽

Chemical Structures ◽

Recent Advances ◽

Catalyst Production

The sugar alcohols and functional sugars have wide applications in food, pharmaceutical, and chemical industries. However, the smaller quantities of natural occurring sugar alcohols and functional sugars restricted their applications. The enzymatic and whole-cell catalyst production is emerging as the predominant alternatives. The properties of Yarrowia lipolytica make it a promising sugar alcohol and functional sugar producer. However, there are still some issues to be resolved. As there exist reviews about the chemical structures, physicochemical properties, biological functions, applications, and biosynthesis of sugar alcohols and/or functional sugars in Y. lipolytica, this mini review will not only update the recent advances in enzymatic and microbial production of sugar alcohols (erythritol, D-threitol, and xylitol) and functional sugars (isomaltulose, trehalose, fructo-oligosaccharides, and galacto-oligosaccharides) by using recombinant Y. lipolytica but also focus on the studies of gene discovery, pathway engineering, expanding substrate scope, bioprocess engineering, and novel breeding methods to resolve the aforementioned issues.

Download Full-text

Characterization of three Yarrowia lipolytica strains in respect to different cultivation temperatures and metabolite secretion

10.1101/645242 ◽

2019 ◽

Author(s):

S Hackenschmidt ◽

F Bracharz ◽

R Daniel ◽

A Thürmer ◽

S Bruder ◽

...

Keyword(s):

Yarrowia Lipolytica ◽

Academic Research ◽

Genomic Analysis ◽

Industrial Applications ◽

Sugar Alcohol ◽

Alcohol Production ◽

Physiological Properties ◽

Sequence Variations ◽

Set Up

AbstractDespite the increasing relevance, ranging from academic research to industrial applications, only a limited number of nonconventional, oleaginous Yarrowia lipolytica strains are characterized in detail. Therefore, we analyzed three strains in regard to their metabolic and physiological properties and in respect to important characteristics of a production strains. A flow cytometry method was set up to evaluate their fitness in a rapid manner. By investigating different cultivation conditions and media compositions, similarities and differences between the distinct strain backgrounds could be derived. Especially sugar alcohol production, as well as a agglomeration of cells were found to be connected with growth at high temperatures. In addition, sugar alcohol production was independent of high substrate concentrations under these conditions. To investigate particular traits, including growth characteristics and metabolite concentrations, genomic analysis were performed. We found sequence variations for one third of the annotated proteins but no obvious link to all phenotypic features.

Download Full-text

Gut Microbiota Prevents Sugar Alcohol-Induced Diarrhea

Nutrients ◽

10.3390/nu13062029 ◽

2021 ◽

Vol 13 (6) ◽

pp. 2029

Author(s):

Kouya Hattori ◽

Masahiro Akiyama ◽

Natsumi Seki ◽

Kyosuke Yakabe ◽

Koji Hase ◽

...

Keyword(s):

Gut Microbiota ◽

Gut Bacteria ◽

Food Science ◽

Sugar Alcohol ◽

Processed Food ◽

Phosphotransferase System ◽

Sugar Alcohols ◽

E Coli ◽

The Family ◽

Rdna Analysis

While poorly-absorbed sugar alcohols such as sorbitol are widely used as sweeteners, they may induce diarrhea in some individuals. However, the factors which determine an individual’s susceptibility to sugar alcohol-induced diarrhea remain unknown. Here, we show that specific gut bacteria are involved in the suppression of sorbitol-induced diarrhea. Based on 16S rDNA analysis, the abundance of Enterobacteriaceae bacteria increased in response to sorbitol consumption. We found that Escherichia coli of the family Enterobacteriaceae degraded sorbitol and suppressed sorbitol-induced diarrhea. Finally, we showed that the metabolism of sorbitol by the E. coli sugar phosphotransferase system helped suppress sorbitol-induced diarrhea. Therefore, gut microbiota prevented sugar alcohol-induced diarrhea by degrading sorbitol in the gut. The identification of the gut bacteria which respond to and degrade sugar alcohols in the intestine has implications for microbiome science, processed food science, and public health.

Download Full-text

Simultaneous lipid biosynthesis and recovery for oleaginous yeast Yarrowia lipolytica

Biotechnology for Biofuels ◽

10.1186/s13068-019-1576-7 ◽

2019 ◽

Vol 12 (1) ◽

Cited By ~ 3

Author(s):

Pratik Prashant Pawar ◽

Annamma Anil Odaneth ◽

Rajeshkumar Natwarlal Vadgama ◽

Arvind Mallinath Lali

Keyword(s):

Yarrowia Lipolytica ◽

Oil Recovery ◽

Oleaginous Yeast ◽

Fermentation Broth ◽

Continuous Production ◽

Oil Production ◽

Biofuel Production ◽

Oil Yield ◽

Microbial Oil

Abstract Background Recent trends in bioprocessing have underlined the significance of lignocellulosic biomass conversions for biofuel production. These conversions demand at least 90% energy upgradation of cellulosic sugars to generate renewable drop-in biofuel precursors (Heff/C ~ 2). Chemical methods fail to achieve this without substantial loss of carbon; whereas, oleaginous biological systems propose a greener upgradation route by producing oil from sugars with 30% theoretical yields. However, these oleaginous systems cannot compete with the commercial volumes of vegetable oils in terms of overall oil yields and productivities. One of the significant challenges in the commercial exploitation of these microbial oils lies in the inefficient recovery of the produced oil. This issue has been addressed using highly selective oil capturing agents (OCA), which allow a concomitant microbial oil production and in situ oil recovery process. Results Adsorbent-based oil capturing agents were employed for simultaneous in situ oil recovery in the fermentative production broths. Yarrowia lipolytica, a model oleaginous yeast, was milked incessantly for oil production over 380 h in a media comprising of glucose as a sole carbon and nutrient source. This was achieved by continuous online capture of extracellular oil from the aqueous media and also the cell surface, by fluidizing the fermentation broth over an adsorbent bed of oil capturing agents (OCA). A consistent oil yield of 0.33 g per g of glucose consumed, corresponding to theoretical oil yield over glucose, was achieved using this approach. While the incorporation of the OCA increased the oil content up to 89% with complete substrate consumptions, it also caused an overall process integration. Conclusion The nondisruptive oil capture mediated by an OCA helped in accomplishing a trade-off between microbial oil production and its recovery. This strategy helped in realizing theoretically efficient sugar-to-oil bioconversions in a continuous production process. The process, therefore, endorses a sustainable production of molecular drop-in equivalents through oleaginous yeasts, representing as an absolute microbial oil factory.

Download Full-text

Snf1 Is a Regulator of Lipid Accumulation in Yarrowia lipolytica

Applied and Environmental Microbiology ◽

10.1128/aem.02079-13 ◽

2013 ◽

Vol 79 (23) ◽

pp. 7360-7370 ◽

Cited By ~ 54

Author(s):

John Seip ◽

Raymond Jackson ◽

Hongxian He ◽

Quinn Zhu ◽

Seung-Pyo Hong

Keyword(s):

Yarrowia Lipolytica ◽

Lipid Accumulation ◽

Oleaginous Yeast ◽

De Novo ◽

Lipid Synthesis ◽

Omega 3 ◽

Wild Type ◽

Content Type ◽

Reverse Transcription Pcr ◽

Dry Cell Weight

ABSTRACTIn the oleaginous yeastYarrowia lipolytica,de novolipid synthesis and accumulation are induced under conditions of nitrogen limitation (or a high carbon-to-nitrogen ratio). The regulatory pathway responsible for this induction has not been identified. Here we report that the SNF1 pathway plays a key role in the transition from the growth phase to the oleaginous phase inY. lipolytica. Strains with aY. lipolyticasnf1(Ylsnf1) deletion accumulated fatty acids constitutively at levels up to 2.6-fold higher than those of the wild type. When introduced into aY. lipolyticastrain engineered to produce omega-3 eicosapentaenoic acid (EPA),Ylsnf1deletion led to a 52% increase in EPA titers (7.6% of dry cell weight) over the control. Other components of theY. lipolyticaSNF1 pathway were also identified, and their function in limiting fatty acid accumulation is suggested by gene deletion analyses. Deletion of the gene encoding YlSnf4, YlGal83, or YlSak1 significantly increased lipid accumulation in both growth and oleaginous phases compared to the wild type. Furthermore, microarray and quantitative reverse transcription-PCR (qRT-PCR) analyses of theYlsnf1mutant identified significantly differentially expressed genes duringde novolipid synthesis and accumulation inY. lipolytica. Gene ontology analysis found that these genes were highly enriched with genes involved in lipid metabolism. This work presents a new role for Snf1/AMP-activated protein kinase (AMPK) pathways in lipid accumulation in this oleaginous yeast.

Download Full-text

Stable isotope and chemical inhibition analyses suggested the existence of a non-mevalonate-like pathway in the yeast Yarrowia lipolytica

Scientific Reports ◽

10.1038/s41598-021-85170-0 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Sivamoke Dissook ◽

Tomohisa Kuzuyama ◽

Yuri Nishimoto ◽

Shigeru Kitani ◽

Sastia Putri ◽

...

Keyword(s):

Yarrowia Lipolytica ◽

Oleaginous Yeast ◽

Isoprenoid Biosynthesis ◽

Mep Pathway ◽

Authentic Standard ◽

Chemical Inhibition ◽

Chromatographic Peaks ◽

Methyl Erythritol Phosphate ◽

Limiting Condition ◽

Yeast Yarrowia Lipolytica

AbstractMethyl erythritol phosphate (MEP) is the metabolite found in the MEP pathway for isoprenoid biosynthesis, which is known to be utilized by plants, algae, and bacteria. In this study, an unprecedented observation was found in the oleaginous yeast Yarrowia lipolytica, in which one of the chromatographic peaks was annotated as MEP when cultivated in the nitrogen limiting condition. This finding raised an interesting hypothesis of whether Y. lipolytica utilizes the MEP pathway for isoprenoid biosynthesis or not, because there is no report of yeast harboring the MEP pathway. Three independent approaches were used to investigate the existence of the MEP pathway in Y. lipolytica; the spiking of the authentic standard, the MEP pathway inhibitor, and the 13C labeling incorporation analysis. The study suggested that the mevalonate and MEP pathways co-exist in Y. lipolytica and the nitrogen limiting condition triggers the utilization of the MEP pathway in Y. lipolytica.

Download Full-text