A two-stage fermentation process of erythritol production by yeast Y. lipolytica from molasses and glycerol

Mirończuk, Aleksandra M.; Rakicka, Magdalena; Biegalska, Anna; Rymowicz, Waldemar; Dobrowolski, Adam

doi:10.1016/j.biortech.2015.09.008

Cited by 85 publications

(54 citation statements)

References 28 publications

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“…Thus, we constructed overexpression cassettes containing the genes TKL1 , TAL1 , ZWF1 , and GND1 under a hybrid TEF promoter [27], and transformed the cassettes into the AMM strain, derived from MK1 [28], resulting in strains listed in Table 1.…”

Section: Resultsmentioning

confidence: 99%

Functional overexpression of genes involved in erythritol synthesis in the yeast Yarrowia lipolytica

Mirończuk

Biegalska

Dobrowolski

2017

Biotechnol Biofuels

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BackgroundErythritol, a four-carbon polyol synthesized by microorganisms as an osmoprotectant, is a natural sweetener produced on an industrial scale for decades. Despite the fact that the yeast Yarrowia lipolytica has been reported since the 1970s as an erythritol producer, the metabolic pathway of this polyol has never been characterized. It was shown that erythritol synthesis in yeast occurs via the pentose phosphate pathway (PPP). The oleaginous yeast Y. lipolytica is a good host for converting inexpensive glycerol into a value-added product such as erythritol. Glycerol is a renewable feedstock which is produced on a large scale as a waste product by many branches of industry.ResultsIn this study, we functionally overexpressed four genes involved in the pentose phosphate pathway (PPP): gene YALI0E06479g encoding transketolase (TKL1), gene YALI0F15587g encoding transaldolase (TAL1), gene YALI0E22649g encoding glucose-6-phosphate dehydrogenase (ZWF1), and gene YALI0B15598g encoding 6-phosphogluconate dehydrogenase (GND1). Here, we show that the crucial gene for erythritol synthesis in Y. lipolytica is transketolase. Overexpression of this gene results in a twofold improvement in erythritol synthesis during a shake-flask experiment (58 g/L). Moreover, overexpression of TKL1 allows for efficient production of erythritol independently from the supplied dissolved oxygen. Fermentation conducted in a 5-L bioreactor at low agitation results in almost 70% higher titer of erythritol over the control strain.ConclusionThis work presents the importance of the PPP in erythritol synthesis and the feasibility for economic production of erythritol from glycerol by the yeast Y. lipolytica.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-017-0772-6) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

Functional overexpression of genes involved in erythritol synthesis in the yeast Yarrowia lipolytica

Mirończuk

Biegalska

Dobrowolski

2017

Biotechnol Biofuels

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“…Y. lipolytica was transformed according to the lithium acetate method described previously [28]. The transformants were plated out on selective media [29]. Auxotrophies were restored via excision using the Cre-lox recombinase system following transformation with the replicative plasmid pUB4-Cre1(JME547) [30].…”

Section: Methodsmentioning

confidence: 99%

Characterization of erythrose reductase from Yarrowia lipolytica and its influence on erythritol synthesis

et al. 2017

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BackgroundErythritol is a natural sweetener that is used in the food industry. It is produced as an osmoprotectant by bacteria and yeast. Due to its chemical properties, it does not change the insulin level in the blood, and therefore it can be safely used by diabetics. Previously, it has been shown that erythrose reductase (ER), which catalyzes the final step, plays a crucial role in erythritol synthesis. ER reduces erythrose to erythritol with NAD(P)H as a cofactor. Despite many studies on erythritol synthesis by Yarrowia lipolytica, the enzymes involved in this metabolic pathway have ever been described.ResultsThe gene YALI0F18590g encoding the predicted erythrose reductase from Y. lipolytica was overexpressed, and its influence on erythritol synthesis was studied. The amino acid sequence of the Y. lipolytica ER showed a high degree of similarity to the previously described erythrose reductases from known erythritol producers, such as Candida magnoliae and Moniliella megachiliensis. Here, we found that the gene overexpression results in an enhanced titer of erythritol of 44.44 g/L (20% over the control), a yield of 0.44 g/g and productivity of 0.77 g/L/h. Moreover, on purification and characterization of the enzyme we found that it displays the highest activity at 37 °C and pH 3.0. The effects of various metal ions (Zn2+, Cu2+, Mn2+, Fe2+) on erythrose reductase were investigated. The addition of Zn2+ ions at 0.25 mM had a positive effect on the activity of erythrose reductase from Y. lipolytica, as well as on the erythritol production.ConclusionsIn this study we identified, overexpressed and characterized a native erythrose reductase in Y. lipolytica. Further optimizations of this strain via metabolic pathway engineering and media optimization strategies enabled 54 g/L to be produced in a shake-flask experiment. To date, this is the first reported study employing metabolic engineering of the native gene involved in the erythritol pathway to result in a high titer of the polyol. Moreover, it indicates the importance of environmental conditions for genetic targets in metabolic engineering.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-017-0733-6) contains supplementary material, which is available to authorized users.

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“…The obtained plasmids were digested with MssI to create linear expression cassettes devoid of E. coli DNA and surrounded by Y. lipolytica rDNA for targeted integrations. First, Y. lipolytica AJD [9] was transformed with GUT1 or GUT2 overexpression cassette, according to the lithium acetate method described before [19], resulting in strains AJD pADUTGut1 or AJD pADUTGut2, respectively. The transformants were plated out on selective media [9] and were confirmed via gDNA extraction and three distinct PCR confirmations.…”

Section: Methodsmentioning

confidence: 99%

“…First, Y. lipolytica AJD [9] was transformed with GUT1 or GUT2 overexpression cassette, according to the lithium acetate method described before [19], resulting in strains AJD pADUTGut1 or AJD pADUTGut2, respectively. The transformants were plated out on selective media [9] and were confirmed via gDNA extraction and three distinct PCR confirmations. Next, auxotrophies were restored via excision using the Cre-lox recombinase system following transformation with the replicative plasmid pUB4-Cre1(JME547) [20].…”

Section: Methodsmentioning

confidence: 99%

A novel strain of Yarrowia lipolytica as a platform for value-added product synthesis from glycerol

Mirończuk

Rzechonek

Biegalska

et al. 2016

Biotechnol Biofuels

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BackgroundIncreasing interest of non-conventional yeasts has been observed for many years due to their biochemical characteristics and potential applications. Well-studied, oleaginous yeast Y. lipolytica is an attractive host for converting a low-cost glycerol, into value-added products such as erythritol (sweetener) or citric acid. Glycerol is an important renewable feedstock and is the main co-product of biodiesel production, which is nowadays applied on a large commercial scale. To this end, we engineered the yeast Y. lipolytica to increase the productivity of this strain.ResultsIn this light, we enhanced glycerol assimilation by over-expression of the YALI0F00484g gene encoding glycerol kinase (GK) and gene YALI0B02948g encoding glycerol-3-P dehydrogenase (GDH). The modified strains have been tested for glycerol consumption rate and erythritol and citric acid synthesis under various conditions. Here, we show that the overexpression of GK and GDH, increased glycerol consumption resulting in rapid erythritol and citric acid synthesis. Next, we combined the two genes in the tandem gene construct for the simultaneous co-expression of GK and GDH, which further increased the desired product synthesis. The glycerol consumption was explored in a 5-L bioreactor and the engineered strains were able to utilize 150 g/L glycerol within 44–48 hours. The erythritol productivity for GK overexpression and co-expression of GK and DGH was 24 and 35 %, respectively, over the control strain. Moreover, we established conditions for the production of citric acid at pH 3.0, the engineered strains increased citric acid production 14-fold over the control.ConclusionThis work demonstrates the excellent capacity of the engineered strains as a starting platform for further modification for broad-range value-added product biosynthesis from glycerol. This study presents the highest reported titer citric acid at low pH to date. The process parameters such as productivity and yield of erythritol and citric acid were significantly elevated, what is valuable for industrial applications.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0593-z) contains supplementary material, which is available to authorized users.

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A two-stage fermentation process of erythritol production by yeast Y. lipolytica from molasses and glycerol

Cited by 85 publications

References 28 publications

Functional overexpression of genes involved in erythritol synthesis in the yeast Yarrowia lipolytica

Functional overexpression of genes involved in erythritol synthesis in the yeast Yarrowia lipolytica

Characterization of erythrose reductase from Yarrowia lipolytica and its influence on erythritol synthesis

A novel strain of Yarrowia lipolytica as a platform for value-added product synthesis from glycerol

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