Articles

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-
ducing valuable chemicals, including biofuels, chemi-
cals and pharmaceuticals, from renewable feedstock1. To
increase the efficiency of microbial cell factories, traditional strain
breeding2 and rational metabolic engineering3,4 have been devel-
oped. Recently, Liu et  al. provided a conceptual and technological
framework (DCEO Biotechnology) to manipulate the flow, direction
and rate of carbon flux5, including approaches for discovering and
combining biochemical pathways to produce desired chemicals6,
efficiently and rapidly assembling a metabolic pathway in the host
strain7, precisely determining metabolic bottlenecks by identifying
the difference between ideal and real conditions8 and modifying
metabolic channels for the optimal production of desired products1,9.
Using this system-wide concept and technique, the metabolic capa-
bilities of an industrial strain can be increased noticeably. In fact, the
efficiency of a microbial cell factory is determined by its metabolic
capability, physiological state and environmental conditions.
Aging is a progressive morphological and functional change that
disturbs the ability to maintain homeostasis and increases the prob-
ability of death. Cell aging is one of the most fundamental features
of Saccharomyces cerevisiae (S. cerevisiae)10 and Escherichia coli (E.
coli)11,12, and is classified into replicative and chronological aging13.
According to this classification, the lifespans of S. cerevisiae and E.
coli can be divided into (1) replicative lifespan (RLS), defined by the
number of daughter cells produced before aging11,13, and (2) chrono-
logical lifespan (CLS), defined as the length of time that cells in sta-
tionary phase culture remain viable13–15. As important physiological
parameters, CLS and RLS could be manipulated as a potential strat-
egy to regulate the physiological state of cells14, such as cell size16,
generation time13 and stress tolerance17. For example, most grape
juice fermentation happens when S. cerevisiae has stopped divid-
ing18, and thus a strategy was adopted for successful wine produc-
tion by prolonging CLS to elongate the stationary phase18,19 and by
increasing cell vitality20. On the other hand, the asymmetric segre-
gation of inclusion bodies is associated with RLS12,21; to increase the
accumulation of inclusion bodies, the modulation of phaM (poly(3-
hydroxybutyrate) (PHB)/polyhydroxyalkanoate (PHA) binding
protein) expression can be used to regulate the size and distribution
of PHB granules in Ralstonia eutropha H16 (ref. 22), and thus RLS
manipulation has provided a potential opportunity to regulate the
distribution of inclusion bodies to increase their accumulation.
The recombinase-based state machine (RSM), an important
genetic circuit tool, has provided an efficient strategy to regulate
cell functions and programme cell behaviours23,24. The RSM is com-
posed of site-specific DNA recombinases (SSRs) and an SsrA-tag
mediated protein degradation system. SSRs can recognize a spe-
cific DNA sequence (recognition sites) and perform cleavage and
reunion of DNA25, and can be divided into serine recombinases
(A118 recombinases) and tyrosine recombinases (Cre and Flp
recombinases). Depending upon their relative location or the ori-
entation of recombination sites, three distinct recombination out-
comes, namely excision, integration or inversion, could be realized26
(Supplementary Note 1). On the other hand, the SsrA-tag mediated
protein degradation system can be used to regulate protein lifespan
and engineer protein instability to obtain specific protein concen-
trations in vivo and prevent unwanted protein accumulation27. The
RSM can be applied to record sequential and temporal informa-
tion24, divide complex tasks into different stages28 and programme
cell differentiation to different cell fates24.
In this study, the lifespan of an industrial strain of E. coli was
modulated by genetic manipulation. The cell size of E. coli was
enlarged to enhance poly(lactate-co-3-hydroxybutyrate) (PLH)
production by engineering the RLS by using a two-output recom-
binase-based state machine (TRSM). Additionally, the CLS of
E. coli was engineered to increase butyrate production through the
use of a multi-output recombinase-based state machine (MRSM).
Strains with engineered lifespan exhibited superior applicability for
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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Articles
Results
Genetic manipulation of lifespan. Many studies have demon-
strated that E. coli lifespan is associated with an electron transfer
chain (ETC)15,29 and stress response pathways30,31 (SRP; Fig. 1a).
Therefore, ETC- and SRP-related genes were selected for lifespan
modulation, such as histone-like nucleoid structuring protein (hns),
regulator of rpoS (rssB), succinate dehydrogenase subunit A (sdhA),
lipoic acid synthase (lipA), DNA-binding ATP-dependent protease
(lon), carbon storage regulator (csrA), hypoxia-inducible transcrip-
tion factor (arcA), methyltransferase (ubiG), sigma-38 (rpoS), gly-
cogen synthase (glg) and positive activator (rcsA).
As shown in Fig. 1b, the deletion of lon, hns, rssB, sdhA, lipA and
ubiG from E. coli ATCC 8739 (wild type) led to CLS increases of 33,
66, 66, 33, 33 and −33%, respectively. On the other hand, overex-
pression of glg, rcsA, rpoS, csrA and arcA resulted in CLS increases
of −9, −7, 23, −15 and −19%, respectively. Of these, hns, rssB and
rpoS were selected to further investigate the effect on the half chron-
ological lifespan (HCLS, the time at which E. coli survival reaches
50%); we found that the HCLSs of the Δhns, ΔrssB, and rpoS over-
expression strains were extended by 15, 81 and 68%, respectively.
These results indicate that rssB, ubiG and rpoS can efficiently regu-
late E. coli CLS and HCLS. Furthermore, three different approaches
were used to investigate the regulation extent of CLS and HCLS.
First, to identify the universality of rssB, ubiG and rpoS in E. coli,
the protein sequences of E. coli were downloaded from the UniProt
database (UniProt, 2019_02). The proteins of E. coli K12 were used
as probes for BLAST (basic local alignment search tool). As shown
in Supplementary Table 6 and Supplementary Note 2, ubiG, rssB
and rpoS are universally distributed in different E. coli strains with
different genetic backgrounds. Second, to evaluate rssB, ubiG and
rpoS as universal tools, another eight E. coli variants, namely F0601
(derived from E. coli W3110), GL0002 (derived from E. coli ATCC
8739), B0013 (derived from E. coli K-12), DH5α, JM109, BL21
(derived from E. coli B), HB101 (a hybrid of E. coli K-12 and B) and
W3110, were engineered at the same sites, according to the targeted
genes for modulating the CLS. As shown in Fig. 1c, these strains
achieved CLS modulation ranging from −30 to 70% compared with
that of the control strain. Third, to investigate the effect of rssB,
ubiG and rpoS on the CLS and HCLS in different carbon sources
and oxygenation conditions, two fermentable carbon sources, two
non-fermentable carbon sources and four different oxygenation
conditions were selected. As shown in Fig. 1d, the CLS and HCLS of
E. coli ATCC 8739 exhibited similar changes to those of the control
group (Luria-Bertani (LB) medium and 200 r.p.m.). Overall, ubiG,
rssB and rpoS provided a robust, strain-independent regulation of
the E. coli lifespan.
The effects of the manipulation of the 11 genes on GL0002 RLS
were determined by microscopy. As demonstrated in Fig. 2a–d
and Supplementary Figs. 2–6, only deletion of csrA in GL0002
led to the accumulation of granules at the poles of E. coli cells
and a reduction of cell viability of 31% compared with that of
strain GL0002. Moreover, DNA staining revealed that some cells
of GL0002ΔcsrA appeared dead, indicating that the accumula-
tion of granules by E. coli led to cell death (Supplementary Fig. 7).
Furthermore, four different approaches were used to evaluate
the effect of csrA deletion on RLS. Firstly, HNS is a histone-like
nucleoid structural protein that binds to AT-rich regions on chro-
mosomal DNA32, and thus the HNS-GFP (histone-like nucle-
oid structuring protein and green fluorescent protein fusions)
is used to indicate the location of a chromosome. In this study,
the poles carrying chromosomes were defined as new poles, and
other poles (not carrying chromosomes) were defined as old
poles. As shown in Fig. 2k,l, the HNS-GFP protein was excluded
from older pole cells in GL0002ΔcsrA, and this exclusion phe-
nomenon became apparent at early pole ages (Supplementary
Fig. 9). Compared with that of the GL0002 strain expressing
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GFP alone (Fig. 2e,f), the distribution of HNS-GFP in GL0002
was unchanged (Fig. 2g, h). Secondly, when HNS-GFP was over-
expressed in GL0002ΔcsrA, the cells were elongated, and this
elongation was modulated by HNS-GFP expression
(Supplementary Figs. 10 and 11). Thirdly, to demonstrate that
the accumulation of granules leads to the asymmetric segre-
gation of chromosomal DNA in GL0002ΔcsrA, Glg-mKate2
(glycogen synthase and far-red fluorescent protein fusions) can be
used as a marker to show the location of glycogen accumulation.
The locations of fluorescent protein in strains that simultaneously
carried Glg-mKate2 and HNS-GFP were compared. Strikingly,
almost all Glg-mKate2 was localized at old poles, whereas HNS-
GFP was localized at new poles (Fig. 2m–p). Finally, asymmetric
segregation of chromosomal DNA was observed by expressing
Glg-mKate2 in GL0002 (Fig. 2q–t). Together, these results dem-
onstrate that csrA deletion led to the accumulation of glycogen
granules, resulting in the asymmetric segregation of chromo-
somal DNA and a shortened E. coli RLS (Supplementary Fig. 12
and Supplementary Note 3).
Enhancement of PLH production by engineering the RLS. PLH,
a biodegradable and biocompatible synthetic polymer, is produced
by bacteria in inclusion bodies33. The accumulation of PLH in cells
leads to (1) HNS-GFP fusion protein exclusion from old poles
(Fig. 3a), (2) loss of chromosomal DNA after the last division, as
demonstrated by the lack of a visible HNS-GFP signal in the old pole
of the daughter cell (Fig. 3a) and (3) an increase in generation time
of 24.6% (Fig. 3b). Moreover, previous studies have demonstrated
that the asymmetric segregation of inclusion bodies is associated
with cell aging and rejuvenation12,34. Therefore, an efficient strategy
to enhance PLH production involves the differentiation of one cell
as the aging mother for PLH storage and the other as a rejuvenated
daughter for PLH biosynthesis and cell growth (Fig. 3c). For this,
the bacterial fate is divided into two modes: (1) the SA mode for cell
growth, expressing the carbon storage regulator (csrA), and (2) the
SB mode for PLH production, expressing propionyl-CoA transferase
(pct) and polyhydroxyalkanoate synthase (phaC; Fig. 3d).
To fine-tune these two modes, the TRSM, which consists of a
controller and an actuator, was introduced. The controller expresses
recombinase A118, which is regulated by PTET, the anhydrotet-
racycline (aTc)-inducible promoter (Fig. 3e and Supplementary
Figs. 13–18). The actuator consists of two states, state A and state B,
with each state coinciding with two distinct outputs: far-red (mKate2)
and blue (BFP) fluorescent proteins, respectively. The actuator
remained at state A and expressed mKate2 without A118 (aTc = 0),
and when A118 was present (aTc = 1), the actuator switched from
state A to state B and expressed BFP. Meanwhile, GP44 (recombina-
tion directionality factor) was also expressed with BFP, and when
its concentration reached a certain level, it combined with A118 to
switch the actuator from state B to state A, and then BFP changed
to mKate2 (Fig. 3e). Furthermore, we first implemented an actuator
by using a DNA fragment encoding mKate2, BFP and A118 recom-
binase recognition sites flanking a PJ23119 promoter on a low-copy
plasmid (1–5 per cell). As shown in Fig. 3f, the expression of recom-
binase A118 made state A change to state B (about 99% strains were
BFP). However, when BFP and GP44 were expressed in tandem, the
TRSM system only detected mKate2 (state A), regardless of the pres-
ence or absence of A118 (Fig. 3g), because the switch function medi-
ated by A118 and GP44 acts rapidly in vivo due to the high level of
GP44 protein (Supplementary Fig. 19 and Supplementary Table 7).
Further, to ensure that GP44 could be degraded, a moderate AAV-
ssrA degradation tag (peptide sequence AANDENYAAAV) was
incorporated into GP44 to quickly degrade GP44; as a result, a
weak state B (BFP) was detected, and the oscillatory expression of
mKate2 and BFP was achieved by the bidirectional switch of the
TRSM (Fig. 3h). Finally, a weak ribosome binding site (RBS) was
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Nature Catalysis Articles
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
controlled by the RLS through the TRSM. As shown in Fig. 4a, a
set of plasmids was constructed to connect the SA and SB modes,
and a series of strains were constructed, namely PLH-0, PLH-
1, PLH-2, PLH-3, PLH-4, PLH-5, PLH-6, PLH-7 and PLH-8
(Fig. 4b). It was found that (1) a larger space for PLH accumulation
was observed in strains PLH-4 and PLH-5 compared with that of
the rod-shaped strain PLH-1 (Fig. 4c), (2) almost all the PLH and
HNS-GFP fusion proteins were localized at the old and new poles,
respectively (Supplementary Figs. 24–26), (3) the generation time
of strain PLH-4 decreased by 14.5 and 18.4% compared with those
of strains PLH-1 and PLH-3, respectively (Fig. 3b) and (4) the PLH
content of strain PLH-4 was 100 and 50% higher than the corre-
sponding values of strains PLH-1 and PLH-3, respectively (Fig. 4b).
To ascertain the applicability of the TRSM to bench-top bioreactors,
the performance of strain PLH-4 in a 5-l fermenter was investigated.
Compared with that in shaker flasks, the PLH content increased
by 66% (Fig. 4d). To investigate the extent to which the RLS can
be engineered, other E. coli variants, namely JM109, BL21, B0013,
HB101 and F0601, were engineered at the same sites, according to
strain PLH-4. As shown in Fig. 4e, these strains with engineered
lifespans showed increased PLH content, ranging from 32 to 50%,
compared with that of the control strain. These results indicate that
engineering the RLS provides an efficient strategy for controlling
the physiological state of industrial strains (Supplementary Fig. 27
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.
Butyrate, an important platform chemical, is widely applied in the
food, pharmaceutical and energy industries5. The accumulation of
butyrate in fermentation broth could induce apoptosis and disrup-
tion of cell growth35,36. In this study, butyrate decreased the CLS and
HCLS of F0601 by 45 and 75%, respectively. To further increase
the butyrate titre, a potential strategy is to increase the robustness
towards butyrate. For this, the fate of a butyrate-producing strain
could be divided into four modes according to CLS modulation: (1)
cell growth mode, expressing methyltransferase (ubiG) and regu-
lator of rpoS (rssB), (2) precursor accumulation mode, expressing
rssB, (3) increased CLS mode, expressing sigma-38 (rpoS), and
(4) butyrate production mode, expressing 3-hydroxybutyryl-CoA
dehydrogenase (hbd), 3-hydroxybutyryl-CoA dehydratase (crt) and
trans-enoyl-CoA reductase (ter; Fig. 5a,b, Supplementary Fig. 28
and Supplementary Note 8).
To switch between these four modes to enhance butyrate pro-
duction, an MRSM was introduced. This MRSM is composed of
two systems: a controller and an actuator (Fig. 5c). The controller
expresses two tyrosine recombinases, flippase recombinase (Flp)
and cyclization recombinase (Cre), which are regulated by PTET and
PBAD, an arabinose (Ara)-inducible promoter, respectively (Fig. 5c
and Supplementary Figs. 29–31). As shown in Fig. 5d, the register
of the actuator is modified by the recombinases expressed from the
controller. The register structure consists of an anti-aligned Flp rec-
ognition site pair and two anti-aligned and orthogonal Cre recog-
nition site pairs (state S00). If Ara is first added to the MRSM, the
Flp sites between the edges of the register are excised, leading to a
state switch from S00 to S10. Upon subsequent addition of aTc to the
MRSM, the fragments of DNA between the Cre sites are excised,
leading to a state switch from S10 to S11. On the other hand, if aTc
is first introduced into the MRSM, Cre expression is induced, lead-
ing to the excision of DNA fragments between the recognition sites
and switching from S00 to S01. Upon the subsequent addition of Ara
to the MRSM, Flp expression is induced, leading to the excision
of DNA between aligned recombination sites of the register and

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

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Articles Nature Catalysis

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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Nature Catalysis
cell lifespan is a potential strategy for increasing the efficiency of
microbial cell factories.
The cell size of industrial strains is important for microbial cell
factory efficiency; this is because cell size can affect the production
of inclusion bodies, such as triacylglycerols37, wax esters38 and poly-
hydroxybutyrate39. To increase the titre of target products, a series of
morphological engineering strategies have been developed, includ-
ing the overexpression of genes involved in cell division, such as
ftsZ (ref. 40), the overexpression of genes involved in binary division,
such as sulA and minCD (ref. 40), and regulation of the architecture
of the peptidoglycan cell wall and the actin-like protein MreB cyto-
skeleton39–41, to accelerate growth, increase cell density, simplify
downstream separation, enlarge inclusion body accumulation space
and reduce the bioproduction cost40. In fact, the intracellular dis-
tribution of inclusion bodies is a crucial component of cell mor-
phology. It has previously been reported that modulation of phaM
expression in Ralstonia eutropha can be applied to regulate the size
and intracellular distribution of PHB granules22, and the nucleoid-
associated like protein PhaF drives the intracellular location and
segregation of PHA in Pseudomonas putida KT2442 (ref. 42). In
addition, to extend the logarithmic phase, three different strategies
can be invoked: (1) the carbon source, nitrogen source and metal
ions can be used to modulate the supply of essential components,
(2) fermentation control can be applied to modulate the nutrient
assimilation and mass transfer and (3) genetic manipulation can be
used to regulate cell growth. In this study, a PLH-producing strain
was divided into an aging mother for the storage of PLH and a reju-
venated daughter for PLH biosynthesis and cell growth. To achieve
this, the cell growth and PLH production modes were fine-tuned
with a TRSM by modulating the RLS. Here, we found that overex-
pression of HNS-GFP can be used not only to indicate the location
of chromosomal DNA, but also to enlarge cell morphology for more
inclusion body accumulation. This enlarged morphology is possibly
due to the inhibition of cell division by storage cells and the remod-
elling of the local DNA structure by HNS-GFP (ref. 43). Moreover,
the engineering of the RLS led to extension of the logarithmic phase,
providing an opportunity to enhance growth-coupled production.
As a result, cell growth and PLH content were increased by 17 and
50%, respectively, through the asymmetric segregation of PLH.
Thus, engineering the RLS provides an opportunity to enlarge the
morphology to enhance inclusion body accumulation in E. coli.
Metabolic engineering strategies have been adopted to enhance
butyrate production, such as the deletion of undesired pathways,
extension of metabolic pathways leading to butyrate44–46 and
enhancement of the metabolite conversion rate45. However, the
titre and yield of butyrate are far below the demands of industrial
production. This is because of a poor tolerance of the butyrate-
producing strain to high butyrate concentrations, which is a vital
issue for butyrate production. To improve the acid tolerance of the
producing strain, a series of strategies, such as microbial membrane
engineering47, tolerance engineering48 and the overproduction of
stress response proteins49, have been developed. In this study, we
divided the state of butyrate-producing bacteria into four modes.
To efficiently switch between the four modes for programming cell
differentiation according to the stages of fermentation, an MRSM
that controls the CLS was introduced to increase the butyrate titre:
(1) the MRSM could be applied to redirect carbon flux and increase
butyrate synthesis ability, (2) the deletion of ubiG blocked the gen-
eration of electron carriers and resulted in the enhanced production
of butyrate precursors and cofactors such as NADH and acetyl-CoA
(ref. 50) and (3) microbial consortia were constructed through cell
differentiation programming by the MRSM and enhanced robust-
ness to acid stress. As a result, the highest titre of butyrate was
achieved in a 5-l fermenter. These results indicate that engineering
of the CLS can be used for programming cell differentiation, thus
improving the robustness of cell factories for chemical production.
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Articles
Taken together, lifespan engineering provides a platform tech-
nology for improving microbial cell factory efficiency. Lifespan
engineering not only improves the physiological status of an indus-
trial strain, but also couples metabolic design to industrial strain cell
fate. Despite millions of years of evolution of E. coli, its lifespan is
remarkably plastic, which is expected to facilitate advances in both
metabolic engineering and synthetic biology. An improved under-
standing of cell senescence that remains stable over time will also
enhance our ability to compartmentalize complex tasks and engi-
neer functionalities for bioproduction.
Methods
Strains, media and culture conditions. All bacterial strains and plasmids used
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)
medium containing 50 g l−1 glucose was used for butyrate production. Modified
Rennie (MR) mineral salt medium containing 50 g l−1 glucose was used for
PLH production. Kanamycin (50 mg l−1), ampicillin (100 mg l−1), spectinomycin
(30 mg l−1) or chloramphenicol (30 mg l−1) were added appropriately. To maintain
plasmid stability, antibiotics were added every 12 h.
For butyrate production, seed cultures were grown at 37 °C and 200 r.p.m. in
LB medium for 10 h and then transferred into 50 ml TB medium supplemented
with 50 g l−1 glucose and 10 g l−1 CaCO3 as an acid-neutralizing agent45. When
cells had grown to an OD600 of 0.6–0.8, isopropyl β-d-thiogalactoside (IPTG,
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
butyrate production in batch fermentation, a multiple-stage fermentation process
was chosen that was controlled according to the change in growth rate, as follows:
stage I: when cells had grown to OD600 = 0.6–0.8, IPTG (0.4 mmol l−1) was added to
induce the expression of enzymes (atoB and tesB); stage II: when the cell growth
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
19 h, aTc (100 ng ml−1) was added to induce Cre recombinase expression; stage IV:
when the specific growth rate (μ) decreased to 0 at 36 h, Ara (1,000 mmol l−1) was
added to induce Flp recombinase expression.
The butyrate fed-batch fermentation was carried out in a 5-l bioreactor
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
same way as the batch fermentation, but with different fermentation time: stage I:
when cells had grown to OD600 = 5–6; stage II: when cell growth rate increased to
0.9 at 12 h; stage III: when cell growth rate decreased to 0.9 at 24 h; stage IV: when
the specific growth rate (μ) decreased to 0 at 48 h. The culture pH was controlled
at 7.0 with a mixture of KOH (1.2 mol l−1) and KHCO3 (2.4 mol l−1; Supplementary
Table 8).
For PLH production in batch fermentation, seed cultures were grown at 37 °C
and 200 r.p.m. in LB medium for 10 h and then transferred into 50 ml MR medium
supplemented with 50 g l−1 glucose and 10 g l−1 CaCO3 as an acid-neutralizing
agent. When the cells had grown to an OD600 of 0.6–0.8, IPTG (0.5 mmol l−1) or aTc
(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
3 l MR medium. The seed cultures for fermentation were grown by transferring
fresh colonies to 50 ml LB medium. After 10 h at 37 °C and 200 r.p.m., this culture
was inoculated into 100 ml MR medium supplemented with 20 g l−1 glucose. After
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
(2.4 mol l−1).
DNA manipulation. The Red homologous recombination method was applied
to gene deletions52. All plasmids were constructed using Gibson assembly and
basic molecular cloning techniques53. To construct the butyrate biosynthesis
pathway, atoB and tesB were PCR-amplified from the genomic DNA of E. coli
W3110. Similarly, crt, ter and hbd were PCR-amplified from the genomic DNA
of Clostridium acetobutylicum. To construct the PLH biosynthesis pathway phaA
and phaB (from Ralstonia eutropha), and phaC (from Pseudomonas sp. 61-3) were
artificially synthesized by Suzhou Genewiz Biotechnology, and pct was PCR-
amplified from the genomic DNA of Megasphaera elsdenii. Supplementary Tables
3–6 provide a list of relevant sequences.
Analytical methods. Optical density (OD600) and glucose measurements
were performed as described previously54. Butyrate was determined by high-
performance liquid chromatography using an Aminex HPX-87H column
(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.
Bacterial cells were collected by centrifugation at 8,000g for 10 min. The
supernatant was discarded and the cells were washed twice with 20 ml distilled
water. Dry cell weight (DCW) was assayed after vacuum lyophilization. PLH
contents were quantitatively analysed using gas chromatography (GC-2014,
Articles
SHIMADZU) after methanolysis of lyophilized cells in chloroform55.
Analytical P3HB and P(GA-LA) purchased from Sigma-Aldrich were used as
analytical standards.
Assay of CLS. Survival measurements were carried performed as described
previously15, with minor modifications. In short, cultures were inoculated
1:1,000 using an overnight culture created by inoculating two to three colonies
from LB agar pads into 5 ml LB medium. Cultures were grown overnight in
50 ml LB medium in 250 ml flasks at 200 r.p.m. and 37 °C, at which point the cell
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
medium of the same strain for the long-lived strains with reduced saturation cell
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
cell-free spent medium was supplemented with glucose (5 g l−1), lactose (5 g l−1),
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
to modulate the lifespan, the shaking speed was maintained at 0, 100, 200 or
400 r.p.m. Survival at 0 h after the last dilution was set to 100% survival and was
used to determine the age-dependent mortality. The assay was performed until
less than 1% of the culture was viable. The CLS is the time at which E. coli survival
reaches 1%. Half chronological lifespan (HCLS) is the time at which E. coli survival
reaches 50%.
Growth assay. To determine the effect of RLS on growth, we assessed the growth
rate (R) and generation time (G), obtained from equations (1)–(3):
ðlog Nt1 � log Nt0 Þ
n¼ ð1Þ
log 2
n 400–600 ms), DsRed fluorescence images (TRITC, exposure, 600 ms) and mKate2
R¼ ð2Þ
t1 � t0
ðt1 � t0 Þ
G¼ ð3Þ
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
number of bacteria at the beginning of a time interval, Nt1 is the number of bacteria
at the end of the time interval, t0 is the time in minutes in the beginning, t1 is the
time in minutes at the end, R is the number of generations per unit time and G is
the generation time of the bacteria.
SYTOX Green Dead nucleic acid staining. Cells in 1 ml LB culture were washed
twice with salt solution (0.5% NaCl in deionized water) and re-suspended in 1 ml
salt solution. Next, 3 μmol l−1 of SYTOX Green Dead nucleic acid stain (1 mmol l−1
in DMSO) were added to the cell suspension, which was then incubated at room
temperature, in the dark, for 10 min. Flow cytometry was applied to differentiate
live from dead bacteria on the basis of SYTOX Green stain intensity. SYTOX Green
stain intensity was measured in the fluorescein isothiocyanate (FITC) channel
(488 nm excitation laser (EL), 530/30 nm detection filter (EF)) in a BD FACS Aria
III cell analyser (BD Biosciences).
Time-lapse microscopy and cell death assay. When the cells had grown to an
OD600 of 0.6–0.8, aTc (10 ng ml−1) was added to induce the expression of HNS-GFP.
After 30 min of induction, cells were collected by centrifugation and re-suspended
in fresh LB medium. To assay E. coli colony growth on LB plates supplemented
with chloramphenicol (30 mg l−1), 2.5 μl of re-suspended bacterial culture was
placed on an LB plate in a microscope cavity slide. Time-lapse microscopy was
carried out as described previously56, with incubation at 30 °C during the whole
experiment. Cell death assays were performed on LB agar pads, with incubation
at 37 °C during the whole experiment. After 100 min of incubation, 3 μl of SYTOX
Green Dead nucleic acid stain (1 mmol l−1 in DMSO) was added to the slide
cavities, which were then incubated at room temperature, in the dark, for 5 min.
Nile red staining. Nile red staining measurements were carried out as described
previously57, with minor modifications. In short, the cells (1 ml MR culture)
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
incubated at room temperature, in the dark, for 5 min. The cells were washed
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
cooled CCD digital camera (Nikon digital sight Ds-Ri1).
Methylene blue staining. Cells in 1 ml LB culture were washed once with PBS
and re-suspended in 1 ml PBS. Next, 10 μl methylene blue (1 mg ml−1 in deionized
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
microscope.
Nature Catalysis
Recombination efficiency assay. Strains containing the corresponding plasmids
were plated or streaked on plates containing LB medium and grown overnight at
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
concentration of 100 ng ml−1 for induction at 30 °C. The CFUs were enumerated
over time by removing an aliquot, serially diluting in 0.5% NaCl, followed by
colony enumeration after plating on LB plates that were incubated at 37 °C
(Supplementary Fig. 22). The TRSM recombination efficiency was calculated from
equation (4):
 
Nm
Effciency ¼ 1 � ´ 100% ð4Þ
NT
where Nm is the number of pink colony-forming units on the LB plates and NT
is the total number of colony-forming units on the LB plates.
Light microscopy. To assay the colony growth on plates containing LB medium,
0.5 μl of exponentially growing bacterial culture was placed on an LB agar pad
in a microscope cavity slide. When the cells had grown to an OD600 of 0.6–0.8,
aTc (10 ng ml−1) or IPTG (0.4 mmol l−1) was added to induce the expression of
HNS-GFP or Glg-mKate2 fusion protein. The BFP intensity was measured on a
C-FL-C DAPI (4’,6-diamidino-2-phenylindole) filter cube (excitation filter (EX),
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;
DM, 505 nm; BA, 512–558 nm) and the DsRed and mKate2 intensities were
measured on a C-FL-C TRITC (tetramethylrhodamine isothiocyanate) filter
cube (EX, 527–553 nm; DM, 565 nm; BA, 577–633 nm). Microscopy images
were obtained with a Nikon ECLIPSE 80i microscope equipped with a ×100 oil
immersion objective. Bright-field images (exposure, 100 ms), BFP fluorescence
images (DAPI, exposure, 600 ms), GFP fluorescence images (FITC, exposure,
fluorescence images (TRITC, exposure, 400–600 ms) were analysed using
Image J software.
Fluorescence intensity assay. Strains harbouring the corresponding plasmids
grown overnight at 37 °C. Then, they were inoculated into 20 ml fresh LB medium
at 2% (v/v) and cultured under the same conditions until an OD600 of 0.6. Ara
(1,000 mmol l−1) or aTc (100 ng ml−1) was added for induction at 30 °C. To assay
cell growth and fluorescence intensity, the corresponding data were recorded on a
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
wavelength (EX) of 402 ± 5 nm and an emission wavelength (EM) of 457 ± 10 nm,
the GFP intensity was measured at an EX of 480 ± 5 nm and an EM of 515 ± 10 nm,
and the mKate2 intensity was measured at an EX of 588 ± 10 nm and an EM of
645 ± 10 nm. All fluorescence was normalized with cell density by measuring the
absorbance at 600 nm.
MRSM implementation assay. MRSM measurements were carried out as
described previously24, with minor modifications. In brief, the strain MRSM-1
harbouring controller plasmid (p15A-PTET-CreGTG-AAV-PBAD-FlpGTG) and actuator
plasmid (pColE-FRT-lox2272-BFP-lox2272-GFP-FRT-lox-DsRed-lox-mKate2) was
inoculated into a medium with chloramphenicol and spectinomycin.
Flow cytometry assays of the MRSM were performed as described previously24,
with minor modification. Briefly, the cells were washed three times with PBS and
diluted to an OD600 of 0.3. A BD FACS AriaTM III cell analyser (BD Biosciences)
was used for the detection of fluorescence. GFP, DsRed, mKate2 and BFP were
detected in the FITC channel (488 nm EL, 530/30 nm EF), PE-A Red channel
(561 nm EL, 582/15 nm EF), PE-Cy5-A Red channel (561 nm EL, 670/14 nm EF)
and DAPI channel (405 nm EL, 450/40 nm EF), respectively. We measured 20,000
cells for each sample. As shown in Supplementary Figs. 34 and 35, the fluorescence
threshold was determined by the negative control (E. coli JM109 containing
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
Supplementary Fig. 36. The percentages of cells in Fig. 5f were determined from
the experimental data in Supplementary Fig. 37.
Reporting Summary. Further information on research design is available in the
Nature Research Reporting Summary linked to this article.
Data availability
The data that support the figures within this paper and other findings of this
study are available from the corresponding author upon reasonable request.
Supplementary Table 9 provides a list of the GenBank accession numbers of the 14
key plasmids constructed in this study.
Received: 16 February 2019; Accepted: 28 November 2019;
Published: xx xx xxxx
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Nature Catalysis
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Acknowledgements
This work was supported by the National Key R&D Program of China
(2018YFA0901401), the National Natural Science Foundation of China (21808083,
21878126), the Key Field R & D Program of Guangdong Province (2019B020218001)
and the National First-Class Discipline Program of Light Industry Technology and
Engineering (LITE2018-08).
Articles Nature Catalysis

Author contributions Additional information

L.G. and L.L. designed the project. L.G. and W.D. conducted and analysed the
experiments. C.G., G.H., Q.D. and X.C. provided technical assistance. L.G. analysed the
data and wrote the manuscript with input from C.Y., J.L. and L.L. All authors reviewed
and approved the manuscript.
Competing interests
The authors declare no competing interests.
Supplementary information is available for this paper at https://doi.org/10.1038/
s41929-019-0411-7.
Correspondence and requests for materials should be addressed to L.L.
Reprints and permissions information is available at www.nature.com/reprints.
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