Combinatorial optimization and spatial remodeling of CYPs to control product profile.

Activating inert substrates is a challenge in nature and synthetic chemistry, but essential for creating functionally active molecules. In this work, we used a combinatorial optimization approach to assemble cytochrome P450 monooxygenases (CYPs) and reductases (CPRs) to achieve a target product profile. By creating 110 CYP-CPR pairs and iteratively screening different pairing libraries, we demonstrated a framework for establishing a CYP network that catalyzes six oxidation reactions at three different positions of a chemical scaffold. Target product titer was improved by remodeling endoplasmic reticulum (ER) size and spatially controlling the CYPs' configuration on the ER. Out of 47 potential products that could be synthesized, 86% of the products synthesized by the optimized network was our target compound quillaic acid (QA), the aglycone backbone of many pharmaceutically important saponins, and fermentation achieved QA titer 2.23 g/L.

Dynamic upregulation of the rate-limiting enzyme for valerolactam biosynthesis in Corynebacterium glutamicum.

Valerolactam is a monomer used to manufacture high-value nylon-5 and nylon-6,5. However, the biological production of valerolactam has been limited by the inadequate efficiency of enzymes to cyclize 5-aminovaleric acid to produce valerolactam. In this study, we engineered Corynebacterium glutamicum with a valerolactam biosynthetic pathway consisting of DavAB from Pseudomonas putida to convert L-lysine to 5-aminovaleric acid and ß-alanine CoA transferase (Act) from Clostridium propionicum to produce valerolactam from 5-aminovaleric acid. Most of the L-lysine was converted into 5-aminovaleric acid, but promoter optimization and increasing the copy number of Act were insufficient to significantly improve the titer of valerolactam. To eliminate the bottleneck at Act, we designed a dynamic upregulation system (a positive feedback loop based on the valerolactam biosensor ChnR/Pb). We used laboratory evolution to engineer ChnR/Pb to have higher sensitivity and a higher dynamic output range, and the engineered ChnR-B1/Pb-E1 system was used to overexpress the rate-limiting enzymes (Act/ORF26/CaiC) that cyclize 5-aminovaleric acid into valerolactam. In glucose fed-batch culture, we obtained 12.33 g/L valerolactam from the dynamic upregulation of Act, 11.88 g/L using ORF26, and 12.15 g/L using CaiC. Our engineered biosensor (ChnR-B1/Pb-E1 system) was also sensitive to 0.01-100 mM caprolactam, which suggests that this dynamic upregulation system can be used to enhance caprolactam biosynthesis in the future.

Programming adaptive control to evolve increased metabolite production.

The complexity inherent in biological systems challenges efforts to rationally engineer novel phenotypes, especially those not amenable to high-throughput screens and selections. In nature, increased mutation rates generate diversity in a population that can lead to the evolution of new phenotypes. Here we construct an adaptive control system that increases the mutation rate in order to generate diversity in the population, and decreases the mutation rate as the concentration of a target metabolite increases. This system is called feedback-regulated evolution of phenotype (FREP), and is implemented with a sensor to gauge the concentration of a metabolite and an actuator to alter the mutation rate. To evolve certain novel traits that have no known natural sensors, we develop a framework to assemble synthetic transcription factors using metabolic enzymes and construct four different sensors that recognize isopentenyl diphosphate in bacteria and yeast. We verify FREP by evolving increased tyrosine and isoprenoid production.

Synthetic pathway for production of five-carbon alcohols from isopentenyl diphosphate.

Synthetic biological pathways could enhance the development of novel processes to produce chemicals from renewable resources. On the basis of models that describe the evolution of metabolic pathways and enzymes in nature, we developed a framework to rationally identify enzymes able to catalyze reactions on new substrates that overcomes one of the major bottlenecks in the assembly of a synthetic biological pathway. We verified the framework by implementing a pathway with two novel enzymatic reactions to convert isopentenyl diphosphate into 3-methyl-3-butenol, 3-methyl-2-butenol, and 3-methylbutanol. To overcome competition with native pathways that share the same substrate, we engineered two bifunctional enzymes that redirect metabolic flux toward the synthetic pathway. Taken together, our work demonstrates a new approach to the engineering of novel synthetic pathways in the cell.

Directed evolution of AraC for improved compatibility of arabinose- and lactose-inducible promoters.

Synthetic biological systems often require multiple, independently inducible promoters in order to control the expression levels of several genes; however, cross talk between the promoters limits this ability. Here, we demonstrate the directed evolution of AraC to construct an arabinose-inducible (P(BAD)) system that is more compatible with IPTG (isopropyl-beta-D-1-thiogalactopyranoside) induction of a lactose-inducible (P(lac)) system. The constructed system is 10 times more sensitive to arabinose and tolerates IPTG significantly better than the wild type. Detailed studies indicate that the AraC dimerization domain and C terminus are important for the increased sensitivity of AraC to arabinose.