Discovery, Engineering, and Application of Transport Systems via Adaptive Laboratory Evolution

The influx and efflux of ions and small molecules across biological membranes is a fundamental process for cellular homeostasis and survival in all domains of life. Not surprisingly, of all sequenced bacterial, archaeal, and eukaryotic genomes, over 10% of the encoded proteins are annotated for transport functions.

A change in the expression levels of membrane transport can have dramatic consequences on cell function. Thus, membrane transporters have been used as potential targets for biotechnological and medical applications. In microbial cell factories, transporters can be engineered to drive the influx toward product or the efflux of product in the extracellular space of fermenters.

In addition, the emergence of efflux-based drug resistance mechanisms causes failure in treatment of infectious diseases. However, harnessing membrane transporters in biotechnology applications or combating them in pathogens involves answering two questions: i) which small molecules use which transporters, and ii) what are the molecular details underlying their transport activities? Despite the methodological progress achieved so far, functional redundancy of transport proteins and their broad specificity (i.e., promiscuity) are two outstanding challenges to answer these questions.

As a result, more than 30% of reported membrane transporter families are either poorly characterized or lack adequate functional annotation. Therefore, methods for both assigning functional roles to membrane transporters and modulating the activities of these transporters in vivo are needed. In this thesis, tolerance adaptive laboratory evolution (TALE) was introduced as an alternative approach to address both challenges in vivo.

TALE was hypothesized to generate tolerance phenotypes based on selection and enrichment of transporter mutants under an external stress environment. Further, the genetic basis underlying these phenotypes was revealed by genome resequencing, highlighting the functionality of transport genes and mutational mechanisms that regulate their activities in the context of a functioning cell.

Throughout the thesis, the TALE approach was utilized to cover the biotechnological and medical relevance of membrane transporters in two separate studies. The first study was conducted to establish the utility of TALE to discover transport systems for a set of amino acids in Escherichia coli. Specific amino acid transport systems (i.e., specific genes) were successfully revealed without requiring a priori knowledge, including uncovering two novel assignments.

The findings show that specific modulating mutations can be uncovered, and reverse engineered for application of the generated phenotype. Additionally, the results show that the mutated strains themselves can be generated with useful tolerant phenotypes. In the second study, functional redundancy of multidrug transporters and their promiscuous nature in E. coli were elucidated under lipophilic cation stress.

The results highlighted the activity of nine redundant multidrug transporters spanning different multidrug transport families. In addition, the identified adaptive mechanisms inferred insightful molecular details of multidrug recognition and transport. Such in vivo demonstration can provide guiding principles for identifying drug export pathways and consequently aid in the rational design of multidrug transporters’ inhibitors to efficiently combat efflux-based resistance mechanisms.

Overall, the results demonstrate the marked efficiency of TALE as a systematic and straightforward tool to enhance the knowledge base of functionally characterized transport systems. The broad applicability of TALE to a range of microbes holds great promise for the identification of transporters for a range of molecules and microbes of interest.

Ultimately, the TALE approach can be used as a tool to drive basic discovery efforts of membrane transporters that have biotechnological and medical relevance.