Systems Metabolic Engineering Strategies: Integrating Systems and Synthetic Biology with Metabolic Engineering.

Metabolic engineering allows development of microbial strains efficiently producing chemicals and materials, but it requires much time, effort, and cost to make the strains industrially competitive. Systems metabolic engineering, which integrates tools and strategies of systems biology, synthetic biology, and evolutionary engineering with traditional metabolic engineering, has recently been used to facilitate development of high-performance strains. The past decade has witnessed this interdisciplinary strategy continuously being improved toward the development of industrially competitive overproducer strains. In this article, current trends in systems metabolic engineering including tools and strategies are reviewed, focusing on recent developments in selection of host strains, metabolic pathway reconstruction, tolerance enhancement, and metabolic flux optimization. Also, future challenges and prospects are discussed.

Markerless gene knockout and integration to express heterologous biosynthetic gene clusters in Pseudomonas putida.

Pseudomonas putida has gained much interest among metabolic engineers as a workhorse for producing valuable natural products. While a few gene knockout tools for P. putida have been reported, integration of heterologous genes into the chromosome of P. putida, an essential strategy to develop stable industrial strains producing heterologous bioproducts, requires development of a more efficient method. Current methods rely on time-consuming homologous recombination techniques and transposon-mediated random insertions. Here we report a RecET recombineering system for markerless integration of heterologous genes into the P. putida chromosome. The efficiency and capacity of the recombineering system were first demonstrated by knocking out various genetic loci on the P. putida chromosome with knockout lengths widely spanning 0.6-101.7 kb. The RecET recombineering system developed here allowed successful integration of biosynthetic gene clusters for four proof-of-concept bioproducts, including protein, polyketide, isoprenoid, and amino acid derivative, into the target genetic locus of P. putida chromosome. The markerless recombineering system was completed by combining Cre/lox system and developing efficient plasmid curing systems, generating final strains free of antibiotic markers and plasmids. This markerless recombineering system for efficient gene knockout and integration will expedite metabolic engineering of P. putida, a bacterial host strain of increasing academic and industrial interest.