In Vitro Expression and Assembly of Nonribosomal Peptide Synthetase for Biosynthesizing D-Phe-L-Pro Diketopiperazine

Nonribosomal peptides (NRPs) are of great interest because they possess significant bioactivities such as antiviral, antimicrobial and anticancer activities. These compounds are natively biosynthesized by large multimodular enzymes called nonribosomal peptide synthetases (NRPSs). Current metabolic engineering and synthetic biology studies have tried to utilize surrogate microbes such as E. coli for the heterologous production of pharmaceutical molecules (e.g. NRPs) by reconstitution of their entire gene clusters in the host cells. Although those in vivo surrogate hosts may seem promising to produce complex NRP compounds, it still remains problematic to obtain high yields, which are mainly due to metabolic burden inhibiting cell growth, unavailability of necessary precursors and toxic products. Alternatively, in this work we demonstrated a novel approach to synthesize natural products based on a completely in vitro, cell-free, platform consisting of enzyme expression and product formation. As a model system, we utilized two NRPSs that are involved in the first step of gramicidin S biosynthesis, namely, GrsA (126 kDa) and GrsB1 (121 kDa, the first module of GrsB). GrsA and GrsB1 can function in together to catalyze the formation of the D-Phe-L-Pro diketopiperazine (DKP). We initially expressed GrsA and GrsB1, respectively, using an E. coli-based cell-free protein expression (CFPS) system. The results indicated that both enzymes were expressed with yields more than 100 μg/mL. Then, we aimed to activate the newly expressed apo enzymes to be functional holo enzymes by addition of the purified phosphopantetheinyl transferase (PPTase, for example, Sfp from Bacillus subtilis). The post-translational modification of GrsA and GrsB1 was confirmed by labeling with a fluorescent dye (BODIPY-CoA) after addition of the PPTase Sfp. We next sought to synthesize D-Phe-L-Pro DKP using the in vitro platform. Instead expression of GrsA and GrsB1 separately, two enzymes were coexpressed in one-pot mixture allowing reconstitution of the full NRPS assembly line for product formation. The results indicated that our in vitro system can produce 12 mg/L of D-Phe-L-Pro DKP, which is higher than the reported in vivo yield at 9 mg/L. Our studies notably demonstrate that in vitro, cell-free, platforms are robust to produce “difficult-to-express” proteins like the very large NRPS enzymes, and more importantly, complex natural products that could be biosynthesized via the in situ CFPS expressed enzymes. We anticipate that our approach could be generalized for the construction of highly efficient cell-free based artificial biosynthetic factories to enable target product discovery and synthesis.