Experiments
The core objective of our project is to introduce the key gene for ε-PLL biosynthesis into Bacillus subtilis. We initially synthesized the DNA sequence encoding the pls gene with coden optimization of the natural ε-PLL-producing strain Streptomyces albulus. The optimized pls gene was then cloned into the high-efficiency expression vector pMA5 plasmid to form the recombinant plasmid pMA5-pls. Subsequently, through chemical tranformation, the constructed pMA5-pls plasmid was efficiently transformed into the B. subtilis host strain, thereby giving B. subtilis the ability to synthesize ε-PLL.
To further enhance the production efficiency of ε-PLL, we introduced the gene encoding polyphosphate kinase 2 (ppk2) into the pMA5-pls plasmid, forming the recombinant plasmid pMA5-pls-ppk2. The ppk2 gene can enhance the regeneration capacity of ATP within the cell, providing stronger energy support for the synthesis of ε-PLL. Ultimately, the pMA5-pls-ppk2 plasmid was then transformed into B. subtilis.
In addition, in order to relieve the substrate limitation of L-lysine in the biosynthetic pathway of B. subtilis, we knocked out the endogenous thrD gene in B. subtilis and replaced it with the lysC gene from Corynebacterium glutamicum by using CRISPR-Cas9. The aspartate kinase encoded by its lysC gene can relieve feedback inhibition in the ε-PLL production process, effectively increasing the synthesis of L-lysine and thus providing sufficient substrate for ε-PLL production.
To meet the requirements for biosafety and plasmid stability in industrial applications, we constructed a self-control system to ensure that the engineered strains can survive and produce only under specific conditions and create a “suicide” mechanism in unintended environments. We knocked out the alrA gene from the genome of B. subtilis ,which can product D-alanine is an essential component for building the tetrapeptide side chain of peptidoglycan.
Simultaneously, we also cloned the lacI gene from the pHT43 plasmid. The repressor protein encoded by this gene can bind to the promoter and inhibit the expression of downstream genes, with its activity being induced by lactose or IPTG. Finally, we combined the lac operon controlled alrA gene with pMA5-pls-ppk2 plasmid and transformed it into the B.subtilis without alrA and replaced with lysC311. Thus, only when IPTG is added to the culture medium, alrA gene is expressed and thus enabling the host strain to synthesize D-alanine and grow normally.
