Production of Succinate from Acetate by Metabolically Engineered

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Production of succinate from acetate by metabolically engineered Escherichia coli Yunjie Li, Bing Huang, Hui Wu, Zhimin Li, Qin Ye, and Y.-H. Percival Zhang ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.6b00052 • Publication Date (Web): 18 Apr 2016 Downloaded from http://pubs.acs.org on April 19, 2016

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ACS Synthetic Biology

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Production of succinate from acetate by metabolically engineered

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Escherichia coli

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Yunjie Li1,4, Bing Huang1, Hui Wu1*, Zhimin Li1, 2*, Qin Ye1, Y-H Percival Zhang3,4

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1. State Key Laboratory of Bioreactor Engineering, East China University of Science

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and Technology, 130 Meilong Road, Shanghai 200237, China

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2. Shanghai Collaborative Innovation Center for Biomanufacturing Technology, 130

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Meilong Road, Shanghai 200237, China

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3. Biological Systems Engineering Department, Virginia Tech, 304 Seitz Hall,

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Blacksburg, Virginia 24061, USA

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4. Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32

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West 7th Avenue, Tianjin Airport Economic Area, Tianjin 300308, China

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* Corresponding author: Zhimin Li

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Telephone: +86-21-64252095

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Fax: +86-21-64252250

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E-mail: [email protected]

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* Corresponding author: Hui Wu

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Telephone: +86-21-64253701

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Fax: +86-21-64252250

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E-mail: [email protected]

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1

ACS Paragon Plus Environment


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Abstract Graphic ACE

FADH2+CO2

×

ATP ackA ADP

poxB

ATP

AcP

FAD

acs ADP

pta ppsA

PEP

pykAF ATP ADP

ADP ppc ATP

pckA

PYR

aceEF

AcCoA

NAD NADH+CO2

CIT

ATP

gltA

acnAB

ADP

×

NAD(P)H+CO2 sfcA/ maeB NAD(P)

OAA

ICT NAD

NADH mdh NAD aceB

icdA aceA

MAL

GOX

fumABC

ICL

×

αKG sucAB

iclR

×

frdABCD

sucCD

FAD

SUC

NAD NADH+CO2

SucCoA

FADH2

FUM sdhABCD

NADH+CO2

ADP

ATP

26 27

2


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Abstract

29

Acetate, a major component of industrial biological waste water and of lignocellulosic

30

biomass hydrolysate, could potentially be a less costly alternative carbon source. Here

31

we engineered Escherichia coli MG1655 strain for succinate production from acetate

32

as the sole carbon source. Strategies of metabolic engineering included the blockage

33

of the TCA cycle, enhancement of acetate utilization, redirection of the

34

gluconeogenesis pathway, and enhancement of the glyoxylate shunt. The engineered

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strain MG03 featuring the deletion of genes: succinate dehydrogenase (sdhAB),

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isocitrate lyase regulator (iclR), and malic enzymes (maeB) accumulated 6.86 mM of

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succinate in 72 h. MG03(pTrc99a-gltA) overexpressing citrate synthase (gltA)

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accumulated 16.5 mM of succinate and the yield reached 0.46 mol/mol, about 92% of

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the maximum theoretical yield. Resting-cell was adopted for the conversion of acetate

40

to succinate and the highest concentration of succinate achieved 61.7 mM.

41 42

Keywords: acetate, succinate, metabolic engineering, Escherichia coli, malic enzyme,

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citrate synthase

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3



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Introduction

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Succinate is a diprotic, dicarboxylic acid with chemical formula C4H6O4. It has wide

48

applications in food, pharmaceutical and chemical industries 1. Besides its use as a

49

surfactant, ion chelator, acidulant, flavor, antimicrobial, paint, coating, and sealant,

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succinate is a precursor to some biodegradable polyesters (e.g., polybutylene

51

succinate) 2. In addition, it is a precursor for 1,4-butanediol and tetrahydrofuran, both

52

of which are used to make engineering plastics, polyurethanes, biodegradable

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polyesters, spandex, and other specialty chemicals. Succinate is traditionally produced

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by hydrogenation of maleic acid, oxidation of 1,4-butanediol, and carbonylation of

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ethylene glycol 3. Recently, its biological production by microbial fermentation of

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renewable carbohydrates receives more attention because of concerns about depletion

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of fossil feed stocks and net emissions of greenhouse gases4. Succinate is a metabolite

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of microbial primary metabolism which usually starts with glucose 5-9, xylose 10 and

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glycerol 11, 12. Very high product yields (e.g. more than one gram of succinate per

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gram of glucose) have been achieved under anaerobic conditions 5, 13. Now the

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biological production of succinate becomes more and more competitive compared to

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traditional chemical catalysis 4, 14.

63 64

Acetate is the second simplest carboxylic acid (after formic acid) with chemical

65

formula C2H4O2. It is produced both chemically and biologically via bacterial

66

fermentation. About 75% of acetate used in the chemical industry are made by 4


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carbonylation of methanol and oxidation of acetaldehyde 15, 16, while the remaining is

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produced via fermentation. Its biological production (vinegar) involves ethanol

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oxidation by the genus Acetobacter and anaerobic fermentation by the genus

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Clostridium or Acetobacterium without ethanol as an intermediate. As a product of

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anaerobic fermentation, it is rich in treated wastewater 17. In addition, acetate is

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available in cellulosic biomass hydrolysate, because hemicellulose of plant cell walls

73

contains acetyl groups. Through a typical dilute acid biomass pretreatment 18, a large

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quantity of acetate is released in the biomass hydrolysate, forming a mixture of

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acetate, glucose, and xylose in the ranges of 13.2-16, 8.8-20.8, and 1.6-5.2 g/L,

76

respectively, together with a small amount of furfural 19. Some microorganisms can

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utilize acetate as an alternative carbon source, for example, Saccharomyces cerevisiae

78

20

79

Cryptococcus curvatus 24, 25, and so on.

, Escherichia coli 21, Corynebacterium glutamicum 22, 23, oleaginous yeast

80 81

The utilization of nontraditional carbon sources, such as acetate 24 , methane 26,

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methanol 27, syngas 28, is becoming one of the most important research areas in

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industry biotechnology because of obviously lower cost, no direct competition with

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food supplies, and less price volatilities. Acetate ($340/metric ton) is less costly than

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glucose in terms of weight and energy content ($/MJ) and is widely available in

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lignocellulosic hydrolysate and industrial waste water. Recently, Tang and his

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coworkers demonstrated that acetate was used to produce free fatty acid by an

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engineered E. coli strain with co-expression of acs and tesA genes and deletion of 5



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fadE gene 19. It is also quite interesting to utilize acetate for production of succinate.

90

To our knowledge, there has been no report pertaining to biosynthesis of succinate

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from acetate.

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92 93

Biotransformation mediated by resting cells that are not actively proliferating is an

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alternative biomanufacturing approach due to potentially high product yield and

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productivity, where the formation of the desired product is not associated with cell

96

mass synthesis. Resting cells can be prepared by placing the cells in a medium lacking

97

key nutrients for cell growth or supplemented with toxic compounds to stop cell

98

growth. Compared to enzyme-based biotransformation, resting-cell based

99

biotransformation does not require protein purification or enzyme immobilization, and

100

ATP and reducing equivalents required by the reactions can be regenerated.

101 102

In this study, the pathways of E. coli strains were modified to convert acetate to

103

succinate, including blockage of the TCA cycle, deletion of a futile cycle, weakening

104

the gluconeogenesis pathway, and enhancing the glyoxylate shunt (Figure 1).

105

Succinate was also produced from acetate by resting engineered cells under aerobic

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conditions.

107 108

Results

109 110

Pathway reconstruction for succinate production from acetate 6


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Succinate can be synthesized from acetate through the glyoxylate shunt and the TCA

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cycle (Figure 1). Acetate can be converted to acetyl-CoA through the ACK-PTA

113

(acetate kinase - phosphotransacetylase) (Equation 1) or ACS (acetyl-CoA synthase)

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pathway, which is driven by ATP. Acetyl-CoA and oxaloacetate (OAA) are combined

115

to form citrate catalyzed by citrate synthase (CS), and then to isocitrate (Equation 2).

116

Isocitrate is further cleaved by isocitratelyase (ICL) to form succinate and glyoxylate

117

(Equation 3), and the former is accumulated when succinate dehydrogenase is

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disrupted. Glyoxylate is condensed with another acetyl-CoA by malate synthase (MS)

119

to synthesize malate (Equation 4) which is further converted to OAA by malate

120

dehydrogenase, together with generating NADH (Equation 5). OAA can further be

121

combined with acetyl-CoA to start the next run of the glyoxylate shunt. The overall

122

reaction is shown in Equation 6, and it means that the maximum theoretical yield is

123

0.5 mol succinate per mol acetate. 2 mol ATP were consumed when 1 mol succinate

124

was produced. In the process of forming 1 mol succinate from 2 mol acetate, 1 mol

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NADH is generated and can be oxidized through the respiratory chain, generating the

126

proton gradient across the cellular membrane and driving ATP synthesis (Equation 7,

127

with a P/O ratio of 2.5). And the formed ATP can be used to convert acetate to

128

acetyl-CoA (Equation 1).

129 130

2 Acetate + 2 ATP + 2 CoA = 2 Acetyl-CoA + 2 ADP + 2 Pi

[1]

131

Acetyl-CoA + Oxaloacetate + H2O = Isocitrate + CoA

[2]

132

Isocitrate = Glyoxylate + Succinate

[3] 7



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Glyoxylate + Acetyl-CoA + H2O = Malate + CoA

[4]

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Malate + NAD+ = Oxaloacetate + NADH + H+

[5]

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2 Acetate + NAD+ + 2 ATP + 2 H2O = Succinate + NADH + H+ + 2 ADP + 2 Pi

[6]

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NADH + H+ + 1/2 O2 + ~2.5 (ADP + Pi) = NAD+ + H2O + ~2.5 ATP

[7]

137 138

Succinate production from acetate

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The inactivation of succinate dehydrogenase (encoded by sdhABCD) blocks the TCA

140

cycle, resulting in succinate accumulation from glucose under aerobic conditions 6, 10,

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29

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SMAC medium (Figure 2). The growth rate of MG1655 was much higher than MG01

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and all acetate was consumed within 12 h but no succinate was produced. MG01

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consumed acetate slowly (only 12.1 mM at 72 h), but was able to produce 2.64 mM

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succinate. Similar phenomenon that the disruption of sdhAB impaired cell growth on

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glucose was reported previously (Lin et al. 2005c). Interestingly, no by-products were

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detected in broth of the both experiments, indicating that all the consumed acetate was

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used for cell growth and succinate production.

. The wild-type strain MG1655 and mutant MG01 (∆sdhAB) were cultured in

149 150

Because the glyoxylate shunt became the main succinate biosynthesis pathway, where

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the IclR repressor of aceBAK operon repressed the glyoxylate shunt, deletion of iclR

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enhanced succinate production from glucose6, 29.Therefore, strain MG02 with iclR

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knockout in MG01 was constructed. MG02 consumed slightly more acetate (12.4 mM

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at 72h) and produced more succinate (2.81 mM) as compared to MG01 (Figure 2).The 8


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specific acetate consumption rate and the specific succinate production rate of MG01

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and MG02 were similar, which indicated that the additional deletion of iclR have little

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positive effect on succinate production here.

158 159

To avoid carbon flux via gluconeogenesis, the gene maeB in strain MG02 was further

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deleted, yielding strain MG03. As shown in Figure 2, strain MG03 consumed more

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acetate (19.9 mM at 72h) and exhibited a drastic increase in succinate titer to 6.86

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mM at 72 h, 2.60-fold of that produced by MG01, suggesting the importance of

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deleting the competing pathway for enhanced succinate yield. The specific acetate

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consumption rate and the specific succinate production rate of MG03 were improved

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to 1.63-fold and 2.55-fold than MG01, respectively.

166 167

There is a futile cycle that converts pyruvate to acetate via pyruvate oxidase (encoded

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by poxB). The poxB gene was interrupted, yielding strain MG04 (∆sdhAB, ∆iclR,

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∆maeB, ∆poxB). However, strain MG04 did not show any advantages over MG03,

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suggesting that the futile cycle via pyruvate oxidase had little function.

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Transcriptome and qPCR analysis of MG03 and MG1655

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To understand the expression of key genes related to acetate utilization and succinate

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formation, the transcriptome sequencing was performed for strains MG1655 (wildtype

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control) and MG03 (∆sdhAB, ∆iclR, ∆maeB). The 676 differentially expressed genes

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were classified into different categories according to Cluster of Orthologous Groups 9



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of proteins (COG). The main down-regulated COG Term were response to amino acid

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transport and metabolism, energy production and conversion, while the main

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up-regulated COG Term were response to inorganic ion transport and metabolism,

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secondary metabolites biosynthesis, transport and catabolism. The wide diversity in

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these COG Terms suggested that the reconstruction strategy has a significant impact

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on the physiology and function of host cells (Figure S1). Table 4 lists the differential

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transcription levels of genes involved in central metabolic pathways which were

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related to succinate biosynthesis [fold change (FC) > 2 or fold change < 0.5, false

185

discovery rate (FDR) < 0.05]. The transcription of aceB encoding malate synthase

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was increased about 4.7-fold, suggesting that the glyoxylate shunt flux was enhanced

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greatly in MG03, thus this regulation benefitted for the production of succinate. The

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transcription level of pckA was down-regulation in MG03 compared with MG1655,

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which is consistent with the poor cell growth rate. Interestingly, the transcription level

190

of fumC encoding fumarase was significantly up-regulated (Log2FC=3.85, about

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14.4-fold).

192 193

The reliability of RNA-sequencing results was validated by RT-PCR (Figure 3). The

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transcriptional levels of aceB and fumC were up-regulated greatly while that of pckA

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was down regulated remarkably, in consistent with the data of RNA-sequencing.

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While the changes in transcriptional levels of gltA, aceE, ackA, acs, ppsA, mdh, icd

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were insignificant (Log2FC < 1 or FDR > 0.05) in RNA-sequencing, the changes

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observed from RT-PCR were also within 2-fold. 10


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199 200

Overexpression of citrate synthase gene

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Since the transcriptional level of gltA between MG03 and MG1655 was similar, and

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citrate synthase (CS) is the rate-limiting step of TCA cycle 30, the citrate synthase

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gene (gltA) was overexpressed by introduction of plasmid pTrc99a-gltA. The specific

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activity of CS was only 0.03 ± 0.00 U/mg protein in the cell lysate of MG1655. In

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contrast, the specific activity of CS in strain MG1655(pTrc99a-gltA) was as high as

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0.62 ± 0.01 U/mg protein, an increase by more than 20 times. The overexpression of

207

CS did not show a negative effect on cell growth of MG1655, and no succinate was

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produced in MG1655(pTrc99a-gltA) (Figure 4). Strain MG03(pTrc99a-gltA) exhibited

209

a drastic increase in succinate production compared to strain MG03: the succinate

210

concentration reached 16.45 mM at 72 h (Figure 4), a 2.40-fold increase. The

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succinate yield of MG03(pTrc99a-gltA) based on consumed acetate also increased to

212

0.46 mol/mol (Figure 5) and the productivity of succinate was 26.98 mg/L/h. The

213

specific acetate consumption rate and the specific succinate production rate of MG03

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(pTrc99a-gltA) were improved to 1.80-fold and 2.45-fold than MG03, respectively.

215 216

Biosynthesis of succinate by resting cells

217

The deletion of sdhAB led to very poor growth on acetate in a minimal medium.

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Therefore, the sdhAB mutants were grown in a complex medium to obtain more cells.

219

In this study, we established a two-stage bioconversion system for synthesis of

220

succinate from acetate. In the first stage, cells were grown in a complex medium and 11



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overexpression of CS was induced. In the second stage, a minimal medium without

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nitrogen source was used, and sodium acetate was the only carbon and energy source.

223

In this system strain MG03(pTrc99a-gltA) produced 44.85 mM of succinate with a

224

molar yield of 0.36 mol/mol (Figure 6) in 12 h at an initial acetate of 122 mM. The

225

succinate production was better than that obtained in flask cultivation, in which the

226

succinate concentration was 16.45 mM in 72 h. The titer of succinate and the

227

productivity were significantly increased, but the yield was decreased in the resting

228

cell system.

229 230

When the initial concentration of sodium acetate was increased to 244 mM, the

231

produced succinate increased to 61.71 mM, but the succinate yield decreased to 0.30

232

mol/mol, suggesting a loss of about 40% carbon. The carbon loss could be attributed

233

to the decarboxylation of isocitrate or gluconeogenesis pathways in the form of

234

carbon dioxide along with energy production. Further increase the initial acetate

235

concentration to 366 mM, the succinate production was seriously suppressed. With

236

the increase in the initial concentration of sodium acetate, the yield of succinate based

237

on consumed acetate was declined, indicating high acetate concentration inhibited

238

succinate formation.

239 240

Discussion

241

The metabolism of acetate and its regulation in E. coli have been well studied over the

242

last 20 years, and the efforts mainly focus on reducing acetate accumulation due to its 12


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negative effects on growth and recombinant proteins production, especially when the

244

microorganism is aerobically cultured on glucose 31, 32. The feasibility of conversion

245

of acetate to oil was verified in the oleaginous yeast C. curvatus 24, 25 and E. coli 19,

246

but generation of succinate from acetate by microorganism has not been reported yet.

247

The present study confirmed the feasibility of this idea. The engineered strain utilized

248

acetate to produce succinate effectively. Strain MG03 (pTrc99a-gltA) produced 16.5

249

mM succinate in a rich medium. When a resting cell conversion system was applied,

250

strain MG03(pTrc99a-gltA) produced 61.71 mM succinate in minimal medium and

251

did not accumulate any byproducts (Figure 5,6).

252

Considering the energy demand in acetate activation, we carried out fermentation

253

aerobically. However, the growth of sdhAB mutant E. coli on acetate was severely

254

impaired due to the incomplete TCA cycle. In MG01, the metabolic rate of acetate

255

was too slow to obtain a high productivity of succinate. Overexpression of acetyl-CoA

256

synthase along with maintaining the native ACK-PTA pathway was considered the

257

best strategy for acetate assimilation in E. coli as the previously report19. To increase

258

the acetate utilization rate, we tried to strengthen the acetate utilization pathways by

259

overexpression of the acetyl-CoA synthase genes from E. coli MG1655 and Bacillus

260

subtilis in MG01, but the growth of both recombinant strains was significantly

261

inhibited (data not shown). As the activation of acetate to acetyl-CoA requires ATP,

262

energy-limitation might be the main reason for the failure of growth.

263 264

To improve the titer and productivity of succinate, partially blocking gluconeogenesis 13



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Page 14 of 35

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pathway was performed by knockout of the maeB gene. In MG03 (∆sdhAB, ∆iclR,

266

∆maeB), the transcription level of pckA encoding the phosphoenolpyruvate

267

carboxykinase (PCK) was down-regulated compared with MG1655. Previous study

268

showed that the growth rate of maeB and pckA double mutant was declined when

269

grown in acetate minimal medium21. It is consistent with the poor cell growth rate of

270

MG03 on acetate. In addition, PCK is responsible for delivering the carbon flux from

271

the TCA cycle to the gluconeogenic pathway when grown on acetate, and competes

272

with citrate synthase for the common substrate oxaloacetate (OAA). This meant that

273

most oxaloacetate was directed to synthesis of citrate, not pyruvate, and could explain

274

why further knockout of poxB in MG03 did not enhance succinate production.

275 276

Additionally, the catabolic repressor/activator protein (Cra) plays an important role

277

in the control of metabolic flux in E.coli, which can active the transcription of gene

278

pckA, ppsA, icdA33. The transcription level of cra was down-regulated to about

279

0.25-fold, which might be one of the reasons why the expression level of pckA was

280

down-regulated33. The expression and regulation of three fumarase were complicated,

281

which was related to oxygen, iron, carbon and superoxide, and FumC was

282

significantly induced when cells encounter oxidative stress conditions34. Fnr and ArcA

283

serve as transcription repressor of fumarase gene expression35，and ArcA-dependent

284

transcription regulation controls TCA cycle flux of E. coli under both aerobic and

285

anaerobic conditions directly or indirectly36. The higher transcription level of fumC

286

may be related to the oxygen level as well as the transcription regulator (such as fnr, 14


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287

arcA), and the exact mechanism was not clear, which needs further investigation.

288 289

Obviously, OAA is a very important node in metabolic flux distribution. Besides

290

deletion of maeB pushed malate to form OAA, the strengthening glyoxylate cycle by

291

overexpression of CS pulled OAA to the glyoxylate shunt was also improved the

292

succinate production dramatically. Combination of these two significant strategies had

293

a direct effect on OAA node. It can be speculated that further increase metabolic flux

294

flow to OAA (for example knockout gene pckA and/or icd) might enhance the

295

production and yield of succinate. In addition, the energy produced by the whole

296

system might be not sufficient to maintain cell growth and product synthesis. Energy

297

limitation could also be the reason of poor cell growth. Follow-up studies will be

298

performed to improve the energy supply and accelerate the glyoxylate cycle.

299 300

In conclusion, this study explores the possibility of using acetate as the new carbon

301

source for succinate production. As we can easily get a lot of cheap acetate from

302

various ways, considering environment-friendly and resource recycling, we try to use

303

acetate as a carbon source to produce high value-added chemicals. However, the

304

current level of succinate titer would not be practical for commercial use, other

305

metabolic engineering strategies need to be explored to co-ordination of acetate

306

utilization, ATP generation, gluconeogenesis pathway, futile cycle and coenzyme

307

levels in the next work.

308 15



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Materials and Methods

310 311

Strains and plasmids

312

All of the strains and plasmids used in this study are listed in Table 1, and the

313

oligonucleotides for gene amplification are listed in Table 2. Mutant strains used in

314

this study were created from the wild type strain MG1655 by using the λ

315

Red-mediated one-step inactivation method37. All mutants were verified by colony

316

PCR and DNA sequencing to ensure that the gene of interest had been disrupted. The

317

citrate synthase gene (gltA) was amplified from E. coli genome, digested with BamHI

318

and Hind III and then ligated to plasmid pTrc99a digested with the same enzymes to

319

form pTrc99a-gltA, which was used to transform the constructed mutant strains.

320 321

Medium and cultivation

322

Luria-Bertani (LB) medium (per liter: tryptone10 g, yeast extract 5 g, sodium chloride

323

10 g) was used for construction of strains and plasmid amplification. During strain

324

and plasmid construction, cultures were grown at 37 °C and 220 rpm. For the

325

cultivation in flasks, the medium SMAC was used which was prepared by

326

supplementing the M9 mineral salts medium with 5 g/L of sodium acetate and 2 g/L

327

of yeast extract. The M9 mineral salts medium consisted of the following components

328

(per liter): Na2HPO4·12H2O (15.12 g), KH2PO4 (3.0 g), NaCl (0.5 g), MgSO4·7H2O

329

(0.5 g), CaCl2 (0.011 g), NH4Cl (1.0 g), 1% (w/v) vitamin B1 (0.2 mL), and trace

330

elements solution (0.1 mL). The stock solution of trace elements contained the 16


Page 17 of 35

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331

following (per liter) in 3 M HCl: FeSO4·7H2O (80 g), AlCl3·6H2O (10 g),

332

ZnSO4·7H2O (2.0 g), CuCl2·2H2O (1.0 g), NaMoO4·2H2O (2.0 g), MnSO4·H2O (10

333

g), CoCl2 (4.0 g), and H3BO4 (0.5 g). Appropriate antibiotics were included as

334

necessary at the following concentrations: 100 µg/mL ampicillin, 50 µg/mL

335

kanamycin, and 35 µg/mL chloramphenicol.

336 337

For fermentation in flasks, the inoculum was prepared by transferring 100 µL of the

338

stock culture in glycerol to 3 mL of LB medium, in which the cells were aerobically

339

incubated at 37 ºC and 220 rpm overnight. 1 mL of the culture was transferred to a

340

250-mL flask containing 50 mL SMAC medium, grown at 37 °C and 220 rpm.

341 342

Production of succinate from acetate by using resting cells was performed in two

343

stages as follows. In the first stage, cells were aerobically grown in a 5-L bioreactor

344

containing 2.5 L of LB medium supplemented with 55 mM glucose. The temperature

345

and agitation speed of the bioreactor were 37 °C and 500 rpm, respectively, and the

346

airflow rate was 1 vvm. The pH was controlled at 7.0 using 25% ammonia. When the

347

cell density reached to 20 (A600), 1 mM IPTG was added to induce the expression of

348

citrate synthase (CS) for 4 h. In the second stage, the cells were harvested by

349

centrifugation and washed once with the M9 medium without NH4Cl, trace elements,

350

and carbon source. Cell pellets were resuspended in 20 mL of fresh NH4Cl-free M9

351

medium containing different concentrations of sodium acetate in a 250-mL flask. The

352

flasks were incubated at 37 °C and 220 rpm. All experiments in shake flasks were 17



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353

Page 18 of 35

performed in triplicate.

354 355

Analytical methods

356

Cell growth was monitored by measuring the absorbance at 600 nm. Before

357

measurement, the culture was diluted to ensure the value of A600 to be less than 0.6.

358

For measuring extracellular metabolites concentrations, cell culture was centrifuged

359

for 10 min at 12,000 rpm and 4 °C, and the supernatant was then filtered through a

360

0.22-µm syringe filter for HPLC analysis. The concentrations of acetate and succinate

361

were analyzed by HPLC with an aminex HPX-87H ion exclusion column (Bio-Rad,

362

USA) 38. Chromatography was performed on an LC Solutions system (Shimadzu

363

Corporation, Kyoto, Japan) with a refractive index detector (RID-10A, Shimadzu

364

Corporation, Kyoto, Japan). The mobile phase was 5 mM H2SO4 solution at a flow

365

rate of 0.6 mL/min. The HPLC column was operated at 65 °C.

366 367

Enzymatic activity assay

368

To determine the citrate synthase (CS) activity, E. coli strains were cultivated in LB

369

medium supplied with 10 g/L sodium acetate, and induced by IPTG when the

370

absorbency at 600 nm (A600) reached 0.4-0.6. After induction for 12 h, 1 mL of cell

371

cultures (about A600 of 7) were harvested by centrifugation at 8000 rpm and 4 °C for

372

10 min, washed twice with 1 mL of 50 mM cold Tris-HCl (pH 7.5). Cells were

373

disrupted by sonication (Scientz JY92-II, China) in an ice bath (output 200 W, a

374

working period of 2 s in each cycle of 5 s, 90 cycles). The cell debris was removed by 18


Page 19 of 35

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375

centrifugation at 12000 rpm and 4 °C for 10 min, and the supernatant was used for the

376

assays as described by Srere39. The activity of CS was determined by measuring the

377

appearance of free SH group of released CoA-SH using Ellman’s reagent (DTNB).

378

We described the step in brief. The reaction system in 1 milliliter contains 100 mM

379

Tris-HCl (pH 7.8), 0.1 mM DTNB, 0.3 mM acetyl-CoA, 0.5 mM oxaloacetate and

380

enzyme solution. Oxaloacetate should be prepared fresh daily. All the components

381

except oxaloacetate were mixed at 30 °C and the absorption at 412 nm is followed for

382

3 minutes to measure possible acetyl-CoA deacylase activity. The reaction was started

383

by the addition of oxaloacetate. Linear rates are obtained to calculate the specific

384

activity. One unit of CS activity was defined as the amount of the enzyme required to

385

produce 1 µmol of Citrate/min.

386 387 388

RNA-sequencing

389

E. coli strains were cultivated for 5 h (strain MG1655) or 24 h (strain MG03) in

390

SMAC medium, and then were harvested by centrifugation for 10 min at 12000 rpm

391

and 4 °C. The cell pellets were treated with liquid nitrogen and were stored at -80 °C

392

prior to RNA extraction. Total RNA was extracted from the cells with Trizol reagent

393

(Invitrogen, Grand Island, NY, USA). The RNA quality was checked by capillary

394

electrophoresis (Bioanalyzer 2200, Aligent, Santa Clara, USA) and the RNA extract

395

was kept at -80 °C. The RNA with RNA Integrity Number (RIN) > 8.0 was suitable

396

for rRNA depletion. The rRNA was depleted by Ribo-ZeroTM rRNA Removal Kits 19



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Page 20 of 35

397

(Gram-Negative Bacteria) (Illumina Inc., San Diego, CA). Sequencing and

398

subsequent bioinformatics analysis were completed at Novel Bioinformatics Co., Ltd,

399

Shanghai, China.

400 401

qRT-PCR

402

The primers used for quantitative real-time PCR analysis are listed in Table 3. The

403

gapA gene encoding glyceraldehyde 3-phosphate dehydrogenase was used as the

404

internal standard in order to standardize the amount of sample DNA added to a

405

reaction 40. For qRT-PCR, total RNA was extracted from cells using the RNAprep

406

Pure Cell/Bacteria Kit (TIANGEN, Beijing, China) according to the manufacturer’s

407

protocol. RNA concentration and purity was assessed by spectrophotometry

408

(Nanodrop 2000, Thermo), and the ratio of absorbance at 260 nm to 280 nm was

409

ensured between 1.8 and 2.1. cDNA was synthesized through reverse transcription

410

reaction using commercial kits (GoScript A5000, Promega). Quantitative real-time

411

PCR was performed by using the GoTaq qPCR Master Mix (Promega) and the

412

reaction was carried out in the Real-Time PCR system (CFX96, BIO-RAD).

413

Amplification of each gene was performed in triplicate. The 2-△△Ct method was used

414

for comparison of the gene transcription in the mutant to that of the wild type strain41.

415 416

Acknowledgements

417

This study was supported by the National Science Foundation for Young Scientist of

418

China (Grant No. 21406065), the Fundamental Research Funds for the Central 20


Page 21 of 35

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419

Universities (Grant No. 222201313007 & 22A201514042), and the National High

420

Technology Research and Development Program of China (Grant No.

421

2012AA022104 & 2012AA021205), the Foundation of Key Laboratory for Industrial

422

Biocatalysis (Tsinghua University), Ministry of Education.

423 424

Competing interests

425

The authors declare that they have no competing interests.

426 427 428

Supporting Information

429

Fig.S1 (A) The down regulated COG Term; (B) The up regulated COG Term.

430 431 432 433

21



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Page 22 of 35

434

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(17) Ni, C., Wu, X., Dan, J., and Du, D. (2014) Facile recovery of acetic acid from waste acids of electronic industry via a partial neutralization pretreatment (PNP) – Distillation strategy, Sep. Purif. Technol. 132, 23-26. (18) Zhu, Z., Sathitsuksanoh, N., Vinzant, T., Schell, D. J., McMillan, J. D., and Zhang, Y. H. P. (2009) Comparative study of corn stover pretreated by dilute acid and cellulose solvent-based lignocellulose fractionation: Enzymatic hydrolysis, supramolecular structure, and substrate accessibility, Biotechnol. Bioeng. 103, 715-724. (19) Xiao, Y., Ruan, Z., Liu, Z., Wu, S. G., Varman, A. M., Liu, Y., and Tang, Y. J. (2013) Engineering Escherichia coli to convert acetic acid to free fatty acids, Biochem. Eng. J. 76, 60-69. (20) Pena, P. V., Glasker, S., and Srienc, F. (2013) Genome-wide overexpression screen for sodium acetate resistance in Saccharomyces cerevisiae, J. Biotechnol. 164, 26-33. (21) Oh, M. K., Rohlin, L., Kao, K. C., and Liao, J. C. (2002) Global expression profiling of acetate-grown Escherichia coli, J. Biol. Chem. 277, 13175-13183. (22) Hayashi, M., Mizoguchi, H., Shiraishi, N., Obayashi, M., Nakagawa, S., Imai, J.-i., Watanabe, S., Ota, T., and Ikeda, M. (2002) Transcriptome analysis of acetate metabolism in Corynebacterium glutamicum using a newly developed metabolic array, Biosci. Biotechnol. Biochem. 66, 1337-1344. (23) Wendisch, V. F., Spies, M., Reinscheid, D. J., Schnicke, S., Sahm, H., and Eikmanns, B. J. (1997) Regulation of acetate metabolism in Corynebacterium glutamicum: transcriptional control of the isocitrate lyase and malate synthase genes, Arch. Microbiol. 168, 262-269. (24) Christophe, G., Deo, J. L., Kumar, V., Nouaille, R., Fontanille, P., and Larroche, C. (2012) Production of oils from acetic acid by the oleaginous yeast Cryptococcus curvatus, Appl. Microbiol. Biotechnol. 167, 1270-1279. (25) Gong, Z., Shen, H., Zhou, W., Wang, Y., Yang, X., and Zhao, Z. K. (2015) Efficient conversion of acetate into lipids by the oleaginous yeast Cryptococcus curvatus, Biotechnol. Biofuels 8, 1-9. (26) Conrado, R. J., and Gonzalez, R. (2014) Envisioning the bioconversion of methane to liquid fuels, Science 343, 621-623. (27) Sonntag, F., Kroner, C., Lubuta, P., Peyraud, R., Horst, A., Buchhaupt, M., and Schrader, J. (2015) Engineering Methylobacterium extorquens for de novo synthesis of the sesquiterpenoid α-humulene from methanol, Metab. Eng. 32, 82-94. (28) Revelles, O., Tarazona, N., García, J. L., and Prieto, M. A. (2016) Carbon roadmap from syngas to polyhydroxyalkanoates in Rhodospirillum rubrum, Environ. Microbiol., 18,708-720. (29) Lin, H., Bennett, G. N., and San, K. Y. (2005) Metabolic engineering of aerobic succinate production systems in Escherichia coli to improve process productivity and achieve the maximum theoretical succinate yield, Metab. Eng. 7, 116-127. (30) Walsh, K., and Koshland, D. E. (1985) Characterization of rate-controlling steps in vivo by use of an adjustable expression vector, Proc. Natl. Acad. Sci. U. S. A. 82, 3577-3581. (31) De Mey, M., De Maeseneire, S., Soetaert, W., and Vandamme, E. (2007) Minimizing acetate formation in E. coli fermentations, J Ind. Microbiol. Biotechnol. 34, 689-700. (32) Gerstmeir, R., Wendisch, V. F., Schnicke, S., Ruan, H., Farwick, M., Reinscheid, D., and Eikmanns, B. J. (2003) Acetate metabolism and its regulation in Corynebacterium glutamicum, J. Biotechnol.104, 99-122. (33) Shimizu, K. (2013) Metabolic regulation of a bacterial cell system with emphasis on Escherichia coli metabolism, ISRN Biochem, 645983. (34) Park, S. J., and Gunsalus, R. P. (1995) Oxygen, iron, carbon, and superoxide control of the 23



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fumarase fumA and fumC genes of Escherichia coli: role of the arcA, fnr, and soxR gene products, J Bacteriol. 177, 6255-6262. (35) Chen, Y. P., Lin, H. H., Yang, C. D., Huang, S. H., and Tseng, C. P. (2012) Regulatory role of cAMP receptor protein over Escherichia coli fumarase genes, J Microbiol. 50, 426-433. (36) Perrenoud, A., and Sauer, U. (2005) Impact of global transcriptional regulation by ArcA, ArcB, Cra, Crp, Cya, Fnr, and Mlc on glucose catabolism in Escherichia coli, J Bacteriol 187, 3171-3179. (37) Datsenko, K. A., and Wanner, B. L. (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products, Proc. Natl. Acad. Sci. U. S. A. 97, 6640-6645. (38) Liu, Y., Wu, H., Li, Q., Tang, X., Li, Z., and Ye, Q. (2011) Process development of succinic acid production by Escherichia coli NZN111 using acetate as an aerobic carbon source, Enzyme Microb. Technol. 49, 459-464. (39) Srere, P. A. (1969) [1] Citrate synthase: [EC 4.1.3.7. Citrate oxaloacetate-lyase (CoA-acetylating)], In Methods in Enzymology (John, M. L., Ed.), pp 3-11, Academic Press. (40) Li, Y., Li, M., Zhang, X., Yang, P., Liang, Q., and Qi, Q. (2013) A novel whole-phase succinate fermentation strategy with high volumetric productivity in engineered Escherichia coli, Bioresour. Technol. 149, 333-340. (41) Schmittgen, T. D., and Livak, K. J. (2008) Analyzing real-time PCR data by the comparative CT method, Nat. Protocols 3, 1101-1108.

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Page 25 of 35

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540 541

Table 1 Strains and plasmids Strains

Genotype

MG1655

wild type

MG01

MG1655, ∆sdhAB::KmR

MG02

MG1655,∆sdhAB,∆iclR::KmR

MG03

MG1655,∆sdhAB,∆iclR,∆maeB::KmR

MG04

MG1655,∆sdhAB,∆iclR,∆maeB,∆poxB::KmR

Plasmids pTrc99a

Cloning vector, ApR

pTrc99a-gltA

Citrate synthase (gltA) from E. coli cloned in pTrc99a, ApR

pKD4

oriR6Kγ, kmR, rgnB (Ter)

pKD46

araBp-gam-bet-exo, bla (ApR), repA101 (ts), oriR101

pCP20

ApR, CmR, FLP recombinance

542 543

25



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544

Page 26 of 35

Table 2 Primers sequences Primers

Sequences（from 5’ to 3’）

F-iclR-check R-iclR-check F-maeB-check R-maeB-check F-poxB-check

ATGAAATTGCCAGTCAGAGAATTTGATGCAGTTGTGATTGGT GCCGGTGGCGTCTTGAGCGATTGTGTAG TTACGCATTACGTTGCAACAACATCGACTTGATATGGCCGAT GGCGCGCGGATGTAACGCACTGAGAAGC GCATGTGGCAGGTGTTGA TACGGGTAATGACTTGTAGGC GAAAACCCGCCGTTGCCACCGCACCAGCGACTGGACAGGTT CAGTCTTTACGTCTTGAGCGATTGTGTAG CAGCGCATTCCACCGTACGCCAGCGTCACTTCCTTCGCCGCT TTAATCACGATGTAACGCACTGAGAAGC CACTTTGCTGCTCACACTTGCTCCCGAC TTGGCGTCAATGCGATTAACAGACACCC CAGGCATGGTATTGCTGGAT TTCGCTGTGGTGCATAAACT TGGTTCTCGCATAATCGC

R-poxB-check

CTCCGTAAACGTCGTCCC

pTrc99a-acs-F (EcoR I)

GCGGAATTCATGAGCCAAATTCACAAACACACCATTCCTG

pTrc99a-acs-R (BamH I)

TATGGATCCTTACGATGGCATCGCGATAGCCTGCTTCTCT

pTrc99a-gltA-F (BamH I)

TTAGGATCCAAGGAGATATAATGGCTGATACAAAAGCAAA

pTrc99a-gltA-R (Hind III)

CGCAAGCTTTTAACGCTTGATATCGCTTTTA

F-sdhAB R-sdhAB F-sdhAB-check R-sdhAB-check F-iclR R-iclR

545

26


Page 27 of 35

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546

Table 3 Primers sequences used in RT-PCR Primers aceA-F aceA-R aceB-F aceB-R aceE-F aceE-R ackA-F ackA-R acs-F acs-R gltA-F gltA-R icd-F icd-R maeA-F maeA-R mdh-F mdh-R pckA-F pckA-R ppsA-F ppsA-R gapA-F-1 gapA-R-1

Sequences（from 5’ to 3’） GGAAGGCATTACTCGCCCAT TTCAATACCCGCTTTCGCCT TGTGTGCCGATTTATGGCCT CATCTCTTCGCCCAGCATCT GGCGACCTGGTTTACTTCCA GTGCGGATAGGAAGAGAGGC ATGCAGCTTCTTTTGCACCG GCAGGGCGTAGAGGTAAGAC CGGTAAGCCAAAAGGTGTGC GCCTTCAAACATCAGCGTGG CTCCTACGCCGGTAACTTCC ATACCTGCTGCGATACAGGC GTACTGCGCCGAAATATGCC AGCAGTTTAGCGCCATCCAT TCGAAGAACAAGCGGAACGA GCCGACGGTTGGGGTATAAA GACCAAACGCATCCAGAACG GTTGAGAGAAGAAACGGGCG AACCCGCAGTGGAAAGAACA GGAGCAGTGCATAGAAGCGA TTTGACGATCAGGAACCGCA AGAGAGACGGACAATGACGC GCTCGTAAACACATCACCGC TAACTTTAGCCAGCGGAGCC

547 548

27



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549 550

Page 28 of 35

Table 4. The transcription levels of differential genes involved in central metabolic pathway in MG03 compared with that in MG1655 GeneID Log2FC FDR Description maeB -6.18 0.00 malic enzyme: putative oxidoreductase/phosphotransacetylase sdhA -5.66 0.00 succinate dehydrogenase, flavoprotein subunit pckA -2.86 0.00 phosphoenolpyruvatecarboxykinase [ATP] aceB 2.22 0.04 malate synthase A sdhC 3.83 0.00 succinate dehydrogenase, membrane subunit, binds cytochrome b556 fumC 3.85 0.00 fumaratehydratase (fumarase C), aerobic Class II sdhD 4.97 0.00 succinate dehydrogenase, membrane subunit, binds cytochrome b556

551 552

28


Page 29 of 35

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553

Figure Captions

554

Figure 1 Metabolic pathways related with acetate utilization and succinate production

555

in E. coli.

556 557

Figure 2 Profiles of cell density (A), acetate (B) and succinate (C) concentrations in

558

cultivation of different E. coli strains: MG1655 (■), MG01 (●), MG02 (◆), MG03

559

(▲), and MG04 (×).

560 561

Figure 3 Relative transcription levels of genes related to succinate biosynthesis in

562

MG03 compared with MG1655.

563 564

Figure 4 Profiles of cell density (A), acetate (B) and succinate (C) concentrations in

565

cultivation of different E. coli strains overexpressing gltA. (×), MG1655(pTrc99a);

566

(▲), MG1655(pTrc99a-gltA); (■), MG02(pTrc99a-gltA); (◆), MG03(pTrc99a-gltA).

567 568

Figure 5 Succinate production and the yield of different metabolic engineered strains.

569

(■) the titer of succinate; (■) the yield of succinate on acetate.

570 571

Figure 6 Effects of different initial acetate concentrations on acetate consumption and

572

succinate production (A) and the yield of succinate (B) by MG03(pTrc99a-gltA)

573

incubated in the resting cell system. The cell density was about 25 (A600). The

574

incubation time was varied depending on the consumption of acetate.

575 576

29



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Page 30 of 35

Gluconeogenesis Pathway ATP ackA FADH2+CO2

ADP

poxB

ATP

AcP

FAD

pfk

pgi

F6P

G6P

fbp

eno

FDP fbaABGAP/DHAP gap BPG

3PG/2PG

X5P

NADP

E4P

gnd

ADP

NAD

CIT

pckA

gltA

Ru5P

OAA NAD(P)H+CO2 sfcA/ maeB

GAP

S7P

MAL

NAD+ icdA NADH+CO2

aceA

α-KG

aceB

GOX

ICL

tktAB

X5P

NAD+ sucAB

fumABC

R5P

rpiAB

FUM

iclR

sdhABCD

frdABCD

577

NADH+CO2

SucCoA

FADH2 sucCD

FAD

Pentose Phosphate Pathway

578

ICT

Glyoxylate Shunt

NADH mdh NAD+

NAD(P)+

rpe

acnAB

ADP

NADPH+CO2 talAB

AcCoA

NADH+CO2

ATP ppc

ATP

aceEF

PYR

pykAF ATP ADP

tktAB NADPH

6PG/PGL

ppsA

PEP

pgk,pgm

NADP

zwf

ADP pta

NAD NADH ADP ATP

ADP

ATP

acs

ATP

ADP

TCA Cycle

Fig. 1

579 580

30


Page 31 of 35

6.00

(A) 5.00

A600

4.00 3.00 2.00 1.00 0.00 0

20

40

60

80

Time (h) Acetate (mM)

80.00

(B)

70.00 60.00 50.00 40.00 30.00 20.00 10.00 0.00 0

20

40

60

80

60

80

Time (h) 8.00

Succinate (mM)

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


7.00

(C)

6.00 5.00 4.00 3.00 2.00 1.00 0.00 0

581 582

20

40

Time (h)

Fig. 2

31



583

18 16 14 12 6

Relative mRNA levels

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

Page 32 of 35

4

2

0

aceB aceE ackA acs gltA pckA ppsA fumC mdh icd

584 585

Fig. 3

586

32


Page 33 of 35

5.00

(A)

A600

4.00 3.00 2.00 1.00 0.00 0

20

40

60

80

Time (h)

Acetate (mM)

70.00

(B)

60.00 50.00 40.00 30.00 20.00 10.00 0.00 0

20

40

60

80

60

80

Time (h) 18.00

Succinate (mM)

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


(C)

16.00 14.00 12.00 10.00 8.00 6.00 4.00 2.00 0.00 0

587 588

20

40

Time (h)

Fig. 4

589 590

33



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

Page 34 of 35

591 592

Fig. 5

593 594 595 596 597 598 599 600 601 602

34


Page 35 of 35

603

250

(A)

Acetate Succinate

80 150 60 100 40 50

Succinate (mM)

Acetate (mM)

120 100

200

20

0

0 100

200

300

400

Initial Conc. of NaAC (mM) 0.4

(B)

0.35 0.3

Yield (mol/mol)

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


0.25 0.2 0.15 0.1 0.05 0 122

604 605

244

366

Initial Conc. of NaAC (mM)

Fig. 6

606 607 608 609 610

35


Production of Succinate from Acetate by Metabolically Engineered

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