A glucose-sensing toggle switch for autonomous, high productivity

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A glucose-sensing toggle switch for autonomous, high productivity genetic control William Henry Bothfeld, Grace Kapov, and Keith Tyo ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.6b00257 • Publication Date (Web): 08 Mar 2017 Downloaded from http://pubs.acs.org on March 8, 2017

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ACS Synthetic Biology

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A glucose-sensing toggle switch for autonomous, high productivity genetic control

William Bothfeld, Grace Kapov, Keith E.J. Tyo*

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Department of Chemical and Biological Engineering E-136, Northwestern University, 2145 Sheridan road, Evanston, IL, USA

Manuscript in preparation for: ACS SynBio

*Corresponding author: Telephone: +1 847 868 0319 Fax: +1 847 491 3728 email: [email protected]

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ACS Paragon Plus Environment


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ABSTRACT

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Many biosynthetic strategies are coupled to growth, which is inherently limited, as (1)

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excess feedstock (e.g., sugar) may be converted to biomass, not product, (2) essential genes must be

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maintained and (3) growth toxicity must be managed. A decoupled growth and production phase

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strategy could avoid these issues. We have developed a toggle switch that uses glucose sensing to

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enable this two-phase strategy. Temporary glucose starvation precisely and autonomously activates

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product expression in rich or minimal media, obviating the requirement for expensive inducers. The

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switch remains stably in the new state even after reintroduction of glucose. In the context of

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polyhydroxybutyrate (PHB) biosynthesis, our system enables shorter growth phases and comparable

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titers to a constitutively expressing PHB strain. This two-phase production strategy, and specifically

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the glucose toggle switch, should be broadly useful to initiate many types of genetic program for

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metabolic engineering applications.

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KEY WORDS [Glucose sensing, genetic toggle switch, PHB production]

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INTRODUCTION

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Great progress has been made in expanding the portfolio of chemicals that can be produced

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biologically1. However, efficient, economical bio-production of these chemicals remains a challenging

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barrier to shifting from a fossil-fuel economy to a renewable one. Currently, many bio-production

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strategies rely on growth-coupled production, because constitutive expression of the product

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pathway is more feasible than inducing production after growth.

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production limits product yield and productivity. Biomass can consume 20-60% of the carbon

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source2 across different cultivation techniques. This biomass consumption could be minimized if an

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extended production phase was maintained after cells reached a target biomass level. Furthermore,

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engineered pathways can cause cell-toxicity slowing growth, and limiting yields, titers and types of

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chemicals considered for production3. Because strains containing these toxic pathways have

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suboptimal growth, escape mutants with mutations that reduce productivity have a growth

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advantage, leading to overall lower batch yields4 (figure 1a).

However, growth-coupled

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Decoupling growth and production phases can address these limitations by preventing

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selection on burdensome pathways and enhancing growth rates in the growth phase, while allowing

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pathway expression levels in the non-growing, production phase that would otherwise inhibit

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growth. Essential enzymes can be eliminated in the production phase, greatly expanding the possible

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engineering targets. Chemicals toxic to certain growth-associated processes of the host could be

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produced using this two-phase strategy. A separate production phase could also alleviate limits of

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oxygen and nutrient uptake as biomass accumulates in growth-coupled platforms. Batch and fed-

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batch cultivations are discontinuous processes that involve repeated reactor prep and growth,

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followed by reactor breakdown and cleaning. A strategy that maintains an extended production

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phase could lessen reactor down-time by enabling longer, robust production within reasonable

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biomass and process parameters.

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A useful tool for decoupling growth and production would, upon transition to production

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phase, precisely activate a designed genetic program to express pathway enzymes and activate or 3



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inactivate genomic targets. On an industrial scale, expensive inducers such as Isopropyl β-D-1-

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thiogalactopyranoside (IPTG) and anhydrotetracycline (aTc) are infeasible. Instead, pathways are

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either (1) constitutive or (2) initiated by nutrient-limitation sensing. Constitutive expression of a

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product pathway causes metabolic burden, slowing growth. This is demonstrated in previous PHB

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studies4. Nutrient-limitation eliminates the slow growth by not expressing the product pathway until

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late in the culture. However, nutrient limitations may require the culture to be maintained in

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suboptimal conditions. For example, nutrient limitation strategies lower glucose uptake rate (GUR)

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by ~ 16, 10, 6, and 2 fold for nitrogen, phosphorus, sulfur and magnesium limitation, respectively5.

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Therefore, production strategies dependent on induction in these limiting regimes will limit carbon

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flux to desired enzyme pathways.

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rapidly switched to an ‘on’ state with complex regulation present6,7.

Finally induction in these systems is often graded, rather than

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Unlike simple induction, a genetic toggle switch could enable complete activation, to a new

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stable state, that would eliminate graded induction. The culture could stay in the production state

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even after the activating condition is gone due to the memory capacity encoded by the switch. The

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“new” switch state could be maintained stably during the entire production phase. A functional

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genetic toggle switch has been demonstrated8 and improved by Collins9, using inducers that are not

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appropriate for industrial scale (IPTG, aTc, arabinose or heat shock). A metabolic switch requiring

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IPTG to induce has been demonstrated using this toggle-switch framework for isopropanol

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production with success10. In production phase, expression of an essential gene (citrate synthase)

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was limited and pathway enzymes were induced to increase titer and yield 3.7 and 3.1 fold to

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~50mM isopropanol10. The same group has subsequently developed an auto-inducible metabolic

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switch using quorum sensing eliminating the need for IPTG in fermentations at industrial scales11.

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This approach is autonomous, but is not flexible. Significant effort would be required to tune the

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auto-inducer system to engage at different cell densities. Furthermore, the inducer was never fully

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removed from the system.

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Glucose levels may be a particularly useful inducer for such a toggle switch. Specifically,

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glucose starvation (1) causes robust gene activation12 (2) has a mode of action that is well studied13 4


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(3) is compatible with common glucose fed-batch systems14 and (4) occurs after a precise amount of

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biomass has been made in a wide range of media types (simple and complex). The glucose-starvation

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promoter would only toggle a switch event, so glucose could be subsequently reintroduced for

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production. This would be an improvement on current strategies that use native nutrient-sensing for

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induction but also drastically reduce metabolic rates.

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The basic architecture of the toggle switch includes regulatory genes and their

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corresponding target promoters, arranged to be mutually inhibitory (figure 1b). The version used in

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this work contains a LacI responsive promoter-(trcp) that expresses TetR and a TetR responsive

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promoter –(tetOp) promoter that expresses LacI; each repressor transcription factor (TF) is under

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control of the opposite promoter9. Reporter proteins mCherry and GFP signal which state the circuit

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is in. Each reporter has a degradation tag to allow observation of dynamic switching events upon

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addition of the chemical inducers IPTG or aTc through fluorescence. For the purposes of the specific

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way we cloned our pathway expression, we refer to LacI/GFP expression as ON and TetR/mCherry

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expression as OFF.

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Functionally, the encoded logic of native transcriptional regulation, such as glucose

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starvation, can be integrated into the toggle switch. Glucose starvation activates the catabolic

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response using cyclic-AMP (cAMP) receptor protein (CRP), one of the best-studied TF’s. Low glucose

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environments cause the cAMP-CRP complex to bind target DNA and regulate hundreds of gene

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targets15. There are 3 classes of CRP promoters, categorized by the number and placement of CRP

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binding sites within each. Class I promoters have one CRP binding site at various distances upstream

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of the -35 box which interact with the α-C Terminal Domain (α-CTD) of RNA Polymerase (RNAP)16.

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Class II promoters have CRP encompassing the -35 box, and interact with α-CTD and α-NTD of RNAP

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for promoter recruiting17.

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multiple interactions with cAMP-CRP are made. Class III promoters contain a mix of multiple Class I

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and II CRP sites16.

This site partially occludes the σ70 subunit of RNAP, and is likely why

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These CRP/glucose-sensitive promoters could be used to switch states in the synthetic

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genetic toggle switch. The switch occurs by using the transient glucose-starvation signal to express

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one of the toggle TF’s (LacI), resulting in a stable output from the genetic switch. The starvation

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event is encoded in the “memory” of the switch and is stable during the desired production phase,

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even after reintroduction of glucose. Importantly, this separates the sensing promoter from the

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production promoter, which isn’t possible using only nutrient-limitation induction of product

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pathways. A separate sensor and production promoter increases flexibility to design different

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expression levels of pathway enzymes without being constrained by the native capacity and kinetics

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of a nutrient sensing induction strategy.

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In this study, we have developed an industrially relevant auto-inducible genetic switch that

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responds to glucose availability to precisely time the expression of burdensome pathway enzymes

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for enhanced bio-production. We characterized the dynamics of a variety of glucose sensitive

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promoters. Select promoters were then integrated into a bi-stable toggle switch to utilize the cell’s

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native capacity to sense and respond to glucose starvation to activate a switching event. The

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resulting state was stable upon re-introduction of glucose (see figure 1c for genetic program). This

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glucose-sensitive switch was then used to autonomously express PHB pathway enzymes at levels

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that would severely limit growth, but when coordinated with initial glucose concentrations designed

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to induce PHB expression at a particular biomass level, improved growth by two-fold with

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comparable PHB production yields to a constitutively expressing system.

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MATERIALS AND METHODS

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STRAINS AND MEDIA

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Cloning was carried out in DH5α Escherichia coli and all experiments were conducted in

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wild-type MG1655 or MG1655 ΔlacI strains as indicated. pSB3C5 from the 2012 iGEM kit was used

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as a source for the p15A origin and chloramphenicol acetyl transferase (CAT). pKDL071 (a gift from

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the Collins Lab) was the source for gfpmut3b and lacI used in cloning and was the basis for the

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genetic toggle switch circuitry9. CRP promoters were from MG1655 gDNA and pAGL20 was the

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source of the phaECAB (PHB) operon18.

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Luria-Bertani Broth (LB)(Difco, New Jersey, USA) was used for cultivation during cloning.

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MOPS minimal (MOPS min) media was prepared according to Neihardt’s recipe19. Supplements for

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EZ rich defined MOPS media were purchased from Teknova (California, USA). Working

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concentrations of antibiotics and inducers used are as follows: kanamycin (25 mg/mL) and

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chloramphenicol (17 mg/L) from Sigma Aldrich, (St Louis, MO); IPTG (1 mM) and aTc (100 μg/L)

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from Promega (Madison, WI). All cloning enzymes were purchased from New England Biolabs

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(Medford, MA).

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CLONING

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The pWB8 base plasmid was created to facilitate swapping in select CRP promoters

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upstream of GFP to assay expression dynamics in response to glucose starvation. PCR products of

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the p15A origin, CAT, RBS:gfpmut3b and a variation of the synthetic promoter CC(-41.5)α(-63)20

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were generated using primers and source DNA as indicated in Table 1 and combined using Gibson

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assembly21. Primers for Gibson assembly were designed using J5 online software22.

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pWB8-crp variants were created using restriction-ligation cloning to insert CRP promoters

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amplified by PCR from MG1655 gDNA, using primers indicated in Table 1. Three versions of each

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promoter were generated: (1) “full length” promoters (crp) starting ~ 500 bp upstream of the open

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reading frame and ending at the transcription start site (2) minimal “truncated” promoters (T-crp)

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containing ~ 50 bp upstream of the transcription start site with care taken to include the full CRP

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binding site, and (3) enhanced truncated promoters (T-αcrp) which include a modified enhancer

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element from the CC(-41.5)α(-63) synthetic elements20 (see supplementary Table 1 for sequence)

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upstream of the T-crp promoters. pWB8-crp and T-crp variants were cloned using HindIII-BamHI

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digestion of the appropriate PCR products and pWB8 backbone, while pWB8Tα-crp variants were 7



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generated using EcoRI HindIII digests of the T-crp PCR product, and ligated into the pWB8 backbone

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cut with the same restriction enzymes.

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Two strategies were pursued to integrate glucose sensing into the toggle switch. First,

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pWB9-crp variants were created to express LacI from crpp promoters on a separate plasmid to

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enable glucose-limitation switching of the toggle switch. The RBS:lacI from pKDL071 was amplified

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by PCR and cloned into select pWB8 variants using HindII-PstI digestion. The resulting pWB9-crp

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variants were co-transformed with plasmid pKDL071 to create the “two plasmid” system. Secondly,

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pWB9 variants were digested with BamHI-SphI and this crpp:RBS:lacI fragment was ligated in place

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of the original RBS:lacI in the toggle switch. This resulted in the tetOp and crpp promoters in series,

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upstream of lacI, to create the “tandem promoter” test system (pKDL071-crp).

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A heterologous pathway under control of the toggle switch was created by inserting the

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phaECAB operon into pKDL071 in place of gfpmut3b. phaECAB was amplified by PCR with SphI-PstI

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overhangs and ligated into the pKDL071 backbone to create pKDL071-phaECAB. The resulting

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plasmid was co-transformed with pWB9 variants to create the two plasmid production system.

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Select pKDL071-crp variants were digested using BamHI-SphI and the crpp:lacI fragment was ligated

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into the pKDL071-phaECAB backbone downstream of tetOp, in place of the existing lacI to create the

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tetOp:crpp:lacI control element in the pKDL071-phaECAB production plasmid, resulting in the

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tandem promoter production system.

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CULTURE CONDITIONS

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Prior to inoculation of all experiments, all strains were streaked out on LB + antibiotic plates

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and single colonies were inoculated into an initial pre-culture of 5 mL of media (type is noted for

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each experiment) in 15 mL culture tubes, and incubated using a New Brunswick Scientific Innova44

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incubator (New Jersey, USA) maintained at 37°C and 250 rpm in a rack angled at 45°. The pre-

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culture was incubated for 12-14 hours and then diluted into a second pre-culture at 1/100 dilution

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into fresh media to return cells to exponential growth. The second pre-culture was then inoculated

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into the experimental culture (LB, LB + glucose, MOPS min + glucose, MOPS min + glucose and 8


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glycerol, MOPS EZ rich + glucose, MOPS EZ rich + glycerol or MOPS EZ rich + gluconate) as indicated.

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Additional culturing steps, as necessary, are described for each experiment.

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CRP-PROMOTER CHARACTERIZATION

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Promoter characterization was conducted in LB media with 0.0 to 0.4% glucose (w/v) as a

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carbon source, and cells were grown in a 96-well plate-reader. Plates with lids were maintained at

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37° C with 300 rpm agitation in an incubator, then manually transferred to the plate reader for

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OD600 and fluorescent measurements. Growth and fluorescence were measured every 30 minutes

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for up to 10 hours.

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TOGGLE SWITCH STABILITY TRIALS

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The stability of the OFF state of glucose toggle switch variants (two promoter and tandem

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promoter) was tested (supplemental figure 1, figure 3). Stable candidates were then tested for OFF

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and ON state stability (figure 3). First, colonies were inoculated in 3 mL of MOPS EZ rich + 0.2 %

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glucose with inducer (1mM IPTG for experimental strains and the OFF control, aTc for the ON

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control). These pre-cultures were incubated at 4° C, then warmed to 37° C and shaken at 250 rpm for

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6 hours to set the OFF state of the genetic switch and generate biomass. Pre-cultures were diluted

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1/100 into fresh media with 0.1 mM IPTG for ~ 2 hours (OFF and ON controls were cultured inducer-

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free from this step on), and these secondary pre-cultures were diluted 1/10 fresh media for another

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2 hours (tertiary pre-cultures) to maintain a glucose-rich environment. Finally, the tertiary pre-

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culture was diluted 1/100 in fresh media lacking inducer, and cells were considered inducer free

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(0.0001 mM IPTG shows no response in a dose-response curve8). This “set” the toggle-state to OFF in

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exponentially growing cultures. Culture was repeatedly transferred into fresh media for 6 hours to

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maintain exponential growth in a glucose rich environment in the absence of inducers. Cultures

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were then spun down at 4000 x g for 5 minutes, resuspended, and grown in LB for 1 hour to test

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glucose-limitation induced toggling of the switch. Culture was then diluted 1/100 in the original

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0.2% MOPS EZ rich media for 10 hours (with re-dilutions) to monitor the transition from the OFF to

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the ON state. Finally, culture was diluted 1/100 into MOPS EZ rich + 0.2 % glucose + 1mM IPTG to

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monitor the switch back to the OFF state (figure 3).

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Other media conditions that initiate a switch from the ON to the OFF state were tested for

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the TaraF tandem promoter glucose toggle variant. Colonies were inoculated in a 96-well plate with

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MOPS EZ rich + 0.1% glucose and 0.1 mM IPTG (aTc only for the ON control) for 3 hours. Culture was

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re-diluted into media without inducers for an additional 4 hours. Cultures were then diluted 1/100 in

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MOPS EZ rich with no extra carbon, 0.1% gluconate, or 0.1% glycerol, grown for 1 hour, then 1 µL of

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40% glucose was added. Cells were then grown for 6 hours in a plate reader to monitor the transition

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from the OFF to the ON state (figure 4).

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PHB PRODUCTION

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PHB production trials were conducted in 50 mL MOPS min with 0.2% or 0.4% glucose in 250

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mL baffled shake-flasks with foam tops for oxygen transfer, and were pre-cultured as described

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previously. 1-2 mL of 400 g/L glucose stock was added to the remaining culture volume at the onset

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of stationary phase (8-10 hours after inoculation of experimental culture, ~42 mL of culture

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remained due to volume lost from sampling and evaporation). Glycerol was added at 0.1% (v/v) to

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the starting media before inoculation, in a select few experiments to optimize PHB production, with

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either 0.05 or 0.1% starting glucose, as noted.

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Chemically induced PHB production experiments were inoculated as described with

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additional steps: 1 mM IPTG was added to the pre-culture to set the OFF state. The secondary pre-

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culture was grown for ~ 2 hours to return cells to exponential phase, then spun down at 4000 x g for

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5 minutes and re-suspended in inducer-free media, and inoculated into 50 mL of media at OD600 =

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0.01 to start each experimental culture. Varied PHB-pathway induction times were investigated:

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induction with aTc upon inoculation (0 h) and at the end of exponential phase (10 h). Un-induced

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controls (always off) and pre-culture induced controls (-14 h, no IPTG in pre-culture, induced with

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aTc) were included for comparison. A genomically integrated, 29-operon copy strain (KS29)23 was

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tested to bench-mark a constitutively expressing strain with near maximal pathway copy number;

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this represents a traditional growth-coupled metabolic engineering design approach.

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Autonomous PHB production experiments were cultured as described above, without

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addition of aTc for induction to express the PHB pathway. To mirror the chemical-induction trials, 2

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mL of stock glucose solution was added for the PHB production phase, at ~ 10, 9, or 5 hours

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(depending on cessation of growth in glycerol trials). In all trials, OD600/fluorescence, dry cell

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weight (DCW), PHB production and organic acid/ glucose consumption were monitored.

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ASSAYS

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OPTICAL DENSITY AND FLUORESCENCE

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OD600 and fluorescence were monitored using a Synergy HI Microplate Reader from Biotek

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using Gen5 V 2.04 software. OD600 was monitored at 600 nm, and GFP and mCHERRY fluorescence

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were monitored at 485nm:525nm and 585nm:615nm excitation:emission, respectively. 200 μL of

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culture was measured for plate-reader growth experiments and 100 μL was measured in triplicate

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for shake-flask cultures in 96-well black-walled Greiner plates (Sigma-Aldrich, St Louis, MO).

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GLUCOSE AND ORGANIC ACID ASSAYS

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Samples for organic acid analysis were collected and centrifuged for 10 minutes at 17000 x

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g. Sodium azide (Sigma-Aldrich, St. Louis, MO) was added at a final concentration of 0.1 mg/L to

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prevent subsequent cell growth. The supernatant was transferred into 1.5 mL auto-sampler vials

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and stored at -20 °C until HPLC analysis. Organic acids and glucose were measured on an HPLC

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Agilent 1200 series with: a Binary pumper with Degasser (G1379B), an 1290 Thermostat (G1330B),

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an hiP-ALS SL+ autosampler (G1367D) maintained at 4 °C, TCC SL (G1316B), DAD SL (G1315C), and

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1260 RID (G1362A). An isocratic method was used with 5mM filtered H2S04 at a 0.6 ml/min flow

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rate through a Biorad Aminex 87-H column maintained at 65 °C. 10 μL of sample was injected.

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Results were monitored and analyzed on Chemstation for LC 3D system Rev B.04.03[16] software.

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Glucose, citrate, pyruvate, succinate, lactate, formate, glycerol and acetate standards (Sigma-Aldrich,

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St. Louis, MO) ranging from 0.05-20 g/L were measured at the beginning and end of each run, and

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used to construct a calibration curve for quantification of organic acids and sugars.

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PHB ANALYSIS

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5-10 mL of culture was collected at indicated times for DCW and PHB quantification. The

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culture was centrifuged at 4000 x g for 10 minutes, washed twice with 1 mL of ddH20 and dried for

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24 hours in an 80 °C oven. PHB was hydrolyzed with 1 mL 95-98% pure H2SO4 (Sigma Aldrich, St

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Louis, MO) and boiled at 95 °C for one hour24. Samples were diluted to 5 mL with water, spun at

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15000 x g for 10 minutes and supernatant was diluted 1/10 or 1/100 in water for HPLC analysis.

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Approximately 5 mg of pure PHB (Sigma Aldrich, St Louis, MO) was processed in parallel, serially

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diluted to precise concentrations and used to construct a standard curve to quantify PHB.

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RESULTS & DISCUSSION

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SELECTING GLUCOSE RESPONSIVE PROMOTERS FOR EVALUATION

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To use glucose as a trigger for the proposed toggle switch, we first needed to identify

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promoters with strong activation due to glucose depletion.

CRP-promoters likely have varied

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dynamic responses to glucose limitation and may be under alternative forms of regulation as well. A

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range of CRP-responsive promoters was chosen to screen for dynamic activity. Candidate promoters

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were identified using the Ecocyc database and were chosen if there was evidence for CRP binding.

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Due to the large abundance of possible CRP promoters, we focused on Class II promoters, which limit

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the necessary size of the promoter, and promoters with few or no known binding sites for other TF’s

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to reduce complexity from other regulation. Each promoter was generated by PCR in three

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variations: (crp contains 500 upstream of the ORF; T-crp contains the CRP binding site ~50 bp

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upstream of the transcription start site; and Tα-crp combines an upstream enhancer element with T-

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crp). These promoters were cloned into the pWB8 plasmid to investigate the effects of limiting

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upstream elements (T-crp) or artificially enhancing expression (Tα-crp). 12


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DIFFERENT GLUCOSE-RESPONSIVE PROMOTERS HAVE DIFFERING ACTIVATATION

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CHARACTERISTICS

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A promoter with both a tight OFF-state and an ON-state with adequate expression during

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glucose-starvation is desirable to integrate sensing with the toggle switch to prevent early switching

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and avoid wasted translation resources, respectively. Therefore, time course trials were carried out

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to assay dynamic GFP activation upon glucose starvation for a variety of CRP-promoters. Strains

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with the different CRP promoters were cultivated in LB + 0.0, 0.05, and 0.4% glucose and GFP

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expression was monitored in the transition from growth to stationary phase (figure 2). Expression

306

varied within ~1 order of magnitude for most of the variants. The amplitude of activated CRP

307

promoters (by GFP expression) varied, ~ 15-fold between low and high expressing promoters (araF

308

vs. TychH), indicating a wide-range of expression capacity among the pool of constructed promoters.

309

Generally, T-crp and Tα-crp variants enhanced GFP expression, though sometimes increased baseline

310

levels (araF variants) and/or disrupted the dynamic range compared to wildtype (T-csiE and Tα-araF

311

promoters). There weren’t any discernable trends across all truncated or all full-length promoters,

312

likely due to complexity in the form of unknown regulation mechanisms. The promoter enhanced Tα-

313

crp variants had variable expression patterns compared to the crp and T-crp promoters, and may be

314

useful to test if trying to enhance promoter activity without altering regulation sites. This promoter

315

characterization should be a useful resource for stationary-phase induction applications.

316

GFP expression is due to glucose limitation and not a general stationary phase response, as

317

the 0.4% glucose condition was chosen to maintain excess glucose throughout the trial. (figure 2).

318

GFP expression is detectable for cultures lacking glucose (LB condition) at ~1.5 hours after

319

inoculation, while there is a lag in GFP expression in LB + 0.05% glucose trials until ~3- 4 hours.

320

Importantly, there is little GFP expression in the LB + 0.4% glucose condition beyond the baseline

321

level, even after cultures reach a stationary phase (growth curves not shown). Notably, growth rates

322

are comparable in all cases, which indicates GFP expression is due to glucose starvation, rather than

13



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Page 14 of 31

323

a general stationary phase response or accumulation of GFP due to slow down of growth. This

324

confirms the role of glucose-starvation in activation of GFP expression.

325

SELECT CRP PROMOTERS ENABLE TOGGLE BEHAVIOR

326

Promoters found to successfully activate GFP production upon glucose limitation were

327

incorporated into the original toggle switch design (the trcp promoter producing mCHERRY is “OFF”

328

and the tetOp producing GFPmut3b is “ON”, Figure 1B). This language mirrors future insertion of

329

metabolic pathways in place of gfpmut3b, so that pathway expression is defined as the ON state. This

330

orientation was chosen because tetOp promoters have a stronger OFF (TetR/mCherry) state, and

331

constitutive LacZ expression would allow the OFF state to be set with lactose rather than IPTG, to

332

further lower process costs. Glucose-sensitive promoters may enable autonomous switching of the

333

circuit with glucose starvation, in a manner analogous to adding aTc. Due to the acceptable dynamic

334

range of the constructed promoters, crp and T-crp variants were both carried forward. crp promoters

335

were placed upstream of LacI in either the two plasmid or tandem promoter system to enable

336

glucose limitation induced switching of the toggle state from OFF to ON (LacI/GFPmut3b) (figure 1b).

337

The stability of the circuit containing glucose-starvation sensing was investigated to ensure

338

switching was due solely to glucose starvation rather than nonspecific LacI expression or circuit

339

imbalance. The addition of new copies of LacI activation may bias the circuit towards the ON state.

340

Only two promoter systems demonstrate a stable OFF state after IPTG removal in a glucose-rich

341

environment (tandem promoter araF and TaraF systems, figure 3 and supplemental figure 1). None

342

of the two plasmid systems could maintain a stable OFF state, despite a wide range of dynamic

343

activity. The tandem TaraF promoter was the only system with a stable OFF state AND a comparable

344

ON state to the control (pKDL071), depicted in figure 3. Furthermore, the full transition to the ON

345

state after induction took ~6-8 hours, while chemical induction takes ~2 hours (figure 3 and 4).

346

Since chemical inducers immediately inhibit their targets, there is immediate repression of the

347

opposing TF. Whereas increased expression of a TF (LacI) needs to be above the stability point of the

348

opposing TF (TetR) to cause a switch in toggle states, and there is an inherent time delay needed for

14


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349

transcription and translation of the TF. Since the toggle switch TF’s do not have degradation tags like

350

the fluorescent reporters, dilution by growth is speculated to be an important mechanism to switch

351

the state of the toggle once newly expressed TF levels are above the threshold for a switching event,

352

ultimately leading to longer switching times.

353

The T-araF tandem glucose toggle switch was able to change states through 4 mechanisms,

354

growth in LB (figure 3), glucose starvation in MOPS EZ rich, and growth in MOPS EZ rich glycerol and

355

gluconate (figure 4). Secondary carbon sources are a cheap way to prevent cells from going into

356

stationary phase, while still enabling a change in the switch states. This strategy proved useful for

357

MOPS min PHB production, described below.

358

THE ORIGINAL GENETIC SWITCH EFFICIENTLY PRODUCES PHB

359

In order to verify the original toggle switch can be used for PHB production and demonstrate

360

the utility of splitting growth and production regimes using a toggle switch, gfpmut3b was replaced

361

by the phaECAB operon for the production of PHB in the toggle switch. Constitutive overexpression

362

of the pathway (stable 29 genomic copies of the PHB pathway in E. coli) leads to cell-burden and

363

reduced growth rates23 18. Decoupling growth and production using a toggle switch could enhance

364

productivity significantly by reducing the burden during growth, allow higher cell densities, suppress

365

low-productivity escape mutants, and allow interrogation of pathway enzyme levels beyond those

366

possible if simultaneous growth is also required.

367

Early induction of the PHB pathway in the cultivation leads to highly variable PHB

368

production titers and reduced growth rates; -14 h induced experiments produced titers from 0.5 g/L

369

to 1.5 g/L of PHB, with growth rates ranging from 0.1-.45 /hour. The relative standard deviation

370

(RSD) for the growth rate among these trials is 38% and for %PHB titer is 30% (figure 5b, green

371

circles). Separate growth and production strategies generally lead to comparable, more consistent

372

titers, and faster growth. Induction time resulted in a range of PHB titers. 0 h and 10 h induced

373

cultures (i.e. at inoculation and 10 h after inoculation, respectively) grew at similar rates (0.55-0.65

374

/h) and produced similar titers (0.8-1.4 g/L PHB)- (figure 5b orange and yellow circles). In

15



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

375

comparison to -14 h pre-culture induced experiments, 0 and 10 h induction cultures had faster

376

growth (up to 5 x faster, 6.5% RSD) and more consistent PHB production (60.9 %PHB, RSD 9%).

377

Non-induced cultures remained tightly off with no detectable PHB production. These results

378

highlight the challenges associated with early induction (-14 h), and the benefits of controlled

379

induction (0 or 10 h).

380

Page 16 of 31

Constitutive, stable expression of PHB is consistent, but at the expense of titer. A strain

381

constitutively expressing 29 copies of the PHB operon (KS29-consitutive) was benchmarked. It grew

382

slightly slower (0.50-0.56) and produced lower titers (0.7-1.0 g/L of PHB) than the induced PHB

383

cultures. While the plasmid induction strategy allows high copy25 (colE1 origin, 50-70 copies) that is

384

induced after growth is complete, the KS29-constitutive strain (29 copies) was developed by

385

selecting for the highest pathway levels compatible with growth4. Therefore, splitting growth and

386

production using induction control appears to enable higher pathway flux in the production phase,

387

while allowing a fast growth phase, which may enable higher biomass-dense cultures to be reached.

388

These results indicate separating growth and production phases using a toggle switch is a promising

389

production strategy.

390

391

A GLUCOSE INDUCED TOGGLE SWITCH ENABLES AUTONOMOUS PHB PRODUCTION

392

In the previous study, the toggle switch was switched to PHB production (ON) by aTc, which

393

was present throughout the production phase. For a successful glucose toggle, the switch to ON

394

should be induced by glucose starvation and should be stably ON after glucose is re-introduced. To

395

test this, successful autonomous, glucose-based switch plasmids were paired with the PHB toggle

396

system. The only stable glucose toggle out of 20 tested was the tandem promoter TaraF system.

397

(Figure 3 and 4). The TaraF tandem system allowed growth rates that were nearly double that of -14

398

h induction trials (0.30 vs. 0.65 h-1), likely due to no growth burden from the PHB pathway.

399

However, this candidate performed poorly from a production standpoint, with %PHB titer at ~ 10%.

16


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400

One distinction between the original toggle and our glucose toggle is the original toggle can

401

continuously grow through the toggling (which dilutes existing TF), while the glucose toggle pauses.

402

This, along with the immediate inhibition of the TF due to inducing chemicals, means the original

403

toggle has a much shorter transition. This was indeed the case, as the time for switching using

404

glucose starvation was ~6-8 hours, while the IPTG induction was closer to two hours (figure 3). To

405

improve the glucose toggle performance, we included a second carbon source, glycerol that would

406

allow the glucose toggle to maintain growth during the transition. Upon glucose exhaustion, glycerol

407

is consumed, allowing growth while not repressing CRP, like glucose. This growth allows more rapid

408

dilution of existing LacI, compared to glucose starved cells that stop growing. We used growth

409

conditions with less glucose (0.05% or 0.1%) and the addition of glycerol 0.1%. %PHB increased 4-

410

fold in these conditions, to ~40%, while maintaining fast growth (.65 h-1) (see figure 5 b and c). We

411

imagine for larger bioprocesses over longer production times, the difference between the chemical

412

and auto induction switch times (2 vs. 6-8 hours) should be less important than the conditions tested

413

in this study (with a maximal growth phase of ~10 hours).

414

Tandem promoters in series (rather than two promoters expressing two different copies of a

415

gene) to encode regulatory function in a genetic circuit proved useful. Previous studies have used

416

repeated tandem promoters to increase pathway expression,26, 27 but we are aware of only one that

417

order promoters in tandem to enable logic functions28 (the repression promoter is upstream of the

418

sensing-promoter to allow response to a transient signal), and it was described as a suboptimal

419

strategy. The benefits of the tandem system is that it requires only one plasmid, was the only stable

420

system, and led to improvements in growth rate even compared to the two plasmid production

421

system. In future studies, mRNA modelling may enhance the design phase, and it may be useful to

422

include mRNA processing in tandem promoter systems to standardize the mRNA resulting from

423

either of the tandem promoters (tetOp or crp)29.

424

This system may be amenable to other transient signals, whether that be other nutrient-

425

limitation, stressors, oxidative, pH stress, etc. However, these other systems may face additional

426

challenges. Sugar consumption is very hierarchical in E. coli due to the overwhelming preference for 17



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Page 18 of 31

427

glucose as the sole carbon source12. Due to this overwhelming preference, the CRP system allows

428

sharp repression and induction. Other nutrients like phosphate, nitrogen, and redox state, and even

429

secondary carbon sources are part of a complex regulatory network, and are often controlled at a

430

level relative to other nutrient cues (i.e. nitrogen sensing is tied to α-ketoglutarate which is a

431

measure of the carbon and nitrogen state of the cell30), rather than an absolute level like glucose.

432

Glucose is convenient, as it is an important energy and biomass constituent.

433

Beyond using the glucose toggle in large scale production, toggles could significantly enable

434

the strain engineering process.

Most strain engineering strategies sequentially require (1)

435

introducing genetic changes (2) growing the cells (typically to a colony and then many generations in

436

liquid culture) and (3) measuring the productivity.

437

opportunities for productive genetic changes to be lost due to slow growth or toxicity and low

438

productivity variants with growth fitness advantages may overtake the culture. This means useful

439

genetic modifications may be passed over and may confound the knowledge-base by implying those

440

are the wrong type of modifications. By generating new genetic constructs with pathways off, then

441

turning them on for the product screening process, it could be possible to recover mutants that

442

would not be viable during constitutive expression of the product pathway.

During the growth phase, there is ample

443

We have developed an autonomous genetic switch that has demonstrated utility in

444

improving growth rates compared to a constitutively expressing system, with a PHB pathway known

445

to cause burden at high enzyme expression levels. We have implemented a tandem promoter system

446

that allows logic to be encoded using different promoters in control of a single-copy of a regulatory

447

gene. Across experiments, regardless of media composition (defined rich, rich, or minimal) the

448

glucose level was the ultimate determination of gene expression, whether it was simple gene

449

expression assays, the stability tests, or the application to PHB production. This glucose sensitive

450

toggle circuit is flexible; it can be used for chemical production in minimal media or protein

451

production in rich media- and is a generally useful synthetic biology tool. This tandem promoter

452

strategy and the glucose-sensitive toggle system are generally useful additions to the field of

18


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453

synthetic biology, and we envision that they can be rapidly implemented into a variety of platforms

454

to push the boundaries of industrially relevant processes.

455

ACKNOWLEDGEMENTS

456

We would like to thank the Collins lab for providing plasmid pKDL071. We thank Karthik

457

Sekar and Jennifer Greene for helpful discussion regarding the design and analysis of experiments.

458

We thank the Northwestern Recombinant Protein Production Core (rPPC) for use of plate readers

459

and the NUSeq core for Sanger sequencing services.

460

SUPPORTING INFORMATION

461

The Supporting Information is available free of charge on the ACS Publications website.

462

Supplementary Table 1

463

Supplementary figure 1.

464

AUTHOR INFORMATION

465

Corresponding Author

466

* E-mail Keith E.J. Tyo: [email protected]

467

Department of Chemical and Biological Engineering E-136, Northwestern University, 2145 Sheridan

468

road, Evanston, IL, USA

469

Author Contributions

470

WHB and GK carried out experiments and WHB designed experiments. WHB and KEJT wrote the

471

manuscript.

472

Notes

473

The authors declare no competing conflicts of interest.

19



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474

Page 20 of 31

FUNDING

475

This work was funded by the National Science Foundation CAREER (1452549), the Institute

476

for Sustainability and Energy at Northwestern (ISEN) Early Career Investigator Award, and funds

477

from the McCormick School of Engineering and Applied Science, Northwestern University.

478

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Maximum Specific Uptake Capacities for Glucose and Oxygen in Glucose-Limited Fed-Batch

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Cultivations of Escherichia coli.

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ABSTRACT FIGURE

556

557 558

559

560

24


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561

FIGURES

562

Figure 1: Strategies for Biosynthesis of Chemicals using Cell Factories

563

Idealized strategies for Biosynthesis of Chemicals using Cell Factories. (a) depicts common biomass

564

(solid line) and product (dashed line) profiles expected from a variety of biosynthesis strategies

565

(green: decoupled growth and production, yellow: constitutive growth and production, red:

566

constitutive pathway overexpression). (b) Shows the integration of glucose sensing into the toggle

567

switch architecture. (c) Depicts Relevant genetic programs followed in this study

568

a.

Growth and Product Profiles for Biosynthesis Strategies

b.

Toggle Switch Architecture with integrated Glucose sensing

569 570

571

25



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572

c.

Page 26 of 31

Program for the glucose-sensing genetic switch

573 574 575 576

26


Page 27 of 31

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577

Figure 2: Glucose-sensitive promoters activate expression over a wide dynamic range only in

578

glucose limited conditions

579

crpp promoters were grown in LB + 0.1% glucose, then transition into three different types of media

580

(LB, LB + 0.1% and 0.4% glucose). GFP expression was due to glucose availability rather than

581

cessation of growth. GFP expression initiation could be titrated depending on the glucose

582

concentration in the media. Red vertical lines indicate time points beyond which fluorescence was

583

above the detection limit of the plate reader.

584 585 586 587

27



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Page 28 of 31

588

Figure 3: Glucose Sensing Autonomously Switches Toggle State from OFF (RFP) to ON (GFP)

589

Successful crpp glucose-starvation sensing promoters were integrated into the toggle switch and

590

tested for the ability to switch the toggle state. After pre-culture (described in the text), each variant

591

was sequentially grown in media conditions and monitored for autonomous toggling of genetic

592

switch states. The araF and TaraF tandem promoter toggle switches were tested. Only the TaraF

593

system was able to remain stably OFF, and activate a switch from the OFF to the ON state upon

594

glucose starvation, while remaining stable upon reintroduction of glucose. The system then returned

595

to the OFF state after IPTG addition.

596 28


Page 29 of 31

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597

Figure 4: The glucose toggle switch can be autonomously activated in varied media conditions

598

The ability of the glucose-sensing toggle switch to activate in a variety of conditions was tested. After

599

pre-culturing to set the switch to the OFF state (described in the text), cells were transferred to 3

600

conditions without glucose (1) no additional carbon- starvation (2) gluconate and (3) glycerol. All

601

three conditions successfully switch the toggle state from OFF to ON. The timescale represents time

602

after starvation and the first time point recorded is after glucose was re-added to the media.

603 604

Figure 5 Production phase glucose consumption and organic acid/PHB production profiles

605

(a) The timeline of chemical and autonomous PHB production is depicted (b) A variety of toggle

606

switch systems for PHB production including pre-culture, chemical and autonomous production. (c)

607

Representative plots of carbon consumption and secretion over time for the glycerol optimized trials.

608

a.

Experimental Timeline

29



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Page 30 of 31

609 610

b.

Growth vs. Production phase for PHB trials

a.

Carbon consumption and secretion over time for glycerol optimized trials

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A glucose-sensing toggle switch for autonomous, high productivity

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