A gene network engineering platform for lactic acid bacteria.
Recent developments in synthetic biology have positioned lactic acid bacteria (LAB) as a major class of cellular chassis for applications. To achieve the full potential of LAB, one fundamental prerequisite is the capacity for rapid engineering of complex gene networks, such as natural biosynthetic pathways and multicomponent synthetic circuits, into which cellular functions are encoded. Here, we present a synthetic biology platform for rapid construction and optimization of large-scale gene networks in LAB. The platform involves a copy-controlled shuttle for hosting target networks and two associated strategies that enable efficient genetic editing and phenotypic validation. By using a nisin biosynthesis pathway and its variants as examples, we demonstrated multiplex, continuous editing of small DNA parts, such as ribosome-binding sites, as well as efficient manipulation of large building blocks such as genes and operons. To showcase the platform, we applied it to expand the phenotypic diversity of the nisin pathway by quickly generating a library of 63 pathway variants. We further demonstrated its utility by altering the regulatory topology of the nisin pathway for constitutive bacteriocin biosynthesis. This work demonstrates the feasibility of rapid and advanced engineering of gene networks in LAB, fostering their applications in biomedicine and other areas.
Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
Wentao
W
Kong
Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
Venkata S
VS
Kapuganti
Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA Department of Physics, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA luting@illinois.edu.
Ting
T
Lu
D001665:Binding Sites; D053898:Biosynthetic Pathways / Q000235:genetics*; D053263:Gene Regulatory Networks; D005818:Genetic Engineering; D019344:Lactic Acid / Q000378:metabolism; D007778:Lactobacillus / Q000235:genetics* / Q000378:metabolism; D012270:Ribosomes / Q000235:genetics / Q000378:metabolism; D058615:Synthetic Biology
D019344:Lactic Acid