Erythritol Production Research Articles

Volemitol production from Yarrowia lipolytica via transaldolase gene (TAL) disruption and erythrose-4-phosphate (E4P) flux regulation

Volemitol (D-glycero-D-manno-Heptitol, C7H16O7 with CAS No.488–38–0), a seven-carbon (C7) sugar-derived alcohol, has the potential to be used as a natural sweetener. The natural scarcity of sugar alcohols restricts their practical uses. However, Yarrowia lipolytica has shown significant promise in industrial production due to its capability to efficiently produce sugar alcohols like erythritol, D-threitol, mannitol, and xylitol by generating key biosynthetic intermediates through glycolysis and the phosphopentose (PPP) pathway. In this study, the transaldolase gene (TAL) in the erythritol synthesis pathway was knocked out in the erythritol-producing Y. lipolytica strain CGMCC7326 to inhibit erythritol production. TAL-deleted strain Y. lipolytica CGMCC7326ΔTAL exhibited a notable decline in erythritol production; however, a novel substance, volemitol, was generated from glucose at a titer of 50 g/L. Volemitol with 99 % purity was obtained as a white microneedle powder crystal through the enzymatic activity of mannitol dehydrogenase (MDH2), which reduces sedoheptulose to volemitol. The proposed biosynthetic pathway in Y. lipolytica CGMCC7326ΔTAL is: sedoheptulose-7-phosphate was converted to sedoheptulose, then was reduced to volemitol. In conclusion, this study is the first to report efficient volemitol production from glucose via fermentation by engineered Y. lipolytica.

Protein rational design and modification of erythrose reductase for the improvement of erythritol production in Yarrowia lipolytica.

Erythritol is a natural non-caloric sweetener, which is produced by fermentation and extensively applied in food, medicine and chemical industries. The final step of the erythritol synthesis pathway is involved in erythritol reductase, whose activity and NADPH-dependent become the limiting node of erythritol production efficiency. Herein, we implemented a strategy combining molecular docking and thermal stability screening to construct an ER mutant library. And we successfully obtained a double mutant ERK26N/V295M (ER*) whose catalytic activity was 1.48 times that of wild-type ER. Through structural analysis and MD analysis, we found that the catalytic pocket and the enzyme stability of ER* were both improved. We overexpressed ER* in the engineered strain ΔKU70 to obtain the strain YLE-1. YLE-1 can produce 39.47 g/L of erythritol within 144 h, representing a 35% increase compared to the unmodified strain, and a 10% increase compared to the strain overexpressing wild-type ER. Considering the essentiality of NADPH supply, we further co-expressed ER* with two genes from the oxidative phase of PPP, ZWF1 and GND1. This resulted in the construction of YLE-3, which exhibited a significant increase in production, producing 47.85 g/L of erythritol within 144 h, representing a 63.90% increase compared to the original chassis strain. The productivity and the yield of the engineered strain YLE-3 were 0.33 g/L/h and 0.48 g/g glycerol, respectively. This work provided an ER mutation with excellent performance, and also proved the importance of cofactors in the process of erythritol synthesis, which will promote the industrial production of erythritol by metabolic engineering of Y. lipolytica.

Economic validation and comparison of microbial tryptophan, erythritol and collagen production in an integrated sugarcane biorefinery

Low sugar prices present a significant challenge to the global sugarcane industry, prompting the exploration of diversification strategies for expanding product portfolios. Techno-economic analyses and environmental sustainability assessments were carried out to evaluate the microbial production of tryptophan, erythritol, and collagen from A-molasses in a biorefinery annexed to an existing sugarcane mill. Tryptophan production exhibited the highest profitability, with a minimum selling price (MSP) at 59.7 % of its current market price, although the achievable production volumes of tryptophan from one sugar mill would oversupply the global market. Due to the larger market size of for collagen the achievable production capacity in the collagen scenario would avoid market saturation, reducing the risk of oversupply and rendering it more economically viable. In contrast, erythritol production was marginally not profitable, with an MSP exceeding the current market price by 1 %, primarily attributed to high operational costs. All scenarios demonstrated relatively low greenhouse gas (GHG) emissions (ranging from 9.1 to 16.5 kg CO2eq/kg product), with tryptophan production emerging as the most environmentally favourable option due to minimal chemical and freshwater usage. When compared with literature-reported data on ethanol and short-chain fructooligosaccharides (scFOS), only collagen and ethanol production were deemed viable, based on their favourable profitability and contribution to the market.

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.

Effect of low frequency alternating magnetic field for erythritol production in Yarrowia lipolytica.

Numerous works have reported that magnetic fields serve as signals capable of influencing microbial metabolism. However, little is known about the effect of magnetic field on erythritol production by the model microorganism Yarrowia lipolytica (Y. lipolytica). Therefore, we investigated the effect of low-frequency alternating magnetic fields (LF-AMF) with different magnetic field intensities (0-1.5mT) and different magnetic field treatment times (1-10 days) on the production of erythritol by Y. lipolytica -JZ204. The optimal treatment condition was 0.5mT for 8 days. As a result, a maximal erythritol yield was achieved 63.74g/L, the biomass was reached 37g/L, and the specific erythritol yield per unit of biomass was 1.7227g/g, which were 60.72%, 32.09%, and 24.85% higher than the control, respectively. We investigated the internal mechanism of magnetic fields impact by using transcriptomics and RT-qPCR technology. This study demonstrated the effectiveness of LF-AMF in enhancing erythritol production by Y. lipolytica JZ-204, providing insights for the application of magnetic field in assisting microbial fermentation and improving the synthesis of beneficial products.

Comparative transcriptomics analysis-guided metabolic engineering of Yarrowia lipolytica for improved erythritol and fructooligosaccharides production

Currently, fructooligosaccharides (FOS) are converted from sucrose by purified enzymes or fungal cells, but these methods are costly and time-consuming. Here, the optimal fermentation conditions for strain E326 were determined through fermentation optimization: initial glucose 200 g/L, NaCl 25 g/L, inoculum volume 20 %, dissolved oxygen 20–30 %, pH 3, and glucose feeding concentration 100 g/L, which increased erythritol titer by 1.5 times. The co-expression of HGT1 and APC11 genes alleviated the erythritol synthesis stagnation, shorten the fermentation time by 16.7 %, and increased the erythritol productivity by 17.2 %. The episomal plasmids based on yeast mitochondrial replication origins (mtORIs) were constructed to surface display fructosyltransferase, effectively utilizing waste yeast cells generated during erythritol fermentation. Under the conditions of 60℃ and pH 6, the FOS yield reached 65 %, which to our best of knowledge is so-far the highest yield of FOS obtained. These findings will contribute to the industrial production of erythritol and FOS.

Exploration of Trichoderma reesei as an alternative host for erythritol production

BackgroundErythritol, a natural polyol, is a low-calorie sweetener synthesized by a number of microorganisms, such as Moniliella pollinis. Yet, a widespread use of erythritol is limited by high production costs due to the need for cultivation on glucose-rich substrates. This study explores the potential of using Trichoderma reesei as an alternative host for erythritol production, as this saprotrophic fungus can be cultivated on lignocellulosic biomass residues. The objective of this study was to evaluate whether such an alternative host would lead to a more sustainable and economically viable production of erythritol by identifying suitable carbon sources for erythritol biosynthesis, the main parameters influencing erythritol biosynthesis and evaluating the feasibility of scaling up the defined process.ResultsOur investigation revealed that T. reesei can synthesize erythritol from glucose but not from other carbon sources like xylose and lactose. T. reesei is able to consume erythritol, but it does not in the presence of glucose. Among nitrogen sources, urea and yeast extract were more effective than ammonium and nitrate. A significant impact on erythritol synthesis was observed with variations in pH and temperature. Despite successful shake flask experiments, the transition to bioreactors faced challenges, indicating a need for further scale-up optimization.ConclusionsWhile T. reesei shows potential for erythritol production, reaching a maximum concentration of 1 g/L over an extended period, its productivity could be improved by optimizing the parameters that affect erythritol production. In any case, this research contributes valuable insights into the polyol metabolism of T. reesei, offering potential implications for future research on glycerol or mannitol production. Moreover, it suggests a potential metabolic association between erythritol production and glycolysis over the pentose phosphate pathway.

Multidimensional combinatorial screening for high-level production of erythritol in Yarrowia lipolytica

Yarrowia lipolytica was successfully engineered to synthesize erythritol from crude glycerol, a cheap by-product of biodiesel production, but the yield remained low. Here, a biosensor-guided adaptive evolution screening platform was constructed to obtain mutant strains which could efficiently utilize crude glycerol to produce erythritol. Erythrose reductase D46A (M1) was identified as a key mutant through whole-genome sequencing of the strain G12, which exhibited higher catalytic activity (1.6-fold of the wild-type). M1 was further modified to obtain a combinatorial mutant with 4.1-fold enhancement of catalytic activity. Finally, the metabolic network was reconfigured to redirect carbon fluxes toward erythritol synthesis. The erythritol titer of the engineered strain G31 reached 220.5 g/L with a productivity of 1.8 g/L/h in a 5-L bioreactor. The study provides valuable guidance for biosensor-based ultra-high-throughput screening strategies in Y. lipolytica, as well as presenting a new paradigm for the sustainable valorization of crude glycerol.

Enhanced Production of Erythritol from Glucose by the Newly Obtained UV Mutant Yarrowia lipolytica K1UV15.

Erythritol is a polyol with a sweet taste but low energy value. Thanks to its valuable properties, as well as growing social awareness and nutritional trends, its popularity is growing rapidly. The aim of this study was to increase the effectiveness of erythritol production from glucose using new UV mutants of the yeast Yarrowia lipolytica obtained in the Wratislavia K1 strain. The ability of the new strains to biosynthesize erythritol and utilize this polyol was examined in shake-flask cultures and fed-batch processes conducted in a stirred tank reactor with a total glucose concentration of 300 and 400 g/L. The Wratislavia K1 strain produced erythritol most efficiently (97.5 g/L; 192 h) at an initial glucose concentration of 250 g/L (total: 300 g/L). New strains were assessed under such conditions, and it was noted that the highest erythritol concentration (145 g/L; 183 h) was produced by the K1UV15 strain. A significant improvement in the erythritol biosynthesis efficiency (148 g/L; 150 h) was achieved upon the increase in (NH4)2SO4 to 3.6 g/L. Further, in the culture with such a concentration of the nitrogen source and increased total glucose level (400 g/L), the K1UV15 strain produced 226 g/L of erythritol within 281 h.

Adaptive responses of erythritol-producing Yarrowia lipolytica to thermal stress after evolution

Elucidation of the thermotolerance mechanism of erythritol-producing Yarrowia lipolytica is of great significance to breed robust industrial strains and reduce cost. This study aimed to breed thermotolerant Y. lipolytica and investigate the mechanism underlying the thermotolerant phenotype. Yarrowia lipolytica HT34, Yarrowia lipolytica HT36, and Yarrowia lipolytica HT385 that were capable of growing at 34 °C, 36 °C, and 38.5 °C, respectively, were obtained within 150 days (352 generations) by adaptive laboratory evolution (ALE) integrated with 60Co-γ radiation and ultraviolet ray radiation. Comparative genomics analysis showed that genes involved in signal transduction, transcription, and translation regulation were mutated during adaptive evolution. Further, we demonstrated that thermal stress increased the expression of genes related to DNA replication and repair, ceramide and steroid synthesis, and the degradation of branched amino acid (BCAA) and free fatty acid (FFA), while inhibiting the expression of genes involved in glycolysis and the citrate cycle. Erythritol production in thermotolerant strains was remarkably inhibited, which might result from the differential expression of genes involved in erythritol metabolism. Exogenous addition of BCAA and soybean oil promoted the growth of HT385, highlighting the importance of BCAA and FFA in thermal stress response. Additionally, overexpression of 11 out of the 18 upregulated genes individually enabled Yarrowia lipolytica CA20 to grow at 34 °C, of which genes A000121, A003183, and A005690 had a better effect. Collectively, this study provides novel insights into the adaptation mechanism of Y. lipolytica to thermal stress, which will be conducive to the construction of thermotolerant erythritol-producing strains.Key points• ALE combined with mutagenesis is efficient for breeding thermotolerant Y. lipolytica• Genes encoding global regulators are mutated during thermal adaptive evolution• Ceramide and BCAA are critical molecules for cells to tolerate thermal stress

Metabolic engineering of erythritol production from glycerol by Yarrowia lipolytica

Metabolic engineering of erythritol production from glycerol by Yarrowia lipolytica

Application of New Yarrowia lipolytica Transformants in Production of Citrates and Erythritol from Glycerol.

Citric acid and erythritol are obtained on an industrial scale using biotechnological methods. Due to the growing market demand for these products, research is underway to improve the process economics by introducing new microorganisms, in particular of the species Yarrowia lipolytica. The aim of this study was to evaluate transformants of Y. lipolytica for growth and ability to overproduce citric acids and erythritol from glycerol. The transformants were constructed by overexpressing glycerol kinase, methylcitrate synthase and mitochondrial succinate-fumarate transporter in the mutant Wratislavia 1.31. Next, strains were assessed for biosynthesis of citrate (pH 5.5; nitrogen limitation) and erythritol (pH 3.0; high osmotic pressure) from glycerol. Regardless of culture conditions strains, 1.31.GUT1/6 and 1.31.GUT1/6.CIT1/3 exhibited high rates of substrate utilization. Under conditions favoring citrate biosynthesis, both strains produced several percent more citrates, accompanied by higher erythritol production compared to the parental strain. During erythritol biosynthesis, the strain 1.31.GUT1/6.CIT1/3.E34672g obtained as a result of co-expression of all three genes stood out, producing 84.0 g/L of erythritol with yield and productivity of 0.54 g/g and 0.72 g/Lh, respectively, which places it in the group of the highest-ranked producers of erythritol among Y. lipolytica species.

The Study and Challenge of Producing Sugar Substitutes via Microorganism Pathway

Nowadays, the ratio of obesity increases continually because of excessive sugar intake with the improvement of living standards, especially for children and teenagers. The obesity, caused by excessive sugar intake, is a main risk to induce cardiovascular disease both in adults and children because of abnormal glucose tolerance, such as hypertension, atherosclerosis and coronary heart disease. Consequently, it’s important to find an effective method to limiting sugar intake, which are welcomed by sweet person, reducing onset of higher sugar intake associated diseases, contains optimistic business opportunities. Therefore, the research and development of sugar alternative products grew rapidly and soon occupied an important position in the market. In comparison with artificial sweeteners, erythritol, as a natural sugar substitutes (NSS), is widely used in good industry to add sweet tastes. But producing erythritol faces problems. One of the main problems is using erythritol is far more expensive by using traditional sugar, like sucrose and fructose. In order to solve this problem, scientists suggest to replace glucose by glycerol. At the same time, change the environment of bacterium or fungi greatly influence the efficiency. In this review, the author aims to summarize the production of erythritol, compare its advantages and limitations, and provide some references for its development as an important sugar substitute in future.

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.

Mechanism and evolutionary analysis of Yarrowia lipolytica CA20 capable of producing erythritol with a high yield based on comparative genomics.

Combined mutagenesis is widely applied for the breeding of robust Yarrowia lipolytica used in the production of erythritol. However, the changes of genome after mutagenesis remains unclear. This study aimed to unravel the mechanism involved in the improved erythritol synthesis of CA20 and the evolutionary relationship between different Y. lipolytica by comparative genomics analysis. The results showed that the genome size of Y. lipolytica CA20 was 20,420,510 bp, with a GC content of 48.97%. There were 6330 CDS and 649 ncRNA (non-coding RNA) in CA20 genome. Average nucleotide identity (ANI) analysis showed that CA20 genome possessed high similarity (ANI > 99.50%) with other Y. lipolytica strains, while phylogenetic analysis displayed that CA20 was classified together with Y. lipolytica IBT 446 and Y. lipolytica H222. CA20 shared 5342 core orthologous genes with the 8 strains while harbored 65 specific genes that mainly participated in the substrate and protein transport processes. CA20 contained 166 genes coding for carbohydrate-active enzymes (CAZymes), which was more than that found in other strains (108－137). Notably, 4, 2, and 13 different enzymes belonging to glycoside hydrolases (GHs), glycosyltransferases (GTs), and carbohydrate esterases (CEs), respectively, were only found in CA20. The enzymes involved in the metabolic pathway of erythritol were highly conserved in Y. lipolytica, except for transaldolase (TAL1). In addition, the titer and productivity of erythritol by CA20 were 190.97 g/L and 1.33 g/L/h, respectively, which were significantly higher than that of WT5 wherein 128.61 g/L and 0.92 g/L/h were obtained (P< 0.001). Five frameshift mutation genes and 15 genes harboring nonsynonymous mutation were found in CA20 compared with that of WT5. Most of these genes were involved in the cell division, cell wall synthesis, protein synthesis, and protein homeostasis maintenance. These findings suggested that the genome of Y. lipolytica is conserved during evolution, and the variance of living environment is one important factor leading to genome divergence. The varied number of CAZymes existed in Y. lipolytica is one factor that contributes to the performance difference. The increased synthesis of erythritol by Y. lipolytica CA20 is correlated with the improvement of the stability of cell structure and internal environment. The results of this study provide a basis for the directional breeding of robust strains used in erythritol production.

Microbial ecology and metabolite dynamics of backslopped triticale sourdough productions and the impact of scale

Triticale (X Triticosecale Wittmack) is a hybrid of wheat (Triticum aestivum L.) and rye (Secale cereale L.), combining the positive attributes of both cereals. However, it has not been exploited for sourdough production yet. Further, the effect of scale on sourdough production has not been investigated systematically up to now. The aims of the present study were to assess the microbial ecology and metabolomic output of eleven spontaneously fermented, backslopped sourdough productions made with triticale flour on a scale of 100, 200, 500, and 1000 g. The acidification profile [pH and total titratable acidity (TTA)], microbial diversity (culture-dependent and culture-independent), metabolite dynamics, and appropriate correlations were determined. After ten fermentation steps, different species of Lactobacillaceae were prevalent in the mature sourdoughs, in particular Latilactobacillus curvatus, Limosilactobacillus fermentum, and Pediococcus pentosaceus. The microbial diversity could be traced back to the grains and was also present in the milling fractions (flour, bran, and shorts). Furthermore, thanks to the use of Illumina-based high-throughput sequencing and an amplicon sequence variant (ASV) approach, the presence of undesirable bacterial groups (bacilli, clostridia, and enterobacteria) during the initial steps of the backslopping cycle was revealed, as well as a finetuned taxonomic diversity of the LAB genera involved. Small sourdough productions (100 and 200 g) selected for a lower species diversity and reached a stable consortium faster than large ones (500 and 1000 g). Although a comparable final pH of 3.6–4.0 was obtained, the TTA of small sourdoughs was lower than that of large ones. Regarding the metabolic output, the simultaneous production of mannitol and erythritol, beyond ethanol and glycerol, could be linked to sourdoughs in which Liml. fermentum was the sole LAB species present. Further, the use of the arginine deiminase pathway by P. pentosaceus and Liml. fermentum was obvious. An appropriate extraction method followed by liquid injection gas chromatography coupled to triple quadrupole tandem mass spectrometry allowed the quantification of interesting volatile organic compounds, such as ethyl lactate. These findings support the inclusion of triticale as a viable alternative to wheat or rye for the production of sourdoughs that can be integrated into bread-making production schemes.

Construction of a xylose metabolic pathway in Trichosporonoides oedocephalis ATCC 16958 for the production of erythritol and xylitol.

Erythritol is a valuable compound as sweetener and chemical material however cannot be fermented from the abundant substrate xylose. The strain Trichosporonoides oedocephalis ATCC 16958 was employed to produce polyols including xylitol and erythritol by metabolic engineering approaches. The introduction of a substrate-specific ribose-5-phosphate isomerase endowed T. oedocephalis with xylose-assimilation activity to produce xylitol, and eliminated glycerol production simultaneously. A more value-added product, erythritol was produced by further introducing a homologous xylulose kinase. The carbon flux was redirected from xylitol to erythritol by adding high osmotic pressure. The production of erythritol was improved to 46.5g/L in flasks by fermentation adjustment, and the process was scaled up in a 5-L fermentor, with a 40g/L erythritol production after 120h, and a time-space yield of 0.56g/L/h. This study demonstrated the potential of T. oedocephalis in the synthesis of multiple useful products from xylose.

Investigations for a Yarrowia-Based Biorefinery: In Vitro Proof-of-Concept for Manufacturing Sweetener, Cosmetic Ingredient, and Bioemulsifier

Yarrowia lipolytica is a widely used microorganism in biotechnology since it is capable of producing a wide range of products (lipase, citric acid, polyols). A less-studied related strain is Y. divulgata, which is also capable of erythritol production in even higher concentration than most Y. lipolytica wild strains from glycerol as renewable feedstock. Thus, the aim of this work was to investigate Y. divulgata’s complex utilisation based on erythritol fermentation from glycerol to establish a Yarrowia-based biorefinery in which both the fermentation broth and separated cells are converted into high added-value products (erythritol, bioemulsifier, cosmetic ingredient, i.e., skin moisturizer). An important parameter of erythritol fermentation is an adequate oxygen level, so both the constant oxygen level and oxygen absorption rate were investigated regarding the three target products. DO (dissolved oxygen) = 10, 20, 30, 40% was examined in the bioreactor, and a KLa range of 18–655 h−1 was investigated in both the bioreactor and in different types of shaking flasks, applying two different glycerol levels (100–150 g/L). The results showed that the Yarrowia divulagata NCAIM 1485 strain could produce one of the highest amounts of erythritol (44.14 ± 1 g/L) among wild-type yeasts from 150 g/L glycerol beside a KLa value of 655 h−1. Cell-lysates skin hydrating activity was the highest (12%) when DO = 20% (KLa 26.4 h−1) was applied. In all cases, the collected samples had an emulsification index above 69% which did not decrease below 54% after 24 h, showing good stability. Since Y. divulgata fermentations resulted in three high added-value products at the same time from a renewable raw material (glycerol), we concluded that it is suitable for complex utilisation in a microbial biorefinery, since the fermentation broth can be used for the isolation of a sweetener and bioemulsifier; meanwhile, the separated cells can be processed for cosmetic application as a skin moisturizer.

Utilization of Diluted Distillery Spent Wash with Different Feedstocks for Production of Erythritol by Moniliella pollinis

Utilization of Diluted Distillery Spent Wash with Different Feedstocks for Production of Erythritol by Moniliella pollinis

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.