Sugar substitution is one of the methods to alleviate the health problems caused by excessive use of sugar
The rate of obesity and the incidence of related diseases are rising at the same time. This is a serious threat to human health. According to China's national inspector, as early as 2020, the rate of overweight or obesity among adults over the age of 18, who make up the largest group of the population base, is already more than half (50.7%). Several studies have shown that reducing excessive intake of high-calorie sugars can reduce the risk of obesity, diabetes, hypertension and cancer [1]! In 2015, the World Health Organization made a call to reduce sugar intake in children and adults [2]. Sugar substitutes are a group of substances that have a sweet flavor but few or no calories compared to sucrose and glucose. They can provide sweetness while reducing calorie intake, which is of great significance for diabetics and people who need to lose weight [3]. Sugar substitutes can be categorized into synthetic and natural sugar substitutes; synthetic sugar substitutes (also known as artificial sweeteners) only provide sweetness, contain no calories, and do not raise blood sugar. However, many studies have shown that various synthetic sugar substitutes have potential cancer risks[4]. Therefore, more and more people are focusing on natural sugar substitutes. Among the natural sugar substitutes, erythritol and xylitol are two typical representatives [5].
Figure 1. Health problems caused by sugar overuse- Diabetes, Obesity and Tooth decay.
Synthesis of sugar substitute by microbial fermentation is a green and sustainable way
Traditional production methods for sugar substitutes are flawed. Traditional production methods for xylitol and erythritol are limited by a number of factors [6]. Currently, the two main production methods for erythritol and xylitol are plant extraction and semi-chemical synthesis. The plant extraction method is limited by a variety of factors, such as low product content in the raw material, long harvesting time, and long plant growth cycle. The result is a cumbersome extraction process, low yield and high cost. The semi-chemical synthesis method, on the other hand, is based on raw material extracts and further obtains the target products through chemical reactions. This is also limited by the problem of low content in the plant. The chemical reaction also brings a series of problems such as wastewater and exhaust gas, which cause environmental pollution. These problems limit the large-scale production of xylitol and erythritol.
Figure 2. Conventional and microbial production of sugar substitutes.
Many wild-type strains have been isolated and proven to have the ability to produce erythritol. Most of them are eukaryotes, such as Yarrowia, Candida, Torula, Pseudozyma and Moniliella [7] [6]. Currently, the following genera are used for the production of erythritol: Moniliella pollinis, Yarrowia lipolytica, Trichosporonoides oedocephalis [8, 9]. Microbial production of xylitol can be divided into two main types: whole-cell conversion of xylose as a substrate to produce xylitol and synthesis of xylitol from scratch using glucose or glycerol as a substrate. In previous studies, Enterobacter liquefaciens 553 [7] and Corynebacterium sp. B-4247 [10] were both able to produce xylitol by using xylose as a substrate for conversion.
Choosing Y. lipolytica as host for erythritol production
Y. lipolytica is an oil-producing yeast [11]. As a "Generally Recognized as Safe" (GRAS) organism, Y. lipolytica has several significant attributes, such as a broad substrate spectrum, relatively straightforward inherited backgrounds, and available genetic manipulation methods which provides many advantages for erythritol production [11-13].
The synthetic pathway of erythritol in Y. lipolytica
Usually, industrial production of erythritol by Y. lipolytica mainly utilizes glucose and glycerol as substrates. The metabolic pathways for the synthesis of erythritol with different substrates are also different. The synthetic pathway for the production of erythritol with glucose as substrate is shown in Figure 3 [14, 15] .
Figure 3. The synthetic pathway of erythritol in Y. lipolytica [14, 15]. Erythritol biosynthesis pathway from glucose and glycerol in yeast or non-yeast organisms. GK, glycerol kinase; GDH, glycerol 3-P dehydrogenase; TKL1 (Transketolase); PFK (Phosphofructokinase); E4PK, erythrose-4-phosphate kinase; ER, Erythrose reductase; Ru5P, ribulose 5-phosphate; Xu5P, xylulose 5-phosphate; R5P, ribose 5-phosphate; S7P, sedoheptulose 7-phosphate; DHAP, dihydroxyacetone phosphate; E4PP: erythrose-4P phosphatase; ER: erythrose reductase [14, 15].
Erythritol is synthesized in Y. lipolytica as follows: glucose 6-phosphate is dehydrodecarboxylated to ribulose 5-phosphate; ribulose 5-phosphate is isomerized to ribulose 5-phosphate and xylulose 5-phosphate by pentose phosphate isomerase and differential enzyme; ribulose 5-phosphate and xylulose 5-phosphate are converted to sedoheptulose 7-phosphate by the transketolase; sedoheptulose 7-phosphate transfer the three-carbon unit to 3-phosphoglycerol aldehyde on the C1 to produce erythrose 4-phosphate under the action of transaldolase . Erythrose 4-phosphate is phosphorylated by erythrose-4P phosphatase, named E4PP or Yida, to obtain erythrose, and erythrose reductase (ER) reduces erythrose to synthesize erythritol [14, 15].
Choosing E.coli as host for xylitol production
E. coli is one of the most commonly engineered strains due to its rapid propagation, clear genetic background and high expression rate [16]. Wild-type E. coli does not have the ability to convert xylose to produce xylitol. Introducing the xylose reductase gene into E. coli cells allows xylitol production by further fermentation.
The production of xylitol by one-step conversion
A one-step conversion for the production of xylitol from xylose as a substrate can be achieved under the catalytic action of xylose reductase (Fig. 4) [17].
Figure 4. A one-step conversion for the production of xylitol from xylose [17]
Goal
We constructed xylose reductase-expressing strains with different enzyme sources to catalyze xylose for xylitol production using fermentation with Escherichia coli(E. coli). The strategy of promoter engineering was utilized to improve the efficiency of conversion. We overexpressed the erythritol synthesis pathway enzyme in Y. lipolytica. Based on a high-throughput screening strategy, we constructed fluorescent reportor system for characterizing erythritol production and improving erythritol production in Y. lipolytica.
Figure 5. a. Promoter engineering and xylose reductase screening; b. Construction of a fluorescent expression system for characterizing erythritol production
Reference
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