unstructuredio/SCORE-Bench · Datasets at Hugging Face

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arc-shaped installing groove; first slideways corresponding to the first arc-shaped sliding blocks are arranged in the arc-shaped installing portion;
the connecting plate is inserted into a movable support, and the movable support is located in the fixed support;


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a second arc-shaped installing groove is formed in the movable support, and the connecting plate is inserted into the second arc-shaped installing groove; second arc-shaped sliding blocks are arranged in the second arc-shaped installing groove, and second slideways


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corresponding to the second arc-shaped sliding blocks are arranged on the connecting plate; and
two torsion gaskets that are arranged between each side of the movable support and the fixed support, one side of each of the two torsion gaskets is slidably arranged on




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the fixed support, and the other side of each of the two torsion gaskets and the movable support are in hinged connection.
2. The overturning and supporting rotating shaft according to claim 1, wherein kidney-shaped holes are formed in


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the fixed support, and sliding blocks are slidably arranged in the kidney-shaped holes and are fixedly arranged on the two torsion gaskets.
3. The overturning and supporting rotating shaft according to claim 1, wherein a connecting fixed shaft is fixedly


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arranged between the two torsion gaskets.
4. The overturning and supporting rotating shaft according to claim 1, wherein a fixed shaft for supporting the fixed support is fixedly arranged between both ends of the fixed support.
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US 9,493,801 B2
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17
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2.7.7.4 coded by cysNcysD). The APS is then converted to PAPS by APS kinase (EC 2.7.1.25 encoded by cysC). This step requires one ATP. PAPS is converted to sulfite by a PAPS reductase (EC 1.8.4.8 coded by cysH) and sulfite is reduced to sulfide by NADPH-sulfite reductase (EC 1.8.1.2 coded by cysIcysJcysG). The alternate pathway, shown on the right side of FIG. 6, converts APS directly to sulfite using an adenylyl sulfate reductase (EC 1.8.9.92 or 1.8.4.9). One of ordinary skill in the art will appreciate that any adenylyl sulfate reductase that can convert APS to sulfite will work. For example, the adenylyl sulfate reductase from Bacillus subtilis (Accession number CAA04409), or from Pseudomonas aeruginosa (Accession number NP_250447).
Adenylyl sulfate reductase encoding nucleic acid sequences can be introduced into any microorganism used to produce methionine. For example the strains described herein, as well as the strains described in WO2005/108561 and WO2006138689 by Metabolic Explorer, and those described by Kumar and Gomes, Biotechnology Advances 23:41-61, 2005, can benefit from the disclosed route bypassing PAPS and thus requiring one less ATP molecule for sulfate assimilation.
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EXAMPLES
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Example 1
Multiple Methionine Production Pathways, One of which Utilizes Direct Sulfhydrylation, Using Exogenously Expressed Nucleic Acid Sequences
A. Construction of a Microorganism Having Both metABC (Transsulfuration) and metAZ (Direct Sulfhydrylation)
As described before, endogenous production of methionine in E. coli occurs mainly through the transulfuration reaction. This Example describes the engineering of E. coli to increase direct sulfhydrylation while also maintaining the endogenous metABC pathway.
Direct sulfhydrylation was increased by cloning O-succinylsulfhydrylase (EC 4.2.99.-) which converts O-succinylhomoserine to homocysteine by reacting with hydrogen sulfide. This enzyme is codified by metZ and can be found in some Pseudomonas species (Vermeij and Kertesz, J Bacteriol. 181:5833-5837, 1999 and Inoue et al., J. Bacteriol. 179:3956-3962, 1997).
More specifically, metZ from Pseudomonas aeruginosa was cloned into methionine auxotrophs of strain TF4076BJF, which was derived from threonine-producing strain TF4076, (additionally modified by the deletion of thrB and metJ, and the insertion of metF under the control of the pTrc promoter, further described in Example 3, below). These auxotrophs have deletion of either the metB or the metB and metC genes. metZ from Pseudomonas aeruginosa enhanced the growth of the metB and the metBC deletion mutants in minimal medium. Even though in flask cultures methionine production was not fully recovered, metZ expression induces the methionine production up to ~100 mg/L in metBC deletion mutant, as shown in Table 1. This indicates that metZ is responsible for the production of homocysteine in the cell.
Low methionine production of the deletion mutants transformed with metZ may be due to the limitation of sulfide in the intracellular fraction (methods of increasing sulfide concentration are provided below). This is supported by the finding that the growth of the metBC deletion strain transformed with metZ was enhanced in M9 media in the


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presence of 2 mM sodium sulfide. In in-vitro assays, the O-succinylsulfhydrylase had low sulfide affinity. Through directed evolution, it is possible to develop improved O-succinylsulfhydrylases with higher sulfide affinity and also
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higher activity. A highly active O-succinylsulfhydrylase could replace metB and metC in the methionine pathway, or could complement the pathway to increase the carbon flux to methionine.
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TABLE 1
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arc-shaped installing groove; first slideways corresponding to the first arc-shaped sliding blocks are arranged in the arc-shaped installing portion;

the connecting plate is inserted into a movable support, and the movable support is located in the fixed support;

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40

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a second arc-shaped installing groove is formed in the movable support, and the connecting plate is inserted into the second arc-shaped installing groove; second arc-shaped sliding blocks are arranged in the second arc-shaped installing groove, and second slideways

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45

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corresponding to the second arc-shaped sliding blocks are arranged on the connecting plate; and

two torsion gaskets that are arranged between each side of the movable support and the fixed support, one side of each of the two torsion gaskets is slidably arranged on

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50

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the fixed support, and the other side of each of the two torsion gaskets and the movable support are in hinged connection.

2. The overturning and supporting rotating shaft according to claim 1, wherein kidney-shaped holes are formed in

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55

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the fixed support, and sliding blocks are slidably arranged in the kidney-shaped holes and are fixedly arranged on the two torsion gaskets.

3. The overturning and supporting rotating shaft according to claim 1, wherein a connecting fixed shaft is fixedly

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60

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arranged between the two torsion gaskets.

4. The overturning and supporting rotating shaft according to claim 1, wherein a fixed shaft for supporting the fixed support is fixedly arranged between both ends of the fixed support.

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US 9,493,801 B2

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17

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2.7.7.4 coded by cysNcysD). The APS is then converted to PAPS by APS kinase (EC 2.7.1.25 encoded by cysC). This step requires one ATP. PAPS is converted to sulfite by a PAPS reductase (EC 1.8.4.8 coded by cysH) and sulfite is reduced to sulfide by NADPH-sulfite reductase (EC 1.8.1.2 coded by cysIcysJcysG). The alternate pathway, shown on the right side of FIG. 6, converts APS directly to sulfite using an adenylyl sulfate reductase (EC 1.8.9.92 or 1.8.4.9). One of ordinary skill in the art will appreciate that any adenylyl sulfate reductase that can convert APS to sulfite will work. For example, the adenylyl sulfate reductase from Bacillus subtilis (Accession number CAA04409), or from Pseudomonas aeruginosa (Accession number NP_250447).

Adenylyl sulfate reductase encoding nucleic acid sequences can be introduced into any microorganism used to produce methionine. For example the strains described herein, as well as the strains described in WO2005/108561 and WO2006138689 by Metabolic Explorer, and those described by Kumar and Gomes, Biotechnology Advances 23:41-61, 2005, can benefit from the disclosed route bypassing PAPS and thus requiring one less ATP molecule for sulfate assimilation.

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EXAMPLES

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Example 1

Multiple Methionine Production Pathways, One of which Utilizes Direct Sulfhydrylation, Using Exogenously Expressed Nucleic Acid Sequences

A. Construction of a Microorganism Having Both metABC (Transsulfuration) and metAZ (Direct Sulfhydrylation)

As described before, endogenous production of methionine in E. coli occurs mainly through the transulfuration reaction. This Example describes the engineering of E. coli to increase direct sulfhydrylation while also maintaining the endogenous metABC pathway.

Direct sulfhydrylation was increased by cloning O-succinylsulfhydrylase (EC 4.2.99.-) which converts O-succinylhomoserine to homocysteine by reacting with hydrogen sulfide. This enzyme is codified by metZ and can be found in some Pseudomonas species (Vermeij and Kertesz, J Bacteriol. 181:5833-5837, 1999 and Inoue et al., J. Bacteriol. 179:3956-3962, 1997).

More specifically, metZ from Pseudomonas aeruginosa was cloned into methionine auxotrophs of strain TF4076BJF, which was derived from threonine-producing strain TF4076, (additionally modified by the deletion of thrB and metJ, and the insertion of metF under the control of the pTrc promoter, further described in Example 3, below). These auxotrophs have deletion of either the metB or the metB and metC genes. metZ from Pseudomonas aeruginosa enhanced the growth of the metB and the metBC deletion mutants in minimal medium. Even though in flask cultures methionine production was not fully recovered, metZ expression induces the methionine production up to ~100 mg/L in metBC deletion mutant, as shown in Table 1. This indicates that metZ is responsible for the production of homocysteine in the cell.

Low methionine production of the deletion mutants transformed with metZ may be due to the limitation of sulfide in the intracellular fraction (methods of increasing sulfide concentration are provided below). This is supported by the finding that the growth of the metBC deletion strain transformed with metZ was enhanced in M9 media in the

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18

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presence of 2 mM sodium sulfide. In in-vitro assays, the O-succinylsulfhydrylase had low sulfide affinity. Through directed evolution, it is possible to develop improved O-succinylsulfhydrylases with higher sulfide affinity and also

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5

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higher activity. A highly active O-succinylsulfhydrylase could replace metB and metC in the methionine pathway, or could complement the pathway to increase the carbon flux to methionine.

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10

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TABLE 1

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