Xue, F; (2021) A synthetic biology toolbox for the screening of outer membrane proteins involved in n-alkane uptake in Escherichia coli. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Advances in synthetic biology enable redesigning microbial cell factories to produce value-added compounds thus creating the basis for a sustainable future. Achieving this goal requires the engineering of the mass transfer process, the availability of substrate and the effective efflux of the products. The transport across the membrane has been identified to be one of the principal bottlenecks in both whole-cell alkane biocatalysis and biosynthesis. However, the mechanisms involved have not been elucidated. A synthetic biology toolbox based on genetically encoded biosensors represents a promising approach for the non-invasive high-throughput detection and quantification of compounds transported across the cell membrane. Here, a novel experimental strategy that uses biosensors to identify outer membrane proteins relevant to n-alkanes uptake in Escherichia coli was suggested. Firstly, two transcription factor-based biosensors, namely as alkS-PalkB and alkR-PalkM biosensor, were characterised to demonstrate the functionality in n-alkane detection both in vivo and in vitro. The performance of the alkS-PalkB biosensor priginated from Pseudomonas putida was highlighted in a rapid response (within 2 hours) and high sensitivity to C8 (at 0.1 µM) yet a rather limited substrate range (C6 to C11). The alkR-PalkM biosensor Acinatobacter baylyi was superior in its broad substrate range (C7 to C16) yet showed a slow response (14 hours) and a lower sensitivity to C8 (at 5 µM). The comparison of the substrate profiles of the biosensors in vivo and in vitro also enabled the identification of permeability through the membrane of E. coli for n-alkanes (C16). The biosensor was then deployed in screening the selected single-gene deletion mutants by comparing the induction fold of the alkane-responsive elements in the biosensor with that of the parental strain. Deletion of fadL, ompC, ompF and ompN ORFs coding for outer membrane proteins led to a drop in the signal intensity, implying a lower alkane bioavailability in those gene-deletion mutants. The up take function was then restored by the complementation with the candidate genes encoded on a plasmid with a controlled expression level. The identified genes coding for the outer membrane proteins were then deleted from the chromosome of E. coli to develop a host cell with a reduced n-alkane uptake. The triple outer membrane protein deletion strain (E. coli ∆fadL ∆ompC ∆ompF) showed a largely reduced alkanes uptake, exhibiting an induction ratio to C8 and C12 from the alkR-PalkM biosensor at nearly 1. The host cell with a reduced n-alkane uptake was then applied in the screening of 10 putative outer membrane proteins from the OmpW family for n-alkane uptake. Among the 10 putative outer membrane proteins, three were capable of transporting all the n-alkanes tested from C8 to C14, namely BSF79 from Litorimicrobium taeanense, AB from Alcanivorax borkumensis and 2×27 from Pseudomonas aeruginosa. NQ from Pseudomonas putida also showed to be able to transport the n-alkanes from C8 to C13. SHE from Shewenalla sp. and MARHY from Marinobacter hydrocarbonoclasticus were found to be only accepting n-alkanes from C8 to C10 as substrates. Results from this work demonstrated how biosensors can allow the identification and elucidation of the transport process of alkanes in E. coli. The strategy can be tailored to understand the transport process of other compounds in any other bacterial chassis as well as screening effective transporters for certain compounds given the availability of large numbers of natural biosensors.

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