Diabetes is a chronic non-communicable lifelong disease that poses a lifelong threat to global health. According to estimates from the Global Burden of Diseases, Injuries, and Risk Factors Study (GBD), there were 529 million (95% UI 500–564) people of all ages, worldwide, living with diabetes in 2021. Type 2 diabetes (T2D) accounts for over 95% of all diabetes cases^[1]. At the current pace, WHO estimates that more than 1.31 billion people will be living with diabetes by 2050, most of whom will have type 2 diabetes^[2]. The prevalence of T2D has placed a heavy economic burden on the world (Figure 1). The primary clinical indicator of T2D is dysfunctional fasting blood glucose levels exceeding 7.0 mmol/L, coupled with insulin resistance triggered by hyperglycemia. The disease is typically characterized by "three polys and one less": increased thirst, urination, appetite, and concurrent weight loss over a short period. As T2D progresses, symptoms such as fatigue, malaise, memory loss, blurred vision, and limb numbness may also occur^[3,4]. Although numerous medications have been approved to control blood glucose levels, they cannot cure the disease and have serious side effects when used over a long period, presenting significant challenges for both physicians and patients^[5,6].

Figure 1. Total diabetes-related health expenditure (USD) for adults (20–79 years) with diabetes, 2021

What is the underlying cause of Type 2 Diabetes (T2D)? There are two main factors involved. First, the functional deficiency of pancreatic islet β-cells leads to insufficient insulin secretion, resulting in elevated blood glucose levels. Second, even when insulin secretion is normal, the body may develop insulin resistance. Therefore, there is an urgent need to develop new oral hypoglycemic drugs to manage this condition^[6].

Vglycin (Vg), a novel orally administrated hypoglycemic peptide derived from soybeans, contains three pairs of disulfide bonds within the molecules (Figure 2), that has been shown to improve blood glucose and lipid disorders in diabetic mice^[7,8]. Long-term administration of Vg has not been associated with any side effects in animals^[9]. Currently, Vg is under development as a new drug for the prevention and treatment of metabolic syndrome, including diabetes and hyperlipidemia by Wuhan Gexin Biotechnology Co., Ltd. However, direct extraction from soybeans is problematic due to low yields, long cycles, and complex processes, which are not conducive to large-scale production.

Figure 2. Amino acid sequence and disulfide bonding mode of Vg. Three pairs of disulfide bonds are formed inside the molecule, which is stable in structure and can be absorbed orally.

HBUT-Wuhan has proposed using synthetic biology to produce Vg, aiming to overcome the low yield issues associated with soybean extraction. Currently, synthetic biology is being utilized for the metabolic engineering of microorganisms to successfully produce various plant metabolites.In contrast to chemical synthesis, heterologous expression of the metabolic pathway offers a more sustainable solution . Expressing large quantities of Vg in Pichia pastoris could reduce costs and facilitate the expansion of manufacturing techniques^[10,11].

Pichia pastoris

In order to select a proper expression vector, our team gathered information on various microorganisms (including Escherichia coli, Saccharomyces cerevisiae, mammalian cells...) and ultimately chosen Pichia pastoris as the expression host. Pichia pastoris is a kind of yeast that can utilize methanol as its sole carbon and energy source. It offers significant advantages over other expression systems in terms of protein processing, secretion, post-translational modification, and glycosylation^[12]. As one of the most widely protein expression systems, Pichia pastoris is recognized as a prominent host in molecular biology for the purpose of generating recombinant proteins. The advantages of utilizing the Pichia pastoris system for protein production encompass the proper folding process occurring within the endoplasmic reticulum^[13].

pPIC9K Vector Design

The vector we selected is pPIC9K, which codes a signaling peptide that makes the expressed protein secret into the culture environment. There is an AOX 1 promoter on the pPIC9K vector, encoding an alcohol oxidase that breaks down methanol as a carbon source^[14]. After the secretion signal, we inserted the fusion expression genes of nattokinase, a soybean kinase with molecular weight of 28 kDa, and 3 Vg in tandem. At the 3' end of the fusion gene, we added a His-tag to facilitate subsequent isolation and purification of the expressed fusion protein. In addition, the acid-sensitive sites (Asp-Pro) were inserted between Vg and Vg to facilitate the release of Vg monomers from fusion proteins.

Figure 3. Visualization of the pPIC9K-NK-3Vg for overexpression.

AOX Promoter

The methylotrophic yeast Pichia pastoris (Komagataella phaffi) has been commonly used for the production of heterologous protein alcohol oxidase I (AOX1) promoter, which is remarkably strong and tightly regulated by methanol turns out to be most popular. The AOX promoter is tightly regulated by methanol induction. High cell density can be achieved by simple culture, and foreign protein can be expressed stably and at high level^[14].

α Factor Secretion Signal

The α-mating factor (MF-α) is a common signal peptide, which is derived from the α-mating factor protein of Saccharomyces cerevisiae. This signal peptide is widely used in Pichia pastoris to guide the secretion and expression of foreign proteins. MF-α signal peptide is popular because it can efficiently guide the target protein into the secretory pathway, thus making the target protein accumulate in the Pichia pastoris culture medium, which is convenient for downstream separation and purification^[15].

NK

Nattokinase (NK) is an alkaline serine protease secreted by Bacillus subtilis (natto). It has the advantages of long half-life, strong specificity, small side effects, and direct oral administration. It can dissolve thrombus, reduce blood viscosity, and prevent and improve cardiovascular and cerebrovascular diseases. It can directly degrade fibrin, dissolve the thrombus and restore the normal flow of blood^[16]. It helps to dilate blood vessels and increase their elasticity, thereby improving blood circulation. The introduction of NK and the expression of three Vg fusions can extend the size of the fusion expression fragments to some extent, facilitating the detection of late proteins.

Acid Sensitive Sites (asp-pro)

To increase the expression yield of the target peptide and simplify subsequent purification, we concatenated three coding Vg genes in the design plasmid. During the later stages of product enrichment, the peptides will be treated with an acidic solution at a pH of approximately 5, causing the acid-sensitive sites to disconnect automatically, resulting in three separated Vg molecules^[17].

His-tag

The His-tag is a commonly used marker in protein recombination technology, consisting of six histidine residues. It is small, with a molecular weight of less than 0.84 KD, and does not alter the biological structure or solubility of the protein. Most importantly, it simplifies protein purification. The imidazole ring on histidine can bind to divalent Ni ions, which aids in further purification and detection. Based on this principle, we have attached a His-tag to the carboxylic end of the Vg chain to facilitate peptide purification.