Rapid and Simultaneous Screening of Pathway Designs and Chassis Organisms, Applied to Engineered Living Materials
Abstract
Achieving a high product titer through pathway optimization often requires screening many combinations of enzymes and genetic parts. Typically, a library is screened in a single chassis that is a model or production organism. Here, we present a technique where the library is first introduced into B. subtilis XPORT, which has the ability to transfer the DNA to many Gram-positive species using an inducible integrated conjugated element (ICE). This approach is demonstrated using a two-gene pathway that converts tyrosine to melanin, a pigment biopolymer that can serve as a protective coating. A library of 18 pathway variants is conjugated by XPORT into 18 species, including those isolated from soil and industrial contaminants. The resulting 324 strains are screened and the highest titer is 1.2 g/L in B. amyloliquefaciens BT16. The strains were evaluated as co-cultures in an industrial process to make mycelia-grown bulk materials, where the bacteria need to be productive in a stressful, spatially non-uniform and dynamic environment. B. subtilis BGSC 3A35 is found to perform well under these conditions and make melanin in the material, which can be seen visually. This approach enables the simultaneous screening of genetic designs and chassis during the build step of metabolic engineering.
Document Details
- Document Type
- Technical Report
- Publication Date
- Jan 16, 2021
- Accession Number
- AD1193837
Entities
People
- Christopher Voigt
- Damen D. Schaak
- Dana Neitzey
- Dechuan Meng
- Matthew Lucht
- Nikita Mukhitov
Organizations
- University of California, Santa Barbara