Enhanced Erythritol Productivity Research Articles

Improving an Alternative Glycerol Catabolism Pathway in Yarrowia lipolytica to Enhance Erythritol Production.

Engineering the glycerol-3-phosphate pathway could enhance erythritol production by accelerating glycerol uptake. However, little work has been conducted on the alternative dihydroxyacetone (DHA) pathway in Yarrowia lipolytica. Herein, this route was identified and characterized in Y. lipolytica by metabolomic and transcriptomic analysis. Moreover, the reaction catalyzed by dihydroxyacetone kinase encoded by dak2 was identified as the rate-limiting step. By combining NHEJ-mediated insertion mutagenesis with a push-and-pull strategy, Y. lipolytica strains with high-yield erythritol synthesis from glycerol were obtained. Screening of a library of insertion mutants allows the identification of a mutant with fourfold increased erythritol production. Overexpression of DAK2 and glycerol dehydrogenase GCY3 together with gene encoding transketolase and transaldolase from the nonoxidative part of the pentose phosphate pathway led to a strain with further increased productivity with a titer of 53.1 g/L and a yield 0.56 g/g glycerol, which were 8.1- and 4.2-fold of starting strain.

Systematic metabolic engineering of Yarrowia lipolytica for the enhanced production of erythritol

In recent times, there has been a growing interest in exploring microbial strains that exhibit enhanced erythritol productivity. Nonetheless, the lack of advanced synthetic biology tools has limited rapid strain development. In this study, the CRISPR/Cas9 system was employed to genetically modify Yarrowia lipolytica at the chromosomal level, which could improve the production of erythritol while saving the time required to markers recovery, and realizing the rapid construction of high-erythritol strains. Firstly, the basic strain E004 was generated by increasing the efficiency of homologous recombination and regulating the erythritol degradation pathway. Secondly, eleven key gene targets and a strong promoter 8UAS1BXPR2-PTEFin was obtained by target screening and promoter engineering. Finally, based on modular pathway engineering and morphological engineering, the high production of erythritol was achieved successfully. The best-engineered strain E326 produced 256 g/L erythritol in a 5-L bioreactor, which is the highest production level reported so far in Y. lipolytica.

Strain improvement for enhanced erythritol production by Moniliella pollinis Mutant-58 using jaggery as a cost-effective substrate.

Erythritol has been produced by various microorganisms including Yarrowia, Moniliella, Aureobasidium, and Candida strains. Due to its relatively high price, erythritol sweetener is used lesser than other polyols despite having many advantages. Therefore, in this study, Moniliella pollinis strain was improved for erythritol production by chemical mutagenesis and subsequently screening for cost-effective carbon sources for the enhanced erythritol yield. M. pollinis was subjected to N-methyl N-nitro N-nitroso guanidine (NTG), ethyl methyl sulfonate (EMS), and UV mutagenesis for improved erythritol production. The fmutant strains were evaluated for enhanced erythritol production medium optimization by using different carbon substrates at the shake flask level. To enhance the production of erythritol and statistical media, optimization was carried out using a central composite design (CCD). Among 198 isolated mutants, Mutant-58 strain generated by EMS mutagenesis was selected for further assessment. The Mutant-58 strain showed significant morphological changes as compared to the parent strain. Furthermore, statistically optimized media composition resulted in the higher production of erythritol (91.2 ± 3.4 g/L) with a yield of 40.7 ± 3.4 % in shake flask experiments. The optimized medium composition for erythritol production constitutes (g/L) 225 jaggery, 4.4 yeast extract (YE), 4.4 KH2PO4, 0.31 MgSO4, and pH 5.5. The present study demonstrated strain improvement, media, and process optimization resulting in a 30% increase in the erythritol production in the Mutant-58 as compared to the parent strain. This is also the first instance where jaggery has been used as a cost-effective carbon source alternative to glucose for industrial-scale erythritol production.

One-step solid-state fermentation for efficient erythritol production from the simultaneous saccharified crop wastes by incorporating immobilized cellulase

Crop wastes are advantaged feedstocks for low-cost erythritol production via Yarrowia procedure, whereas a high amount of cellulose remains in the fermentative medium due to the lack of cellulase. The present study proposed a one-step solid-state fermentation (SSF) for erythritol production from oil crop waste that was simultaneous saccharified by the incorporated exogenous cellulase. Cellulase was immobilized in alginate-chitosan beads, and the outstanding performance of the immobilized cellulase in erythritol cultivation was proven based on its tolerance to pH and osmotic pressure, with high stability and reusability (83% of activity maintained after five cycles of utilization). Under the optimal condition of pH 6.0 and 0.01 g/g dry substrate with the immobilized cellulase addition of 1.5% (w/w), persistent cellulase activity accompanied by the efficient consumption of total sugar was detected during the cultivation. Enhanced erythritol production was achieved with the highest titer of 226.8 mg/g dry substrate in scale-up batch fermentation, when the fed-batch cultivation contributed to a considerable total amount of erythritol. Cost analysis suggests that the present process leads toward the development of eco-friendly and economically feasible erythritol production.

Enhancing erythritol production by wheat straw biochar-incorporated solid-state fermentation of agricultural wastes using defatted Schizochytrium sp. biomass as supplementary feedstock

Defatted Schizochytrium residue was used as a supplementary feedstock for efficiency erythritol production in this study. Microalgae are attractive feedstocks for the microbial process. However, proper enzymatic hydrolysis and toxicity alleviation must be considered for efficient fermentation. A strategy was proposed herein for producing erythritol from enzymatic hydrolysate of the defatted Schizochytrium residue (SRH), resorting to the wheat (Triticum aestivum L.) straw biochar-incorporated solid-state fermentation (SSF) of agricultural wastes to relieve the toxicant inhibition. Enhanced erythritol production (190.5 mg/gds) was achieved in batch SSF supplemented with 4% biochar and 20 % concentrated SRH, which was obtained from the cellulose–protease (1:1) system performed under pH 5.0 and 45 °C for 24 h with 0.8 % (m/v) of enzyme addition. Fed- and repeated-batch fermentation demonstrated enduring high cell viability and improved erythritol yield of 223.2 and 205.4 mg/gds, respectively. Cost analysis suggested high availabilities of raw substrates for erythritol cultivation, which was energy conserving and operable. Moreover, SRH was proven to be an economic alternative feedstock for low-cost erythritol production via the biochar-incorporated SSF.

Multiple gene integration to promote erythritol production on glycerol in Yarrowia lipolytica.

Erythritol (1,2,3,4-butanetetrol) is a 4-carbon sugar alcohol that occurs in nature as a metabolite or storage compound. In this study, a multiple gene integration strategy was employed to enhance erythritol production in Y. lipolytica. The effects on the production of erythritol in Y. lipolytica of seven key genes involved in the erythritol synthesis pathway were evaluated individually, among which transketolase (TKL1) and transaldolase (TAL1) showed important roles in enhancing erythritol production. The combined overexpression of four genes (GUT1, TPI1, TKL1, TAL1) and disruption of the EYD1 gene (encoding erythritol dehydrogenase), resulted in produce approximately 40g/L erythritol production from glycerol. Further enhanced erythritol synthesis was obtained by overexpressing the RKI1 gene (encoding ribose 5-phosphate isomerase) and the AMPD gene (encoding AMP deaminase), indicating for the first time that these two genes are also related to the enhancement of erythritol production in Y. lipolytica. A combined gene overexpression strategy was developed to efficiently improve the production of erythritol in Y. lipolytica, suggesting a great capacity and promising potential of this non-conventional yeast in converting glycerol into erythritol.

Metabolic engineering of Yarrowia lipolytica for thermoresistance and enhanced erythritol productivity

BackgroundFunctional sugar alcohols have been widely used in the food, medicine, and pharmaceutical industries for their unique properties. Among these, erythritol is a zero calories sweetener produced by the yeast Yarrowia lipolytica. However, in wild-type strains, erythritol is produced with low productivity and yield and only under high osmotic pressure together with other undesired polyols, such as mannitol or d-arabitol. The yeast is also able to catabolize erythritol in non-stressing conditions.ResultsHerein, Y. lipolytica has been metabolically engineered to increase erythritol production titer, yield, and productivity from glucose. This consisted of the disruption of anabolic pathways for mannitol and d-arabitol together with the erythritol catabolic pathway. Genes ZWF1 and GND encoding, respectively, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were also constitutively expressed in regenerating the NADPH2 consumed during erythritol synthesis. Finally, the gene RSP5 gene from Saccharomyces cerevisiae encoding ubiquitin ligase was overexpressed to improve cell thermoresistance. The resulting strain HCY118 is impaired in mannitol or d-arabitol production and erythritol consumption. It can grow well up to 35 °C and retain an efficient erythritol production capacity at 33 °C. The yield, production, and productivity reached 0.63 g/g, 190 g/L, and 1.97 g/L·h in 2-L flasks, and increased to 0.65 g/g, 196 g/L, and 2.51 g/L·h in 30-m3 fermentor, respectively, which has economical practical importance.ConclusionThe strategy developed herein yielded an engineered Y. lipolytica strain with enhanced thermoresistance and NADPH supply, resulting in a higher ability to produce erythritol, but not mannitol or d-arabitol from glucose. This is of interest for process development since it will reduce the cost of bioreactor cooling and erythritol purification.

Enhanced erythritol production by a Snf1-deficient Yarrowia lipolytica strain under nitrogen-enriched fermentation condition

Erythritol is a new polyol used as a bulk sweetener in food industries, and the bioconversion of agricultural or industrial wastes to erythritol by Yarrowia lipolytica at high productivity is of great cost-effectiveness. In the Yarrowia-erythritol procedure, erythritol synthesis is triggered only under nitrogen starvation condition (or a high carbon-to-nitrogen ratio, C/N), causing an inefficient product yield and the unavailability of nitrogen-rich wastes. Sucrose non-fermenting protein kinase (Snf1) plays an important role in the nitrogen starvation-triggered process. In this study, a Snf1-deficient strain Y. lipolytica M53-S was obtained, and the calculated carbon source uptake (Qs) and erythritol formation (QEry) rates were respectively increased by 46% and 49% when a lower C/N ratio of 20/1 was imposed on M53-S cells than that during nitrogen limitation (C/N=250/1). Moreover, loss of Snf1 abolished the reliance on high osmotic pressure and supplemental nutrition. Results of qRT-PCR suggested that the metabolic enzymes in M53-S were u P-R egulated at transcriptional level under low C/N ratio. The increased enzyme activitiesf also suggested a carbon overflow into the erythritol biosynthetic pathway. During the scaled-up fermentation process, 72.5g/L of erythritol was produced by M53-S within a greatly shortened period. This work confirms the critical role of Snf1 in coupling between nitrogen starvation and product synthesis, which also presents a new perspective in developing a cost-effective process for efficient production of secondary metabolites from nitrogen-rich wastes.

Oil crop wastes as substrate candidates for enhancing erythritol production by modified Yarrowia lipolytica via one-step solid state fermentation

Oil crop wastes are attractive feedstocks in microbial processes due to their low cost. However, the product yields can be limited by their undesirable nitrogen surplus. Present study proposed a one-step solid state fermentation (SSF) method for producing erythritol from unrefined oil crop wastes using a modified strain Y. lipolytica M53-S. Enhanced erythritol production (185.4 mg/gds) was obtained from peanut press cake mixed with 40% sesame meal and 10% waste cooking oil. The process was performed at pH 4.0 in 5 L flasks, with initial moisture content, NaCl addition, and inoculum size of 70%, 0.02 g/gds, and 7.5 × 104 cells/gds, respectively. This procedure showed advantages in terms of lower material cost than that of submerged fermentation and shorter culture cycle (96 h) than other SSF processes. In repeated-batch fermentation, erythritol was continuously produced for seven cycles. This study presents a feasible approach in developing an efficient erythritol cultivation from nitrogen-rich wastes.

Enhancing the erythritol production by Yarrowia lipolytica from waste oil using loofah sponge as oil-in-water dispersant

Waste oil is an economical alternative carbon resource in Yarrowia-based erythritol production, but the water insolubility limits its further utilization. Loofah sponge (LS), a natural polymer material and accessible agricultural waste, was selected as oil-in-water dispersant in this study. In the oil–erythritol cultivation system with LS addition, efficient oil dispersion and enhanced erythritol production (by ˜30%) were achieved, with high biomass and scarce residual oil in the medium. Up to 60.0 g/L of waste oil could be completely consumed under the effect of LS, resulting to an erythritol yield of 0.76 ± 0.03 g/g oil. Scanning electron microscopy (SEM) images and reusability experiments showed that the LS fiber was well preserved and could be stable for more than five continuous cycles in this process. The changes in activities of the metabolic enzymes suggested an enhanced carbon flux in erythritol synthesis pathway, which was accompanied with an improved oxygen consumption. Moreover, with LS addition, scale-up and fed-batch cultivations was well conducted, up to 114.3 ± 1.8 g/L of erythritol was produced from waste oil when the two cultivation strategies were combined. This study demonstrated an efficient and eco-friendly method for the utilization of waste oily substrates in production of microbial metabolites.

Enhancement of erythritol production in Trichosporonoides oedocephalis by regulating cellular morphology with betaine

Erythritol is a four-carbon sugar alcohol produced by microorganisms and is used as a natural sweetener in food industry. The present study aimed to examine the impact of various surfactants, including betaine, Tween 20, Tween 80, Span 20, and Triton X-100, on the production of erythritol by Trichosporonoides oedocephalis ATCC 16958. Finally, after 120 h culture via agitation, the supplementation with 0.5 g/L betaine increased the erythritol production by 50.38%, which was best among the five surfactants. Therefore, betaine was chosen for further research. Subsequently, the activity of erythrose reductase (ER) with the addition of 0.5 g/L betaine (0.211 U/mg protein) was slightly higher than that of the culture without betaine (0.192 U/mg protein) at 48 h of culture. However, no significant changes were observed in the ER activity with betaine addition during the fermentation stages. Interestingly, further observation with scanning electron microscope indicated that the disruption of cell was found in the culture without betaine, while that of the culture with 0.5 g/L betaine addition remained intact at 120 h post-cultivation. The enhanced erythritol production with betaine addition affected the cellular morphology that could be regulated effectively by zwitterionic surfactant betaine. Finally, the scale-up fermentation in 5-L bioreactor, erythritol production with initial 0.5 g/L betaine and 30 mg/L CuSO4·5H2O addition was up to 59.34 g/L, which was 19.59% higher than the control with only 30 mg/L CuSO4·5H2O supplementation (49.62 g/L). Thus, the present study demonstrated the mechanisms underlying the betaine-affected erythritol production and provided a feasible approach for improving the erythritol production by T. oedocephalis.

An Effective Method of Continuous Production of Erythritol from Glycerol by Yarrowia lipolytica MK1.

This study demonstrates the potential applicability of the UV mutant Yarrowia lipolytica MK1 for the valorisation of glycerol and erythritol production in a chemostat culture. The aim of this research is to investigate the optimal C:N ratio in the feeding medium in order to enhance erythritol production. The highest erythritol concentration, at 113.1 g/L with a volumetric erythritol production rate of 1.1 g/(L·h) and a yield of 0.57 g/g, was obtained in the feeding medium with a C:N ratio of 80:1. Moreover, no residual glycerol was observed in the culture broth during cultivation. The chemical composition of the biomass was analysed. The contents of lysine and threonine in the biomass protein amino acid profile were higher than those required by the FAO/WHO for fodder yeast.

Newly isolated mutant of Yarrowia lipolytica MK1 as a proper host for efficient erythritol biosynthesis from glycerol

Mutants of Yarrowia lipolytica Wratislavia K1 were generated by ultraviolet mutagenesis to enhance erythritol production and to minimize by-products formation. One of the mutants named as MK1 was selected for further investigation. In batch culture strain Y. lipolytica MK1 was able to produce up to 82.2gl−1 erythritol with 0.55gg−1 yield and productivity of 0.84gl−1h−1. The concentration of by-products was below 5% of all synthesized metabolites. In order to enhance the production of erythritol and to inhibit the production of by-products the repeated batch culture (RBC) was performed. In RBC strain Y. lipolytica MK1 gave maximum erythritol production 224gl−1 with 0.77gg−1 yield and productivity of 0.54gl−1h−1. The concentration of by-products was below 2.3% of all synthesized metabolites. Moreover, the lipid and fatty acid content in the biomass was determined. This is the first study on newly isolated Y. lipolytica mutant. It demonstrates that Y. lipolytica MK1 is able to produce significantly high erythritol with strong inhibition of by-products formation.

Erythritol production with minimum By-product using Candida magnoliae mutant

In order to enhance erythritol production, mutants of Candida magnoliae DSM70638 were generated by ultraviolet and chemical mutagenesis. Erythritol productivity of samples was analyzed by TLC and HPLC with the refractive index detector. One of the mutants named mutant 12-2 gave a 2.4-fold increase in erythritol (20.32 g/L) and a 5.5-fold decrease in glycerol production compared to the wild strain. A sequence-based map of erythrose reductase gene in this mutant showed a replacement of the A321 by G321 that did not cause any amino acid exchange in protein structure. Therefore, the reason of higher erythritol production in C. magnoliae mutant 12-2 is probably the increase in expression of the open reading frame gene. This study revealed that a mutation or minor change in the sequence of genes involved in a production pathway can lead to a significant increase in protein translation.

Improvement of Erythrose Reductase Activity, Deletion of By-products and Statistical Media Optimization for Enhanced Erythritol Production from Yarrowia lipolytica Mutant 49

The purpose of the present investigation was to produce erythritol by Yarrowia lipolytica mutant without any by-products. Mutants of Y. lipolytica were generated by ultra-violet for enhancing erythrose reductase (ER) activity and erythritol production. The mutants showing the highest ER activity were screened by triphenyl tetrazolium chloride agar plate assay. Productivity of samples was analyzed by thin-layer chromatography and high-performance liquid chromatography equipped with the refractive index detector. One of the mutants named as mutant 49 gave maximum erythritol production without any other by-products (particularly glycerol). Erythritol production and specific ER activity in mutant 49 increased to 1.65 and 1.47 times, respectively, in comparison with wild-type strain. The ER gene of wild and mutant strains was sequenced and analyzed. A general comparison of wild and mutant gene sequences showed the replacement of Asp(270) with Glu(270) in ER protein. In order to enhance erythritol production, we used a three component-three level-one response Box-Behnken of response surface methodology model. The optimum medium composition for erythritol production was found to be (g/l) glucose 279.49, ammonium sulfate 9.28, and pH 5.41 with 39.76 erythritol production.

A novel osmotic pressure control fed-batch fermentation strategy for improvement of erythritol production by Yarrowia lipolytica from glycerol

The effect of osmotic pressure on erythritol and mannitol production by an osmophilic yeast strain of Yarrowia lipolytica CICC 1675 using glycerol as the sole carbon source was investigated. Appropriately high osmotic pressure was found to enhance erythritol production and inhibit mannitol formation. A novel two-stage osmotic pressure control fed-batch strategy based on the kinetic analysis was developed for higher erythritol yield and productivity. During the first 96 h, the osmotic pressure was maintained at 4.25 osmol/kg by feeding glycerol to reduce the inhibition of cell growth. After 132 h, the osmotic pressure was controlled at 4.94 osmol/kg to maintain a high dp(ery)/dt. Maximum erythritol yield of 194.3g/L was obtained with 0.95 g/L/h productivity, which were 25.7% and 2.2%, respectively, improvement over the best results in one-stage fed-batch fermentation. This is the first report that a novel osmotic pressure control fed-batch strategy significantly enhanced erythritol production.

Strain improvement and statistical media optimization for enhanced erythritol production with minimal by-products from Candida magnoliae mutant R23

Mutants of Candida magnoliae NCIM 3470 were generated by ultra-violet and chemical mutagenesis to enhance erythritol production. The mutants were screened for higher reductase activity on agar plates containing high concentration of glucose and 2,3,5-triphenyl tetrazolium chloride (TTC). One of the mutants named as R23 gave maximum erythritol production, 60.3 g L −1, compared to 14 g L −1 of the parent strain. Glucose and yeast extract were identified as critical medium components which decide the ratio of polyols produced, mainly erythritol, mannitol and glycerol. In order to enhance the production of erythritol and to minimize the production of mannitol and glycerol, a four component-five level-three response central-composite-rotatable-design (CCRD) of response surface methodology (RSM) model was used. The optimum medium composition for erythritol production was found to contain (g L −1) glucose 238, yeast extract 9.2, KH 2PO 4, 5.16 and MgSO 4 0.23. Moreover, erythritol production was studied in a 10 L fermentor in batch and fed-batch mode using RSM optimized medium. In fed-batch fermentation, 87.8 g L −1 erythritol was produced with 31.1% yield, without formation of any other polyols. Thus present study involving strain improvement followed by media and process optimization resulted in 6.2-fold increase in erythritol production and 3.4-fold increase in the yield.