The logics of metabolic regulation in bacteria challenges biosensor-based metabolic engineering

Abstract

Synthetic Biology (SB) aims at the rational design and engineering of novel biological functions and systems. By facilitating the engineering of living organisms, SB promises to facilitate the development of many new applications for health, biomanufacturing, and the environment. Over the last decade, SB promoted the construction of libraries of components enabling the fine-tuning of genetic circuits expression and the development of novel genome engineering methodologies for many organisms of interest. SB thus opened new perspectives in the field of metabolic engineering, which was until then mainly limited to (over)producing naturally synthesized metabolic compounds. To engineer efficient cell factories, it is key to precisely reroute cellular resources from the central carbon metabolism (CCM) to the synthetic circuitry. This task is however difficult as there is still significant lack of knowledge regarding both the function of several metabolic components and the regulation of the CCM fluxes for many industrially important bacteria. Pyruvate is a pivotal metabolite at the heart of the CCM and a key precursor for the synthesis of several commodity compounds and fine chemicals. Numerous bacterial species can also use it as a carbon source when present in the environment but bacterial, pyruvate-specific uptake systems were to be discovered. This is an issue for metabolic engineering as one can imagine to make use of pyruvate transport systems to replenish synthetic metabolic pathways towards the synthesis of chemicals of interest. Here we describe a recent study (MBio 8(5): e00976-17), which identified and characterized a pyruvate transport system in the Gram-positive (G^+ve) bacterium Bacillus subtilis, a well-established biotechnological workhorse for the production of enzymes, fine chemicals and antibiotics. This study also revealed that the activity of the two-component system (TCS) responsible for its induction is retro-inhibited by the level of pyruvate influx. Following up on the open question which is whether this retro-inhibition is a generic mechanism for TCSs, we will discuss the implications in metabolic engineering.

Keywords: Bacillus subtilis; biosensor; catabolite repression; malate; metabolic engineering; pyruvate transport; synthetic Biology; two-component systems.

Figure 1. FIGURE 1: The bacterial pyruvate transport system PftAB and its complex regulation by the two-component system LytST.

(A) The pyruvate facilitated transporter PftAB of B. subtilis can either import or export pyruvate depending on the concentration gradient of pyruvate across the cell membrane. (B) E. coli cell death or cell growth inhibition increases with the level of heterologous expression of pftAB. (C) Expression of pftAB in WT (grey), ΔpftAB mutant (blue), and pftAB over-expressing (red) B. subtilis cells grown in minimal medium with glutamate and succinate as carbon source, and pyruvate concentrations ranging from 0.1 to 100 mM (left panel). Extracellular pyruvate acts as the signal molecule for LytST, which induces expression of pftAB. However, when the pyruvate influx is high, LytST activity is drastically retro-inhibited. Consistently, in the ΔpftAB mutant, the level of induction is maximal as there is no influx of pyruvate (right panel). (D) In metabolic engineering, the expression of one (or more) gene(s) of interest (GOI) is (are) under the control of promoter(s) that can be activated by the use of inducer metabolite(s) (M). The activity of a heterologously expressed TCS may be retro-inhibited by the inducer or derivative metabolites (M or M') if naturally present in the host cell (left panel). As a result, the TCS-induced expression of a synthetic circuit will not exhibit a log-linear dose response as M increases. The distortion between the expected (wanted) and effective (unwanted) induction challenges the rational design of novel nature-inspired sensors (right panel).