With the increasing prevalence of diabetes in the country, the pursuit of low-sugar green life has become the mainstream lifestyle. On December 6, 2021, the International Diabetes Federation ( IDF) officially released the 10th edition of the ' Global Diabetes Map '. In 2021, there will be 537 million ( 10.5% ) diabetics among adults aged 20-79. The total number of diabetes is expected to increase to 643 million (11.3%) by 2030 and 783 million (12.2%) by 2045[1] (https://diabetesatlas.org/).Therefore, the topic of sugar substitution has gradually been recognized. Various sugar substitutes, such as stevioside and erythritol, are also on the market[2-3]. These traditional sweeteners have the disadvantages of high calories and high blood sugar. Excessive intake can lead to chronic diseases, such as diabetes, obesity and cardiovascular disease. Therefore, the development of new low-calorie functional sweeteners has become a research hotspot in international food science. D-psicose, discovered in recent years, is a new functional sweetener [3].
D-psicose is a rare sugar, its sweetness is about 70 % of sucrose, but the calorie is 0.3 % of sucrose. It is considered to be an ideal new low-calorie functional sweetener and can be used as an ideal substitute for sucrose [4].D-allulose has many beneficial physiological functions to human body, including reducing postprandial blood glucose response, enhancing glucose tolerance and improving insulin sensitivity by stimulating the translocation of glucokinase in liver cells, thereby inhibiting the increase of blood glucose level. It has lipid-lowering function by regulating adipogenic transcription factors in vivo, thereby inhibiting preadipocyte differentiation and lipid accumulation in vivo [4-5] ; The antioxidant capacity of D-psicose-protein conjugates is higher than that of ordinary protein, which has antioxidant effect [5] ;
D-allulose can effectively inhibit the deformation of dopamine neurons caused by ROS, and has a significant protective effect on nerve cells. In nature, D-psicose is present in a very small amount in some plants, such as Virginia stings, wheat, etc., and cannot be directly extracted to achieve large-scale production [4-6]. The absorption rate of D-psicose in the human body is very low. D-psicose can compete with glucose, fructose and other transporters on the surface of the cell membrane to reduce the absorption of glucose and fructose in the human body, thereby enhancing insulin resistance, reducing body fat accumulation and reducing the potential risk of diabetes [6]. In 2020, the global market size of D-allulose has reached USD 220 million, 54 % is used in the food industry, and 36 % is used in beverages. It is expected that industrial scale will reach USD 430 million in 2030. D-allulose is added to foods, such as desserts, beverages, jelly, and formulated milk. Therefore, D-allulose has great market development prospects.
At present, there are two main methods for the synthesis of D-psicose, namely chemical synthesis and biosynthesis. The chemical method refers to the catalytic formation of D-psicose by D-fructose [7]. However, there are many shortcomings in the chemical synthesis of D-psicose. It requires protection and deprotection reactions, harsh reaction conditions, complex separation and purification processes, generation of unnecessary by-products, chemical waste pollution, etc. In contrast, the biological enzyme method is the most important method for the synthesis of D-psicose.Under the catalysis of D-psicose-3-epimerase, D-fructose is converted to D-psicose. Biological enzymatic production has become a hot spot due to its advantages of mild conditions, environmental friendliness and so on. Izumoring provides researchers with a new method for the enzymatic synthesis of D-psicose, which is the main method for the production of rare sugars. [8] D-psicose 3-epimerase is already the main catalyst for D-psicose, and its unique catalytic mechanism is significantly different from other epimerase [9]. In the industrial production of D-psicose, the higher reaction temperature can improve the solubility of the substrate and the product, thereby reducing the viscosity and making the reaction more stable. In addition, the reaction rate can also be increased and increased. In addition, it can also increase the reaction rate and increase the diffusion rate, thereby improving the reaction activity of the enzyme. For the endothermic reaction, the higher reaction temperature can make the equilibrium move in the direction of the generated product, improve the conversion rate, and greatly reduce the probability of bacterial infection. However, the reported D-psicose 3-epimerase has poor thermal stability, rapid inactivation at high temperature, and low expression level, which cannot meet the needs of industrial production [10-14 ].
In order to solve the problem of poor thermal stability and low expression level of D-psicose 3-epimerase in industrial production. Liu Weiwei et al.used genetic engineering to construct a strain of Escherichia coli to express D-allulose 3-epimerase (DAE) to catalyze fructose production, and obtained the best medium combination [15]. Zhang et al.studied the protein expression of DPEase (NtDPEase) from Novibacillus thermophilus in Pichia pastoris and studied the enzymatic properties [16]. Zhang et al.reported that the recombinant D-allulose 3-epimerase derived from Dorea sp.CAG317 is a metal-dependent enzyme, the cofactor is Co2+, and the auxiliary concentration of 1 mM Co2+ is optimal. The maximum activity of the enzyme is 803 U/mg [17]. Chen et al.studied the DAEase from T.caenicola.The optimal concentration of metal-assisted was also 1 mM Co2+, and D-fructose produced 139.8 g/L D-psicose with a conversion rate of 28.0% [18]. Xu etal.studied the presence of Co2+ in Novibacillus thermophilus DAE (NtDAE). The conversion yield of 500 g/L d-fructose to d-allulose at 60° C achieved 29.7 % [19]. Hongbin Q studied TtDAE from Thermogutta terrifontis with a half-life of 32 h at 70°C, but did not study its yield [20](Talbe 1).
Table 1: Conversion rates of D-psicose in TcDAE, TtCAE, NtDAE, and DsDAE in current literature
| Name | Conversion rates(100%) |
| TcDAE | 28.0% |
| TtCAE | unknown |
| NtDAE | 29.7% |
| DsDAE | unknown |
Therefore, we selected the above four sources of DAE, compared the four sources of DAE enzymes, studied the properties and expression levels of the four enzymes, and further studied their conversion rate of D-fructose to D-psicose(Figure 1).
Figure 1. DAE converts fructose into D-alloulose
In the choice of chassis organisms, we also did research. Pichia pastoris is a yeast that can use methanol as the sole carbon source. It not only has the basic properties of the yeast expression system, but also integrates heterologous genes into the genome through homologous recombination, thus showing a high degree of genetic stability and having an efficient and stable gene expression system [21]. The expression system of Pichia pastoris has great advantages in the production of functional proteins, chemicals and natural product synthesis. As a model organism using single carbon resources, Pichia pastoris has excellent performance in the secretion of exogenous proteins, low glycosylation and high-density fermentation, and is an ideal host for the expression of exogenous genes. In addition, Pichia pastoris can perform high-density fermentation, which can easily increase the expression of recombinant proteins[22].
D-pscose-3-epimerase (DAE) plays an important role in the biotransformation of D-psicose, but the thermal stability of D-psicose-3-epimerase is poor and the expression level is low, which cannot meet the requirements of industrial production. In this study, we choosed four origin of DAE, is respectively Thermoclostridium caenicola(TcDAE)，Novibacillus thermophilus(NtDAE), Thermogutta terrifontis(TtDAE), and Dorea sp. CAG 317(DsDAE). Then we ligated the coding genes into the expression vectors, and transformed them into Pichia pastoris GS115 strain. The target protein was induced and purified, and tested the yield of D-psicose (Figure 2). Based on the yield of D-alloulose, it can be screened which source of DAE has a higher yield and higher thermal stability.
On this basis, the liquid PTVA method was used to obtain high-copy strains by Zeocin antibiotic screening to further improve the expression level of D-psicose-3-epimerase. This study will provide a new strategy for the industrial production of D-psicose.
Figure 2. The engineering schematic diagram of the project design