Substrate‐activated expression of a biosynthetic pathway in Escherichia coli

Abstract

BackgroundMicrobes can facilitate production of valuable chemicals more sustainably than traditional chemical processes in many cases: they utilize renewable feedstocks, require less energy intensive process conditions, and perform a variety of chemical reactions using endogenous or heterologous enzymes. In response to the metabolic burden imposed by production pathways, chemical inducers are frequently used to initiate gene expression after the cells have reached sufficient density. While chemically inducible promoters are a common research tool used for pathway expression, they introduce a compound extrinsic to the process along with the associated costs. We developed an expression control system for a biosynthetic pathway for the production of D-glyceric acid that utilizes galacturonate as both the inducer and the substrate, thereby eliminating the need for an extrinsic chemical inducer. Methods and ResultsActivation of expression in response to the feed is actuated by a galacturonate-responsive transcription factor biosensor. We constructed variants of the galacturonate biosensor with a heterologous transcription factor and cognate hybrid promoter, and selected for the best performer through fluorescence characterization. We showed that native E. coli regulatory systems do not interact with our biosensor and favorable biosensor response exists in the presence and absence of galacturonate consumption. We then employed the control circuit to regulate the expression of the heterologous genes of a biosynthetic pathway for the production D-glyceric acid that was previously developed in our lab. Productivity via substrate-induction with our control circuit was comparable to IPTG-controlled induction and significantly outperformed a constitutive expression control, producing 2.13±0.03 g/L D-glyceric acid within six hours of galacturonate substrate addition. ConclusionThis work demonstrated feed-activated pathway expression to be an attractive control strategy for more readily scalable microbial biosynthesis.

Biosensor development for single-cell detection of glucuronate

“…1 b). This range was comparable by order of magnitude to a number of other TF-based biosensor systems (Hanko et al, 2018 ; Liu C. et al, 2018 ; Ni et al, 2021 ; Siedler et al, 2014 ). The operational range of the biosensor spanned from 0.01 to 1 g/L glucuronate.…”

“…To improve glucuronate yields in E. coli , we aimed to develop an HTS tool to aid the search through a library of MIOX enzymes for an improved variant. This was facilitated by previous work in our lab in which a genetic circuit using ExuR from Bacillus subtilis was constructed to regulate pathways in response to the presence of galacturonate (Ni et al, 2021 ). Though Ni et al characterized the biosensor in response to galacturonate, in published literature there is evidence that this TF also responds to glucuronate (Mekjian et al, 1999 ), making it an ideal candidate for a biosensor for improved glucuronate yields from MIOX.…”

“…The ExuR biosensor as designed by Ni et al ( 2021 ), P1OO, constitutively expresses exuR using promoter BBa_J23117 from the Anderson promoter library ( http://parts.igem.org/Promoters/Catalog/Anderson ) and expresses sfgfp using a hybrid promoter based on the insulated proD promoter (Davis et al, 2011 ) with two ExuR binding sites (Fig. 1 a).…”

“…One operator is located in between the −10 and −35 σ 70 RNA polymerase recognition sites, and the other is downstream of the −10 site. This configuration was found to have the lowest leakiness and the highest dynamic range (Ni et al, 2021 ). Upon binding to these two operators, ExuR will repress the sfgfp expression.…”

Low-Temperature Production of Glyceric Acid from Biomass-Based Sugar via the Cooperative Roles of MgO and NaBF4

“…Glycerol , and biomass-based monosaccharides , are the main raw materials for the current production of glyceric acid. For glycerol transformation to glyceric acid, chemo-catalysis with the use of a noble-metal catalyst achieves a satisfactory yield.…”