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Promoter Sequence Determinants of Transcription Initiation Rates in Zymomonas mobilis
Author(s) -
Schiffman Allison,
Ghosh Indro,
Vera Jessica,
Landick Robert
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.05268
Subject(s) - zymomonas mobilis , isobutanol , promoter , computational biology , biology , transcription (linguistics) , synthetic biology , genetics , gene , biochemistry , gene expression , fermentation , linguistics , philosophy , ethanol fuel , ethanol
A modification to a naturally occurring ethanologenic pathway in a gram‐negative bacterium called Zymomonas mobilis has been engineered to produce isobutanol, an advantageous biofuel. However, low isobutanol yields remain an issue. Optimizing this pathway can be aided by a selective use of strong and weak promoters, but the determinants of promoter strength in Z. mobilis are unknown. Thus, a comprehensive database of promoter strengths will enable more accurate engineering of the isobutanol pathway and other synthetic pathways in Z. mobilis . Here, two libraries based on the rpoB ‐P1 promoter with modifications to the −10 and −35 elements were designed. One of the libraries contained modifications at the bases that are highly conserved and the other contained modifications at the bases that are less conserved. In total, 20,480 different promoter sequences were assembled into plasmids and transformed into Z. mobilis where they drive expression of a synthetic RNA containing a randomized 15‐base pair barcode. DNA sequencing data confirm that the libraries have assembled as expected and that ~70% of transformants into Z. mobilis are from the designed libraries. This result has established the database of promoter sequences and corresponding barcodes. The quantity of barcoded‐RNA produced will now be used as a readout for strength of the associated promoter. Promoters selected from the complete library of promoter strengths will then be used to optimize the isobutanol pathway in Z. mobilis .