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Metabolic Engineering of Escherichia coli for the Production of 3Hydroxypropionic Acid and Malonic Acid through beta-Alanine Route Chan Woo Song, Je Woong Kim, In Jin Cho, and Sang Yup Lee ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.6b00007 • Publication Date (Web): 29 Feb 2016 Downloaded from http://pubs.acs.org on March 2, 2016

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ACS Synthetic Biology

Research Article

1 2 3

Metabolic Engineering of Escherichia coli for the Production of 3-

4

Hydroxypropionic Acid and Malonic Acid through

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β-Alanine Route

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Chan Woo Song,† Je Woong Kim,† In Jin Cho,† Sang Yup Lee†,‡,*

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†

Metabolic and Biomolecular Engineering National Research Laboratory, Department of

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Chemical and Biomolecular Engineering (BK21 plus program), Center for Systems and

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Synthetic Biotechnology, Institute for the BioCentury, Korea Advanced Institute of Science

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and Technology (KAIST), Daejeon 34141, Republic of Korea.

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‡

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Daejeon 34141, Republic of Korea.

BioInformatics Research Center and BioProcess Engineering Research Center, KAIST,

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*Corresponding author at: Department of Chemical and Biomolecular Engineering, KAIST,

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Daejeon 34141, Republic of Korea. Fax: +82 42 350 3910.

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E-mail address: [email protected]

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ACS Paragon Plus Environment


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ABSTRACT

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Escherichia coli was metabolically engineered to produce industrially important platform

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chemicals, 3-hydroxypropionic acid (3-HP) and malonic acid (MA), through the β-alanine

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(BA) route. First, various combinations of downstream enzymes were screened and BA

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pyruvate transaminase (encoded by pa0132) from P. aeruginosa was selected to generate

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malonic semialdehyde (MSA) from BA. This platform strain was engineered by introducing

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E. coli MSA reductase (encoded by ydfG) to reduce MSA to 3-HP. Replacement of native

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promoter of the sdhC gene with the strong trc promoter in the genome increased 3-HP

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production to 3.69 g/L in flask culture. Introduction of E. coli semialdehyde dehydrogenase

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(encoded by yneI) into the platform strain resulted in the production of MA, and additional

11

deletion of the ydfG gene increased MA production to 0.450 g/L in flask culture. Fed-batch

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cultures of final engineered strains resulted in the production of 31.1 g/L 3-HP or 3.60 g/L

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MA from glucose.

14 15

KEYWORDS: 3-Hydroxypropionic acid, Malonic acid, Metabolic engineering, Fumaric

16

acid, β-Alanine, Escherichia coli

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INTRODUCTION

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Biorefineries for sustainable production of chemicals have been attracting ever increasing

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attention due to our serious concerns on climate change and environmental problems caused

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by consuming fossil resources. Among the top building block chemicals derived from

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biomass selected by the U.S. Department of Energy (DOE) in 2004, 3-hydroxypropionic acid

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(3-HP) was ranked at the third.1 It has bi-functional characteristics due to the presence of both

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carboxyl and hydroxyl groups, and thus can be used as a versatile agent for organic synthesis

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and for the production of various value-added chemicals such as acrylic acid, 1,3-propanediol

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(PDO), methyl acrylate and acrylamide.2-3 Over the last decade, numerous metabolic

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engineering studies have been performed to produce 3-HP.

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Malonic acid (MA) is also listed as one of the top 30 chemicals which can be

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produced from biomass by the US DOE.1 It has been used as a high value specialty chemical

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for a number of manufacturing processes such as electronic, flavor and fragrance, specialty

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solvents, polymer crosslinking, and pharmaceutical industries. Also, MA is used as a building

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block chemical to produce diverse valuable compounds.4 However, research on the biological

16

production of MA has not been much progressed due to the lack of our knowledge on suitable

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enzymes and metabolic pathways.

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Various metabolic pathways have been predicted and used for the production of 3-HP

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from several precursors including glycerol, lactate, malonyl-CoA, and β-alanine (BA).3, 5-9

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BA is also known as 3-aminopropionic acid (3-AP). Traditionally, many studies have been

3



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performed to produce 3-HP using glycerol as a substrate. To produce 3-HP from glycerol, the

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glycerol metabolism in E. coli was engineered and optimized. The engineered E. coli strain

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produced 42.1 g/L 3-HP with an average yield of 0.268 g/g glycerol by optimization of L-

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arabinose inducible expression of glycerol kinase glpK, knock-out of glycerol regulator factor

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glpR repressing glycerol utilization, overexpression of glycerol facilitator glpF and deletion

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of by-products forming pathways.10 The highest 3-HP titer that has been reported from

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glycerol, while glucose was used at the initial stage of fermentation, is 71.9 g/L with a

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productivity of 1.8 g/L/h by a metabolically engineered E. coli. In this study, a mutant

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aldehyde dehydrogenase GabD4_E209Q/E269Q from Cupriavidus necator was developed

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and introduced into E. coli.11 However, vitamin B12 dependent glycerol dehydratase used in

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the glycerol pathway has been recognized as a major problem for industrial production of 3-

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HP. In the case of lactate pathway, unfavorable thermodynamic characteristics led to the

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inefficient production of 3-HP.5 Also, the lack of net ATP generation and inability of malonyl-

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CoA reductase to utilize NADH as a cofactor have been recognized as main bottlenecks for

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the efficient production of 3-HP through malonyl-CoA pathway.3 Furthermore, it was

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reported that the predicted maximum yield of malonyl-CoA route is lower than that of BA

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route because malonyl-CoA route is highly oxygen-dependent due to ATP requirement for

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acetyl-CoA synthesis.7 Recently, the metabolic pathway using BA as a precursor has been

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demonstrated in Saccharomyces cerevisiae for the production of 3-HP and 13.7±0.3 g/L 3-HP

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was achieved in 80 h by using a novel β-alanine pyruvate aminotransferase in Bacillus

4


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cereus.7 Thus, it was reasoned that BA route would be the most attractive pathway for the

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efficient production of 3-HP. Furthermore, it was expected that employing fumaric acid

3

producer having high aspartase activity would be beneficial for the enhanced production of 3-

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HP because BA route requires aspartate as a major precursor.

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In the consecutive two step reactions converting BA to 3-HP, malonic semialdehyde

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(MSA) is used as an intermediate. BA is converted into MSA by β-alanine pyruvate

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transaminase and MSA is further reduced to form 3-HP. If oxidation, instead of reduction, of

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MSA is possible, it is theoretically possible to produce MA from MSA. Although there has

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been no known MSA dehydrogenase to oxidize MSA to MA, one paper published more than

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50 years ago described detection of MSA dehydrogenase activity in the soluble extracts of

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Pseudomonas aeruginosa.12 Based on this literature, we decided to examine whether

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production of MA from MSA would be possible through screening of more diverse candidate

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enzymes having MSA dehydrogenase activity.

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Recently, we reported development of metabolically engineered E. coli strain capable

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of producing BA.13 This BA producing E. coli strain was developed based on fumaric acid

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overproducing E. coli strain by reinforcing the aspartase (ASPA) catalyzed reaction and

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overexpressing the aspartate-α-decarboxylase from C. glutamicum. In this study, this

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engineered E. coli strain was employed as a parent strain to produce 3-HP and MA. To

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efficiently convert BA to 3-HP or to MA, various candidate enzymes were screened and the

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best performing combinatorial enzymes were introduced into the BA producing strain. After

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successful production of 3-HP in one engineered strain and MA in another engineered strain

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in flask cultures, preliminary fed-batch cultures of these engineered strains were also

3

performed to demonstrate the possibility of their efficient production.

4 5

RESULTS AND DISCUSSION

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Introduction of Heterologous Pathway to Produce 3-HP from Glucose. The overall

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metabolic pathway design for the production of BA and its subsequent conversion to 3-HP

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and MA is shown in Figure 1. To construct a synthetic pathway from aspartate to 3-HP, three

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genes encoding L-aspartate-α-decarboxylase (PAND) from C. glutamicum, β-alanine

10

pyruvate

11

hydroxyisobutyrate dehydrogenase (HIBDH) from Bacillus cereus (BC4042) were

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overexpressed in the wild type E. coli W3110 strain. Production of small amount (91.9 mg/L)

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of 3-HP was detected (Figure 2) when three enzymes were overexpressed by introducing

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p100-99A-PDT, while the control wild type strain did not produce it. From this result, it was

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validated that the three step heterologous pathway designed for the conversion of aspartate to

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3-HP is successfully working.

transaminase

(BAPAT)

from

Pseudomonas

putida

(PP0596),

and

3-

17 18

Re-design of Strain to Enhance 3-HP Production. To further increase the metabolic flux to

19

3-HP, the previously engineered E. coli strains producing fumaric acid and BA were

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employed.13 In our previous study, six genes (iclR, fumA, fumB, fumC, ptsG, and lacI) were

6


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deleted to increase the flux to fumaric acid. From this strain, an aspartate overproducer was

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developed by reinforcing the AspA-catalyzed direct amination of fumarate rather than AspC-

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catalyzed transamination from oxaloacetate. For the development of 3-HP producing strain,

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three downstream enzymes, C. glutamicum L-aspartate-α-decarboxylase (PAND), P. putida β-

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alanine pyruvate transaminase (PP0596), and B. cereus 3-hydroxyisobutyrate dehydrogenase

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(BC4042), were additionally overexpressed by introducing p100-99A-PDT into the aspartate

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producer CWF4NA13 (Figure 2). Production of 3-HP increased only slightly to 140 mg/L,

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which was much lower than expected. Since overexpression of PEP carboxylase (PPC) was

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beneficial in enhancing the production of fumaric acid and BA in our previous studies,13-14

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PPC was additionally overexpressed by using another expression vector pTac15kppc.

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Additional overexpression of PPC was indeed effective, and the titer of 3-HP was increased

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to 0.654 g/L (Figure 2). However, the yield of 3-HP was only 0.0420 g 3-HP/g glucose

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mainly due to the accumulation of byproducts, 0.455 g/L of fumaric acid and 0.220 g/L of

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BA. In order to increase and optimize the flux to 3-HP, expression vectors were reconstructed

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and the aspA gene was additionally overexpressed. The expression vector for PAND and

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ASPA, pTac15kPTA, developed in our previous study was used and the ppc gene was

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additionally cloned in this vector to make pTac15kPTAP. Another synthetic expression vector

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p100-99A-DT3 was used to express PP0596 and BC4042. When the two plasmids

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reconstructed were introduced into the CWF4NA strain, 3-HP titer was dramatically

20

increased to 1.29 g/L. However, about 2.13 g/L of BA was still produced as a byproduct

7



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(Figure 2). From these results, it was concluded that the downstream pathway should be

2

reinforced to efficiently convert BA into 3-HP.

3 4

Testing Combinations

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Overexpression of Succinate Dehydrogenase to Increase 3-HP Production. As described

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above, the conversion efficiency of BA to 3-HP by introducing PP0596 from P. putida and

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BC4042 from B. cereus was not high enough. In order to find better downstream enzymes,

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diverse enzymes from various bacteria were examined in combination for more efficient

9

production of 3-HP. Four different β-alanine pyruvate transaminases from Ralstonia eutropha

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H16, B. cereus, P. putida, and P. aeruginosa were combinatorially expressed with three

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different 3-hydroxyisobutyrate dehydrogenases or MSA reductase from B. cereus, P. putida,

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and E. coli. The 12 different expression vectors (p100-99A-DT1 to 12) were introduced into

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the CWF4NA strain harboring pTac15kPTAP. Then, flask cultures were carried out to

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compare 3-HP production performance.

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The results of flask cultures with respect to 3-HP produced, byproducts produced, and 3-HP

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yield are shown in Figure 3. Overall, the most efficient enzyme for the reduction of MSA was

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YDFG from E. coli. The highest 3-HP titer of 3.31 g/L was obtained by combinatorial

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expression of YDFG from E. coli with PA0132 from P. aeruginosa (p100-99A-DT12).

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Accumulation of a major byproduct BA was significantly decreased to 0.585 g/L, which

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suggests that more efficient conversion of BA into 3-HP was successful by the expression of

of Downstream Enzymes from Various Bacteria and

8


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the above two enzymes. The 3-HP yield on glucose was also increased to 0.207 g 3-HP/g

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glucose. Additionally, it was examined whether expression of GABA aminotransferases

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(GABT) encoded by gabT and puuE genes from E. coli together with YDFG is effective for

4

enhanced 3-HP production. It was found that 3-HP was little produced using these

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combinations. After finding the best combination of downstream enzymes, it was aimed to

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further enhance the 3-HP production by enhancing upstream flux. Since overexpression of

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succinate dehydrogenase complex in fumaric acid producer led to increased fumaric acid

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yield,15 the native promoter of sdhC gene in CWF4NA strain was replaced with strong trc

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promoter to make CWF4NAS strain. The CWF4NAS strain harboring p100-99A-DT12 and

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pTac15kPTAP could produce 3.69 g/L of 3-HP in flask culture with a yield of 0.227 g 3-HP/g

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glucose, showing further increase of 3-HP titer and yield.

12 13

Screening of Candidate Semialdehyde Dehydrogenases to Produce MA. After

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successfully producing 3-HP in engineered E. coli through fumarate-aspartate-BA-MSA

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intermediates where reduction of MSA is a final enzymatic conversion step to form 3-HP, we

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came up with an idea of producing MA by oxidation of MSA by introducing an MSA

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dehydrogenase. We found a very old paper demonstrating the detection of MSA

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dehydrogenase activity in the soluble extract of P. aeroginosa.12 Thus, we decided to screen

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more diverse semialdehyde dehydrogenases. For this, 16 candidate semialdehyde

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dehydrogenases (encoded by pa0265, pa5373, pa0219, pa4899, pa3504, pa4123, pa0130,

9



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pa5312, pa4073, pa0265, pa3570, pa1027, pa1253, pa4022, pa0366, and pa1989 genes) in P.

2

aeruginosa were expressed together with PA0132 in the CWF4NAS strain harboring

3

pTac15kPTAP, and production of MA from glucose was examined (data not shown). As a

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result, small amount of MA (0.1 g/L) was produced only when the pa4123 gene was

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employed (Figure 4B). As another strategy, two succinic semialdehyde dehydrogenases,

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encoded by gabD and yneI genes from E. coli, were tested because molecular structures of

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succinic semialdehyde and MSA are similar. Also, conversion of succinic semialdehyde to

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succinic acid using these enzymes has been reported.16 The gabD and yneI genes were

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separately expressed together with PA0132 in the CWF4NAS strain harboring pTac15kPTAP.

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As a result, 0.195 g/L of MA was produced when the yneI gene was employed (Figure 4B).

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This MA titer is about twice of that produced by employing pa4123 gene. However, 1.26 g/L

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of 3-HP was produced as a byproduct when the yneI gene was employed. During the above

13

study on 3-HP production, it was found that E. coli YDFG was efficient in reducing MSA to

14

3-HP. In order to block reduction of MSA to 3-HP, the ydfG gene was deleted in the

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CWF4NAS strain to make the CWF4NASY strain. Then, both plasmids p100-99A-DT16 and

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pTac15kPTAP were introduced into the CWF4NASY strain. Flask culture of this engineered

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strain, 0.45 g/L of MA was produced with much less accumulation of 3-HP (Figure 4B).

18 19

Fed-Batch Cultures. After developing platform strains for the production of 3-HP and MA

20

through fumarate-aspartate-BA-MSA route, fed-batch cultures were carried out to

10


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1

demonstrate the potential of their actual production. Fed-batch culture of the CWF4NAS

2

strain harboring p100-99A-DT12 and pTac15kPTAP was carried out to produce 3-HP. The

3

cell growth dependent production profile was observed and the 3-HP titer continuously

4

increased during the growth phase. Production of 3-HP continued during the stationary phase,

5

but at a lower rate than that in the growth phase. Acetic acid began to accumulate as a major

6

byproduct in the stationary phase. Finally, 22.9 g/L of 3-HP was produced in 43.5 h with a

7

yield of 0.207 g 3-HP/g glucose and 15.5 g/L of acetic acid was produced as a byproduct

8

(Figure 5A).

9

It was thus reasoned that 3-HP production could be further increased by reducing acetic acid

10

formation in the fed-batch culture. As a strategy to reduce acetic acid accumulation, plasmid

11

expression vectors were reconstructed. Since PPC can serve as a key flux valve to derive

12

more flux towards our desired products and reduce acetic acid formation,15 we attempted to

13

increase the expression level of PPC to some extent, but not too much. Thus, the ppc gene in

14

pTac15kPTAP (low copy expression vector) was transferred into p100-99A-DT12 (medium

15

copy expression vector) to generate p100-99A-DT12P. Then, these expression vectors were

16

introduced into the CWF4NAS strain. Fed-batch culture of the CWF4NAS strain harboring

17

pTac15kPTA and p100-99A-DT12P allowed production of 31.1 g/L of 3-HP in 49 h with a

18

yield of 0.423 g 3-HP/g glucose (Figure 5B). Acetic acid production was significantly

19

reduced to less than 3 g/L, while BA accumulation slightly increased at the end of fed-batch

20

culture.

11



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1

Next, the same cultivation condition and strategy were employed for the production of MA.

2

Fed-batch culture of the CWF4NASY strain harboring p100-99A-DT16 and pTac15kPTAP

3

resulted in production of 3.60 g/L MA in 36 h cultivation. However, 12.9 g/L of acetic acid

4

and 12.5 g/L of BA were accumulated as byproducts (Figure 6A). The similar strategy was

5

employed to reconstruct plasmids to reduce acetic acid accumulation. Fed-batch culture of

6

the CWF4NASY strain harboring p100-99A-DT16P and pTac15kPTA, however, resulted in

7

less MA titer of 1.53 g/L, although accumulation of acetic acid was indeed reduced to 6.4 g/L

8

(Figure 6B). Furthermore, 9.98 g/L of BA was still accumulated as a major byproduct. From

9

these results, it was concluded that relatively low activity of semialdehyde dehydrogenase

10

(YNEI) might be a major bottleneck for the high-level production of MA. Thus, further

11

studies are needed to improve the activity of semialdehyde dehydrogenase.

12 13

CONCLUSIONS

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In this study, we developed engineered E. coli platform strains capable of producing 3-HP or

15

MA through BA pathway using glucose as a carbon source. A novel metabolic pathway to 3-

16

HP was constructed by extending the BA production pathway through the introduction of

17

MSA reductase (YDFG) from E. coli and β-alanine pyruvate transaminase (PA0132) from P.

18

aeruginosa. Overexpression of PPC played a key role to enhance 3-HP production. Fed-batch

19

culture of final engineered strains produced 31.1 g/L of 3-HP with an overall yield of 0.423 g

20

3-HP/g glucose in 49 h, demonstrating the potential of the engineered E. coli strain for the

12


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1

efficient production of 3-HP. Furthermore, MA could also be successfully produced by

2

introducing semialdehyde dehydrogenase (YNEI) rather than MSA reductase to oxidize MSA

3

to MA. Although fed-batch culture of the engineered strain allowed production of only 3.60

4

g/L of MA in 36 h with an overall yield of 0.0373 g MA/g glucose, it is expected that

5

improving the activity of YNEI will further enhance MA production. Taken together, we

6

developed platform E. coli strains for the production of key three carbon chemicals, 3-HP and

7

MA. Further improvement in the production of 3-HP and MA will be possible by taking

8

systems metabolic engineering approaches17-18 including system-wide flux optimization,

9

evolutionary engineering, and synthetic small regulatory RNA based target screening,19-20 and

10

also fermentation optimization.21

11 12

METHODS

13

Bacterial Strains and Plasmids. The strains and plasmids used in this study are listed in

14

Table 1. E. coli NEB-10-beta (New England Biolabs, Ipswich, MA) was employed for gene

15

cloning purpose, and E. coli W3110 and CWF4NA13 were used as base strains for 3-HP

16

production. The general molecular biological experiments including PCR, gel electrophoresis,

17

and transformation for strain development were performed based on the standard protocol.22

18

When necessary, 50 µg/mL ampicillin, 17 µg/mL chloramphenicol, and/or 25 µg/mL

19

kanamycin were added into the medium for strain selection.

20

All of the used primers in this study are listed in Supplementary Table 1. To construct

13



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1

vectors expressing downstream enzymes to convert BA to 3-HP, constitutive expression

2

vector p100-99A was employed as a backbone plasmid.13 Four types of β-alanine pyruvate

3

transaminases, the pp0596 gene from P. putida KT2440, the pa0132 gene from P. aeruginosa

4

PAO1, the a0272 gene from R. eutropha H16, and the bc1610 gene from B. cereus ATCC

5

14579, were amplified from each genomic DNA using corresponding cloning primers

6

(Supplementary Table 1) and cloned into p100-99A vector, respectively. Based on these

7

vectors, three types of genes, the bc4042 gene from B. cereus ATCC 14579, the pp4666 gene

8

from P. putida KT2440, and the ydfG gene from E. coli W3110, encoding 3-

9

hydroxyisobutyrate dehydrogenase or MSA reductase were PCR-amplified from each

10

genomic DNA using corresponding cloning primers (Supplementary Table 1) and

11

additionally cloned into the constructed vectors, respectively, to generate 12 combinations of

12

expression vectors (p100-99A-DT1 to 12). The gabT and puuE genes encoding GABA

13

aminotransferases from E. coli were also cloned into p100-99A-DT9 to generate p100-99A-

14

DT13 and 14, respectively, by enzyme digestion with XbaI and SbfI to replace a0272 gene

15

with gabT or puuE. The panD gene from C. glutamicum ATCC 13032 was cloned into SacI

16

site of p100-99A-DT3 by using Gibson assembly procotol23 to construct p100-99A-PDT. The

17

ppc gene encoding phosphoenolpyruvate carboxylase (PPC) from E. coli W3110 with

18

BBa_J23100 constitutive promoter was amplified from pSynPPC7 plasmid using cloning

19

primers and digested with SbfI enzyme, finally cloned into pTac15kPTA13 and p100-99A-

20

DT12 to construct the pTac15kPTAP and p100-99A-DT12P, respectively. For MA production,

14


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1

amplified each insert fragment, pa4123 gene from P. aeruginosa PA01 and yneI gene from E.

2

coli W3110, and vector p100-99A-DT12 were digested with SacI and XbaI sites, and ligated

3

to construct p100-99A-DT15 and p100-99A-DT16, respectively. Additionally, the ppc

4

expression cassette in p100-99A-DT12P was sub-cloned into p100-99A-DT16 by digestion

5

with SbfI to construct p100-99A-DT16P.

6 7

Cultivation Conditions. For gene cloning and manipulation purposes, the Luria-Bertani

8

(LB) liquid or agar (1.5%, w/v) was routinely used. For the production tests, 10 mL of LB

9

medium in 25 mL test tube was inoculated with small aliquot (10 µL) of cell stock and grown

10

for 12 h at 37℃ and 220 rpm in a rotary shaker. And then, 3 mL of seed culture was

11

transferred into culture medium in a 300 mL baffled flask to make 50 mL of working volume.

12

All flask cultures were carried out for 24 h, and the titer and yield of metabolites including 3-

13

HP and MA were analyzed. All the described flask cultivations were performed in duplicate,

14

and the results were expressed as mean ± standard deviation in the graph. Flask cultivation

15

was carried out using MR medium with supplementation of 15 g/L glucose, 3 g/L yeast

16

extract, 3 g/L NaHCO3, and 9 g/L (NH4)2SO4 to make optimal production condition. The MR

17

medium (pH 6.5) contains per L: 6.67 g KH2PO4, 4 g (NH4)2HPO4, 0.8 g MgSO4-·7H2O, 0.8

18

g citric acid, and 5 ml trace metal solution.24 Yeast extract was additionally supplemented to

19

enhance cell growth, NaHCO3 was supplemented as a CO2 source, pH buffering agent, and to

20

adjust starting pH around 7.0, and (NH4)2SO4 was supplemented as an ammonium source to

15



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

1

accelerate the amination step of fumaric acid to aspartic acid. The medium components

2

(glucose, yeast extract, NaHCO3, MgSO4·7H2O, and (NH4)2SO4) were autoclaved separately

3

to prevent unwanted reactions.

4

Fed-batch cultures were performed using a 6.6-L jar fermentor (Bioflo 3000; New Brunswick

5

Scientific Co., Edison, NJ) containing 2 L of total working volume composed of MR medium

6

supplemented with 20 g/L of glucose, 3 g/L of yeast extract, 2 g/L of Na2CO3 , and 9 g/L of

7

(NH4)2SO4. Seed cultures were performed by transferring 3 mL of 12 h cultured cells in 10

8

mL LB medium into 100 mL of the same medium in 250 mL Erlenmeyer flasks and

9

incubating in a rotary shaker to make final OD600 around 2 at 220 rpm and 37°C. The seed

10

culture (200 mL) was inoculated to the fermentor to make the initial OD600 of ca. 0.2. The

11

culture pH was controlled at 7.0 using 28% (w/v) NH4OH. The dissolved oxygen (DO) was

12

controlled at 40% (v/v) by flowing 2 L/min of air and automatically changing the agitation

13

speed from 200 to 1000 rpm. Feeding solution was composed of 700 g/L of glucose, 42.5 g/L

14

of (NH4)2SO4, and 8 g/L of MgSO4·7H2O. When glucose concentration decreased below 5

15

g/L, 50 mL of feeding solution (equivalent to 35 g glucose, 2.125 g (NH4)2SO4, and 0.4 g

16

MgSO4·7H2O) was intermittently added. For each fed-batch culture, more than two

17

independent cultures were performed to confirm the reproducibility, and the representative

18

data were shown in the figures.

19

16


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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


1

Gene Manipulations. For gene manipulation studies, previously engineered E. coli strain,

2

CWF4NA (iclR, fumC, fumA, fumB, ptsG, and lacI genes knock out and aspA gene promoter

3

replacement mutant of W3110 strain), was employed as a parent strain.13 For the promoter

4

replacement of the sdhC gene, plasmid pMtrc9 containing the strong trc promoter with lox71-

5

chloramphenicol resistance gene-lox66 cassette was used as a template and 2-step PCR

6

extension of 100-bp homologous arm to target gene was carried out to generate linear

7

promoter replacement fragments. For additional deletion of the ydfG gene, the linear gene

8

knockout fragments were prepared by the two-step PCR extension of 100-bp homologous

9

arm, using plasmid pECmulox25 as a template. Prepared gene knockout and promoter

10

replacement fragments were electroporated to manipulate target genes with the help of

11

pCW611 using Gene pulser II (Bio-Rad, Hercules, CA; 2.5kV, 200Ω of resistance, and 25 µF

12

of capacitance) and 2 mm electroporation gap cuvettes (Bio-Rad). The pCW611 was

13

generated by integrating the two helper plasmids pKD4626 and pJW16827, and it expresses

14

Red recombinase and Cre recombinase independently to perform gene manipulation cycles.

15

Detailed procedures are described in the previous report. 28

16 17

Analytical Procedures. Cell growth was monitored by measuring the OD600 with Ultrospec

18

3100 spectrophotometer (Amersham Biosciences, Uppsala, Sweden). YSI 2700 select

19

biochemistry analyzer (YSI Life Sciences, OH) was used for calculating glucose

20

concentration. The concentration of organic acids including 3-HP, MA, and acetate was

17



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

1

analyzed using Waters 1515 high performance liquid chromatography (Waters Co., Milford,

2

MA, USA) equipped with Waters 2414 refractive index detectors. A MetaCarb 87H column

3

(300 by 7.8 mm; Agilent) was eluted isocratically with 0.01 N H2SO4 at 35°C at a flow rate

4

of 0.5 mL/min. The analysis results of 3-HP and MA were further validated by GC-MS

5

analysis (Supplementary Figure 1 and 2) at the Korea Basic Science Institute (KBSI, Seoul,

6

Korea). The concentrations of fumaric acid and BA were analyzed under the same conditions

7

with previous reports13-14 using high performance liquid chromatography (1100 Series HPLC,

8

Agilent Technologies, Palo Alto, CA).

9 10

AUTHOR INFORMATION

11

Corresponding Author

12

*Phone: 82-42-350-3930. Fax: 82-42-350-3910. E-mail: [email protected]

13 14

Author Contributions

15

C.W.S. and S.Y.L. designed research; C.W.S., J.W.K., and I.J.C. performed research; and

16

C.W.S. and S.Y.L. wrote the paper.

17 18

ACKNOWLEDGEMENT

19

The work on malonic acid was supported by the Technology Development Program to Solve

20

Climate Changes on Systems Metabolic Engineering for Biorefineries from the Ministry of

18


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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


1

Science, ICT and Future Planning (MSIP) through the National Research Foundation (NRF)

2

of Korea (NRF-2012M1A2A2026556 and NRF-2012M1A2A2026557). The work on 3-

3

hydroxypropionic acid was supported by Hanwha Chemical. Authors declare that they have

4

conflict of interest as the technology described here is patent filed (KR-10-2016-0003228) for

5

potential commercialization.

6

19



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

1

References

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Eliot, D., Lasure, L., and Jones, S. (2004) Top value added chemicals from biomass.

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hydroxypropionic acid from glycerol by acid tolerant Escherichia coli. J Ind Microbiol

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Nielsen, J. (2015) Establishing a synthetic pathway for high-level production of 3-

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production of 3-hydroxypropionic acid by metabolic engineering of the glycerol

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metabolism in Escherichia coli. Metab Eng. 23, 116-122.

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(11) Chu, H. S., Kim, Y. S., Lee, C. M., Lee, J. H., Jung, W. S., Ahn, J. H., Song, S. H.,

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Choi, I. S., and Cho, K. M. (2015) Metabolic Engineering of 3-Hydroxypropionic Acid

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Biosynthesis in Escherichia coli. Biotechnol Bioeng. 112, 356-364.

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(12) Nakamura, K., and Bernheim, F. (1961) Studies on malonic semialdehyde dehydrogenase from Pseudomonas aeruginosa. Biochim Biophys Acta. 50, 147-152.

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Escherichia coli for the production of 3-aminopropionic acid. Metab Eng. 30, 121-129.

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(15) Song, C. W., and Lee, S. Y. (2015) Combining rational metabolic engineering and

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flux optimization strategies for efficient production of fumaric acid. Appl Microbiol

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Biotechnol. 99, 8455-8464.

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(16) Fuhrer, T., Chen, L., Sauer, U., and Vitkup, D. (2007) Computational prediction and

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experimental verification of the gene encoding the NAD+/NADP+-dependent succinate

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semialdehyde dehydrogenase in Escherichia coli. J Bacteriol. 189, 8073-8078.

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metabolic engineering of microorganisms for natural and non-natural chemicals. Nat

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Chem Biol. 8, 536-546.

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(18) Lee, J. W., Kim, T. Y., Jang, Y. S., Choi, S., and Lee, S. Y. (2011) Systems metabolic engineering for chemicals and materials. Trends Biotechnol. 29, 370-378. (19) Yoo, S. M., Na, D., and Lee, S. Y. (2013) Design and use of synthetic regulatory small RNAs to control gene expression in Escherichia coli. Nat Protoc. 8, 1694-1707.

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H. O. (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases.

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Nat Methods. 6, 343-U41.

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(24) Lee, Y., and Lee, S. Y. (1996) Enhanced production of poly(3-hydroxybutyrate) by

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Polym Degr. 4, 131-134.

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(25) Kim, J. M., Lee, K. H., and Lee, S. Y. (2008) Development of a markerless gene

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(26) Datsenko, K. A., and Wanner, B. L. (2000) One-step inactivation of chromosomal

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genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A. 97, 6640-

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G., Valle, F., and Bolivar, F. (2000) A family of removable cassettes designed to obtain

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1

Page 24 of 34

Figure Legends

2 3

Figure 1. The metabolic pathway designed for the production of 3-HP and MA, and key

4

metabolic engineering strategies. Thick arrows indicate overexpression of the corresponding

5

genes through plasmid-based overexpression or promoter replacement. The “X” indicates

6

deletion of the corresponding gene. Enzymes encoded by the genes shown are: fum,

7

fumarase; ptsG, glucose-specific PTS enzyme IIBC components; iclR, isocitrate lyase

8

repressor; lacI, transcriptional repressor of the lac operon; ppc, phosphoenolpyruvate

9

carboxylase;

aspA,

aspartase;

sdh,

succinate

dehydrogenase;

panD,

aspartate-α-

10

decarboxylase; pa0132, β-alanine pyruvate transaminase; ydfG, malonic semialdehyde

11

reductase; yneI, succinic semialdeyde dehydrogenase. Abbreviations of chemicals are: Fum,

12

fumaric acid; Asp, aspartic acid; BA, β-alanine (or 3-aminopropionic acid); MA, malonic

13

acid; MSA, malonic semialdehyde; 3-HP, 3-hydroxypropionic acid; Glu, glucose.

14 15

Figure 2. Comparison of the titers of fumaric acid (blue bar), 3-HP (red bar), and BA (green

16

bar), and the yield of 3-HP on glucose (purple bar) following the introduction of different

17

expression systems in W3110 and CWF4NA strains.

18 19

Figure 3. Comparison of the titers of fumaric acid (blue bar), 3-HP (red bar), and BA (green

20

bar), and the yield of 3-HP on glucose (purple bar) following the introduction of 14 different

21

expression vectors (p100-99A-DT1~14) into the CWF4NA strain harboring pTac15kPTAP.

22

The last column of the bar graphs shows the results obtained with further engineered strain

23

CWF4NAS harboring pTac15kPTAP and p100-99A-DT12.

24 24


Page 25 of 34

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


1

Figure 4. Metabolic pathways and metabolic engineering strategy employed for the

2

production of MA (A). Comparison of 3-HP (red bar) and MA (yellow bar) production

3

following the introduction of p100-99A-DT15 and DT16 in the CWF4NAS strain harboring

4

pTac15kPTAP, respectively. The last column of the bar graphs show the results obtained with

5

further engineered strain CWF4NASY, which is ydfG deleted CWF4NAS strain, harboring

6

pTac15kPTAP and p100-99A-DT16 (B).

7 8

Figure 5. Fed-batch culture profiles of the CWF4NAS strain harboring pTac15kPTAP and

9

p100-99A-DT12 (A) and the CWF4NAS strain harboring pTac15kPTA and p100-99A-

10

DT12P (B) for the production of 3-HP. Symbols are: red filled circle, cell growth; green filled

11

triangle, 3-HP; gray filled diamond, BA; open diamond, fumaric acid; blue filled square,

12

acetic acid; open circle, residual glucose.

13 14

Figure 6. Fed-batch culture profiles of the CWF4NASY strain harboring pTac15kPTAP and

15

p100-99A-DT16 (A) and the CWF4NASY strain harboring pTac15kPTA and p100-99A-

16

DT16P (B) for the production of MA. Symbols are: red filled circle, cell growth; green filled

17

triangle, 3-HP; open triangle, MA; gray filled diamond, BA; open diamond, fumaric acid;

18

blue filled square, acetic acid; open circle, residual glucose.

25



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1

Page 26 of 34

Table 1. E. coli Strains and Plasmids Used in This Study Strain / plasmid

Description

Source or reference

Strains W3110

Coli genetic stock center strain No.4474

CGSCa

NEB-10-beta

araD139 ∆(ara-leu)7697 fhuA lacX74 galK (ϕ80 ∆(lacZ)M15) mcrA galU recA1 endA1 nupG rpsL (StrR) ∆(mrr-hsdRMSmcrBC)

NEBb

CWF4NA

W3110 ∆iclR ∆fumC ∆fumA ∆fumB ∆ptsG ∆lacI PaspA::Ptrc

CWF4NAS

W3110 ∆iclR ∆fumC ∆fumA ∆fumB ∆ptsG ∆lacI PaspA::Ptrc PsdhC::Ptrc

This study

CWF4NASY

W3110 ∆iclR ∆fumC ∆fumA ∆fumB ∆ptsG ∆lacI PaspA::Ptrc PsdhC::Ptrc ∆ydfG

This study

13

Plasmids pEcmuloxC

ApR, CmR, lox66-cat-lox71, 3.5-kb

25

pMtrc9

Modified pECmulox containing trc promoter downstream of lox66-cat-lox71 cassette R

pCW611

Ap , λ-Red recombinase under arabinose-inducible PBAD promoter, Cre-recombinase under IPTG-inducible lacUV5 promoter, ts origin, 9.0kb

p100-99A

ApR, BBa_J23100 constitutive promoter, pBR322 origin, 2.6-kb

pTac15k

KmR, tac promoter, p15A origin, 4.0-kb

pTac15kppc

pTac15k containing the ppc gene from E. coli W3110

p100-99A-PDT pTac15kPTA pTac15kPTAP

Lab stock 28 13

Lab stock 14

p100-99A containing the panD gene from C. glutamicum ATCC 13032, the bc4042 gene from B. cereus ATCC 14579, and the pp0596 gene from P. putida KT2440 pTac15k containing the panD gene from C. glutamicum ATCC 13032 and the aspA gene from E. coli W3110 with strong tac promoter and ptrc RBS sequence pTac15k containing the panD gene from C. glutamicum ATCC 13032, the aspA gene from E. coli W3110 with strong tac promoter and ptrc RBS sequence, and the ppc gene from E. coli W3110 with BBa_J23100 constitutive promoter

This study 13

This study

p100-99A-DT1

p100-99A containing the bc4042 gene from B. cereus ATCC 14579 and the a0272 gene from R. eutropha H16

This study

p100-99A-DT2

p100-99A containing the bc4042 and bc1610 genes from B. cereus ATCC 14579

This study

26


Page 27 of 34

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

1 2 3 4


p100-99A-DT3

p100-99A containing the bc4042 gene from B. cereus ATCC 14579 and the pp0596 gene from P. putida KT2440

This study

p100-99A-DT4

p100-99A containing the bc4042 gene from B. cereus ATCC 14579 and the pa0132 gene from P. aeruginosa PAO1

This study

p100-99A-DT5

p100-99A containing the pp4666 gene from P. putida KT2440 and the a0272 gene from R. eutropha H16

This study

p100-99A-DT6

p100-99A containing the pp4666 gene from P. putida KT2440 and the bc1610 gene from B. cereus ATCC 14579

This study

p100-99A-DT7

p100-99A containing the pp4666 gene from P. putida KT2440 and the pp0596 gene from P. putida KT2440

This study

p100-99A-DT8

p100-99A containing the pp4666 gene from P. putida KT2440 and the pa0132 gene from P. aeruginosa PAO1

This study

p100-99A-DT9

p100-99A containing the ydfG gene from E. coli W3110 and the a0272 gene from R. eutropha H16

This study

p100-99A-DT10

p100-99A containing the ydfG gene from E. coli W3110 and the bc1610 gene from B. cereus ATCC 14579

This study

p100-99A-DT11

p100-99A containing the ydfG gene from E. coli W3110 and the pp0596 gene from P. putida KT2440

This study

p100-99A-DT12

p100-99A containing the ydfG gene from E. coli W3110 and the pa0132 gene from P. aeruginosa PAO1

This study

p100-99A-DT13

p100-99A containing the ydfG and puuE genes from E. coli W3110

This study

p100-99A-DT14

p100-99A containing the ydfG and gabT genes from E. coli W3110

This study

p100-99A-DT15

p100-99A containing the pa4123 gene from P. aeruginosa PAO1 and the pa0132 gene from P. aeruginosa PAO1

This study

p100-99A-DT16

p100-99A containing the yneI gene from E. coli W3110 and the pa0132 gene from P. aeruginosa PAO1

This study

p100-99A containing the ydfG gene from E. coli W3110, the pa0132 gene from P. aeruginosa PAO1, and the ppc gene p100-99A-DT12P from E. coli W3110 with BBa_J23100 constitutive promoter p100-99A containing the yneI gene from E. coli W3110 and the pa0132 gene from P. aeruginosa PAO1, and the ppc gene p100-99A-DT16P from E. coli W3110 with BBa_J23100 constitutive promoter Abbreviations: Ap, ampicillin; Cm, chloramphenicol; Km, kanamycin; R, resistance; and ts, temperature sensitive. a Coli Genetic Stock Center, New Haven, CT. b New England Biolabs, Ipswich, MA.

5

27


This study This study


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Metabolic engineering of E. coli for the production of 3-hydroxypropionic acid and malonic acid via betaalanine pathway 34x25mm (300 x 300 DPI)


Page 28 of 34

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Figure 1. The metabolic pathway designed for the production of 3-HP and MA, and key metabolic engineering strategies. See text for the details. 92x82mm (300 x 300 DPI)



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

Figure 2. Comparison of the titers of fumaric acid (blue bar), 3-HP (red bar), and BA (green bar), and the yield of 3-HP on glucose (purple bar) following the introduction of different expression systems in W3110 and CWF4NA strains. 76x45mm (300 x 300 DPI)


Page 30 of 34

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Figure 3. Comparison of the titers of fumaric acid (blue bar), 3-HP (red bar), and BA (green bar), and the yield of 3-HP on glucose (purple bar) following the introduction of 14 different expression vectors (p10099A-DT1~14) into the CWF4NA strain harboring pTac15kPTAP. The last column of the bar graphs shows the results obtained with further engineered strain CWF4NAS harboring pTac15kPTAP and p100-99A-DT12. 75x45mm (300 x 300 DPI)



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Figure 4. Metabolic pathways and metabolic engineering strategy employed for the production of MA (A). Comparison of 3-HP (red bar) and MA (yellow bar) production following the introduction of p100-99A-DT15 and DT16 in the CWF4NAS strain harboring pTac15kPTAP, respectively. The last column of the bar graphs show the results obtained with further engineered strain CWF4NASY, which is ydfG deleted CWF4NAS strain, harboring pTac15kPTAP and p100-99A-DT16 (B). 81x34mm (300 x 300 DPI)


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Figure 5. Fed-batch culture profiles of the CWF4NAS strain harboring pTac15kPTAP and p100-99A-DT12 (A) and the CWF4NAS strain harboring pTac15kPTA and p100-99A-DT12P (B) for the production of 3-HP. Symbols are: red filled circle, cell growth; green filled triangle, 3-HP; gray filled diamond, BA; open diamond, fumaric acid; blue filled square, acetic acid; open circle, residual glucose. 53x21mm (300 x 300 DPI)



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Figure 6. Fed-batch culture profiles of the CWF4NASY strain harboring pTac15kPTAP and p100-99A-DT16 (A) and the CWF4NASY strain harboring pTac15kPTA and p100-99A-DT16P (B) for the production of MA. Symbols are: red filled circle, cell growth; green filled triangle, 3-HP; open triangle, MA; gray filled diamond, BA; open diamond, fumaric acid; blue filled square, acetic acid; open circle, residual glucose. 55x20mm (300 x 300 DPI)


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Metabolic Engineering of Escherichia coli for the ... - ACS Publications

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