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https://doi.org/10.1038/s41929-019-0411-7

Engineering Escherichia coli lifespan for enhancing


chemical production
Liang Guo   1,2, Wenwen Diao1,2, Cong Gao1,2, Guipeng Hu1,2, Qiang Ding1,2, Chao Ye1,2, Xiulai Chen1,2,
Jia Liu1,2 and Liming Liu   1,2,3*

Industrial chemical production from renewable feedstocks by microbial cell factories provides a promising avenue towards
sustainability. However, the small size of bacterial cells and environmental stress significantly affect microbial cell factory per-
formance. Here, we engineered the Escherichia coli lifespan to improve the chemical production of poly(lactate-co-3-hydroxy-
butyrate) and butyrate. The replicative lifespan was shortened by deleting a carbon storage regulator, and the chronological
lifespan was extended by deleting a response regulator and overexpressing sigma-38 in Escherichia coli. The replicative lifespan
was fine-tuned using a two-output recombinase-based state machine, and the cell size was enlarged 13.4-fold. The highest
poly(lactate-co-3-hydroxybutyrate) content of 52 wt% was achieved in a 5-l fermenter. The chronological lifespan was modu-
lated through a multi-output recombinase-based state machine, resulting in the highest butyrate titre of 29.8 g l−1, by program-
ming cell differentiation according to different fermentation stages. These results highlight the applicability of engineering the
bacterial lifespan to increase microbial cell factory performance.

M
icrobial cell factories offer an alternative strategy to pro- gation of inclusion bodies is associated with RLS12,21; to increase the
ducing valuable chemicals, including biofuels, chemi- accumulation of inclusion bodies, the modulation of phaM (poly(3-
cals and pharmaceuticals, from renewable feedstock1. To hydroxybutyrate) (PHB)/polyhydroxyalkanoate (PHA) binding
increase the efficiency of microbial cell factories, traditional strain protein) expression can be used to regulate the size and distribution
breeding2 and rational metabolic engineering3,4 have been devel- of PHB granules in Ralstonia eutropha H16 (ref. 22), and thus RLS
oped. Recently, Liu et  al. provided a conceptual and technological manipulation has provided a potential opportunity to regulate the
framework (DCEO Biotechnology) to manipulate the flow, direction distribution of inclusion bodies to increase their accumulation.
and rate of carbon flux5, including approaches for discovering and The recombinase-based state machine (RSM), an important
combining biochemical pathways to produce desired chemicals6, genetic circuit tool, has provided an efficient strategy to regulate
efficiently and rapidly assembling a metabolic pathway in the host cell functions and programme cell behaviours23,24. The RSM is com-
strain7, precisely determining metabolic bottlenecks by identifying posed of site-specific DNA recombinases (SSRs) and an SsrA-tag
the difference between ideal and real conditions8 and modifying mediated protein degradation system. SSRs can recognize a spe-
metabolic channels for the optimal production of desired products1,9. cific DNA sequence (recognition sites) and perform cleavage and
Using this system-wide concept and technique, the metabolic capa- reunion of DNA25, and can be divided into serine recombinases
bilities of an industrial strain can be increased noticeably. In fact, the (A118 recombinases) and tyrosine recombinases (Cre and Flp
efficiency of a microbial cell factory is determined by its metabolic recombinases). Depending upon their relative location or the ori-
capability, physiological state and environmental conditions. entation of recombination sites, three distinct recombination out-
Aging is a progressive morphological and functional change that comes, namely excision, integration or inversion, could be realized26
disturbs the ability to maintain homeostasis and increases the prob- (Supplementary Note 1). On the other hand, the SsrA-tag mediated
ability of death. Cell aging is one of the most fundamental features protein degradation system can be used to regulate protein lifespan
of Saccharomyces cerevisiae (S. cerevisiae)10 and Escherichia coli (E. and engineer protein instability to obtain specific protein concen-
coli)11,12, and is classified into replicative and chronological aging13. trations in vivo and prevent unwanted protein accumulation27. The
According to this classification, the lifespans of S. cerevisiae and E. RSM can be applied to record sequential and temporal informa-
coli can be divided into (1) replicative lifespan (RLS), defined by the tion24, divide complex tasks into different stages28 and programme
number of daughter cells produced before aging11,13, and (2) chrono- cell differentiation to different cell fates24.
logical lifespan (CLS), defined as the length of time that cells in sta- In this study, the lifespan of an industrial strain of E. coli was
tionary phase culture remain viable13–15. As important physiological modulated by genetic manipulation. The cell size of E. coli was
parameters, CLS and RLS could be manipulated as a potential strat- enlarged to enhance poly(lactate-co-3-hydroxybutyrate) (PLH)
egy to regulate the physiological state of cells14, such as cell size16, production by engineering the RLS by using a two-output recom-
generation time13 and stress tolerance17. For example, most grape binase-based state machine (TRSM). Additionally, the CLS of
juice fermentation happens when S. cerevisiae has stopped divid- E. coli was engineered to increase butyrate production through the
ing18, and thus a strategy was adopted for successful wine produc- use of a multi-output recombinase-based state machine (MRSM).
tion by prolonging CLS to elongate the stationary phase18,19 and by Strains with engineered lifespan exhibited superior applicability for
increasing cell vitality20. On the other hand, the asymmetric segre- increasing chemical production through a microbial cell factory.

1
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, China. 2Key Laboratory of Industrial Biotechnology, Ministry of
Education, Jiangnan University, Wuxi, China. 3National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi, China.
*e-mail: mingll@jiangnan.edu.cn

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Results GFP alone (Fig. 2e,f), the distribution of HNS-GFP in GL0002


Genetic manipulation of lifespan. Many studies have demon- was unchanged (Fig. 2g, h). Secondly, when HNS-GFP was over-
strated that E. coli lifespan is associated with an electron transfer expressed in GL0002ΔcsrA, the cells were elongated, and this
chain (ETC)15,29 and stress response pathways30,31 (SRP; Fig. 1a). elongation was modulated by HNS-GFP expression
Therefore, ETC- and SRP-related genes were selected for lifespan (Supplementary Figs. 10 and 11). Thirdly, to demonstrate that
modulation, such as histone-like nucleoid structuring protein (hns), the accumulation of granules leads to the asymmetric segre-
regulator of rpoS (rssB), succinate dehydrogenase subunit A (sdhA), gation of chromosomal DNA in GL0002ΔcsrA, Glg-mKate2
lipoic acid synthase (lipA), DNA-binding ATP-dependent protease (glycogen synthase and far-red fluorescent protein fusions) can be
(lon), carbon storage regulator (csrA), hypoxia-inducible transcrip- used as a marker to show the location of glycogen accumulation.
tion factor (arcA), methyltransferase (ubiG), sigma-38 (rpoS), gly- The locations of fluorescent protein in strains that simultaneously
cogen synthase (glg) and positive activator (rcsA). carried Glg-mKate2 and HNS-GFP were compared. Strikingly,
As shown in Fig. 1b, the deletion of lon, hns, rssB, sdhA, lipA and almost all Glg-mKate2 was localized at old poles, whereas HNS-
ubiG from E. coli ATCC 8739 (wild type) led to CLS increases of 33, GFP was localized at new poles (Fig. 2m–p). Finally, asymmetric
66, 66, 33, 33 and −33%, respectively. On the other hand, overex- segregation of chromosomal DNA was observed by expressing
pression of glg, rcsA, rpoS, csrA and arcA resulted in CLS increases Glg-mKate2 in GL0002 (Fig. 2q–t). Together, these results dem-
of −9, −7, 23, −15 and −19%, respectively. Of these, hns, rssB and onstrate that csrA deletion led to the accumulation of glycogen
rpoS were selected to further investigate the effect on the half chron- granules, resulting in the asymmetric segregation of chromo-
ological lifespan (HCLS, the time at which E. coli survival reaches somal DNA and a shortened E. coli RLS (Supplementary Fig. 12
50%); we found that the HCLSs of the Δhns, ΔrssB, and rpoS over- and Supplementary Note 3).
expression strains were extended by 15, 81 and 68%, respectively.
These results indicate that rssB, ubiG and rpoS can efficiently regu- Enhancement of PLH production by engineering the RLS. PLH,
late E. coli CLS and HCLS. Furthermore, three different approaches a biodegradable and biocompatible synthetic polymer, is produced
were used to investigate the regulation extent of CLS and HCLS. by bacteria in inclusion bodies33. The accumulation of PLH in cells
First, to identify the universality of rssB, ubiG and rpoS in E. coli, leads to (1) HNS-GFP fusion protein exclusion from old poles
the protein sequences of E. coli were downloaded from the UniProt (Fig. 3a), (2) loss of chromosomal DNA after the last division, as
database (UniProt, 2019_02). The proteins of E. coli K12 were used demonstrated by the lack of a visible HNS-GFP signal in the old pole
as probes for BLAST (basic local alignment search tool). As shown of the daughter cell (Fig. 3a) and (3) an increase in generation time
in Supplementary Table 6 and Supplementary Note 2, ubiG, rssB of 24.6% (Fig. 3b). Moreover, previous studies have demonstrated
and rpoS are universally distributed in different E. coli strains with that the asymmetric segregation of inclusion bodies is associated
different genetic backgrounds. Second, to evaluate rssB, ubiG and with cell aging and rejuvenation12,34. Therefore, an efficient strategy
rpoS as universal tools, another eight E. coli variants, namely F0601 to enhance PLH production involves the differentiation of one cell
(derived from E. coli W3110), GL0002 (derived from E. coli ATCC as the aging mother for PLH storage and the other as a rejuvenated
8739), B0013 (derived from E. coli K-12), DH5α, JM109, BL21 daughter for PLH biosynthesis and cell growth (Fig. 3c). For this,
(derived from E. coli B), HB101 (a hybrid of E. coli K-12 and B) and the bacterial fate is divided into two modes: (1) the SA mode for cell
W3110, were engineered at the same sites, according to the targeted growth, expressing the carbon storage regulator (csrA), and (2) the
genes for modulating the CLS. As shown in Fig. 1c, these strains SB mode for PLH production, expressing propionyl-CoA transferase
achieved CLS modulation ranging from −30 to 70% compared with (pct) and polyhydroxyalkanoate synthase (phaC; Fig. 3d).
that of the control strain. Third, to investigate the effect of rssB, To fine-tune these two modes, the TRSM, which consists of a
ubiG and rpoS on the CLS and HCLS in different carbon sources controller and an actuator, was introduced. The controller expresses
and oxygenation conditions, two fermentable carbon sources, two recombinase A118, which is regulated by PTET, the anhydrotet-
non-fermentable carbon sources and four different oxygenation racycline (aTc)-inducible promoter (Fig. 3e and Supplementary
conditions were selected. As shown in Fig. 1d, the CLS and HCLS of Figs. 13–18). The actuator consists of two states, state A and state B,
E. coli ATCC 8739 exhibited similar changes to those of the control with each state coinciding with two distinct outputs: far-red (mKate2)
group (Luria-Bertani (LB) medium and 200 r.p.m.). Overall, ubiG, and blue (BFP) fluorescent proteins, respectively. The actuator
rssB and rpoS provided a robust, strain-independent regulation of remained at state A and expressed mKate2 without A118 (aTc = 0),
the E. coli lifespan. and when A118 was present (aTc = 1), the actuator switched from
The effects of the manipulation of the 11 genes on GL0002 RLS state A to state B and expressed BFP. Meanwhile, GP44 (recombina-
were determined by microscopy. As demonstrated in Fig. 2a–d tion directionality factor) was also expressed with BFP, and when
and Supplementary Figs. 2–6, only deletion of csrA in GL0002 its concentration reached a certain level, it combined with A118 to
led to the accumulation of granules at the poles of E. coli cells switch the actuator from state B to state A, and then BFP changed
and a reduction of cell viability of 31% compared with that of to mKate2 (Fig. 3e). Furthermore, we first implemented an actuator
strain GL0002. Moreover, DNA staining revealed that some cells by using a DNA fragment encoding mKate2, BFP and A118 recom-
of GL0002ΔcsrA appeared dead, indicating that the accumula- binase recognition sites flanking a PJ23119 promoter on a low-copy
tion of granules by E. coli led to cell death (Supplementary Fig. 7). plasmid (1–5 per cell). As shown in Fig. 3f, the expression of recom-
Furthermore, four different approaches were used to evaluate binase A118 made state A change to state B (about 99% strains were
the effect of csrA deletion on RLS. Firstly, HNS is a histone-like BFP). However, when BFP and GP44 were expressed in tandem, the
nucleoid structural protein that binds to AT-rich regions on chro- TRSM system only detected mKate2 (state A), regardless of the pres-
mosomal DNA32, and thus the HNS-GFP (histone-like nucle- ence or absence of A118 (Fig. 3g), because the switch function medi-
oid structuring protein and green fluorescent protein fusions) ated by A118 and GP44 acts rapidly in vivo due to the high level of
is used to indicate the location of a chromosome. In this study, GP44 protein (Supplementary Fig. 19 and Supplementary Table 7).
the poles carrying chromosomes were defined as new poles, and Further, to ensure that GP44 could be degraded, a moderate AAV-
other poles (not carrying chromosomes) were defined as old ssrA degradation tag (peptide sequence AANDENYAAAV) was
poles. As shown in Fig. 2k,l, the HNS-GFP protein was excluded incorporated into GP44 to quickly degrade GP44; as a result, a
from older pole cells in GL0002ΔcsrA, and this exclusion phe- weak state B (BFP) was detected, and the oscillatory expression of
nomenon became apparent at early pole ages (Supplementary mKate2 and BFP was achieved by the bidirectional switch of the
Fig. 9). Compared with that of the GL0002 strain expressing TRSM (Fig. 3h). Finally, a weak ribosome binding site (RBS) was

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a b
2

lon rcsA hns rssB Degradation


of σs

Relative CLS
1

arcA rpoS σs csrA glg

Stress reponse pathway


0
Sod 2
Cell aging Cell damage O2– H2O2 H2O

ROS

Relative HCLS
2H++½O2
PYR FADH FAD+
1

H2O
lipA
NADH NAD+ ubiG
H+ Q8
Cytoplasm

2e– 2e– 0
Q8 Q8H2

sdhA
lipA

csrA
arcA
rcsA
lon

ubiG
hns
rssB

rpoS
glg
ct

pColE
SDH Cytochrome
H+ NAD
Periplasm oxidase Knockout Expression
complex Electron transfer chain
E. coli ATCC 8739

c
ct
Knockout

1.75
rssB

Relative HCLS,
relative CLS
ubiG

1.25
rpoS pColE
Expression

0.75

0.25
1 2 3 α 9 1 1 10 1 2 3 α 9 1 1 10
60 00 01 H5 10 L2 10 31 60 00 01 H5 10 L2 10 31
F0 G
L0 B0 D JM B HB W F0 G
L0 B0 D JM B HB W
Relative HCLS Relative CLS

d
1.75
ct
Knockout
rpoS pColE ubiG rssB
E. coli ATCC 8739

Relative HCLS,
relative CLS
1.25

0.75
Expression

0.25
s e se rol te LB s e se rol te m
.
m
.
m
.
m
.
m
.
m
.
m
.
m
.
LB

co a a . . . . . . . .
u cto y ce tyr u co cto y ce tyr r.p r.p r.p r.p r.p r.p r.p r.p
Gl La Gl Bu Gl La Gl Bu 0 0 0 0 0 0 0 0
10 20 40 10 20 40
Relative HCLS Relative CLS Relative HCLS Relative CLS

Fig. 1 | Characterization of CLS regulation by genetic manipulation. a, Overview of the modulation of cell lifespan by regulating the electron transfer chain and
stress response pathways in E. coli. b, Effect of age-specific genes on CLS and HCLS in E. coli ATCC 8739. ct, as the control group for gene deletion; pColE, E. coli
harboring the empty plasmid as the control group for gene expression. Values and error bars represent the mean values and standard deviations of biological
triplicates. c, Comparison of HCLS and CLS for different strains and the corresponding engineered strains with ΔubiG, ΔrssB and rpoS overexpression. d, Effect
of various carbon sources and oxygenation conditions on the genetic manipulation to regulate lifespan. The shaking speed was maintained at 400, 200, 100
or 0 r.p.m. Each gene encodes the following: lon, DNA-binding ATP-dependent protease; hns, histone-like nucleoid structuring protein; rssB, regulator of rpoS;
ubiG, methyltransferase; sdhA, subunit A of succinate dehydrogenase; lipA, lipoic acid synthase; rpoS, sigma-38; glg, glycogen synthase; csrA, carbon storage
regulator; arcA, hypoxia-inducible transcription factor; rcsA, positive activator. Abbreviations: σs, sigma-38; PYR, pyruvate; Q8, ubiquinone-8; NAD complex,
NADH dehydrogenase complex 1; SDH, succinate dehydrogenase complex; ROS, reactive oxygen species; Sod, superoxide dismutase.

used to further reduce the GP44 expression level, and we found that that the TRSM can be used as a bidirectional switch for balancing
the accumulation of BFP increased and the position and amplitude two cell physiological states (Supplementary Figs. 20, 21 and 23 and
of the TRSM were regulated (Fig. 3i). These results demonstrate Supplementary Notes 4 and 5).

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GL0002 GL0002 GFP GL0002ΔcsrA GFP GL0002 ΔcsrA HNS-GFP,Glg-mKate2 GL0002 HNS-GFP,Glg-mKate2
a DIC e DIC i DIC m DIC q DIC

GL0002ΔcsrA GL0002 GFP GL0002ΔcsrA GFP GL0002 ΔcsrA HNS-GFP,Glg-mKate2 GL0002 HNS-GFP,Glg-mKate2
b DIC f GFP j GFP n GFP r GFP

GL0002 GL0002 HNS-GFP GL0002ΔcsrA HNS-GFP GL0002 ΔcsrA HNS-GFP,Glg-mKate2 GL0002 HNS-GFP,Glg-mKate2
c DIC g DIC k DIC o mKate2 s mKate2

GL0002ΔcsrA GL0002 HNS-GFP GL0002ΔcsrA HNS-GFP GL0002 ΔcsrA HNS-GFP,Glg-mKate2 GL0002 HNS-GFP,Glg-mKate2
d DIC h GFP l GFP p Merge t Merge

Fig. 2 | Characterization of RLS regulation by genetic manipulation. a,b, Representative phase contrast images of the GL0002 (a) and GL0002ΔcsrA
(b) strains after treatment with methylene blue (0.1 mg·ml−1 in deionized water) for 3 min. Red arrows indicate the accumulation of granules. c,d,
Representative phase contrast images of GL0002 (c) and GL0002ΔcsrA (d) strains after treatment with methylene blue (1 mg·ml−1 in deionized water)
for 30 min. e–h, GL0002 cells containing a plasmid encoding the GFP protein (e,f) or the HNS-GFP fusion protein (g,h). i–l, GL0002ΔcsrA cells containing
a plasmid encoding the GFP protein (i,j) or the HNS-GFP fusion protein (k,l). White arrows indicate the loss of chromosomes. m–p, Phase-contrast (m)
and fluorescence (GFP (n), mKate2 (o) and Merge (p)) images of GL0002ΔcsrA with HNS-GFP and Glg-mKate2 fusion proteins. q–t, Phase-contrast (q)
and fluorescence (GFP (r), mKate2 (s) and Merge (t)) images of GL0002 with HNS-GFP and Glg-mKate2 fusion proteins. White arrows indicate the loss
of chromosones, blue arrows indicate the accumulation of glycogen synthase, and green arrows indicate the pole carrying chromosomes. The images are
representative of three independent experiments. Scale bar, 5 µm.

The balance between cell growth and PLH production can be sponding values of strains PLH-1 and PLH-3, respectively (Fig. 4b).
controlled by the RLS through the TRSM. As shown in Fig. 4a, a To ascertain the applicability of the TRSM to bench-top bioreactors,
set of plasmids was constructed to connect the SA and SB modes, the performance of strain PLH-4 in a 5-l fermenter was investigated.
and a series of strains were constructed, namely PLH-0, PLH- Compared with that in shaker flasks, the PLH content increased
1, PLH-2, PLH-3, PLH-4, PLH-5, PLH-6, PLH-7 and PLH-8 by 66% (Fig. 4d). To investigate the extent to which the RLS can
(Fig. 4b). It was found that (1) a larger space for PLH accumulation be engineered, other E. coli variants, namely JM109, BL21, B0013,
was observed in strains PLH-4 and PLH-5 compared with that of HB101 and F0601, were engineered at the same sites, according to
the rod-shaped strain PLH-1 (Fig. 4c), (2) almost all the PLH and strain PLH-4. As shown in Fig. 4e, these strains with engineered
HNS-GFP fusion proteins were localized at the old and new poles, lifespans showed increased PLH content, ranging from 32 to 50%,
respectively (Supplementary Figs. 24–26), (3) the generation time compared with that of the control strain. These results indicate that
of strain PLH-4 decreased by 14.5 and 18.4% compared with those engineering the RLS provides an efficient strategy for controlling
of strains PLH-1 and PLH-3, respectively (Fig. 3b) and (4) the PLH the physiological state of industrial strains (Supplementary Fig. 27
content of strain PLH-4 was 100 and 50% higher than the corre- and Supplementary Notes 6 and 7)

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a b c d
45 New pole Old pole csrA
12 h
SA mode: cell growth

Generation time (min)


35

A
S
PLH-2

B
S
24 h

25
PLH-0 PLH-1 PLH-3 PLH-4
PLHP – + + +
RLS – – + + SB mode: PLH production
TRSM – – – + Daughter Mother pct phaC
(rejuvenated cell) (storage cell)

e Logic gate Truth table


In Out
Name Symbol Architecture aTc
BFP mKa Recombined architecture
(A118)

PTET
SB checkpoint Controller
Two-output recombinase-based

0 0 1
GP44
state machine

Actuator
State A

GP44 1 0
SB
SA A118 SET RESET +
A118 1
SA checkpoint State B
A118 0 1

f g h i

3,000 100 3,000 100 3,000 100 3,000 100


Fluorescence (a.u.)
Fluorescence (a.u.)

Fluorescence (a.u.)
Fluorescence (a.u.)

Efficiency (%)
Efficiency (%)

Efficiency (%)
Efficiency (%)

75 75 75 75
2,000 2,000 2,000 2,000
50 50 50 50
1,000 1,000 1,000 1,000
25 25 25 25

0 0 0 0 0 0 0 0
0 2 4 6 8 10 12 14 0 2 4 6 8 10 12 14 0 2 4 6 8 10 12 14 0 2 4 6 8 10 12 14
Time (h) Time (h) Time (h) Time (h)

Key: A118 GP44 mKate2 BFP attB*attP Terminator RBS1 RBS6 RBS8 BFP fluorescence
A118+GP44 GP44 off mKate2 off BFP off attL*attR Promoter AAV Efficiency mKate2 fluorescence

Fig. 3 | Overview of the design and validation of the TRSM. a, Effect of PLH accumulation on asymmetric segregation of the chromosome. The location of
the chromosome is indicated by the HNS-GFP fusion protein and the PLH granule is stained with Nile red. PLH-2, GL0002 introducing PLH biosynthesis
pathway and expressing HNS-GFP. Scale bar, 5 µm. b, Effect of PLH accumulation on generation time. PLHP, biosynthesis pathway of PLH; PLH-0, GL0002
harbouring empty plasmid; PLH-1, GL0002 introducing PLH biosynthesis pathway; PLH-3, GL0002ΔcsrA introducing PLH biosynthesis pathway; PLH-4,
GL0002ΔcsrA introducing PLH biosynthesis, modulated SA and SB by the TRSM. c, Schematic of storage and rejuvenated cells in the functionally asymmetric
division in E. coli. Green shading indicates the new pole elements, which contain chromosomal DNA. Red shading indicates the old poles, which contain
PLH-filled granules. These PLH granules accumulate in a group of cells, resulting in their aging and death. The dashes indicate old poles. d, Schematic of
the balancing of SA and SB for PLH production. e, Design of the TRSM. The DNA inversion TRSM module is driven by one generic transcription input signal,
set and reset. mKa, mKate2. f, Implementation of the one-output recombinase-based state machine using A118 recombinases. g, Effect of the tandem
expression cassette (BFP and GP44) on the TRSM. h, Effect of GP44 and moderate AAV degradation tag fusion proteins on the TRSM. i, Effect of reducing
the GP44 expression level on the TRSM. attB*attP, attachment site encoded within the host chromosome and attachment site on the infecting phage
chromosome; attL*attR, left attachment site and right attachment. Values and error bars represent the mean values and standard deviations of
biological triplicates.

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a b DCW (g l –1)
phaC
TRSM RLS phaAB csrA Strain 0 1 2 3
-pct
Plasmids
– – – – + PLH-0
phaA hns phaB A118 csrA phaC pct GP44
PTac PTET PJ23119 – – + + + PLH-1

DCW
Kan ColE1 Amp pSC101 – + + + – PLH-3

pct
+ + + + – PLH-4
LA LACoA phaC

PLH content
+ – + + + PLH-6
Glc PYR phaA phaB PLH
adhE + – + + – PLH-7
AcCoA AceCoA 3HCoA State B

HNS State A –
+ + + + PLH-8
ackA
csrA RLS and cell elongated 0 10 20 30 40
PLH content (wt%)

c d e
75 Glucose PLH content PLH content (%)
PLH-1 PLH-4 PLH-5 50
DCW 30 25 20 15

PLH content (wt%)


60 40
Glucose (g l–1)

DCW (g l–1)
45 30 RLS

30 20 Control

15 10

JM109

BL21

B0013

HB101

F0601

GL0002
0 0
0 24 48 72
Time (h)

Fig. 4 | Overview of engineering RLS for PLH production. a, Schematic of the PLH biosynthetic pathway. A red × indicates that the corresponding gene
was knocked out. Green arrows denote the biosynthesis pathway of PLH. The upper box depicts the introduced plasmids. b, PLH production obtained with
engineered strains in shaken flasks. PLH-0, GL0002 harbouring empty plasmid; PLH-1, GL0002 introducing PLH biosynthesis pathway; PLH-3, GL0002ΔcsrA
introducing PLH biosynthesis pathway; PLH-4, GL0002ΔcsrA introducing PLH biosynthesis pathway, modulated SA and SB by the TRSM; PLH-6, GL0002
introducing PLH biosynthesis pathway, modulated SB by the TRSM; PLH-7, GL0002ΔcsrA introducing PLH biosynthesis pathway, modulated SB by the TRSM;
PLH-8, GL0002ΔcsrA introducing PLH biosynthesis pathway, modulated SA by the TRSM. DCW, dry cell weight. Minus (−) indicates that the corresponding
gene was lacking. Plus (+) indicates that the corresponding gene was introduced. Plus sign in a circle (⊕) indicates that the corresponding gene was
introduced and regulated by the TRSM. Values and error bars represent the mean values and standard deviations of biological triplicates. c, Effect of RLS
manipulation on asymmetric segregation of PLH. PLH-5, GL0002ΔcsrA introducing PLH biosynthesis pathway, expressing HNS-GFP, modulated SA and
SB by the TRSM. Scale bar, 5 µm. d, pH-stat fed-batch cultures of strain PLH-4 in a 5-l bioreactor. e, PLH content of different strains and the corresponding
engineered strains with modulation of RLS by the TRSM system (72 h). Experiments were performed in duplicate. amp, ampicillin-resistance gene; ColE1,
replication origin; kan, kanamycin-resistance gene; pSC101, replication origin; PTac, IPTG-inducible promoter; PTET, aTc-inducible promoter; PJ23119, constitutive
promoter. Each gene encodes the following: phaA, β-ketothiolase; phaB, acetoacetyl-CoA reductase; phaC, polyhydroxyalkanoate synthase; pct, propionyl-
CoA transferase; ackA, acetate kinase A; adhE, alcohol dehydrogenase; hns, histone-like nucleoid structuring protein; csrA, carbon storage regulator; a118,
recombinases; gp44, recombination directionality factor; phaAB, β-ketothiolase and acetoacetyl-CoA reductase. Abbreviations: Glc, glucose; PYR, pyruvate;
LA, lactate; LACoA, lactyl-CoA; AcCoA, acetyl-CoA; AceCoA, acetoacetyl-CoA; 3HCoA, 3-hydroxybutyryl-CoA; PLH, poly(lactate-co-3-hydroxybutyrate).

Enhancement of butyrate production by engineering the CLS. two systems: a controller and an actuator (Fig. 5c). The controller
Butyrate, an important platform chemical, is widely applied in the expresses two tyrosine recombinases, flippase recombinase (Flp)
food, pharmaceutical and energy industries5. The accumulation of and cyclization recombinase (Cre), which are regulated by PTET and
butyrate in fermentation broth could induce apoptosis and disrup- PBAD, an arabinose (Ara)-inducible promoter, respectively (Fig. 5c
tion of cell growth35,36. In this study, butyrate decreased the CLS and and Supplementary Figs. 29–31). As shown in Fig. 5d, the register
HCLS of F0601 by 45 and 75%, respectively. To further increase of the actuator is modified by the recombinases expressed from the
the butyrate titre, a potential strategy is to increase the robustness controller. The register structure consists of an anti-aligned Flp rec-
towards butyrate. For this, the fate of a butyrate-producing strain ognition site pair and two anti-aligned and orthogonal Cre recog-
could be divided into four modes according to CLS modulation: (1) nition site pairs (state S00). If Ara is first added to the MRSM, the
cell growth mode, expressing methyltransferase (ubiG) and regu- Flp sites between the edges of the register are excised, leading to a
lator of rpoS (rssB), (2) precursor accumulation mode, expressing state switch from S00 to S10. Upon subsequent addition of aTc to the
rssB, (3) increased CLS mode, expressing sigma-38 (rpoS), and MRSM, the fragments of DNA between the Cre sites are excised,
(4) butyrate production mode, expressing 3-hydroxybutyryl-CoA leading to a state switch from S10 to S11. On the other hand, if aTc
dehydrogenase (hbd), 3-hydroxybutyryl-CoA dehydratase (crt) and is first introduced into the MRSM, Cre expression is induced, lead-
trans-enoyl-CoA reductase (ter; Fig. 5a,b, Supplementary Fig. 28 ing to the excision of DNA fragments between the recognition sites
and Supplementary Note 8). and switching from S00 to S01. Upon the subsequent addition of Ara
To switch between these four modes to enhance butyrate pro- to the MRSM, Flp expression is induced, leading to the excision
duction, an MRSM was introduced. This MRSM is composed of of DNA between aligned recombination sites of the register and

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a b c
Ara
Active Flp
recombination ubiG rssB cre flp
i
Cell growth PTET PBAD
flp
i Controller
ted

ii rssB

In
cumula

cre
aTc

ase
ii iii Active Cre 2:4
2:4
r ac

d CLS
recombination iii rpoS Register
so
ur

Pr
ec
iv
cre PJ23119
Product butyrate iv
hbd crt ter
Actuator

d S00: e
Key:
S00
95 Inputs: {Ara, aTc}
Ara aTc
Ara aTc States: {S00, S10, S01, S11}
S10: S01:
Recombinases:
S10 S01
99 96 Cre Flp

aTc Ara Addresses:


aTc Ara
BFP
S11: S11:
S11 S11 DsRed DsRed off
95 93
GFP GFP off
f State diagram Recombined architecture mKate2 mKate2 off
Performance
S00 S00: BFP Recombinase recognition sites:
(94)
= unrecombined Cre sites*
Ara
= unrecombined Flp sites
S10 S00 Red + BFP
S10: S00: (98) = recombined Cre sites

= recombined Flp sites


aTc aTc
mKa + GFP *Recognition sites with different symbol
S11 S01 (95)
shapes do not cross react
S11: S01:
= % of cells in expected state
Ara Ara mKa
(96) = % of cells not in expected state
S11 S11
S11: S11:

Fig. 5 | Overview of the design and validation of the MRSM. a, Strategies used for microbial consortia to enhance butyrate production. b, The small molecules
aTc and Ara were applied to induce Cre and Flp recombinase expression, respectively. The actuator system contains four modes of protein group output.
c, Schematic of the two-plasmid system in the MRSM. d, Schematic of the resulting register was accomplished by recombining the two inputs (Ara, yellow
arrow; aTc, cyan arrow). e, Performance of strain MRSM-1. Numbers indicate the expected state of the cell population (corresponding to d) induced by
recombining the inputs Ara (yellow arrow) and aTc (cyan arrow). f, Effect of inducer dose on the switch of the MRSM. The gene regulation programmes of the
MRSM are described in the left pillar, with each sphere having a different colour corresponding to which fluorescent protein (BFP, blue; RFP, red; GFP, green;
mKa, far-red) is expressed in that state. The corresponding MRSM state schematic is depicted in the middle pillar, with non-expressed fluorescent protein
(off) depicted by a trapezoid outline and expressed fluorescent protein (on) depicted by a shaded trapezoid. In the right pillar, numbers indicate the expected
state of the cell population induced by recombining the inputs Ara (yellow arrow) and aTc (cyan arrow). cre, cyclization recombinase; flp, flippase recombinase.

switching from S01 to S11 (Fig. 5d). These four states, blue (BFP, S00), tern of recombinase induction (Supplementary Figs. 38 and 39). As
red (Red, S10), green (GFP, S01) and far-red (mKate2, S11) corre- a result, 94% of the expected state cells can switch to the correspond-
spond to the four outputs of the MRSM (Supplementary Fig. 33). ing states. These results demonstrate that the MRSM can be used to
Furthermore, the performance of the MRSM in E. coli was evaluated precisely regulate the cell physiological state (Supplementary Fig. 32
by flow cytometry, as demonstrated in Fig. 5e and Supplementary and Supplementary Note 5).
Figs. 34–36, and at least 93% of cells adopted the expected expres- To enhance butyrate production, two plasmids, pColE-AT and
sion profiles. Moreover, the effect of the inducer dose on the switch pCloDF13-URRrthc, were built to regulate the four modes (Fig. 6a
of the four states was investigated and the results are shown in Fig. and Supplementary Figs. 40 and 41). These plasmids were intro-
5f and Supplementary Fig. 37. It was found that (1) if a low level of duced into F0601, and a series of strains were produced, namely
Ara (100 mmol l−1) was first added to the MRSM, Flp was expressed BUT-0, BUT-1, BUT-2, BUT-3, BUT-4, BUT-5 and BUT-6 (Fig. 6b).
at a low level and a portion of S00 cells switched to S10, (2) upon It was found that (1) the CLS and HCLS of strain BUT-6 increased
subsequent addition of aTc (150 ng ml−1) to the MRSM, almost all by 25 and 20%, respectively, compared with the corresponding val-
S10 and S00 cells switched to S11 and S01, respectively, and (3) the ues in strain BUT-1 (Supplementary Fig. 42) and (2) the butyr-
addition of Ara (1000 mmol l−1) led to a switch of all S01 cells to S11. ate titre of strain BUT-6, which regulated the four modes using
Furthermore, similar results can be achieved by regulating the pat- the MRSM, was 15.5 g l−1 in the shaken flask culture, which is

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Plasmids

cre flp atoB tesB ubiG rssB rssB rpoS ter hbd crt
PTET PBAD PTac PJ23119

Cm p15A Kan ColE1 Spe CloDF13

O O OH O O O
atoB hbd crt ter
CoA CoA CoA CoA
S S S S
Glc G6P G3P PYR AcCoA
AceCoA HyCoA CrCoA BuCoA

tesB
adhE
ackA pta

ubiG
poxB

O
ldhA
pflB

ETC
OH
NADH ATP Growth rssB rpoS CLS
BUT

b 16 c d
Glucose OD600 Butyrate E. coli BUT (g l–1)
60 50
15
12 JM109
50
Butyrate (g l–1)

40

OD600, Butyrate (g l–1)


BL21
Glucose (g l–1)

8 40
30
30 B0013 10
4
20
20 HB101
0 10
Strain BUT-0 BUT-1 BUT-2 BUT-3 BUT-4 BUT-5 BUT-6 10 GL0002
BUTP – + + + + + +
0 5
ubiG + + – + – – – 0
rssB + + + – – – – F0601
0 12 24 36 48 60 72
rpoS – – – – – + +
Stage II Stage III Stage IV
Stage I Control CLS
MRSM – – – – – – +
Time (h)

Fig. 6 | Overview of engineering CLS for butyrate production. a, Schematic of the butyrate biosynthetic pathway. The black dashed arrow represents
a multi-step pathway. Solid arrows represent central each gene encodes pathways. A red × indicates that the corresponding gene was knocked out.
Green arrows denote the biosynthesis pathway of butyrate. The upper box depicts the introduced plasmids. b, Butyrate production obtained with
engineered strains in shaken flasks. BUTP, biosynthesis pathway of butyrate; BUT-0, F0601 harbouring empty plasmid; BUT-1, F0601 introducing
butyrate biosynthesis pathway; BUT-2, F0601ΔubiG introducing butyrate biosynthesis pathway; BUT-3, F0601ΔrssB introducing butyrate biosynthesis
pathway; BUT-4, F0601ΔubiGΔrssB introducing butyrate biosynthesis pathway; BUT-5, F0601ΔubiGΔrssB introducing butyrate biosynthesis pathway
and expressing rpoS; BUT-6, F0601ΔubiGΔrssB introducing butyrate biosynthesis pathway and containing the MRSM. Values and error bars represent
the mean values and standard deviations of biological triplicates. c, pH-stat fed-batch cultures of BUT-6 in a 5-l bioreactor. Stages I, II, III and IV are
multiple-stage fermentations. d, The titre of different strains and the corresponding engineered strains with modulation of the CLS by the MRSM system
(72 h). Experiments were performed in duplicate. cm, chloramphenicol-resistance gene; p15A, replication origin; kan, kanamycin-resistance gene; ColE1,
replication origin; spe, spectinomycin-resistance gene; CloDF13, replication origin; PTET, aTc-inducible promoter; PBAD, Ara-inducible promoter; PTac,
IPTG-inducible promoter; PJ23119, constitutive promoter. Each gene encodes the following: atoB, acetoacetyl-CoA thiolase; hbd, 3-hydroxybutyryl-CoA
dehydrogenase; crt, 3-hydroxybutyryl-CoA dehydratase; ter, trans-enoyl-CoA reductase; tesB, acyl-CoA thioesterase II; ubiG, methyltransferase; rssB,
regulator of rpoS; rpoS, sigma-38; cre, cyclization recombinase; flp, flippase recombinase; ackA, acetate kinase A; adhE, alcohol dehydrogenase; ldhA,
lactate dehydrogenase; poxB, pyruvate oxidase; pflB, pyruvate-formate lyase; pta, phosphate acetyltransferase. Abbreviations: Glc, glucose; G6P, glucose
6-phosphate; G3P, glyceraldehyde 3-phosphate; PYR, pyruvate; AcCoA, acetyl-CoA; AceCoA, acetoacetyl-CoA; HyCoA, 3-hydroxybutyryl-CoA; CrCoA,
crotonyl-CoA; BuCoA, butyryl-CoA; BUT, butyrate; ETC, electron transfer chain.

1.5- and 2.1-fold higher than those of strain BUT-5 (10.3 g l−1) ate titres 1.27- to 2.6-fold compared with that of the control strain.
and strain BUT-1 (7.5 g l−1), respectively (Fig. 6b, Supplementary These results demonstrate that engineering of the CLS provides a
Figs. 43–53 and Supplementary Note 9). To ascertain the applica- potential strategy for controlling the physiological state of indus-
bility of the MRSM to bench-top bioreactors, the performance of trial strains.
strain BUT-6 in a 5-l fermenter was investigated. Compared with
that of shaker flasks, a 1.92-fold higher butyrate titre was achieved Discussion
(Fig. 6c and Supplementary Fig. 54). To investigate the extent to In this study, we found that E. coli lifespan is controlled by three
which the CLS can be engineered, other E. coli variants, namely genes: rssB, rpoS and csrA. Deletion or overexpression of these genes
JM109, BL21, B0013, HB101 and GL0002, were engineered at the could manipulate the RLS and CLS of E. coli, leading to the highest
same sites, according to strain BUT-6. As shown in Fig. 6d, these content of PLH (52 wt%) and the highest titre of butyrate (29.8 g l−1)
strains with the engineered lifespans achieved increased the butyr- in a 5-l fermenter. These results demonstrate that engineering

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cell lifespan is a potential strategy for increasing the efficiency of Taken together, lifespan engineering provides a platform tech-
microbial cell factories. nology for improving microbial cell factory efficiency. Lifespan
The cell size of industrial strains is important for microbial cell engineering not only improves the physiological status of an indus-
factory efficiency; this is because cell size can affect the production trial strain, but also couples metabolic design to industrial strain cell
of inclusion bodies, such as triacylglycerols37, wax esters38 and poly- fate. Despite millions of years of evolution of E. coli, its lifespan is
hydroxybutyrate39. To increase the titre of target products, a series of remarkably plastic, which is expected to facilitate advances in both
morphological engineering strategies have been developed, includ- metabolic engineering and synthetic biology. An improved under-
ing the overexpression of genes involved in cell division, such as standing of cell senescence that remains stable over time will also
ftsZ (ref. 40), the overexpression of genes involved in binary division, enhance our ability to compartmentalize complex tasks and engi-
such as sulA and minCD (ref. 40), and regulation of the architecture neer functionalities for bioproduction.
of the peptidoglycan cell wall and the actin-like protein MreB cyto-
skeleton39–41, to accelerate growth, increase cell density, simplify Methods
downstream separation, enlarge inclusion body accumulation space Strains, media and culture conditions. All bacterial strains and plasmids used
and reduce the bioproduction cost40. In fact, the intracellular dis- in this study are listed in Supplementary Tables 1 and 2. LB medium and plates
were used for the cultivation of E. coli for DNA manipulations. Terrific broth (TB)
tribution of inclusion bodies is a crucial component of cell mor- medium containing 50 g l−1 glucose was used for butyrate production. Modified
phology. It has previously been reported that modulation of phaM Rennie (MR) mineral salt medium containing 50 g l−1 glucose was used for
expression in Ralstonia eutropha can be applied to regulate the size PLH production. Kanamycin (50 mg l−1), ampicillin (100 mg l−1), spectinomycin
and intracellular distribution of PHB granules22, and the nucleoid- (30 mg l−1) or chloramphenicol (30 mg l−1) were added appropriately. To maintain
plasmid stability, antibiotics were added every 12 h.
associated like protein PhaF drives the intracellular location and
For butyrate production, seed cultures were grown at 37 °C and 200 r.p.m. in
segregation of PHA in Pseudomonas putida KT2442 (ref. 42). In LB medium for 10 h and then transferred into 50 ml TB medium supplemented
addition, to extend the logarithmic phase, three different strategies with 50 g l−1 glucose and 10 g l−1 CaCO3 as an acid-neutralizing agent45. When
can be invoked: (1) the carbon source, nitrogen source and metal cells had grown to an OD600 of 0.6–0.8, isopropyl β-d-thiogalactoside (IPTG,
ions can be used to modulate the supply of essential components, 0.4 mmol l−1), aTc (100 ng ml−1) or Ara (100 or 1,000 mmol l−1) was added to induce
the expression of enzymes. To evaluate the modulation of the CLS for enhancing
(2) fermentation control can be applied to modulate the nutrient butyrate production in batch fermentation, a multiple-stage fermentation process
assimilation and mass transfer and (3) genetic manipulation can be was chosen that was controlled according to the change in growth rate, as follows:
used to regulate cell growth. In this study, a PLH-producing strain stage I: when cells had grown to OD600 = 0.6–0.8, IPTG (0.4 mmol l−1) was added to
was divided into an aging mother for the storage of PLH and a reju- induce the expression of enzymes (atoB and tesB); stage II: when the cell growth
venated daughter for PLH biosynthesis and cell growth. To achieve rate increased to 0.4 at 9 h, Ara (100 mmol l−1) was added to induce a low-level
expression of Flp recombinase; stage III: when cell growth rate decreased to 0.4 at
this, the cell growth and PLH production modes were fine-tuned 19 h, aTc (100 ng ml−1) was added to induce Cre recombinase expression; stage IV:
with a TRSM by modulating the RLS. Here, we found that overex- when the specific growth rate (μ) decreased to 0 at 36 h, Ara (1,000 mmol l−1) was
pression of HNS-GFP can be used not only to indicate the location added to induce Flp recombinase expression.
of chromosomal DNA, but also to enlarge cell morphology for more The butyrate fed-batch fermentation was carried out in a 5-l bioreactor
inclusion body accumulation. This enlarged morphology is possibly containing 3 l TB medium. The seed cultures and inoculum concentration were the
same as indicated above, and the multiple-stage fermentation was controlled in the
due to the inhibition of cell division by storage cells and the remod- same way as the batch fermentation, but with different fermentation time: stage I:
elling of the local DNA structure by HNS-GFP (ref. 43). Moreover, when cells had grown to OD600 = 5–6; stage II: when cell growth rate increased to
the engineering of the RLS led to extension of the logarithmic phase, 0.9 at 12 h; stage III: when cell growth rate decreased to 0.9 at 24 h; stage IV: when
providing an opportunity to enhance growth-coupled production. the specific growth rate (μ) decreased to 0 at 48 h. The culture pH was controlled
As a result, cell growth and PLH content were increased by 17 and at 7.0 with a mixture of KOH (1.2 mol l−1) and KHCO3 (2.4 mol l−1; Supplementary
Table 8).
50%, respectively, through the asymmetric segregation of PLH. For PLH production in batch fermentation, seed cultures were grown at 37 °C
Thus, engineering the RLS provides an opportunity to enlarge the and 200 r.p.m. in LB medium for 10 h and then transferred into 50 ml MR medium
morphology to enhance inclusion body accumulation in E. coli. supplemented with 50 g l−1 glucose and 10 g l−1 CaCO3 as an acid-neutralizing
Metabolic engineering strategies have been adopted to enhance agent. When the cells had grown to an OD600 of 0.6–0.8, IPTG (0.5 mmol l−1) or aTc
butyrate production, such as the deletion of undesired pathways, (100 ng ml−1) was added to induce the expression of enzymes51.
The PLH fed-batch fermentation was carried out in a 5-l bioreactor containing
extension of metabolic pathways leading to butyrate44–46 and 3 l MR medium. The seed cultures for fermentation were grown by transferring
enhancement of the metabolite conversion rate45. However, the fresh colonies to 50 ml LB medium. After 10 h at 37 °C and 200 r.p.m., this culture
titre and yield of butyrate are far below the demands of industrial was inoculated into 100 ml MR medium supplemented with 20 g l−1 glucose. After
production. This is because of a poor tolerance of the butyrate- 10 h at 37 °C and 200 r.p.m., this culture was inoculated into a 5-l bioreactor. The
culture pH was controlled at 7.0 with a mixture of KOH (1.2 mol l−1) and KHCO3
producing strain to high butyrate concentrations, which is a vital
(2.4 mol l−1).
issue for butyrate production. To improve the acid tolerance of the
producing strain, a series of strategies, such as microbial membrane DNA manipulation. The Red homologous recombination method was applied
engineering47, tolerance engineering48 and the overproduction of to gene deletions52. All plasmids were constructed using Gibson assembly and
stress response proteins49, have been developed. In this study, we basic molecular cloning techniques53. To construct the butyrate biosynthesis
pathway, atoB and tesB were PCR-amplified from the genomic DNA of E. coli
divided the state of butyrate-producing bacteria into four modes. W3110. Similarly, crt, ter and hbd were PCR-amplified from the genomic DNA
To efficiently switch between the four modes for programming cell of Clostridium acetobutylicum. To construct the PLH biosynthesis pathway phaA
differentiation according to the stages of fermentation, an MRSM and phaB (from Ralstonia eutropha), and phaC (from Pseudomonas sp. 61-3) were
that controls the CLS was introduced to increase the butyrate titre: artificially synthesized by Suzhou Genewiz Biotechnology, and pct was PCR-
(1) the MRSM could be applied to redirect carbon flux and increase amplified from the genomic DNA of Megasphaera elsdenii. Supplementary Tables
3–6 provide a list of relevant sequences.
butyrate synthesis ability, (2) the deletion of ubiG blocked the gen-
eration of electron carriers and resulted in the enhanced production Analytical methods. Optical density (OD600) and glucose measurements
of butyrate precursors and cofactors such as NADH and acetyl-CoA were performed as described previously54. Butyrate was determined by high-
(ref. 50) and (3) microbial consortia were constructed through cell performance liquid chromatography using an Aminex HPX-87H column
differentiation programming by the MRSM and enhanced robust- (7.8 × 300 mm; Bio-Rad) at 45 °C with 0.05 mmol l−1 sulfuric acid as the mobile
phase. The injection volume was 20 μl and the flow rate was 0.6 ml min−1.
ness to acid stress. As a result, the highest titre of butyrate was Bacterial cells were collected by centrifugation at 8,000g for 10 min. The
achieved in a 5-l fermenter. These results indicate that engineering supernatant was discarded and the cells were washed twice with 20 ml distilled
of the CLS can be used for programming cell differentiation, thus water. Dry cell weight (DCW) was assayed after vacuum lyophilization. PLH
improving the robustness of cell factories for chemical production. contents were quantitatively analysed using gas chromatography (GC-2014,

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Articles Nature Catalysis
SHIMADZU) after methanolysis of lyophilized cells in chloroform55. Recombination efficiency assay. Strains containing the corresponding plasmids
Analytical P3HB and P(GA-LA) purchased from Sigma-Aldrich were used as were plated or streaked on plates containing LB medium and grown overnight at
analytical standards. 37 °C. Then, it was inoculated into 20 ml fresh LB at 2% (v/v) and cultured under
the same conditions until an OD600 of 0.6. Next, aTc was added to give a final
Assay of CLS. Survival measurements were carried performed as described concentration of 100 ng ml−1 for induction at 30 °C. The CFUs were enumerated
previously15, with minor modifications. In short, cultures were inoculated over time by removing an aliquot, serially diluting in 0.5% NaCl, followed by
1:1,000 using an overnight culture created by inoculating two to three colonies colony enumeration after plating on LB plates that were incubated at 37 °C
from LB agar pads into 5 ml LB medium. Cultures were grown overnight in (Supplementary Fig. 22). The TRSM recombination efficiency was calculated from
50 ml LB medium in 250 ml flasks at 200 r.p.m. and 37 °C, at which point the cell equation (4):
density was adjusted to about 1.5 × 109 colony-forming units (CFU) per ml by  
re-suspending a pellet containing the desired number of cells in cell-free spent Nm
Effciency ¼ 1 � ´ 100% ð4Þ
medium of the same strain for the long-lived strains with reduced saturation cell NT
density (Supplementary Figs. 1 and 8). To assay the impact of the carbon source
on the performance of the genetic manipulation to modulate the lifespan, the where Nm is the number of pink colony-forming units on the LB plates and NT
cell-free spent medium was supplemented with glucose (5 g l−1), lactose (5 g l−1), is the total number of colony-forming units on the LB plates.
glycerol (5 g l−1) and butyrate (5 g l−1) for survival measurements. To assay the
impact of oxygenation conditions on the performance of the genetic manipulation Light microscopy. To assay the colony growth on plates containing LB medium,
to modulate the lifespan, the shaking speed was maintained at 0, 100, 200 or 0.5 μl of exponentially growing bacterial culture was placed on an LB agar pad
400 r.p.m. Survival at 0 h after the last dilution was set to 100% survival and was in a microscope cavity slide. When the cells had grown to an OD600 of 0.6–0.8,
used to determine the age-dependent mortality. The assay was performed until aTc (10 ng ml−1) or IPTG (0.4 mmol l−1) was added to induce the expression of
less than 1% of the culture was viable. The CLS is the time at which E. coli survival HNS-GFP or Glg-mKate2 fusion protein. The BFP intensity was measured on a
reaches 1%. Half chronological lifespan (HCLS) is the time at which E. coli survival C-FL-C DAPI (4’,6-diamidino-2-phenylindole) filter cube (excitation filter (EX),
reaches 50%. 361–389 nm; dichroic mirror (DM), 415 nm; barrier filter (BA), 430–490 nm),
the GFP intensity was measured on a C-FL-C FITC filter cube (EX, 465–495 nm;
Growth assay. To determine the effect of RLS on growth, we assessed the growth DM, 505 nm; BA, 512–558 nm) and the DsRed and mKate2 intensities were
rate (R) and generation time (G), obtained from equations (1)–(3): measured on a C-FL-C TRITC (tetramethylrhodamine isothiocyanate) filter
cube (EX, 527–553 nm; DM, 565 nm; BA, 577–633 nm). Microscopy images
ðlog Nt1 � log Nt0 Þ were obtained with a Nikon ECLIPSE 80i microscope equipped with a ×100 oil
n¼ ð1Þ
log 2 immersion objective. Bright-field images (exposure, 100 ms), BFP fluorescence
images (DAPI, exposure, 600 ms), GFP fluorescence images (FITC, exposure,
n 400–600 ms), DsRed fluorescence images (TRITC, exposure, 600 ms) and mKate2
R¼ ð2Þ fluorescence images (TRITC, exposure, 400–600 ms) were analysed using
t1 � t0
Image J software.

ðt1 � t0 Þ
G¼ ð3Þ Fluorescence intensity assay. Strains harbouring the corresponding plasmids
n were plated or streaked on LB agar pads containing antibiotics of interest and
where n is the number of generations in a given amount of time, Nt0 is the grown overnight at 37 °C. Then, they were inoculated into 20 ml fresh LB medium
number of bacteria at the beginning of a time interval, Nt1 is the number of bacteria at 2% (v/v) and cultured under the same conditions until an OD600 of 0.6. Ara
at the end of the time interval, t0 is the time in minutes in the beginning, t1 is the (1,000 mmol l−1) or aTc (100 ng ml−1) was added for induction at 30 °C. To assay
time in minutes at the end, R is the number of generations per unit time and G is cell growth and fluorescence intensity, the corresponding data were recorded on a
the generation time of the bacteria. SpectraMax M3 plate reader (Molecular Devices) using 96-well plates. Each well
was filled with 200 μl cell culture. The BFP intensity was measured at an excitation
SYTOX Green Dead nucleic acid staining. Cells in 1 ml LB culture were washed wavelength (EX) of 402 ± 5 nm and an emission wavelength (EM) of 457 ± 10 nm,
twice with salt solution (0.5% NaCl in deionized water) and re-suspended in 1 ml the GFP intensity was measured at an EX of 480 ± 5 nm and an EM of 515 ± 10 nm,
salt solution. Next, 3 μmol l−1 of SYTOX Green Dead nucleic acid stain (1 mmol l−1 and the mKate2 intensity was measured at an EX of 588 ± 10 nm and an EM of
in DMSO) were added to the cell suspension, which was then incubated at room 645 ± 10 nm. All fluorescence was normalized with cell density by measuring the
temperature, in the dark, for 10 min. Flow cytometry was applied to differentiate absorbance at 600 nm.
live from dead bacteria on the basis of SYTOX Green stain intensity. SYTOX Green
stain intensity was measured in the fluorescein isothiocyanate (FITC) channel MRSM implementation assay. MRSM measurements were carried out as
(488 nm excitation laser (EL), 530/30 nm detection filter (EF)) in a BD FACS Aria described previously24, with minor modifications. In brief, the strain MRSM-1
III cell analyser (BD Biosciences). harbouring controller plasmid (p15A-PTET-CreGTG-AAV-PBAD-FlpGTG) and actuator
plasmid (pColE-FRT-lox2272-BFP-lox2272-GFP-FRT-lox-DsRed-lox-mKate2) was
Time-lapse microscopy and cell death assay. When the cells had grown to an inoculated into a medium with chloramphenicol and spectinomycin.
OD600 of 0.6–0.8, aTc (10 ng ml−1) was added to induce the expression of HNS-GFP. Flow cytometry assays of the MRSM were performed as described previously24,
After 30 min of induction, cells were collected by centrifugation and re-suspended with minor modification. Briefly, the cells were washed three times with PBS and
in fresh LB medium. To assay E. coli colony growth on LB plates supplemented diluted to an OD600 of 0.3. A BD FACS AriaTM III cell analyser (BD Biosciences)
with chloramphenicol (30 mg l−1), 2.5 μl of re-suspended bacterial culture was was used for the detection of fluorescence. GFP, DsRed, mKate2 and BFP were
placed on an LB plate in a microscope cavity slide. Time-lapse microscopy was detected in the FITC channel (488 nm EL, 530/30 nm EF), PE-A Red channel
carried out as described previously56, with incubation at 30 °C during the whole (561 nm EL, 582/15 nm EF), PE-Cy5-A Red channel (561 nm EL, 670/14 nm EF)
experiment. Cell death assays were performed on LB agar pads, with incubation and DAPI channel (405 nm EL, 450/40 nm EF), respectively. We measured 20,000
at 37 °C during the whole experiment. After 100 min of incubation, 3 μl of SYTOX cells for each sample. As shown in Supplementary Figs. 34 and 35, the fluorescence
Green Dead nucleic acid stain (1 mmol l−1 in DMSO) was added to the slide threshold was determined by the negative control (E. coli JM109 containing
cavities, which were then incubated at room temperature, in the dark, for 5 min. pTetR-1 and pJ-lox with no fluorescent reporter genes) population in each channel.
The percentages of cells in Fig. 5e were determined from the experimental data in
Nile red staining. Nile red staining measurements were carried out as described Supplementary Fig. 36. The percentages of cells in Fig. 5f were determined from
previously57, with minor modifications. In short, the cells (1 ml MR culture) the experimental data in Supplementary Fig. 37.
were washed once with PBS and re-suspended in 1 ml PBS. Next, 5 μl Nile
red (0.1 mg ml−1 in acetone) was added to the cell suspension, which was then Reporting Summary. Further information on research design is available in the
incubated at room temperature, in the dark, for 5 min. The cells were washed Nature Research Reporting Summary linked to this article.
twice with absolute ethanol and re-suspended in 1 ml PBS. Microscopy images
were obtained with a Nikon upright fluorescence microscope (Nikon ECLIPSE
80i) equipped with water emersion objectives (CFI Plan Fluor) and connected to a Data availability
cooled CCD digital camera (Nikon digital sight Ds-Ri1). The data that support the figures within this paper and other findings of this
study are available from the corresponding author upon reasonable request.
Methylene blue staining. Cells in 1 ml LB culture were washed once with PBS Supplementary Table 9 provides a list of the GenBank accession numbers of the 14
and re-suspended in 1 ml PBS. Next, 10 μl methylene blue (1 mg ml−1 in deionized key plasmids constructed in this study.
water) were added to the cell suspension, which was then incubated at 37 °C, in
the dark, for 3 or 30 min. Images of the cells were taken with a Nikon ECLIPSE 80i Received: 16 February 2019; Accepted: 28 November 2019;
microscope. Published: xx xx xxxx

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Articles Nature Catalysis

Author contributions Additional information


L.G. and L.L. designed the project. L.G. and W.D. conducted and analysed the Supplementary information is available for this paper at https://doi.org/10.1038/
experiments. C.G., G.H., Q.D. and X.C. provided technical assistance. L.G. analysed the s41929-019-0411-7.
data and wrote the manuscript with input from C.Y., J.L. and L.L. All authors reviewed Correspondence and requests for materials should be addressed to L.L.
and approved the manuscript.
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The authors declare no competing interests. © The Author(s), under exclusive licence to Springer Nature Limited 2020

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