What Are the Source of Erythritol Powder?

In recent years, with the accelerated pace of life and changes in lifestyle, people's eating habits have undergone tremendous changes. The accompanying health problems such as obesity, diabetes, and cardiovascular disease have caused serious problems and inconvenience in people's lives [1]. Excessive intake of sugar in the diet is a major factor contributing to this phenomenon, and excessive consumption of sugar can also lead to an increase in the incidence of oral diseases such as tooth decay [2]. The discovery and use of sweeteners has helped reduce the addition of high-calorie sugar in foods, thereby reducing sugar intake. Sweeteners are a class of compounds that provide sweetness but have low calories. They can be divided into natural sweeteners and synthetic sweeteners according to their sources [1]. Early synthetic sweeteners such as stevia, abas sweet and saccharin, although highly sweet, have been found to cause intestinal flora disorders and are not beneficial to health when consumed frequently [3]. Compared to synthetic sweeteners, the use of natural sweeteners, such as mannitol, erythritol, xylitol and sorbitol, is more acceptable to people. These sugar alcohols have low metabolic energy, hypoglycemic and safe properties [4].

Erythritol, chemically (2R, 3S) butane 1,2,3,4-tetrol, is a white, odorless, non-hygroscopic, optically inactive, thermally stable, and water-soluble four-carbon alcohol that is widely found in fruits, vegetables, and fermented foods [5]. Erythritol has attracted particular attention due to its properties, which include being largely unused by the body and intestinal microorganisms, not changing blood glucose concentrations or insulin levels, and not causing diarrhea [6]. Since its first discovery in 1848, erythritol has been approved for direct use as a food ingredient and sweetener in Japan, the United States, and some European countries in the 1990s. In China, an official announcement was made in 2008 allowing erythritol to be used in food in moderation as needed [ 7-8]. In 2019, the global market volume of erythritol was 70,400 tons, and by 2026, the market demand for erythritol is expected to increase by 1.5 times [9]. The rising market demand for erythritol has placed new requirements on the production of erythritol.

Erythritol can be synthesized by chemical and microbial fermentation methods. However, chemical synthesis has disadvantages such as low production efficiency, high cost and operational hazards, and therefore has not been industrialized [1]. The production of erythritol by microbial fermentation solves the adverse effects of chemical synthesis. Over the years, researchers have done a lot of work on the fermentation production process of erythritol. The results show that the composition of the fermentation medium (including carbon source, nitrogen source, inorganic salts, etc.), fermentation conditions (including temperature, pH, dissolved oxygen, etc.) and fermentation methods (including continuous fermentation, batch fermentation and batch feeding fermentation, etc.) have a significant impact on the yield and production of erythritol. The relevant content [1, 9] has been well described recently, so I will not repeat it here.

The production of erythritol by microbial fermentation has low yields and conversion rates compared to the fermentation production of other sugar alcohols. The mutagenesis and metabolic pathway modification of microbial strains provides a new direction for improving erythritol production. Based on this, the author summarizes the latest research on the microbial fermentation synthesis of erythritol, including the main strains and erythritol metabolic pathways, the synthesis of erythritol from renewable resources, and the metabolic engineering of the strain. The potential routes to increase erythritol production are discussed, with a view to providing new research ideas for the future breeding and construction of high-yield erythritol strains.

1 Main microorganisms for the fermentation production of erythritol and their breeding

1. 1 Bacteria

Bacteria can use glucose as a substrate to synthesize erythritol through the action of glucose kinase, glucose-6-phosphate isomerase, phosphoketolase, erythritol-4-phosphate dehydrogenase and phosphatase (Fig. 1). 4-phosphate dehydrogenase (erythritol-4-phosphate dehydrogenase) and phosphatase (phosphatase) to synthesize erythritol (Figure 1) [1, 10]. At present there are few known bacteria that can directly synthesize erythritol, mainly Lactobacillus and Oenococcus (Table 1). Tyler et al. [11] found that Lactobacillus florum 2F can synthesize 2.04 g/L erythritol using glucose as a substrate, while the final concentration of erythritol is lower when fructose is used as a substrate. final concentration was lower; at the same time, the study analyzed the production of erythritol by 22 other strains of bacteria, including Lactobacillus and Oenococcus, and found that strains including genera such as Leuconostoc, Oenococcus and Weissella were able to synthesize 0.02 to 0.45 g/L of erythritol (Table 1). n der Woude et al. [12] used Synechocystis sp. PCC6803 as the starting strain, and overexpressed erythritol 4-phosphate phosphatase and erythritol reductase in the bacteria through genetic engineering to obtain a dominant strain SEP024, which can synthesize 0. 256 g / L erythritol. Overall is low, and the industrial production of erythritol cannot be achieved.

1. 2. Fungi

Compared with bacteria, fungi can metabolize and synthesize erythritol through the pentose phosphate pathway using glucose, glycerol, fructose and other substrates. The main strain used in the industrial production of erythritol is yeast [9]. In yeast glucose, glycerol and fructose as substrates to synthesize erythritol. Among the yeasts used in industrial production of erythritol, the main species is Saccharomyces cerevisiae[9]. In yeast, when glucose is used as the carbon source, glucose is first converted to glucose-6-phosphate in the cell by glucose kinase, glucose-6-phosphate dehydrogenase, 6-phosphogluconolactonase, phosphogluconate dehydrogenase, ribulose phosphate 3-epimerase and ribulose-5-phosphate isomerase catalyze the formation of ribulose-5-phosphate and ribose-5-phosphate, which are then converted by transketolase, transaldolase, erythrose-4-phosphate kinase and erythrose-4-phosphate reductase ( transketolase), transaldolase, erythrose-4-phosphate kinase and erythrosereductase (Figure 2) [13-14].

When glycerol as the substrate, glycerol can be converted to fructose 1,6-diphosphate by glycerol kinase, glycerol-3-phosphate dehydrogenase, triose phosphate isomerase isomerase and aldolase, and then enters the pentose phosphate pathway via glucose 6 phosphate. Recently , Niang et al. [15] found that erythritol can be further degraded to erythrose and erythrose phosphate and utilized by cells. During this process, erythritol dehydrogenase  erythrulose dehydrogenase, erythrulose kinase and erythrulose phosphate isomerase play a key role in this process.

At present yeasts with high erythritol production have been reported [16-25], including species such as Candida, Yarrowia, and Torula, which can synthesize 25-245 g/L erythritol (Table 2). Among them , Yarrowia lipolytica is the most widely used strain in the industrial production of erythritol. Yarrowia lipolytica is a type of unconventional oleaginous yeast and is also considered a safe microorganism (GRAS), so it has important application value in industrial production.

In addition in addition, Yuill et al. [26] first reported in 1948 that Aspergillus niger can metabolize and synthesize erythritol, but other molds, including Aspergillus, have not been used in the industrial production of erythritol.

1. 3. Strain selection

Strains isolated from nature have the problem of low erythritol synthesis efficiency. Therefore, in order to increase the initial erythritol synthesis of the strain, the traits of the strain need to be modified. Commonly  methods include physical mutagens such as ultraviolet mutagenesis, radiation mutagenesis and room temperature plasma mutagenesis, as well as chemical mutagens such as the use of diethyl sulphate, ethyl methanesulfonate, nitroso guanidine, ethyleneimine and sodium azide to treat the target target strains. Moon et al. [10] obtained a mutant strain with an erythritol yield increased by 47.6% by treating Aureobasidium sp. SN124A with ultraviolet mutagenesis and nitrosoguanidine. Dong Hai et al. [27] subjected the isolated Saccharomyces cerevisiae ERY237 to ultraviolet and chemical mutagenesis to obtain a dominant strain that produced 87.8 g/L erythritol under optimal fermentation conditions. Wang Feng Wei et al. [28] obtained a strain of the yeast Saccharomyces cerevisiae JunA 6 that produces erythritol by screening samples rich in high concentrations of sugars in nature. After combined mutagenesis with ultraviolet light, LiCl and diethyl sulfate (DES), mutagenesis treatment, the strain JunA 27 was obtained. The erythritol production of this strain reached 67.5 g/L, which is 4.2 times that of the initial strain JunA 6.

Gh Gh ezelbash et al. [29] obtained mutant 49, a mutant strain that can synthesize 39.76 g/L erythritol, by irradiating the yeast Yarrowia lipolytica DSM70562 with ultraviolet light, which is 65 . 7%; further research found that the increase in erythritol production was related to the increase in erythritol reductase activity. Ultraviolet irradiation caused the replacement of aspartic acid at position 270 of the enzyme with glutamic acid, which ultimately led to a 1. 47-fold increase in the enzyme activity. In addition fermentation of the original strain produces 6.37 g/L of the by-product glycerol, while mutant 49 does not synthesize glycerol.

Similarly, Ghezelbash et al. [30] treated Candida magnolia DS M70638, obtained mutant 12, a mutant strain with excellent traits. The yield of erythritol in this strain reached 20.32 g/L, which is a 2.4-fold increase compared to the starting strain, while the concentration of the by-product glycerol decreased by 5.5 times. Qiu et al. [31] used ultraviolet mutagenesis and room temperature plasma mutagenesis combined with ultraviolet mutagenesis to treat the yeast BBE 18. A primary and secondary screening of the 1,152 mutant strains obtained was carried out, and finally the high-yield mutant strain yliUA8 was obtained. After fermentation conditions were optimized, it could produce 148 g/L erythritol, which was much higher than the 43 g/L of the starting strain. Thus shows that conventional mutagenesis and screening is still an important way to obtain strains with excellent traits. Further improvement of erythritol production requires the combination of other methods such as fermentation process optimization and metabolic pathway modification.

2 Fermentation of renewable resources or waste as raw materials to synthesize erythritol

2. 1 Using crude glycerin as raw material

Traditional The liquid fermentation production of erythritol mainly uses commercial glucose and glycerol as substrates. However, the synthesis of erythritol from pure glycerol or glucose not only leads to increased costs, but also contradicts the sustainable development concept of a green energy economy. In recent years, researchers have conducted a large amount of research on the synthesis of erythritol from renewable resources, among which crude glycerol is a more widely used raw material. Crude glycerin is a by-product of the production process of biodiesel, and its main components include glycerin (80%), residual oil, free fatty acids and sodium salt [32]. Tomaszewska et al. [33] investigated the synthesis of erythritol by Yarrowia lipolytica under conditions of pure glycerin and crude glycerin as carbon sources . Tomaszewska et al. [33] investigated the synthesis of erythritol by Yarrowia lipolytica using pure glycerol and crude glycerol as carbon sources. The results showed that Yarrowia lipolytica can use crude glycerol to synthesize erythritol with a maximum yield of 80.5 g/L, which is slightly lower than the 84.1 g/L obtained using pure glycerol. This indicates that crude glycerol can be used as the main raw material in the industrial production of erythritol.

Mi Mironczuk et al. [34] optimized repeated batch fermentation to enable the yeast Yarrowia lipolytica to synthesize 155.5 g/L of erythritol using crude glycerol as a raw material. Although this is lower than the 208 g/L obtained using pure glycerol in the control group, the yield reached 0.56 g/g, which is higher than the 0.41 g/g in the control group group's 0. 41 g / g. Kobayashi et al. [24] confirmed that the zygosporella yeast can ferment and synthesize erythritol using crude glycerol as a substrate, with a conversion rate of 60%, which is higher than the 50% conversion rate when pure glucose is used as a carbon source. This also shows that crude glycerol can replace glucose as a raw material for fermenting and synthesizing erythritol. In addition Rakicka et al. [21] tested the synthesis of erythritol by Yarrowia lipolytica using crude glycerol from two sources (83% glycerol from a biodiesel production line and 76% glycerol from a soap production line) in a two-stage fermentation process. The results showed that the maximum yields of erythritol were 162 and 116 g/L, respectively. This yield is lower than the 199.4 g/L obtained using pure glycerol, but considering the economic cost of raw materials, it is better to use crude glycerol as a raw material for the synthesis of erythritol.

2. 2 Using molasses and waste cooking oil as raw materials

In addition to crude glycerin, Mironczuk et al. [35] used molasses, a by-product of agricultural processing, as a raw material for fermenting and synthesizing erythritol. The main components of molasses are sucrose (55%), other sugars, organic acids and salts. In this study, after two-stage fermentation using molasses as a raw material, the yeast Arthrospira lipolytica AMM could synthesize 70 g/L erythritol. H hijosa Valsero et al. [23] studied the use of Moniliella pollinis MUCL 40570, M. pollinis MUCL 28141, Pseudozyma fusiformata DSM 27425 and P. tsukubaensis NRRL Y 7792 to synthesize erythritol using sugarcane molasses, beet molasses, red grape must and rose grape must. It was found that MUCL 40570 and MUCL 28141 can synthesize 50 to 97 g/ L erythritol; DSM 27425 and NRRL Y 7792 cannot metabolize sucrose molasses and beet molasses to synthesize erythritol, but can use grape juice as a raw material to synthesize 14 to 30 g/L erythritol. Thus it can be seen that different strains have different abilities to metabolize raw materials.

Kitchen waste oil contains a large amount of oil and chemical organic matter. Discarding it carelessly can cause serious environmental pollution, so it is of great significance to reuse kitchen waste oil. Liu et al. [36] found that the yeast M53 can metabolize and synthesize erythritol using kitchen waste oil as a raw material . When 30 g/L of waste oil was added to the medium and cultivated in a 5 L fermenter for 72 h, 22.1 g/L of erythritol was obtained, with a yield of 0.74 g/g. Subsequently, the Liu et al. [37] optimized the process for the production of erythritol from waste oils and fats. First, the loofah sponge was cleaned and dried, and then cut into particles with a size of 1 cm × 1 cm × 0.5 cm and added to the fermentation broth to improve the utilization of the oil by the cells as an oil-in-water dispersant. It was found that at at least 60 g/L of substrate can be completely utilized, and the yield of erythritol can reach 0.76 g/g of oil. By batch feeding and scale-up fermentation, the yield of erythritol can be further increased to 114.3 g/L.

2.3 Using agricultural waste as raw material

Agricultural waste contains a large amount of organic matter that can be utilized by microorganisms. Liu et al. [38] attempted to synthesize erythritol using soybean residue as raw material. Since Yarrow lipase cannot directly decompose soybean residue, so the residue was first pre-fermented using Mucor flavus and Trichoderma reesei, and then the pre-fermented product was used as a raw material for further fermentation with yeast was used to further ferment and synthesize erythritol. It was found that in a 5 L fermenter, the yeast could metabolize the substrate to synthesize 14. 7 g/L erythritol, with a yield of 0. 49 g/g.

The process of synthesizing erythritol from renewable resources using solid-state fermentation has made breakthroughs in recent years. Compared with traditional  traditional liquid deep fermentation, solid-state fermentation has the characteristics of low production cost, more stable production and higher yield [39]. Liu et al. [39] first used a two-stage solid-state fermentation method to achieve the production of erythritol from soybean residue as a substrate. The first stage was fermentation by Aspergillus niger, and after 72 h, the second stage of erythritol fermentation was carried out by in situ inoculation of Yarrowia lipolytica. In addition, in order to add dry loofah pulp, bran, corncobs and buckwheat husks to the raw materials to loosen the soybean residue in order to solve the problem of internal hypoxia caused by the agglomeration of the substrate soybean residue in the actual fermentation process.

It was found that the yield of erythritol was 143.3 mg/g (based on 1 g of dry substrate) when bran was used as a bulking agent and solid-state fermentation was carried out for 192 h. In another study, Liu et al. [40] attempted to synthesize erythritol from oil crop waste by a one-step solid fermentation method. Because oil crop waste contains high levels of nitrogen, which inhibits the synthesis of erythritol, they used a modified strain of Yarrowia lipolytica M53 S. This strain has a knockout of the snf1 gene (encoding sucrose non-fermenting protein kinase) was knocked out, which can be used to synthesize erythritol from the substrate under conditions of sufficient nitrogen source. It was found that M53 S can be used to synthesize erythritol by fermenting a mixture of peanut filter cake, 40% sesame meal and 10% kitchen waste oil, with a yield of 185.4 mg/g.

Biochar can provide a place for cell colonization, while also promoting cell growth and metabolism. To further increase the yield of erythritol, Liu et al. [41] introduced biochar into the solid-state fermentation system. First,  different substrates, including rice bran, wheat straw, mushroom residue and pig manure, were carbonized at high temperatures and then ground into carbon particles. The substrate was then added to a substrate consisting of soybean meal residue, sesame meal and kitchen waste oil (in a mass ratio of 5:4:1). It was finally found that the biocarbon particles made from wheat straw had the most significant effect on promoting the synthesis of erythritol by Yarrowia lipolytica the most effective in promoting the synthesis of erythritol by Yarrowia lipolytica, and the yield of erythritol increased from 182.4 mg/g without biocarbon particles to 199.7 mg/g. Subsequently, through continuous batch fermentation optimization, the yield of erythritol could reach 222.5 mg/g.

2.4 Using microalgae residue as raw material

Microalgae are considered an important sustainable raw material because they do not require land for cultivation and therefore do not compete with food crops. Liu et al. [42] showed that Yarrowia lipolytica can use Schizochytrium sp. zjut8 residue (remaining after oil extraction), soybean meal residue cake and sesame meal as raw materials to synthesize erythritol. After fed-batch fermentation, the yield of the product can reach 223.2 mg/g (based on 1 residues), soybean meal residue cake and sesame meal. The yield of the product after batch fermentation with supplements can reach 223.2 mg/g (based on 1 g of dry substrate). However, in the research, because the microalgae residue contains cellulose components, it cannot be directly utilized by Yarrowia lipolytica, so protease and cellulase need to be used for pretreatment, which to some extent increases the production cost. In future research, the direct utilization of raw materials containing cellulose components by Yarrowia lipolytica can be achieved by metabolic pathway modification.

It can be concluded that that the use of microorganisms to synthesize erythritol from renewable resources through liquid or solid fermentation and its industrial production is the main research direction in the future. This will not only greatly reduce production costs, but also achieve the recycling and reuse of waste, which is also of great significance for environmental protection and energy conservation.

3 Erythritol metabolic pathway modification

The rapid development of genetic engineering technology and the rapid development of omics technology provide a guarantee for the targeted modification of microbial metabolic pathways. Compared with traditional mutagenesis and breeding and fermentation process optimization, metabolic engineering methods aimed at improving the yield of target microbial products by targeted modification of metabolic pathways have the advantages of short cycle, high efficiency and more targeted advantages. At present, the use of metabolic pathway modification to improve the production of erythritol, research has mainly focused on the yeast Yarrowia lipolytica, including improving the metabolism of substrates glucose and glycerol, improving the efficiency of the pentose phosphate pathway and erythritol synthesis, and blocking the catabolism of erythritol (Figure 3). In addition, the  The gene knockout methods involved in metabolic pathway modification mainly include homologous recombination, Cre Lox-based homologous recombination, and the CRISPR Cas knockout system, and the plasmid vectors used are mostly chromosomally integrated plasmids (Table 3).

Carl et al. [43] discovered and identified the gene EYK1, which encodes the erythrose kinase that catalyzes the synthesis of erythrose 1-phosphate from erythrose (Fig. 2). When EYK1 was knocked out in the Yarrowia lipolytica W29, the erythritol increased from 30.7 g/L to 35.7 g/L (Table 3). The erythritol yield of the knockout strain was 0.49 g/g, which was higher than that of the original strain (0.39 g/g). Car ly et al. [44] found that when glycerol kinase (GUT1), glycerol 3-phosphate dehydrogenase (GUT2), phosphopyruvate isomerase (TPI1), transketolase (TKL1), erythrose 4-phosphate phosphatase (E4 PP) and erythrose reductase (ER), it was found that when GUT1 and TKL1 or GUT1 and ER were overexpressed at the same time, the concentration of erythritol in the fermentation broth was significantly higher than that of the control strain.

Among them, the yield of erythritol increased from 0.46 g/g to 0.61 g/g (Table 3), higher than the yield of other single gene overexpression; and knocking out EYK1 and combining it with the overexpression of GUT1 and TKL1 can further increase the production of erythritol in the fermentation broth to 80.6 g/L. Mironczuk et al. [13] overexpressed phosphoglucomutase (GND1), glucose-6-phosphate dehydrogenase (ZWF1), TKL1 and transaldolase (TAL1) in the yeast Arthrospira mutabilis AMM, respectively, to investigate their effects on the biosynthesis of erythritol. The results showed that compared with the fermentation synthesis of erythritol by the control strain MK1, glucose 6-phosphate dehydrogenase (ZWF1), TKL1 and transaldolase (TAL1), and examined their effect on the synthesis of erythritol by the cell. The results showed that compared with the control strain MK1, which fermented and synthesized 25.30 g/L of erythritol, overexpressing any of these genes could cause the cell to synthesize more than 40 g/L of erythritol.

Chen g et al. [45] analyzed the endogenous erythritol reductase in the yeast CGMCC 7326 and overexpressed ER10, ER25 and ER27. The results showed that the overexpression of each of the three genes alone could improve the erythritol synthesis rate of CGMCC 7326 to a certain extent rate. Among them, the effect of overexpressing ER27 was the most obvious. The erythritol in the fermentation broth produced by the original bacteria increased from 154 g/L to 182 g/L in the recombinant bacteria, and the production intensity increased from 1.6 to 2.2 g/(L·h) (Table 3). On this basis they further overexpressed GND1 and ZWF1 in this strain.

The erythritol yield and production intensity of the obtained strain were increased by 23.5% and 50% compared to the original strain, reaching 0.63 g/g and 2.4 g/(L·h), respectively, while the erythritol reached 190 g/L. Jagtap et al. [46] overexpressed the phosphoenolpyruvate carboxylase (PEP) from Saccharomyces cerevisiae in the Yarrowia lipolytica Po1f, which improved the utilization efficiency of glycerol substrates and the production of erythritol. erythritol production, erythritol production intensity, erythritol yield and glycerol utilization rate in the fermentation broth increased from 10.70 g/L, 0.09 g/(L·h), 0.11 g/g and 0.41 g/(L·h) to 18.60 g/L, 0.16 g/(L·h), 0.19 g/g and 0.41 g/(L·h), respectively. g and 0. 41 g / ( L · h) to 18. 60 g / L, 0. 16 g / ( L · h) , 0. 19 g / g and 0. 56 g / ( L · h). In addition, they further overexpressed GUT1 and TKL1 in this recombinant bacterium, and after optimization of fed-batch fermentation, the final fermentation broth could reach 58.8 g/L of erythritol. Also using  the starting strain of the Yarrowia lipolytica Polf, Zhang et al. [14] knocked out the The encoded gene EYD1, the obtained strain MY11 can ferment and synthesize 40 g/L erythritol from glycerol as a substrate, which is a significant increase compared to the 18 g/L of the control strain. In addition, in order to further increase the production of erythritol, they overexpressed ribose 5-phosphate isomerase (RKI1) on the basis of MY11, and the yield of erythritol in the obtained strain could reach 0.52 g/g, with a yield of 52 g/L.

In summary, metabolic pathway engineering is an effective way to improve the initial strain's erythritol production. However, at present, most of the erythritol metabolic pathway engineering focuses on laboratory model strains with low initial erythritol production, such as Yarrowia lipolytica W29, Polf and MK1. There has been relatively little metabolic pathway engineering of industrialized strains. Therefore, the author believes that metabolic pathway modification based on industrial strains with high initial erythritol production, combined with fermentation process optimization, will be an effective way to improve the bottleneck of erythritol production.

4 Conclusions and prospects

At present, erythritol is mainly produced by yeasts through solid-state fermentation or liquid fermentation, of which Yarrowia lipolytica is the main strain. In recent years, researchers have combined strain mutagenesis and selection, fermentation process parameter optimization, and erythritol synthesis pathway modification to achieve the goal of increasing the erythritol production of the starting strain. At the same time, there have been examples of research on the synthesis of erythritol from renewable resources or waste materials through mixed microbial fermentation processes. However, there are still many problems to be explored in the process of microbial metabolism to synthesize erythritol. ①Currently, the strain most commonly used is the Saccharomyces cerevisiae, which is an internationally recognized safe microorganism. However, its growth environment requires a temperature of around 30 C, which can cause contamination and back-contamination during summer production, and there is also the problem of high cooling energy consumption.

For this reason, in the future, consideration could be given to the targeted modification of the heat resistance of existing industrial strains, or the isolation of strains from the environment that can withstand high temperature stress and have excellent traits. (2) For the yeast Yarrowia lipolytica, it is currently known that environmental osmotic pressure plays an important regulatory role in its metabolism and synthesis of erythritol, and the underlying molecular mechanism is not yet clear. Among them, the transcriptional regulation of key enzymes in the erythritol synthesis pathway and the temporal characteristics of erythritol synthesis deserve further research and exploration. ③ For the metabolic pathway modification of erythritol, it is mainly based on overexpressing the genes involved in the substrate metabolic process, the pentose phosphate pathway and the key steps of erythritol synthesis, as well as knocking out the genes in the erythritol degradation pathway.

However, research on modifying the substrate transport system to improve substrate transport and the export of erythritol has not been carried out in depth, which will help further improve the yield of erythritol. 4. If erythritol is synthesized using renewable resources or waste as raw materials, the strain used must be able to decompose and utilize cellulose or hemicellulose as a carbon source, which poses a new challenge for the fermentation of erythritol using Yarrowia lipolytica. Although this problem can be solved by pretreating the raw materials or through mixed fermentation, it undoubtedly increases the production cycle and cost. At the same time, the acids and hydroxymethylfurfural present in the cellulosic hydrolysate mixture inhibit the growth of Yarrowia lipolytica.

Therefore, in the future, the targeted modification of Yarrowia lipolytica is expected to improve its direct utilization of renewable resources and enhance its ability to cope with environmental stress, which will lead to the sustainable production of erythritol. ⑤ Finally, the increase in strain yield caused by mutagenesis is related to the increase in enzyme activity caused by mutations in key enzymes in the metabolic pathway. In the future, it is worth studying whether the activity and stability of key enzymes can be improved through in vitro directed evolution, and then the evolved enzymes can be expressed intracellularly through reverse metabolic engineering technology, so as to achieve the purpose of increasing the yield of target products.

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