Introduction

Aim

To express the NADPH cofactor in order to create a better environment for the activity of the 3α-HSD enzyme.

The Cofactor?

Co-factors such as NADH and NADPH are essential for most enzymatic reactions, 3α-hydroxysteroid dehydrogenase (3α-HSD) among them. 3α-HSD enzyme catalyzes the reaction between 5α-dihydrotestosterone (DHT) and 3α-androstanediol (3α-Diol) using NADH and NADPH as co-factors; also studies show that this enzyme has higher affinity to NADPH

Expression in B. subtilis

As stated above, our goal was to express the 3α-HSD protein, originally from rat liver, and check whether it is able to fold properly and break down DHT.

For this purpose, we cloned 3α-HSD into B.subtilis under the inducible P_hyper-spank promoter.

pDR111 is a shuttle vector for E.coli and B.subtilis. When in E.coli, an ampicillin resistance gene is expressed and when in B.subtilis, a spectinomycin resistance is expressed. The plasmid contains homologous regions with the B. subtilis genome, enabling gene introduction by recombination into the nonessential amyE locus of the chromosomal DNA as a single copy.³

The plasmid also contains lacI gene for the inducible promoter P_hyper-spank, making our inserted genes inducible with the addition of IPTG.

In the lab, we inserted our gene in the multiple cloning site between the NheI and SalI restriction sites, which can be seen in the basic pDR111 plasmid below.

In order to predict the target gene expression, we cloned mCherry into B.subtilis, since it is easier to track reporter genes. The simple, planned circuit of the gene in the shuttle vector is featured in the image below.

Secretion

Secretion of the 3α-HSD enzyme was an extremely important component of our project. The secretion would enable the bacteria to live on the consumer’s scalp, as opposed to being lysed in order to recover the enzyme. This could reduce the production costs and improve product efficiency over time.

As mentioned previously, B. subtilis, as well as some other gram-positive bacteria, has protein secretion mechanisms and regularly secretes proteins such as various proteases.

In order to engineer the bacteria to meet our purposes, we fused our target gene coding for 3α-HSD to the signal peptide (SP) for the gene aprE, which encodes to extracellular alkaline-serine protease (subtilisin E), the most abundant protease secreted to the medium in wildtype B. subtilis. ⁴

By doing so, the 3α-HSD enzyme could be recognized by the secretion system of the protease and could be excreted into the extracellular medium.

The circuit can be seen below.

Click here to see our results.