An automated Design-Build-Test-Learn pipeline for enhanced microbial production of fine chemicals

The microbial production of fine chemicals provides a promising biosustainable manufacturing solution that has led to the successful production of a growing catalog of natural products and high-value chemicals. However, development at industrial levels has been hindered by the large resource investments required. Here we present an integrated Design–Build-Test–Learn (DBTL) pipeline for the discovery and optimization of biosynthetic pathways, which is designed to be compound agnostic and automated throughout. We initially applied the pipeline for the production of the flavonoid (2S)-pinocembrin in Escherichia coli, to demonstrate rapid iterative DBTL cycling with automation at every stage. In this case, application of two DBTL cycles successfully established a production pathway improved by 500-fold, with competitive titers up to 88 mg L−1. The further application of the pipeline to optimize an alkaloids pathway demonstrates how it could facilitate the rapid optimization of microbial strains for production of any chemical compound of interest.

library was generated through a DoE approach by varying promoters, gene arrangement and plasmid copy number. Plasmids were assembled by ligase cycling reaction, transformed into E. coli DH5α, then grown in triplicate in TBsb media (0.4% glycerol) at 30 °C. Cultures were quenched and processed for analysis 24 h after pathway induction by IPTG.
Supplementary Data 4. Quantified pinocembrin titers and cinnamic acid peak values (mean, standard deviation, standard error of the mean, median, interquartile range), and mean OD600 values at induction and harvest points, for the combinatorial plasmid library of the second DBTL round. The library is a full factorial focused library for selected factors that were identified as having the highest effect on pinocembrin titers in the first DBTL round. Plasmids were assembled by ligase cycling reaction, transformed into E. coli DH5α, then grown in triplicate in TBsb media (0.4% glycerol) at 30 °C. Cultures were quenched and processed for analysis 24 h after pathway induction by IPTG.

Supplementary Data 5.
Quantified pinocembrin titers and cinnamic acid peak values (mean, standard deviation, standard error of the mean, median, interquartile range), and mean OD600 values at induction and harvest points, for chassis selection experiments. The three best performing constructs from the second DBTL cycle (plasmids 3353, 3382, and 3391) were screened in triplicate in a library of E. coli strains (Table 1) grown at 30 °C in TBsb media supplemented with 0.4% glycerol.
Cultures were quenched and processed for analysis 24 h after pathway induction by IPTG.
Supplementary Data 6. Quantified pinocembrin and cinnamic acid titers (mean, standard deviation, standard error of the mean, median, interquartile range), and mean OD600 values at induction and harvest points, for media selection experiments. The best performing construct (plasmid 3382) was screened in quadruplicate in E. coli MG1655 and MDS42 strains grown at 30 °C in the indicated media supplemented with 0.4% glycerol. Cultures were quenched and processed for analysis 24 h after pathway induction by IPTG.

Supplementary Data 7.
Quantified pinocembrin titers and cinnamic acid titers (mean, standard deviation, standard error of the mean, median, interquartile range), and mean OD600 values at induction and harvest points, for titer optimization experiments. The chromosomal fabF gene was knocked out in E. coli MG1655 and MDS42, then these strains were transformed with the best performing construct (plasmid 3382). To elucidate the optimal induction point for pinocembrin production, cultures were screened in quadruplicate following induction at different OD600 values (Odi). Cultures were quenched and processed for analysis 24 h after pathway induction by IPTG.
Supplementary Data 8. Quantified reticuline and scoulerine titers (mean, standard deviation, standard error of the mean, median, interquartile range), and mean OD600 values at induction and harvest points, for the combinatorial plasmid library. The library was generated through a DoE approach by varying promoters, gene arrangement and plasmid copy number. Plasmids were assembled by ligase cycling reaction, transformed into E. coli DH5α, then grown in triplicate in phosphate-buffered TB media (0.4% glycerol) at 30 °C. Cultures were quenched and processed for analysis 24 h after THP substrate was added to IPTG induced cells.