Toward industrial production of isoprenoids in Escherichia coli: Lessons learned from CRISPR‐Cas9 based optimization of a chromosomally integrated mevalonate pathway

Abstract

Escherichia coli has been the organism of choice for the production of different chemicals by engineering native and heterologous pathways. In the present study, we simultaneously address some of the main issues associated with E. coli as an industrial platform for isoprenoids, including an inability to grow on sucrose, a lack of endogenous control over toxic mevalonate (MVA) pathway intermediates, and the limited pathway engineering into the chromosome. As a proof of concept, we generated an E. coli DH1 strain able to produce the isoprenoid bisabolene from sucrose by integrating the cscAKB operon into the chromosome and by expressing a heterologous MVA pathway under stress-responsive control. Production levels dropped dramatically relative to plasmid-mediated expression when the entire pathway was integrated into the chromosome. In order to optimize the chromosomally integrated MVA pathway, we established a CRISPR-Cas9 system to rapidly and systematically replace promoter sequences. This strategy led to higher pathway expression and a fivefold improvement in bisabolene production. More interestingly, we analyzed proteomics data sets to understand and address some of the challenges associated with metabolic engineering of the chromosomally integrated pathway. This report shows that integrating plasmid-optimized operons into the genome and making them work optimally is not a straightforward task and any poor engineering choices on the chromosome may lead to cell death rather than just resulting in low titers. Based on these results, we also propose directions for chromosomal metabolic engineering.

Mechanistic Modeling of Biochemical Systems Without A Priori Parameter Values Using the Design Space Toolbox v.3.0

“…This decreases strain fitness and can ultimately lead to cellular death (Dahl et al 2013, George et al 2014, Alonso-Gutierrez et al 2017. DST3 is able to identify regions in the parameter space leading to metabolic imbalances and to provide clues to rectify these phenotypes.…”

“…Imbalances are frequently encountered in the metabolism of engineered microbial strains (Dahl et al 2013, George et al 2014, Alonso-Gutierrez et al 2017) and in inborn metabolic diseases such as phenylketonuria (Levy 1999) and maple syrup urine disease (Haymond et al 1973), often by the excretion of some metabolite. Consider the simplest example of a metabolic pool as shown in Fig.…”

Techniques for chromosomal integration and expression optimization in Escherichia coli

“…The replacement of each of the native promoters with a strong promoter in the spectinabilin gene cluster from Streptomyces orinoci resulted in the successful activation of the spectinabilin pathway, as well as the improved production of spectinabilin in a heterologous host from an undetectable amount to a high level (Shao et al, ). Thus, the set of strong promoters, such as T7 (Alonso‐Gutierrez et al, ; Borgeaud & Blokesch, ; Studier & Moffatt, ), ParaB , PrhaB , PrhaS , Ptac , Psyn1 , or Psyn4 (Haldimann & Wanner, ), that are suitable for the RNA polymerase in different bacterial cell hosts can be changed to optimize expression levels. For instance, a set of insulated bacterial promoters (Davis, Rubin, & Sauer, ) has been designed for stimulatory or repressive effects, which are likely to be useful when designing expression cassette elements.…”

Characterization and repurposing of the endogenous Type I-F CRISPR-Cas system ofZymomonas mobilisfor genome engineering

“…In particular, they have enabled rapid, easy yet accurate construction of strains for some production platform microorganisms, e.g. Saccharomyces cerevisiae and Escherichia coli (15)(16)(17)(18), contributing significantly to the bio-based economy. For the bio-based economy, other alternative production microorganisms are also essentially required to generate sustainable alternatives for yielding green chemicals and biofuels from environmentally friendly resources, and become increasingly important.…”