Investigation of the Synergetic Effect of Xylose Metabolic Pathways on

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Investigation of the synergetic effect of xylose metabolic pathways on the production of glutaric acid Jia Wang, Xiaolin Shen, Yuheng Lin, Zhenya Chen, Yaping Yang, Qipeng Yuan, and Yajun Yan ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.7b00271 • Publication Date (Web): 25 Sep 2017 Downloaded from http://pubs.acs.org on September 26, 2017

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ACS Synthetic Biology

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Investigation of the synergetic effect of xylose metabolic pathways on the production of

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glutaric acid

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Jia Wang a,b, Xiaolin Shena,b, Yuheng Linc, Zhenya Chen a,b, Yaping Yangd,

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Qipeng Yuana,b,*, Yajun Yand,*

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a

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Technology, Beijing 100029, China

State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical

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b

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University of Chemical Technology, Beijing 100029, China

Beijing Advanced Innovation Center for Soft Matter Science and Engineering, Beijing

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c

BiotecEra Inc., Athens, GA 30602, USA

d

College of Engineering, The University of Georgia, Athens, GA 30602, USA

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* Corresponding authors:

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Qipeng Yuan

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15 Beisanhuan East Road, Chaoyang District, Beijing 100029, China

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E-mail: [email protected]; telephone: +86-10-64437610

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Yajun Yan

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146 Riverbend Research Lab South, The University of Georgia, Athens, GA 30602, USA

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E-mail: [email protected]; telephone: +1-706-542-8293

1 ACS Paragon Plus Environment


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Abstract

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Efficient utilization of lignocellulose is pivotal for economically converting renewable

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feedstocks into value-added products. Xylose is the second most abundant sugar in

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lignocellulose, but it is quite challenging to ferment xylose as efficiently as glucose by

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microorganisms. Here, we investigated the metabolic potential of three xylose catabolic

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pathways (isomerase, Weimberg and Dahms pathways) and illustrated the synergetic effect

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between isomerase pathway and Weimberg pathway for the synthesis of chemicals derived from

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2-ketoglutarate and acetyl-CoA. When using glutaric acid as the target product, employment of

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such synergetic pathways in combination resulted in an increased glutaric acid titer (602 mg/L)

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compared with using each pathway alone (104 or 209 mg/L) and this titer even outcompetes that

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obtained from glucose catabolic pathway for glutaric acid synthesis (420 mg/L). This work

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validates a novel and powerful strategy for xylose metabolic utilization to overcome the

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inefficiency of using single xylose metabolic pathway for the synthesis of TCA cycle derived

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chemicals.

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Keywords: Lignocellulose, Xylose Metabolism, Synergetic Pathways, Glutaric Acid


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Lignocellulose is an attractive renewable feedstock for the production of chemicals owing to its

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low cost and abundance 1. Xylose, as the second most abundant sugar in lignocellulose,

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constitutes as much as 30% of the total sugar in lignocellulosic hydrolysates 2. However,

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utilization of xylose as efficiently as glucose by microorganisms is still quite challenging so far.

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Thus, development of efficient metabolic pathways and rational optimization strategies for the

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conversion of xylose into desired products is very crucial for complete utilization of

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lignocellulose.

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There are at least three pathways for assimilation of xylose into the tricarboxylic acid (TCA)

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cycle in nature (Fig 1). The isomerase pathway is the native xylose metabolic pathway in wild

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type E. coli. Xylose is first isomerized and phosphorylated into xylulose-5P. Xylulose-5P is then

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diverted into the pentose phosphate pathway to generate fructose-6P and glyceraldehyde-3P,

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which are further processed through the Embden-Meyerhof-Parness pathway to enter the TCA

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cycle (Fig 1).

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enzymatic steps to reach 2-ketoglutarate (2-KG), a key intermediate in TCA cycle, with a

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theoretical molar yield of 83%. The Weimberg pathway is another xylose metabolic pathway

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where the xylose is oxidized into xylonic acid. Xylonic acid is then dehydrated two water

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molecules to form 2-keto-glutarate semialdehyde, which is subsequently converted into 2-KG 3.

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This pathway is nonphosphorylative and more straightforward to generate 2-KG with five

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enzymatic steps and a 100% theoretical molar yield (Fig 1). The third xylose metabolic pathway

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is Dahms pathway, which starts as the Weimberg pathway but cleaves the intermediate 2-keto-3-

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deoxy-xylonate into glycolaldehyde and pyruvate 4, the latter is decarboxylated into acetyl-CoA

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to enter the TCA cycle (Fig 1). Each of the three pathways has been successfully engineered to

This pathway produces a lot of intermediates and requires more than ten



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produce many high value compounds in E. coli, including muconic acid 5, 1,4-butanediol 6, 7, 3,4-

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dihydroxybutyric acid 8, mesaconate 9, poly(lactate-co-glycolate)

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glycol

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production of TCA cycle-derived products so far.

11

10

, glycolic acid and ethylene

. However, no one has investigated which pathway is the most favorable route for the

67 68

Frequently, a desired product can be synthesized through more than one metabolic route.

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Synergetic effect among these pathways may be formed when intermediate employment,

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cofactor demand and ATP requirement are complementary to each other. In this case,

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combination of such pathways may result in improved product titer and yield compared with

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utilization of each single pathway along

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pathway are co-utilized to divert xylose into the TCA cycle. However, this metabolism contains

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multiple reaction steps and suffers complicated regulations, which limit the production titer and

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yield. In order to overcome those issues, novel and efficient xylose metabolic utilization

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strategies need to be developed.

12

. Traditionally, glycolysis and pentose phosphate

77 78

In this work, we investigated the metabolic potential and illustrated the synergetic effect among

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abovementioned three xylose catabolic pathways for producing the compounds derived from

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TCA cycle. We chose glutaric acid as our target product for the following reasons. First, glutaric

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acid is an important dicarboxylic acid with a wide range of applications in polymer industry for

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the synthesis of polyamides and polyesters 13. It is of great industrial significance to pursue high-

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level biosynthesis of glutaric acid. Second, we previously established a novel glutaric acid

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biosynthetic pathway using glucose as the carbon source, yielding 420 mg/L glutaric acid by

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employment of a carbon chain elongation pathway from acetyl-CoA and 2-KG 14, which are two


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intermediates produced by glycolysis and TCA cycle, respectively (Fig. 1). The high-level

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production of glutaric acid requires sufficient amounts of both acetyl-CoA and 2-KG. Those

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reasons render glutaric acid as an ideal product for demonstration of the synergetic effect

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between different xylose metabolic pathways.

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In order to test the production potential of xylose catabolic pathways for glutaric acid

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biosynthesis, E. coli native xylose isomerase pathway was first investigated independently. We

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calculated its maximum theoretical molar yield (See the supporting information). The xylose

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isomerase pathway has the potential to produce 0.556 mol glutaric acid per mol xylose. The

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reason for the poor yield of this pathway is that generation of acetyl-CoA and 2-KG causes

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carbon loss due to the pyruvate decarboxylation reaction. Then, to test the glutaric acid

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production in vivo, the plasmids pJW87 carrying kivD and gabD with pZE-HCS-HA-HICDH

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were introduced into E. coli BW25113 (F’), generating strain JW11. The resulting strain was

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cultivated in LB medium containing 10 g/L xylose. As shown in Fig. 2A, JW11 produced 104

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mg/L glutaric acid with an OD600 value of 2.11 at 60 hours. This is the first time to achieve

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biosynthesis of glutaric acid using xylose as the carbon source. However, the low titer obtained

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in xylose isomerase pathway indicated that the E. coli native xylose metabolic pathway is less

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efficient for the production of compounds derived from TCA cycle due to lengthy enzymatic

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steps and complex regulations involved in this pathway.

105 106

Compared with xylose isomerase pathway, Weimberg pathway is a promising alternative

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metabolic pathway that can generate 2-KG with only 5 enzymatic steps in a nonphosphorylated

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and carbon-conserved manner. It has been reported that introduction of Weimberg pathway to



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generate a 2-KG pool, coupled with over-expression of downstream mesaconate biosynthetic

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pathway yielded 12.5 g/L mesaconate from xylose [9], demonstrating the high efficiency of

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Weimberg pathway for the production of 2-KG derived compounds. We speculated that

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employment of Weimberg pathway would enhance the 2-KG availability thereby to improve the

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glutaric acid titer. However, we believe that Weimberg pathway alone could not provide enough

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acetyl-CoA to support the high-level glutaric acid production since no intermediate could be

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directly used for acetyl-CoA supply in this pathway. Branching off from Weimberg pathway,

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Dahms

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glycolaldehyde and pyruvate, the latter is the direct precursor for acetyl-CoA formation.

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Therefore, simultaneous use of Weimberg pathway and Dahms pathway was conducted so that

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the former mainly generates 2-KG and the latter offers acetyl-CoA via decarboxylation of

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pyruvate. As calculated in the supporting information, the maximum theoretical molar yield of

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glutaric acid using this strategy is only 0.5 mol per mol xylose. The lower theoretical yield

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obtained in this way is caused by the by-product formation, glycoaldehyde was produced as 0.5

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mol per mol xylose. In order to verify our hypothesis, strain JW271 was constructed by deletion

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of xylA gene encoding xylose isomerase to block the pentose phosphate pathway. The plasmids

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pJW88 carrying xylD, kivD and gabD and pJW31 carrying HCS, HA, HICDH, xylB, xylC, xylX,

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xylA were introduced into JW271 to generate strain JW12. As we expected, the glutaric acid titer

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was increased by 2-fold compared with JW11 and reached 209 mg/L with a comparable OD600

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value of 2.03 at 60 hours (Fig. 2B), which indicates the superiority of Weimberg pathway for the

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production of 2-KG derived glutaric acid. However, this titer is still far from our previous

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feeding experiment which produced 600 mg/L glutaric acid by feeding 4 g/L 2-KG

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noticed that there is a trade-off relationship between acetyl-CoA formation and 2-KG generation

pathway

cleavages

the

common

intermediate


2-keto-3-deoxy-xylonate

14

into

. We

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in Weimberg-Dahms pathway since Dahms pathway is branching off from Weimberg pathway.

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In this condition, the production of the acetyl-CoA is at the expense of 2-KG formation. Thus,

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though the Weimberg pathway could supply more 2-KG, the Dahms pathway is not an ideal

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route to provide enough acetyl-CoA. More efficient optimization strategies need to be developed

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to overcome this issue.

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In some cases, complementary characteristics of different pathways may result in synergetic

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effect when producing one desired product, which improves the production titer and yield. For

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instance, both E. coli native threonine pathway and heterologous citramalate pathway are able to

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produce 1-propanol. Those two pathways are complementary to each other in redox demands.

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Utilization of the dual pathway for 1-propanol production achieved higher productivity and yield

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than individual pathway alone

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TCA cycle and glyoxylate shunt for the succinic acid synthesis has also been revealed

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previously15. Utilization of the synergetic pathways in combination led to increased succinic acid

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titer. In our case, the isomerase pathway is inefficient for 2-KG generation but provides

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sufficient amount of acetyl-CoA, While Weimberg pathway or Weimberg-Dahms pathway are

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more straightforward for the synthesis of 2-KG but less efficient for the production of acetyl-

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CoA. The complementary precursors supply between xylose isomerase pathway and Weimberg

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pathway or Weimberg-Dahms pathway may lead to a synergetic effect for the production of

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chemicals derived from acetyl-CoA and 2-KG. In order to verify our hypothesis, we first

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combined xylose isomerase pathway with Weimberg-Dahms pathway to explore the synergetic

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effect between those pathways for the de novo production of glutaric acid. All of the three xylose

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metabolic pathways were co-expressed in E. coli. The maximum theoretical molar yield of

12

. Similarly, the synergetic effect between oxidative branch of



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glutaric acid using this strategy is increased to 0.528 mol per mol xylose (Supporting

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information). To experimentally verify it, plasmids pJW88 and pJW31 were co-introduced into

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BW25113 (F’) to generate JW13 which carries xylose Weimberg, Dahms, isomerase pathway

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and downstream glutaric acid biosynthetic pathway together. Remarkably, glutaric acid titer was

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further improved to 484 mg/L with a comparable OD600 value of 2.24 after 60 hours (Fig. 3).

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Although this titer is still lower than that of the direct feeding of 2-KG as the precursor, it is

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higher than our previously constructed glutaric acid de novo biosynthetic pathway which

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produced 420 mg/L glutaric acid using 20 g/L glucose as the substrate

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that combination of those three pathways increased glutaric acid titer by 380% to 130%

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compared with utilization of isomerase pathway or Weimberg-Dahms pathway alone, which

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demonstrates the synergetic effect between xylose isomerase and Weimberg-Dahms pathway in

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glutaric acid biosynthesis.

14

. It is exciting to note

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Next, we hypothesized that Dahms pathway is not essential for maintaining of such a synergetic

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effect, because: 1) the supply of acetyl-CoA could be achieved by expression of xylose

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isomerase pathway alone; and 2) The trade-off relationship between acetyl-CoA formation and

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2-KG generation in Dahms pathway reduced the 2-KG generation efficiency in Weimberg

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pathway. Thus, we speculated that deletion of Dahms pathway would keep the synergetic effect

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and furthermore can conserve more 2-KG to support the improvement of glutaric acid titer. We

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also calculated the maximum theoretical molar yield of glutaric acid after deletion of Dahms

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pathway. Co-utilization of xylose isomerase pathway and Weimberg pathway has the potential to

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produce 0.611 mol glutaric acid per mol xylose (Supporting information). In order to verify our

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hypothesis, Dahms pathway was blocked in BW25113 (F’) by deletion of yagE and yjhH which


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encoding aldolase, generating strain JW285. Plasmids pJW88 and pJW31 were co-introduced

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into JW285 to yield strain JW14. Remarkably, the glutaric acid titer was enhanced to 602 mg/L

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with a higher OD600 value of 3.09 (Fig. 4A). This titer was improved by 480% and 188%

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compared with utilization of isomerase pathway or Weimberg-Dahms pathway alone, which

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revealed that the synergetic effect still exists when only co-utilization of xylose isomerase

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pathway and Weimberg pathway. Furthermore, the increased titer compared with using all of

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three pathways demonstrated that deletion of Dahms pathway led to more pronounced synergetic

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effect due to conserved 2-KG availability. Therefore, we concluded that xylose isomerase

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pathway and Weimberg pathway are synergetic pathways for glutaric acid synthesis. It is worth

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noting that this titer is comparable with our previous feeding experiment (600 mg/L) and higher

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than the de novo production titer (420 mg/L) that obtained by using glucose as the substrate

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coupled with down-regulation of 2-KG dehydrogenase (SucAB), which indicated the efficiency

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of the constructed xylose synergetic pathways outcompetes the glucose metabolic pathway for

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glutaric acid synthesis. In addition, we are wondering that whether the synergetic effect could be

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further enhanced by inactivation of the citrate synthase (encoded by gltA), which is responsible

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for diverting acetyl-CoA into the TCA cycle. By doing so, the xylose isomerase pathway

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conserves more acetyl-CoA and no longer offers 2-KG and Weimberg pathway is the sole

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pathway to produce 2-KG. As we calculated, the maximum theoretical molar yield of glutaric

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acid is increased to 0.625 mol per mol xylose in this condition (Supporting information). To

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experimentally verify it, strain JW287 was constructed by deletion of gltA in strain JW285 to

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create a ∆yjhH ∆yagE∆gltA triple knockout strain. Plasmids pJW88 and pJW31 were co-

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introduced into JW287 to yield strain JW15. As shown in Fig. 4B, the glutaric acid titer

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decreased to 250 mg/L and the OD600 value reach to 1.62 reduced by nearly 50% compared with



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JW14. We concluded that deletion of gltA impairs the synergetic effect between xylose

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isomerase pathway and Weimberg pathway, probably due to reduced growth caused by the

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disruption of the TCA cycle (∆gltA).

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In summary, we investigated the biosynthesis potential of different xylose metabolic pathways

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and illustrated a novel synergetic effect between the xylose isomerase pathway and Weimberg

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pathway for glutaric acid synthesis in E. coli. The two pathways are complementary with each

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other in precursors supply. The host strain with the dual pathways for glutaric acid production

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yielded higher titer than utilization of each pathway alone. The efficiency of the constructed

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xylose synergetic pathways is even higher than that of glutaric acid biosynthesis from glucose.

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We believe that the glutaric acid titer could be further improved by increase of the acetyl-CoA

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supply. The recently developed genome-scale models are promising to predict the gene over-

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expression or deletion targets to enhance the acetyl-CoA availability16,

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pathway efficiency could also be improved by precisely controlling the carbon flux distribution

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between the xylose isomerase and Weimberg pathway to maximize the glutaric acid titer via the

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library-based combinatorial pathway optimization18, 19.

17

. Additionally, the

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This work presented here provides a novel and powerful strategy for xylose metabolic utilization

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to overcome the inefficiency of single xylose metabolic pathway. Besides glutaric acid, acetyl-

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CoA and 2-KG are the precursors for biosynthesis of many other high-value chemicals, such as

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1,4-BDO

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poly(D-lactate-co-glycolate-co-D-2-hydroxybutyrate)

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, five-, six and seven carbon lactams

21

, nylon-6 10, 23

22

, poly(lactate-co-glycolate) and

. Therefore, the applicability of our


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constructed xylose synergetic pathways can be expanded to synthesize other value-added

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compounds to achieve production improvement.



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Methods

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Media, Strains and Plasmids

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Luria-Bertani (LB) medium containing 10 g/L tryptone, 5 g/L yeast extract and 10 g/L NaCl was

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used for cell inoculation and plasmid propagation. The modified LB medium (LB medium added

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with 10 g/L of xylose) was used for de novo biosynthesis of glutaric acid. The antibiotics

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Ampicillin (100 µg/mL) and kanamycin (50 µg/mL) were supplemented into the medium if

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necessary. E. coli BW25113 (F’) was used as the host for glutaric acid production and E. coli

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strain XL1-Blue for DNA manipulation experiments. Knock-out strains of E. coli BW25113 (F’)

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were generated by disrupting target gene(s) using P1 transduction 24. The high-copy number and

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medium-copy number plasmids pZE12-luc and pCS-27 were used for pathway construction. All

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of used strains and plasmids in this study were listed in Table 1.

236 237

DNA manipulations

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Genes kivD from L. lactis and gabD from P. putida were cloned into the backbone of pCS27 to

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create pJW87 by amplifying kivD-gabD from pZE-kivD-gabD and inserting into pCS27 between

240

AvrII and XhoI. Genes xylD from Caulobacter crescentus, kivD and gabD were cloned into the

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backbone of pZE12-luc using Acc65I, PstI, sphI and XbaI to create pJW77. To create pJW88,

242

the fragment xylD-kivD-gabD was amplified from pJW77 and inserting into pCS27 between

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AvrII and XhoI. Genes xylB, xylC, xylX and xylA from C. crescentus were cloned into the

244

backbone of pZE12-luc using Acc65I, BamHI, NdeI, HindIII and XbaI to create pJW19. To

245

create pJW31, fragment xylB-xylC-xylX-xylA was amplified from pJW19 and inserting into pZE-

246

HCS-HA-HICDH between SpeI and SacI.

247


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De novo biosynthesis of glutaric acid

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Shake flask experiments were conducted using LB medium supplemented with 10 g/L xylose.

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Production colonies were inoculated in 3 ml LB medium (37ºC, 270 rpm) for overnight. 200 µL

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of the pre-inoculum along with appropriate antibiotics was added into screw cap bottles (125 mL)

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consisting of 20 mL LB xylose medium and grown for 3 hours (37ºC, 270 rpm). Then 0.5 mM

253

IPTG were added to the cultures and the cultures were grown till 60 hours at 30ºC, 270 rpm. 1

254

mL samples were collected every 12 hours for HPLC analysis.

255 256

Analytical procedures

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Genesys 10S UV-Vis Spectrophotometer (Thermo Scientific, Waltham, MA) was used to

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measure the optical density at 600 nm. To analyze the samples, 1 mL samples were collected

259

every 12 hours during the shake flask experiments and centrifuged at 13,000 rpm for 10 min,

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following which the supernatant was filtered and analyzed by HPLC-RID (Shimadzu) equipped

261

with a Coregel-64H column (Transgenomic). The eluent used was 4 mN H2SO4 with a flow rate

262

of 0.6 mL/min. The column temperature was set at 55ºC.



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AUTHOR INFORMATION

264

Corresponding Authors

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*E-mails: [email protected] (Q. Yuan), [email protected] (Y. Yan).

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Author Contributions

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JW conceived the study and wrote the manuscript. JW and XS performed the experiments. QY

269

and YYan directed the research. JW, YL, XS and YYan revised the manuscript. ZC and YYang

270

participated the research.

271 272

Notes

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The authors declare no competing financial interest.

274 275

ACKNOWLEDGMENTS

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This work was supported by National Natural Science Foundation of China (21376017,

277

21406010, 21636001), the Program of Introducing Talents of Discipline to Universities (“111”

278

project, B13005), the Program for Changjiang Scholars and Innovative Research Team in

279

Universities in China (No. IRT13045), the National High Technology Research and

280

Development Program of China (863 Program, No. NO.2015AA021001). We would also like to

281

thank the College of Engineering, The University of Georgia, Athens.

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284 285


Table 1. List of strains and plasmids used in this study. Strain

Genotype

Reference

E. coli BW25113 (F’)

rrnBT14 ∆lacZWJ16 hsdR514 ∆araBADAH33

25

∆rhaBADLD78 F‫[ ׳‬traD36 proAB lacIqZ∆M15 Tn10(Tetr)] E. coli XL1-Blue

recA1 endA1gyrA96thi-1hsdR17supE44relA1lac

Stratagene

[F’ proAB lacIqZDM15Tn10 (TetR)] JW3537-1

BW25113 ∆xylA::kan

Yale CGSC

JW5775-2

BW25113 ∆yjhH::kan

Yale CGSC

JW0261-1

BW25113 ∆yagE::kan

Yale CGSC

JW271

BW25113 (F’) ∆xylA

This study

JW285

BW25113 (F’) ∆yjhH ∆yagE

This study

JW287

BW25113 (F’) ∆yjhH ∆yagE∆gltA

This study

JW11

BW25113 (F’) harboring pJW87 and pZE-HCS-

This study

HA-HICDH JW12

JW271 harboring pJW88 and pJW31

This study

JW13

BW25113 (F’) harboring pJW88 and pJW31

This study

JW14


This study

JW15


This study

Plasmid

Description

Reference

pZE12- luc

pLlacO-1; luc; ColE1 ori; AmpR

26

pCS27

pLlacO-1; p15A ori; KanR

27

kivD from Lactococcus lactis and gabD from



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pZE-kivD-gabD

Pseudomonas putida cloned into pZE12-luc

14

pJW87

kivD from Lactococcus lactis and gabD from

This study

Pseudomonas putida cloned into pCS27 pZE-HCS-HA-HICDH

HCS, HA and HICDH from Saccharomyces

14

cerevisiae cloned into pZE12-luc pJW77

xylD from Caulobacter crescentuss, kivD from

This study

Lactococcus lactis and gabD from Pseudomonas putida cloned into pZE12-luc pJW88

xylD from Caulobacter crescentus, kivD from

This study

Lactococcus lactis and gabD from Pseudomonas putida cloned into pCS27 pJW19

xylB, xylC, xylX and xylA from C. crescentus

This study

cloned into pZE12-luc pJW31

HCS, HA, HICDH and xylB, xylC, xylX, xylA from C. crescentus cloned into pZE12-luc

286


This study

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287


Figure Legends

288 289

Fig. 1. Schematic representation of three different xylose metabolic pathways and glutaric acid

290

biosynthetic pathway. GA: Glycolaldehyde. Genes: EcxylA: encoding xylose isomerase; CcxylB:

291

encoding xylose dehydrogenase; CcxylC: encoding xylonolactonase; CcxylD: encoding xylonate

292

dehydratase; CcxylX: encoding 2-keto-3-deoxy-xylonate dehydratase; CcxylA: encoding 2-

293

ketoglutarate semialdehyde dehydrogenase; yagE or yjhH: encoding aldolase; gltA: encoding

294

citrate synthase; HCS: encoding homocitrate synthase; HA: encoding homoaconitase; HICDH:

295

encoding homoisocitrate dehydrogenase; kivD: encoding alpha-ketoisovalerate decarboxylase;

296

gabD: encoding succinate semialdehyde dehydrogenase.

297 298

Fig. 2. Results of shake flask studies for glutaric acid production from two different xylose

299

metabolic pathways. (A) Glutaric acid produced from xylose isomerase pathway; (B) Glutaric

300

acid produced from xylose Weimberg-Dahms pathway. The data were generated from three

301

independent experiments (n=3; s.d. represented by ±).

302 303

Fig. 3. Results of shake flask studies for glutaric acid production from xylose synergetic

304

pathways by utilization of xylose isomerase pathway and Weimberg-Dahms pathway in

305

combination. The data were generated from three independent experiments (n=3; s.d. represented

306

by ±).

307 308

Fig. 4. Results of shake flask studies for glutaric acid production from re-constructed xylose

309

synergetic pathways. (A) Glutaric acid produced from xylose synergetic pathways by utilization



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310

of xylose isomerase pathway and Weimberg pathway in combination; (B) Glutaric acid produced

311

from xylose synergetic pathways by utilization of xylose isomerase pathway and Weimberg

312

pathway in combination, but the TCA cycle was disrupted by deletion of gltA in this case. The

313

data were generated from three independent experiments (n=3; s.d. represented by ±).


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314


Supporting Information

315 316

The maximum theoretical molar yields of glutaric acid from different xylose metabolic pathways

317



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Page 20 of 27

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(3) Weimberg, R. (1961) Pentose oxidation by Pseudomonas fragi, J. Biol. Chem. 236, 629-635.

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Stephanopoulos, G. (2016) Efficient utilization of pentoses for bioproduction of the

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and Lee, S. Y. (2013) Metabolic engineering of Escherichia coli for the production of 5-

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(23) Choi, S. Y., Kim, W. J., Yu, S. J., Park, S. J., Im, S. G., and Lee, S. Y. (2017) Engineering

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57x32mm (300 x 300 DPI)

ACS Paragon Plus Environment


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

Schematic representation of three different xylose metabolic pathways and glutaric acid biosynthetic pathway. GA: Glycolaldehyde. Genes: EcxylA: encoding xylose isomerase; CcxylB: encoding xylose dehydrogenase; CcxylC: encoding xylonolactonase; CcxylD: encoding xylonate dehydratase; CcxylX: encoding 2-keto-3-deoxy-xylonate dehydratase; CcxylA: encoding 2-ketoglutarate semialdehyde dehydrogenase; yagE or yjhH: encoding aldolase; gltA: encoding citrate synthase; HCS: encoding homocitrate synthase; HA: encoding homoaconitase; HICDH: encoding homoisocitrate dehydrogenase; kivD: encoding alpha-ketoisovalerate decarboxylase; gabD: encoding succinate semialdehyde dehydrogenase. 84x67mm (300 x 300 DPI)


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Results of shake flask studies for glutaric acid production from two different xylose metabolic pathways. (A) Glutaric acid produced from xylose isomerase pathway; (B) Glutaric acid produced from xylose WeimbergDahms pathway. The data were generated from three independent experiments (n=3; s.d. represented by ±). 143x49mm (300 x 300 DPI)



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Results of shake flask studies for glutaric acid production from xylose synergetic pathways by utilization of xylose isomerase pathway and Weimberg-Dahms pathway in combination. The data were generated from three independent experiments (n=3; s.d. represented by ±). 70x49mm (300 x 300 DPI)


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Results of shake flask studies for glutaric acid production from re-constructed xylose synergetic pathways. (A) Glutaric acid produced from xylose synergetic pathways by utilization of xylose isomerase pathway and Weimberg pathway in combination; (B) Glutaric acid produced from xylose synergetic pathways by utilization of xylose isomerase pathway and Weimberg pathway in combination, but the TCA cycle was disrupted by deletion of gltA in this case. The data were generated from three independent experiments (n=3; s.d. represented by ±). 140x49mm (300 x 300 DPI)


Investigation of the Synergetic Effect of Xylose Metabolic Pathways on

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