Feedforward growth rate control mitigates gene activation burden

Abstract

Heterologous gene activation causes non-physiological burden on cellular resources that cells are unable to adjust to. Here, we introduce a feedforward controller that actuates growth rate upon activation of a gene of interest (GOI) to compensate for such a burden. The controller achieves this by activating a modified SpoT enzyme (SpoTH) with sole hydrolysis activity, which lowers ppGpp level and thus increases growth rate. An inducible RelA+ expression cassette further allows to precisely set the basal level of ppGpp, and thus nominal growth rate, in any bacterial strain. Without the controller, activation of the GOI decreased growth rate by more than 50%. With the controller, we could activate the GOI to the same level without growth rate defect. A cell strain armed with the controller in co-culture enabled persistent population-level activation of a GOI, which could not be achieved by a strain devoid of the controller. The feedforward controller is a tunable, modular, and portable tool that allows dynamic gene activation without growth rate defects for bacterial synthetic biology applications.

A blueprint for a synthetic genetic feedback optimizer

“…Closed loop performance is evaluated in the presence of stochastic noise impacting the kinetics of all species. Cellular stress is modulated via β p 63 . a Growth rate is negatively impacted by rising levels of the alarmone (p)ppGpp ( p ) as it downregulates the production of ribosomes ( z ).…”

“…Cellular stress results in elevated RelA ( r ) expression, which upregulates the synthesis of (p)ppGpp via the increased production rate constant β p . Conversely, (p)ppGpp concentration can be decreased via SpoTH ( s ) by activating its expression either exogenously 63 or by placing its promoter under the control of the regulator x . The dashed red flat headed arrows from SpoTH represent the load that SpoTH expression places on ribosomes as its mRNA is translated.…”

“…Leveraging this, the feedforward controller developed in ref. 63 and outlined in Fig. 8a can be deployed to exogenously adjust growth rate via SpoTH.…”

“…While expression of SpoTH has a positive impact on growth rate at moderate levels as a result of (p)ppGpp hydrolysis 64 , its overexpression can result in considerable metabolic burden, thus giving rise to the non-monotonic relationship between SpoTH expression and growth rate observed in ref. 63 and illustrated in Fig. 8b .…”

Microbial cell factory optimisation using genome-wide host-pathway interaction screens

“…The importance of host:pathway interactions has long been recognised (Cardinale and Arkin, 2012; de Lorenzo, 2011) and has motivated the development of a “host-aware” concept in synthetic biology (Boo et al, 2019). Focusing on the expression constructs themselves, successful strategies have sought to isolate them (Darlington et al, 2018; Lou et al, 2012; Meyer et al, 2015) or to minimise the burden they create on their host (Barajas et al, 2022; Ceroni et al, 2018). The importance of the host and its impact on the behaviour of engineered sequences is also increasingly being recognised (Calero and Nikel, 2019; Chan et al, 2023; Khan et al, 2020; Tas et al, 2021).…”