At present, the problem of appearance anxiety is becoming more and more common. According to a questionnaire survey conducted by domestic media on "appearance anxiety", nearly 60% of people have some degree of appearance anxiety.

Some surveys show that nowadays, every vacation as well as graduation season, there will be a phenomenon of students getting together for plastic surgery, and the "post-00s" have become the main force of medical beauty consumption. Reporters tried to search on multiple social and short video apps, and found that "summer vacation plastic surgery", "summer vacation beauty", "summer vacation thinning", etc., were all hot topics during the summer vacation.

"If only I could have a pointed chin like……", people around me often touch their double chins and say words like these.

According to Grand View Research, the global aesthetic medicine market was valued at $112 billion in 2022 and is expected to grow at a compound annual growth rate (CAGR) of 14.7% from 2023 to 2030. According to McKinsey, the aesthetic injectable market is growing by more than 10% per year.

Fullness under the chin or "double chin" is a common concern for both men and women. At present, surgical procedures such as liposuction seem to have become a major way for them to pursue beauty. However, the risks are likely to be pain and complications that will last a lifetime. Therefore, people prefer well-tolerated alternative methods, and "Double chin Buster" deoxycholic acid injection has entered our sights in this way.

Deoxycholic acid (DCA) is a bile acid that promotes bile secretion, eliminates bile blockade, and aids fat digestion and absorption.

Deoxycholic acid (DCA) has been authorized by Federal Drug Agency for the cosmetic reduction of redundant submental fat. DCA has gained popularity in recent years because of its proven efficacy, fewer side effects, and limited downtime^[1]. When injected into submental fat, deoxycholic acid helps destroy (adipocytes) fat cells, which will be metabolized by the body for several months^[2].

Although the risk of injecting fat-dissolving injections is smaller and better tolerated, compared with the immediate treatment effect and liposuction surgery, the weakness of it is that takes at least 2 months for patients to see significant results. Therefore, our project hopes to continue to optimize deoxycholic acid on this basis.

Numerous studies have shown that bile acids are enhanced in function and value when they are hydroxylated. Take the hydroxylation of lithocholic acid to ursodeoxycholic acid (UDCA) and murideoxycholic acid (MDCA) for illustration. UDCA can treat various cholestasis^[3] and intestinal environmental changes and related diseases^[4]-[5]. MDCA can effectively dissolve stones, prevent and inhibit the formation of gallstones^[6]-[7]. In a word, the value of hydroxylated bile acids has been greatly improved compared to bile acids.

Given the significant effects of hydroxylation, we hope to obtain 3a, 6b, 12a-trihydroxy-5b-cholan-24-oic acid (6β-OH DCA) to improve the solubility and pharmaceutic properties of DCA for further applications through hydroxylation modification of deoxycholic acid. However, at present, the synthesis of 6β-OH DCA mainly adopts chemical synthesis and animal extraction methods, which have some problems such as complex steps, low efficiency, and high toxicity. In addition, the animal extraction method is also very cruel.

In the course of extensive reading of the literature on the hydroxylation of sterols, we found that UDCA has been synthesized by expressing the P450 monooxygenase CYP107D1 (Olep) hydroxylated in E. coli to modify lithophanic acid LCA^[9], significantly enhancing the efficiency of the biocatalytic method for the preparation of UDCA. In a further review of the literature on Olep, we learned that Olep can also hydroxylate testosterone at the positions 6β, 7β, 12β, and 15β^[10], bile acids like LCA and deoxycholic acid (DCA) are hydroxylated exclusively at the 6β-position, forming MDCA and 3α-, 6β-, 12α-trihydroxy-5β-cholan-24-oic acid, respectively^[9].

Cytochrome P450 is an extensively studied class of heme-dependent oxidative enzymes. Using molecular oxygen as an oxidant, P450s are capable of stereo- and regioselective hydroxylation reactions of a wide variety of substrates^[8], which gives them great potential for the hydroxylation of steroids.

Herein, a combinatorial strategy was applied to construct a powerful Cytochrome P450 biocatalyst (CYP107D1, Olep) to convert DCA to 6β-OH DCA with higher solubility, and economic value. We successfully expressed Olep in E. coli to hydroxylate LCA to produce MDCA and DCA to 6β-OH DCA. However, the low catalytic efficiency remains a greater challenge. Currently, a major challenge limiting the efficiency of Olep hydroxylating sterols is the incompatibility of redox partners. In addition, the expression of Olep is also limited by the low intracellular heme content and the low soluble expression in E. coli, which generally plagues teams working on Olep.

Researchers in China and abroad have been modifying P450 enzymes to improve their efficiency or alter their catalytic specificity for the hydroxylation of steroid drugs through rational design and screening of redox partners. Our experiment was designed to improve the catalytic efficiency of P450 enzymes through the following four strategies, respectively:

Because whole-cell catalytic systems are simpler and less costly than conventional enzymatic reactions, we ultimately planned to construct an optimal whole-cell catalytic system by integrating the three strategies.