Pathway optimization and balancing for the improved microbial production of high-value chemicals

Abstract

Moving forward, the work contained here lays a solid framework for the optimization of chemical production pathways in E. coli. The techniques presented below, both genetic and fermentation-based, highlight the methods available to probe the sensitivity of microbial systems allowing for more in-depth analysis to be undertaken. It is clear that efforts in pathway optimization and balancing will be key to the smooth transition of microbially based chemical production platforms from the laboratory to the marketplace.

There is no doubt that the development of microbial cell factories for the renewable production of a wide variety of chemicals will prove to be ever more important as the demand for natural products surpass the supply from their traditional sources. This motivates the work presented below which focuses on methods to optimize and improve microbial production hosts to achieve higher titers, yields, and productivities for a variety of natural products. In Chapter 1, we review the methods for pathway optimization and balancing from both a genetic and fermentation perspective. In subsequent chapters, we demonstrate proof of principle studies on how these tools and techniques can be used to improve natural product titers in E. coli.

In chapter 2, we begin with a case study for the production of the flavonoid natural product, catechin, from eriodictyol. In this work we combined several modes of pathway balancing including gene homolog screening, DNA-copy number optimization, and post-translational balancing, ultimately resulting in near gram-scale production of catechin, an 19-fold improvement in titer over previous efforts in the literature. Although impressive, the bulk of the improvement from this study was achieved through the selection of new gene sources, while the increases resulting from other forms of balancing, although statistically significant, were moderate in comparison.

Building upon this work, in chapter 3 we developed a method for the combinatorial construction of transcriptionally varied pathways, named ePathOptimize. We began by developing a library of mutant T7 promoters using site-directed mutagenesis targeted to the binding strength region of the consensus T7 promoter. Using five promoters of varied strength, and the previously developed ePathBrick cloning platform, we developed a protocol for the combinatorial cloning of entire pathways on a single plasmid such that a promoter of different strength controls each gene. As a proof of principle, we applied this method to the five-gene violacein pathway and after genetic and fermentation optimization were able to demonstrate titers up to 1.83 g/L, a 2.6-fold improvement in titer and a 30-fold improvement in productivity over previously published efforts.

In an attempt to demonstrate the wide applicability of the ePathOptimize system, we then used it to improve titers from p-coumaric acid to naringenin (Chapter 4) and to improve growth of E. coli on methanol and subsequently improve ¹³C labeling from methanol to the high-value flavonoid product, naringenin (Chapter 6). Although we found that many pathways can benefit from transcriptional optimization, we did see that the application of ePathOptimize to improve the hydroxylation of naringenin resulted in no improvements (Chapter 5). This demonstrates that the proverbial ‘silver bullet’ does not exist and each pathway and system will require a tailored optimization approach.

Taking advantage of the individually optimized modules for flavonoid production, we were able to develop a mixed culture fermentation approach to improve and extend upon flavonoid production in E. coli. Combining the pFlavoᵒᵖᵗ module from Chapter 4 and the p168 module from Chapter 2 we were able to demonstrate the production of afzelechin from p-coumaric acid with 970-fold improvement over previous published titers. Further extending upon this idea, we developed a phenylpropanoic acid production module and an anthocyanin production module that when combined with the previous co-culture, enabled the de novo production of flavan-3-ols and anthocyanidin-3-glucosides for the first time in a microbial host (Chapter 7).