iGEM 2023 — Team njtech-china-b

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2023.02

1.Formed experimental group.

2.Conducted literature research.

3.Entered the lab for relevant skills training.

2023.03

1.Recruited team members and determine the grouping of our practice sections(HP group, art group ,model group, wiki group).

2.Conducted project research organised project presentations.

3.Had a brainstorm and finalized our project idea.

2023.04

Experiment

1.Developed an experimental programme and confirmed the feasibility of the experiment.

2.The *Escherichia coli* NT1003 which could produce high lysine was used as the chassis strain, and designed and constructed a portion of the metabolic pathway for 1,5-pentanediol(1,5-PDO).

3.The *Escherichia coli* NT1003 which could produce high lysine was used as the chassis strain, and designed and constructed a portion of the metabolic pathway for 1,5-pentanediol(1,5-PDO).

4.Confirmed the selection of the carboxylic acid reductase pathway as the synthetic pathway for the subsequent study.

HP

1.Determined the human practice.

2.Interviewed with Professor Bingzhi Li of Tianjin University and he approved our modular design of metabolic pathway in synthetic biology .

2023.05

Experiment

1.The plasmids pTrc99a-davB-davA-GabT and pACYCDuet-sfp-MmCAR-YahK were constructed.

2.These plasmids were then transferred into NT1003 to construct strain NT1003-P1. Through fermentation,the production pathway of glucose to 1,5-PDO was successfully verified.

HP

1.Interviewed Professor Yingxiu Cao of Tianjin University and she suggested us to assess whether engineering of branched pathways could enhance the product titers. Furthermore, She emphasized the importance of creating a high-quality wiki and HP sections.

2.Professor Liya Liang was interviewed and she suggested replacing plasmids with higher copy numbers to enhance the expression of the 1,5-PDO synthesis module.

3.Interviewed with expert from Sinopec Beijing Research Institure Of Chemical Industry and discussed prospects for 1,5-pentanediol synthesis and other related topics.

4.Talked with high school team Nanjing-BioXstem and shared team case to help them better understand igem.

5.Participated in Nanjing iGEM association.

2023.06

By metabolic flux analysis, the maximum theoretical yield of 1, 5-PDO can reach 0.847 mol/mol Glu via the lysine pathway using glucose as substrate.

Experiment

1.Constructed the plasmid pRSFDuet-sfp-MmCAR-YahK.

2.Constructed the strain NT1003-P2: pTrc99a-davB-davA-GabT + pRSFDuet-sfp-MmCAR-YahK.

Through fermentation,it showed that the yield of 1,5-PDO was nearly 4-fold higher in NT1003-P2 than in NT1003-P1.

HP

1.Interviewed with Professor Xinkun Ren of Nanjing University, and suggested that We can mutate the rate-limiting enzyme MmCAR to enhance the production.

2.Organized a science popularization event with the theme of iGEM for students majoring in Bioengineering at Nanjing Tech University.

3.Provided a demonstration on the usage of software such as SnapGene and GraphPad to the high school team Nanjing-BioXstem.

4.Conducted a preaching activity for middle school students.

2023.07

Model

Based on the model analysis, it had been predicted that the knockout of genes ackA and pta can enhance the production of 1,5-PDO.

Experiment

1.By utilizing CRISPR-Cas9 technology, the genes ackA and pta were knocked out.

2.Using CRISPR-Cas9 technology, we had knocked out the genes ackA and pta and successfully constructed the strain NT1003-P2-ΔackA-pta.

3.Based on previous research in the laboratory, knocking out the gene YcjQ was identified and the strain NT1003-P2-ΔYcjQ.

Based on fermentation result, we decided to continue the modification using NT1003-P2-ΔYcjQ in subsequent experiments.

HP

1.Interviewed Professor Jiaxin Xu, who suggested enzyme assembly as a strategy to enhance the yield of 1,5-PDO.

2.Participated in the 10th Conference of the China iGEMer Community (CCiC).

3.Participated in the Environmental Protection and Sustainability Summit (EPSS) hosted by Nanjing Normal University.

4.Took part in the 15th Training Course of Coating Formulation Design and the 10th Training Course of Waterborne Technology.

5.Participated in the National Chemical Engineering and Biochemical Annual Conference.

6.Held an online networking meeting with the NNU-CHINA team.

2023.08

Model

Based on iteration results,several genetic targets that can be used to optimize the fluxes to produce the production of 1, 5-pentadiol are predicted.

Experiment

1.To enhance the carboxylic acid reductive efficiency of MmCAR enzyme, we conducted molecular docking and identified the mutation sites.

2.Constructed the plasmid PRSFDuet-sfp-MmCARQ302E-Yahk and PRSFDuet-sfp-MmCART390E-Yahk

These plasmids were then transferred into *E. coli* BL21.Through the Whole-cell catalysis, it determined that the mutation at position 302 yielded the best effect.

2023.09

Experiment

1.Constructed strain NT1003-P3-ΔYcjQ:pTrc99a-davB-davA-GabT+pRSFDuet-sfp-MmCARQ302E-YahK.

As fermentation was performed, the mutated MmCAR moderately increased the yield of 1,5-PDO

2.Constructed the plasmid pRSFDuet-sfp-MmCARQ302E-SpyTag-YahK-EutM-SpyCatcher.

3.Constructed strain NT1003-P4-ΔYcjQ: pTrc99a-davB-davA-GabT +pRSFDuet-sfp-MmCARQ302E-SpyTag-YahK-EutM-SpyCatcher.

Fermentation was carried out and the final pentylene glycol yield was boosted to 23 mM (2.4 g/L).

HP

1.Came to Qiliqiao Community of Dingshan Street for a science popularization propaganda activity.

2.NJTech-China-B and College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University successfully held an activity called “Synthetic Biology Carnival”.

2023.10

Experiment

To further identify the performance of the *E. coli* NT1003-P4-ΔYcjQ, we performed a fed-batch fermentation using a 5-liter bioreactor. After fermentation of 80 h, the 1,5-PDO production was reached to 48 mM (5.0 g/L).

HP

In order to promote the project, we visited Nanjing Shenghu New Material Technology Co. Ltd. Jing Wang, Yang Liao and Yue Yang provided suggestions on technology implementation and project promotion.

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Overview

We designed an artificial 1,5-pentanediol (1,5-PDO) biosynthetic pathway, and identified its theoretical feasibility through a metabolic network model simulation. After screening the functional enzymes in the pathway, we assembly the whole pathway in a lysine-producing strain *Escherichia coli* NT1003, and successfully fulfill the pathway for 1,5-PDO production from glucose. In addition, in conjunction with dry experiment of metabolic network model simulation, several engineering strategies were employed to enhance the 1,5-PDO production, including optimizing the gene expression plasmid, inhibiting acetic acid production pathway and 1,5-PDO degradation pathway, mutating the limiting enzymes and also assembling the limiting enzymes. The related parts have been registered and tested, and most of them worked well to perform their functions as we expected.

Cycle 1

### Screening the enzymes for constructing the functional modules to synthesize 1,5-PDO

Design

Because none of natural pathways for 1,5-PDO biosynthesis is present, we first designed an artificial 1,5-PDO biosynthetic pathway in our work. In the designed pathway, lysine is used as a director precursor, which is then converted into 5-hydroxyvalerate (5-HV) and 1,5-PDO via two synthetic modules (5-HV synthesis module and 1,5-PDO synthesis module). The 5-HV synthesis module converts lysine into 5-HV via the oxidase, hydrolase, transaminase, and aldehyde reductase, while 1,5-PDO synthesis module converts 5-HV into 1,5-PDO via carboxylate reductase and aldehyde reductase. *E. coli* lacks some related enzymes in our designed 1,5-PDO biosynthetic pathway. We added several heterologous enzymes into the iML1515 metabolic network model of *E. coli* to establish the metabolic model for 1,5-PDO synthesis. Through a flux balance analysis (FBA), the maximum theoretical yield for 1,5-PDO production based on our designed pathway was determined to be 0.847 mol/molglucose, indicating the feasibility of our designed pathway for 1,5-PDO biosynthesis.

![](https://static.igem.wiki/teams/4800/wiki/main/engineering-success/engineering-success-211.jpg)

Figure 1. A proposed biosynthesis route for de novo production of 1,5-PDO from glucose.

Subsequently, to establish the synthetic pathway for 1,5-PDO production, the screening of desired enzymes to construct the functional modules was designed. To establish a functional 1,5-PDO synthesis module, two enzymes of carboxylate reductase and aldehyde reductase from different organisms were screened. The carboxylate reductases originated from *Mycobacteroides abscessus* (MAB3367) *Mycolicibacterium smegmatis* (MSMEG5739), *Mycobacterium abscessus* (MAB4714), *Mycobacterium avium* (MpCAR), and *Mycobacterium marinum* (MmCAR) were selected as candidate genes. For aldehyde reductases, the enzymes originated from *Oenococcus oeni* (Adh1), *Oenococcus alcoholitolerans* (Hsd36), *Escherichia coli* (AdhP) and *E. coli* (YahK) were selected. To establish a functional 5-HV synthesis module, the key enzyme of transaminase originated from *E. coli* (GabT and PatA), *Paracoccus denitrificans* (QLH), *Streptomyces avermitilis* (SAV2585), *Ruegeria pomeroyi* (Spo3471) and *Pseudomonas aeruginosa* (SpuC) were selected as candidates for screening.

![](https://static.igem.wiki/teams/4800/wiki/main/
engineering-success/engineering-success-212.png)

Figure 2. The screening of enzymes for the establishment of functional modules for 1,5-PDO bioproduction.

Build

Screening of carboxylate reductase and aldehyde reductase for 1,5-PDO synthesis module

To obtain the desired 1,5-PDO synthesis module, several carboxylate reductases and aldehyde reductases were evaluated. Through literature research, five carboxylate reductases (MAB3367 from *M. abscessus*
[BBa\_K4800020](http://parts.igem.org/wiki/index.php?title=Part:BBa_K4800020), MpCAR from *M. avium* [BBa\_K4800021](http://parts.igem.org/wiki/index.php?title=Part:BBa_K4800021), MSMEG5739 from *M. smegmatis* [BBa\_K4800022](http://parts.igem.org/wiki/index.php?title=Part:BBa_K4800022), MmCAR from *M. marinum* [BBa\_K4800017](http://parts.igem.org/wiki/index.php?title=Part:BBa_K4800017) and MAB4714 from *M. abscessus* [BBa\_K4800023](http://parts.igem.org/wiki/index.php?title=Part:BBa_K4800023)), and four aldehyde reductases (YahK from *E. coli* [BBa\_K4800027](http://parts.igem.org/wiki/index.php?title=Part:BBa_K4800027), Hsd36 from *O. alcoholitolerans* [BBa\_K4800025](http://parts.igem.org/wiki/index.php?title=Part:BBa_K4800025), Adh1 from *O. oeni* [BBa\_K4800024](http://parts.igem.org/wiki/index.php?title=Part:BBa_K4800024) and Adhp from *E. coli* [BBa\_K4800026](http://parts.igem.org/wiki/index.php?title=Part:BBa_K4800026)) were codon-optimized and synthesized. Among them, MmCAR and sfp is the component of an existing part of [BBa\_K1655000](http://parts.igem.org/wiki/index.php?title=Part:BBa_K1655000). The gene fragments of carboxylate reductase were subcloned into the linear pRSFDuet-1 together with its activating factor phosphopantetheinyl transferase (sfp) from *Bacillus subtilis*, yielding the plasmids of pRSFDuet-MAB3367 [BBa\_K4800033](http://parts.igem.org/wiki/index.php?title=Part:BBa_K4800033), pRSFDuet-MpCAR [BBa\_K4800034](http://parts.igem.org/wiki/index.php?title=Part:BBa_K4800034), pRSFDuet-MSMEG5739 [BBa\_K4800035](http://parts.igem.org/wiki/index.php?title=Part:BBa_K4800035), pRSFDuet-MmCAR [BBa\_K4800060](http://parts.igem.org/wiki/index.php?title=Part:BBa_K4800060), and pRSFDuet-MAB4714 [BBa\_K4800036](http://parts.igem.org/wiki/index.php?title=Part:BBa_K4800036). The gene fragments of aldehyde reductase were subcloned into the linear pTrc99a by the enzyme digestion and ligation, yielding the plasmids of to pTrc99a-YahK [BBa\_K4800040](http://parts.igem.org/wiki/index.php?title=Part:BBa_K4800040), pTrc99a-Hsd36 [BBa\_K4800038](http://parts.igem.org/wiki/index.php?title=Part:BBa_K4800038), pTrc99a-Adh1 [BBa\_K4800037](http://parts.igem.org/wiki/index.php?title=Part:BBa_K4800037) and pTrc99a-Adhp [BBa\_K4800039](http://parts.igem.org/wiki/index.php?title=Part:BBa_K4800039). The plasmids were transformed into *E. coli* BL21 respectively, and corrected colonies were verified by PCR, sequencing, and SDS-PAGE analysis. As shown in Figure 3, all five carboxylate reductases and four aldehyde reductases have soluble expression in *E. coli* BL21.

![](https://static.igem.wiki/teams/4800/wiki/main/
engineering-success/engineering-success-2211.png)

Figure 3. (a) Constructing expression vectors of different carboxylate reductases. (b) SDS-PAGE analysis of of different carboxylate reductases in *E. coli*. (c) Constructing expression vectors of different aldehyde reductases. (d) SDS-PAGE analysis of of different aldehyde reductases in *E. coli*.

To obtain functional 1,5-PDO synthesis module, plasmids of pRSFDuet-MAB3367, pRSFDuet-MpCAR, pRSFDuet-MSMEG5739, pRSFDuet-MmCAR, and pRSFDuet-MAB4714 were transferred into *E. coli* BL21 together with pTrc99a-YahK to compare the activity of different carboxylate reductases. The plasmids of pTrc99a-Yahk, pTrc99a-Hsd36, pTrc99a-Adh1 and pTrc99a-Adhp were transferred into *E. coli* BL21 together with pRSFDuet-MmCAR to compare the activity of different aldehyde reductases.

![](https://static.igem.wiki/teams/4800/wiki/main/
engineering-success/engineering-success-2212.png)

Figure 4. (a) Plasmid transformation for carboxylate reductase screening assay. (b) Plasmid transformation for aldehyde reductase screening assay.

Screening of transaminase for 5-HV synthesis module

In 5-HV systhesis module, DavB and DavA have been identified capable of efficiently converting lysine to 5-aminovalerate (5-AVA) in the reported reference. To obtain the efficient 5-HV synthesis module, we turned out our attention to screen different transaminases. Six transaminases, including GabT from *E. coli* [BBa\_K4800015](http://parts.igem.org/wiki/index.php?title=Part:BBa_K4800015), PatA from *E. coli* [BBa\_K4800028](http://parts.igem.org/wiki/index.php?title=Part:BBa_K4800028), Spo3471 from *R. pomeroyi* [BBa\_K4800029](http://parts.igem.org/wiki/index.php?title=Part:BBa_K4800029), SAV2585 from *S. avermitilis* [BBa\_K4800030](http://parts.igem.org/wiki/index.php?title=Part:BBa_K4800030), SpuC from *P. aeruginosa* [BBa\_K4800032](http://parts.igem.org/wiki/index.php?title=Part:BBa_K4800032), and QLH from *P. denitrificans* [BBa\_K4800031](http://parts.igem.org/wiki/index.php?title=Part:BBa_K4800031) were codon-optimized and synthesized. The gene fragments of transaminases were subcloned into the linear pTrc99a, yielding the plasmids of pTrc99a-GabT [BBa\_K4800046](http://parts.igem.org/wiki/index.php?title=Part:BBa_K4800046), pTrc99a-PatA [BBa\_K4800041](http://parts.igem.org/wiki/index.php?title=Part:BBa_K4800041), pTrc99a-Spo3471 [BBa\_K4800042](http://parts.igem.org/wiki/index.php?title=Part:BBa_K4800042), pTrc99a-SAV2585 [BBa\_K4800043](http://parts.igem.org/wiki/index.php?title=Part:BBa_K4800043), pTrc99a-SpuC [BBa\_K4800045](http://parts.igem.org/wiki/index.php?title=Part:BBa_K4800045), and pTrc99a-QLH [BBa\_K4800044](http://parts.igem.org/wiki/index.php?title=Part:BBa_K4800044).

The plasmids were transformed into *E. coli* BL21 respectively, and corrected colonies were verified by PCR, sequencing, and SDS-PAGE analysis. As shown in Figure 5, all six transaminases show soluble expression in *E. coli*.

![](https://static.igem.wiki/teams/4800/wiki/main/
engineering-success/engineering-success-2221.png)

Figure 5. (a) Constructing expression vectors of different transaminases. (b) SDS-PAGE analysis of different transaminases in *E. coli*.

To compare the activity of different transaminases, the DNA fragments of Ptrc-RBS-YahK were amplified and ligated into the plasmids of pTrc99a-GabT, pTrc99a-PatA, pTrc99a-Spo3471, pTrc99a-SAV2585, pTrc99a-SpuC, and pTrc99a-QLH, yielding the desired plasmids of pTrc99a-YahK-GabT [BBa\_K4800047](http://parts.igem.org/wiki/index.php?title=Part:BBa_K4800047), pTrc99a-YahK-PatA [BBa\_K4800048](http://parts.igem.org/wiki/index.php?title=Part:BBa_K4800048), pTrc99a-YahK-Spo3471 [BBa\_K4800049](http://parts.igem.org/wiki/index.php?title=Part:BBa_K4800049), pTrc99a-YahK-SAV2585 [BBa\_K4800050](http://parts.igem.org/wiki/index.php?title=Part:BBa_K4800050), pTrc99a-YahK-SpuC [BBa\_K4800051](http://parts.igem.org/wiki/index.php?title=Part:BBa_K4800051), and pTrc99a-YahK-QLH [BBa\_K4800052](http://parts.igem.org/wiki/index.php?title=Part:BBa_K4800052). The plasmids were transferred into *E. coli* BL21 to compare the activity of different transaminases by detecting 5-HV production from 5-AVA.

![](https://static.igem.wiki/teams/4800/wiki/main/
engineering-success/engineering-success-2222.png)

Figure 6. Constructing expression vectors of different transaminases to compare their activity.

Test

By using a whole-cell catalysis process, the enzyme activity was compared. The activity of carboxylate reductase was determined by detecting the 5-HV consumption and 1,5-PDO production with HPLC. By comparing the 5-HV consumption, MmCAR and MpCAR displayed the higher substrate consumption activity. The further detection of 1,5-PDO production showed that MmCAR resulted in the highest activity for 1,5-PDO production (11.5mM). Therefore, MmCAR was selected as the carboxylate reductase in the following strain construction.

![](https://static.igem.wiki/teams/4800/wiki/main/
engineering-success/engineering-success-231.png)

Figure 7. Comparing the activity of carboxylate reductases from different organisms.

After identifying the efficient carboxylate reductase for 1,5-PDO synthesis module, we performed the synthesis of 1,5-PDO by whole-cell catalysis to compare the activities of the four aldehyde reductases by detecting 1,5-PDO production with HPLC. The results confirmed that YahK from *E. coli* exhibited the highest activity for 1,5-PDO accumulation. Thus, YahK was selected to assembly the 1,5-PDO synthesis module in the following work.

![](https://static.igem.wiki/teams/4800/wiki/main/
engineering-success/engineering-success-232.png)

Figure 8. Comparing the activity of aldehyde reductases from different organisms.

The activity of transaminase was determined by detecting the 5-HV production from 5-AVA with HPLC. The results showed the 6 transaminases all exhibit ammonia-transamination activity. Among them, GabT from *E. coli* exhibited the highest ammonia-transamination efficiency. Thus, GabT was selected to assembly the 5-HV synthesis module in the following work.

![](https://static.igem.wiki/teams/4800/wiki/main/
engineering-success/engineering-success-233.png)

Figure 9. Comparing the activity of transaminases from different organisms.

Learn

Production of chemicals without natural synthetic pathways is a big challenge for synthetic biology. We attempted to engineer *E. coli* to produce 1,5-PDO via an artificial synthetic pathway. To assembly the functional pathways, we screened different candidate enzymes in designed synthesis modules, and efficient carboxylate reductase, aldehyde reductase and transaminase were obtained.

In this cycle, we learned that different sources of enzymes exhibit the different activity, and would affect the titer of the targeted products. In addition, the metabolic network model also shows that the enzymes in the 5-HV synthesis module and 1,5-PDO synthesis module are important for increasing the final 1,5-PDO yield. Before the construction of the producing strain, the identification of efficient parts with higher activity is an effective strategy, which provides a good basis for us to construct the 1,5-PDO-producing strain in the following work. This was the first time that the team members designed and completed the experiment independently by themselves, and everyone in the team gained a lot in the whole process.

Cycle 2

### Constructing the 1,5-PDO producing strain

Design

Through preliminary experiments, we have identified the suitable carboxylate reductase, aldehyde reductase and transaminase in the metabolic pathway, and obtain the functional modules. Subsequently, we expressed the genes of the two modules in two plasmids and introduced them into a lysine-producing *E. coli* NT1003 to construct a cell factory capable of synthesizing 1,5-pentanediol from glucose. Subsequently, we used a direct fermentation to validate the feasibility to produce 1,5-PDO of our engineered strain.

![](https://static.igem.wiki/teams/4800/wiki/main/
engineering-success/engineering-success-311.jpg)

Figure 10. Constructing the 1,5-PDO producing strain.

Build

In order to produce 1,5-PDO by using *E. coli* NT1003, the genes of 5-HV synthesis module including davA, davB, and GabT were subcloned into the plasmid pTrc99a together under the control of the trc promoter to yield plasmid pTtrc99a-davB-davA-GabT [BBa\_K4800054](http://parts.igem.org/wiki/index.php?title=Part:BBa_K4800054). In detail, using the laboratory conserved plasmid pET22b-T7-davB-T7-davA as templates, the fragments of davB and davA were amplified by PCR with a consensus RBS and Ptrc respectively. Subsequently, the Ptrc-RBS-davB-Ptrc-RBS-davA fragment was obtained by overlap PCR, which was then ligated into the linearized vector pTrc99a-GabT by In-fusion cloning method to obtain plasmid pTtrc99a-davB-davA-GabT. The plasmid was transformed into *E. coli* Trans-T1, and corrected colonies were verified by PCR, and sequencing.

![](https://static.igem.wiki/teams/4800/wiki/main/
engineering-success/engineering-success-321.png)

Figure 11. (a) The schematic of plasmid pTrc99a-davB-davA-GabT. (b) The colony PCR validation diagram.

The genes of 1,5-PDO synthesis module including MmCAR and YahK were subcloned into the plasmid pACYCDuet together under the control of trc promoter to yield plasmid pACYCDuet-sfp-MmCAR-YahK [BBa\_K4800053](http://parts.igem.org/wiki/index.php?title=Part:BBa_K4800053). In detail, based on the sequence of an existing part of [BBa\_K1655000](http://parts.igem.org/wiki/index.php?title=Part:BBa_K1655000), it was codon-optimized and synthesized to obtain the fragment of sfp-MmCAR, which was then ligated into the pACYCDuet plasmid under the control of trc promoter. The Ptrc-YahK fragment was amplified with plasmid pTrc99a-YahK as the template, and In-Fusion cloning was then used to ligate into the above vectors to yield plasmid pACYCDuet-sfp-MmCAR-YahK. The plasmid were transformed into *E. coli* Trans-T1, and corrected colonies were verified by PCR, and sequencing.

![](https://static.igem.wiki/teams/4800/wiki/main/
engineering-success/engineering-success-322.png)

Figure 12. (a) The schematic of plasmid pACYCDuet-sfp-MmCAR-YahK. (b) The colony PCR validation diagram.

The plasmid of pTrc99a-davB-davA-GabT and pACYCDuet-sfp-MmCAR-YahK were co-transformed into a lysine producing strain *E. coli* NT1003 to construct an engineered 1,5-PDO producing strain NT1003-P1.

Test

The 1,5-PDO production of the engineered strain *E. coli* NT1003-P1 was determined by the HPLC analysis. After the fermentation of 72 hours, the fermentation broth contained 2.82 mM (0.293g/L) of 1,5-pentanediol, which indicated that the synthetic pathway we designed was experimentally feasible, and the cell factory that could produce 1,5-PDO from glucose has been successfully developed. However, the 1,5-PDO titer was still lower than we expected, and the intermediate of 5-HV was largely accumulated.

![](https://static.igem.wiki/teams/4800/wiki/main/
engineering-success/engineering-success-331.png)

Figure 13. The 1,5-pentanediol production by the engineered strain of *E. coli* NT1003-P1.

Learn

In this cycle, we successfully constructed an engineered strain of *E. coli* NT1003-P1 to produce 1,5-PDO from glucose. We learned that the assembly of multiple genes in pathway into different plasmids with different replicon could make the pathway functional work in the strain. However, the 1,5-PDO titer is far below than we expected. Based on the dry expriment group, several potential gene targets are predicted for the strain optimization. We designed the experiment protocol and discussed with other professors, and they gave us some advices.

Prof. Liang Liya from Dalian University of Technology advised us to change a plasmid with high copy number to enhance the expression of the 1,5-PDO synthesis module, as the plasmid pACYCDuet-sfp-MmCAR-YahK contained a replicon with low copy number.

Prof. Cao Yingxiu, from Tianjin University, exhibited keen interest in our modular designing idea, and she advised us to adjust the metabolic pathway to improve the product titer.

In view of dry experiment simulation results, as well as the professor’s advice, team members designed the following two cycles to modify the strain of *E. coli* NT1003-P1, hoping to further improve 1, 5-PDO production. One is the optimization of expression plasmid, and another one is the deletion of branched genes.

Cycle 3

### The optimization of 1,5-PDO production by changing plasmid with high copy number

By combining FSEOF, and OptKnock algorithms in the metabolic network model, several gene overexpression targets that potentially involved in the improvement of 1,5-PDO production were predicted. As a large amount of 5-HV was accumulated in the fermentation broth in our wet experiment, we focused on the genes in the 1,5-PDO synthesis module among these overexpression targets. After consulting with experts, we recognized that using pACYCDuet plasmid to express the 1,5-PDO synthesis module might not be the optimal choice, After in-depth discussions within the team, we decided to adopt a high-copy number plasmid of pRSFDuet to express the 1,5-PDO synthesis module. The resulting plasmid was co-transformed with 5-HV synthesis module into the *E. coli* NT1003 to evaluate the effect on 1,5-PDO production.

Build

We used pACYCDuet-sfp-MmCAR-YahK as a template and amplified the PT7-YciA-Ptrc-sfp-MmCAR-Yahk fragment by PCR, and homologous recombination of PT7-YciA-Ptrc-sfp-MmCAR-Yahk with the linearized pRSFDuet vector was carried out by the In-fusion cloning method to yield plasmid of pRSFDuet-sfp-MmCAR-YahK [BBa\_K4800055](http://parts.igem.org/wiki/index.php?title=Part:BBa_K4800055). The plasmids were transformed into *E. coli* Trans-T1, and corrected colonies were verified by PCR, and sequencing. Subsequently, the plasmids of pTrc99a-davB-davA-GabT and pRSFDuet-sfp-MmCAR-YahK were co-transformed into a lysine producing strain *E. coli* NT1003 to construct an engineered 1,5-PDO producing strain NT1003-P2.

![](https://static.igem.wiki/teams/4800/wiki/main/
engineering-success/engineering-success-421.png)

Figure 14. (a) The schematic of plasmid pRSFDuet-sfp-MmCAR-YahK. (b) The colony PCR validation diagram.

Test

The yield of 1,5-PDO produced by the engineered strain NT1003-P2 was determined by the HPLC analysis. After a total fermentation period of 84 hours, 8.09 mM (0.843g/L) 1,5-PDO was produced by the engineered strain *E. coli* NT1003-P2, which is 3.5-fold higher than that in *E. coli* NT1003-P1.

![](https://static.igem.wiki/teams/4800/wiki/main/
engineering-success/engineering-success-431.png)

Figure 15. Fermentation of the engineered strain *E. coli* NT1003-P2 to produce 1,5-PDO from glucose.

Learn

In this cycle, we have learned how to optimize gene expression for the increased product titer. Due to the difference in the replicon, different plasmid has different copy number in the strain, which would affect the gene expression level. Therefore, the selection of appropriate plasmid is also an effective strategy for improving strain producing ability.

Cycle 4

### Knockout of genes in the branched pathway to improve 1,5-PDO production

Design

By using OptKnock algorithms in the metabolic network model, several gene deletion targets that potentially involved in the improvement of 1,5-PDO production were predicted. Among them, the ackA-pta gene encodes enzymes that convert pyruvate to acetic acid, the main by-product during the 1,5-PDO fermentation. After discussions with the experts, we also attempt to evaluate the effect of deleting these two genes on the 1,5-PDO production. In addition, in the previous study of the laboratory, it was found that the dehydrogenase of YcjQ was involved in the 1,5-PDO degradation. Therefore, in order to increase the yield, we also decided to knock out the YcjQ gene. Eventually, we decided to construct two strains NT1003-ΔackA-pta and NT1003-ΔYcjQ by the CRISPR/Cas9.

![](https://static.igem.wiki/teams/4800/wiki/main/
engineering-success/engineering-success-511.jpg)

Figure 16. Metabolic pathway for 1,5-PDO production in NT1003. The genes marked red would be knocked out.

Build

Firstly, we extracted the pCas9 and pTarget-sgRNA plasmids from laboratory preserved strains. In order to knock out these three genes, the upstream and downstream sequences (about 1000 bp) flanking ackA-pta and YcjQ were overlap PCR-amplified ([BBa\_K4800010](http://parts.igem.org/wiki/index.php?title=Part:BBa_K4800010) and [BBa\_K4800011](http://parts.igem.org/wiki/index.php?title=Part:BBa_K4800011)). Next, we designed the N20 sequence of ackA-pta and YcjQ by using the CHOPCHOP website (<http://chopchop.cbu.uib.no>), synthesized them on primers and ligated the N20 into the pTarget-sgRNA plasmid by PCR. With these tools, we used CRISPR/Cas9 strategy to knock out target genes of ackA-pta and YcjQ by homologous recombination. Finally, the corrected colonies were verified by PCR to determine the deletion of ackA-pta and YcjQ.

The plasmid of pTrc99a-davB-davA-GabT, and pRSFDuet-sfp-MmCAR-YahK were co-transformed into *E. coli* NT1003-ΔackA-pta and *E. coli* NT1003-ΔYcjQ to construct the engineered 1,5-PDO producing strain NT1003-P2-ΔackA-pta, NT1003-P2-ΔYcjQ respectively.

![](https://static.igem.wiki/teams/4800/wiki/main/
engineering-success/engineering-success-521.png)

Figure 17. Schematic diagram of CRISPER/Cas9.

Test

The yield of 1,5-PDO produced by these strains was analyzed by HPLC. In comparison to engineered strain NT1003-P2, the knockout of ackA-pta increased the 1,5-PDO titer by 16%, and the titer in strain NT1003-P2-Δacka-pta reached 9.3 mM (0.97 g/L). Meanwhile, the deletion of YcjQ gene markedly improved the final 1,5-PDO titer, 1.75-fold higher than that in the strain NT1003-P2. These results demonstrated that NT1003-P2-ΔYcjQ performed better, and we had decided to adopt this strain in the following experiments.

![](https://static.igem.wiki/teams/4800/wiki/main/
engineering-success/engineering-success-531.png)

Figure 18. The production of 1,5-PDO. (a) Comparison of production between NT1003-P2 and NT1003-P2-ΔackA-pta. (b) Comparison of production between NT1003-P2 and NT1003-P2-ΔYcjQ. (c) Comparison of production between NT1003-P2, NT1003-P2-ΔackA-pta and NT1003-P2-ΔYcjQ.

Learn

In this cycle, we learned that metabolic network model simulation is very helpful for guiding strain engineering, which can reduce the workload of the experiment. This also has reference significance for the work of other teams. In addition, we realized that knockout of genes in the branched pathway was a very effective strategy in microbial metabolic engineering for the improvement of strain performance.

After the optimization of expression plasmid and also deletion of branched pathways, the production 1,5-PDO was increased from 8 mM (0.83 g/L) to 14 mM (1.46 g/L). However, the accumulation of 5-HV was still detected. We thought that the activity of 1,5-PDO synthesis module was required to be further improved. Therefore, we further discussed our idea with some professors. Prof. Ren Xinkun from Nanjing university proposed that the engineering of limiting enzyme of MmCAR can be employed to improve the final production. Prof. Xu Jiaxing from Huaiyin Normal University advised us an strategy of enzyme assembly based on the protein scaffold, which might also be benefit for increasing enzyme activity. Based on their suggestion, we further designed the following two cycles of experiment.

Cycle 5

### Engineering carboxylate reductase for improved 1,5-PDO production

Design

As the suggestion by Prof. Ren Xinkun, we attempted to improve the catalytic activity of MmCAR by a rational engineering strategy. After reviewing relevant literature and consulting with Prof. Ren, we decided to carry out targeted mutagenesis of MmCAR by rational design and verified the effect of mutantion on its catalytic activity. The A-domain crystal structure of carboxylate reductase (MmCAR) from *Mycobacterium marinum* was not available in the PDB database. Therefore, we submitted amino acid sequences of MmCAR including A-domain and PCP arm to the phyre2 website (<http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index>) to build a protein model. After obtaining the PDB file of the protein structure, we used Autodock software to dock the protein structure with the 5-hydroxyvaleric acid (at least 50 docking) to find the 10th result with the lowest binding energy. For each docking result, the orientation of small molecules binding to the pocket was analyzed to see if it corresponded to the actual orientation. From structure of MmCAR binding to AMP and 5-hydroxyvaleric acid, it was found that the amino acids Q302 and T390 were on the side of 5-hydroxyvaleric acid and AMP away from the active center site, respectively. As both substrates are small polar molecules, we decided to mutant Q302 and T390 of MmCAR to acidic amino acid of glutamate (E) to change the polarity of the residues, and evaluated the effect of these mutants on MmCAR catalytic activity.

![](https://static.igem.wiki/teams/4800/wiki/main/
engineering-success/engineering-success-611.png)

Figure 19. The targeted mutant amino acid sites of Q302 and T390 in MmCAR.

Build

Based on the plasmid of pRSFDuet-sfp-MmCAR-YahK, we designed point mutation primers and cloned the whole plasmid using PCR. The linearized plasmid was isolated and purified by agarose gel electrophoresis, and then transferred into *E. coli* Trans-T1 to obtain the targeted plasmids of pRSFDuet-sfp-MmCARQ302E-YahK [BBa\_K4800059](http://parts.igem.org/wiki/index.php?title=Part:BBa_K4800059) and pRSFDuet-sfp-MmCART390E-YahK [BBa\_K4800057](http://parts.igem.org/wiki/index.php?title=Part:BBa_K4800057). The corrected colonies were verified by sequencing. The plasmids of pRSFDuet-sfp-MmCARQ302E-YahK and pRSFDuet-sfp-MmCART390E-YahK were transferred into *E. coli* BL21 respectively to evaluate the effect of mutation on the activity of 1,5-PDO synthesis module. The mutant plasmid with higher activity was further transformed into *E. coli* NT1003-ΔYcjQ together with pTrc99a-davB-davA-GabT to obtain the engineered strain NT1003-P3-ΔYcjQ for 1,5-PDO production from glucose.

![](https://static.igem.wiki/teams/4800/wiki/main/
engineering-success/engineering-success-621.png)

Figure 20. (a) The pRSFDuet-sfp-MmCARQ302E-YahK plasmid. (b) The pRSFDuet-sfp-MmCART390E-YahK plasmid. (c) Sequencing results indicate the successful mutation of pRSFDuet-sfp-MmCARQ302E-YahK plasmid. (d) Sequencing results indicate the successful mutation of pRSFDuet-sfp-MmCART390E-YahK plasmid.

Test

The effect of MmCAR mutation on the activity of 1,5-PDO synthesis module was determined by detecting the level to convert 5-HV to 1,5-PDO with HPLC analysis. The 1,5-PDO production of the engineered strain NT1003-P3-ΔYcjQ was determined by the HPLC analysis. From the catalysis results, we found that the 1,5-PDO synthesizing activity of the 1,5-PDO synthesis module could be increased by 50% when the amino acid site at position 302 was mutant to glutamic acid (E) from glutamine (Q) in MmCAR. However, when the amino acid site at position 390 was changed to glutamic acid (E) from threonine (T) in MmCAR, the activity of 1,5-PDO sysnthesis module was moderately affected. Therefore, we selected mutant MmCARQ302E for the following strain construction, and obtained the engineered strain of *E. coli* NT1003-P3-ΔYcjQ. Through a fermentation experiment, the mutation of MmCARQ302E could moderately increase the final 1,5-PDO production, and 14 mM (1.45 g/L) of 1,5-PDO in the engineered *E. coli* NT1003-P3-ΔYcjQ was finally obtained from glucose.

![](https://static.igem.wiki/teams/4800/wiki/main/
engineering-success/engineering-success-631.png)

Figure 21. (a) Whole-cell catalytic results of mutant 1,5-PDO sysnthesis module to convert 5-HV to 1,5-PDO. (b) The 1,5-pentanediol production by the engineered strain of *E. coli* NT1003-P3-ΔYcjQ.

Learn

In this cycle, we learned that the types of the amino acid sites that make up enzymes have a critical influence on enzyme activity. Rational engineering of enzymes is an effective strategy to improve enzyme activity, which is also a powerful tool to improve the limiting enzyme activity in a metabolic pathway. In addition, we learned that the cultivation condition could also affect the enzyme activity. Although the mutation of enzyme significantly improve the activity of 1,5-PDO synthesis module, it did not significantly improve the 1,5-PDO production in the final fermenting strain. As a next plan, the optimization of cultivation conditions, as well as the further engineering MmCAR can be considered.

Cycle 6

### The immobilization of carboxylate reductase by a self-assembling protein scaffold system

Design

In our communication with Prof. Xu Jiaxing, we learned that certain protein scaffolds can be used to enhance the catalytic efficiency of enzymes. After reviewing the literature and discussing within the group, we focused on the EutM from *Salmonella enterica*. EutM is known to self-assemble as filament-like structure in vivo, and has been developed as a powerful protein scaffolding system to rapidly co-immobilize enzymes for improving the biocatalysis efficiency. Therefore, we chose the EutM protein scaffold to immobilize the mutated MmCAR in vivo. At the same time, we intended to use the SpyTag-SpyCatcher system to help MmCAR and EutM bind better. The whole-cell conversion experiment and fermentation experiment were subsequently designed to verify whether the immobilization of the mutated MmCAR by the protein scaffold can further improve the 1,5-PDO production.

Build

The genes of certain protein scaffolds including SpyTag, EutM and SpyCatcher were subcloned into the plasmid pRSFDuet-sfp-MmCARQ302E-YahK together to yield plasmid pRSFDuet-sfp-MmCARQ302E-SpyTag-EutM-SpyCatcher-YahK [BBa\_K4800062](http://parts.igem.org/wiki/index.php?title=Part:BBa_K4800062). In detail, using the laboratory conserved plasmid pETDuet-T7-EutM-SpyCatcher as templates, the fragments of EutM and SpyCatcher were amplified by PCR with a consensus RBS and trc promoter, which was then ligated into the linearized vector pRSFDuet-sfp-MmCARQ302E-YahK by In-fusion cloning method. Subsequently, the MmCARQ302E-Spytag fragment was obtained by modifying the 5' end of the primer, which was then ligated into the linearized vector pRSFDuet-sfp-EutM-SpyCatcher-YahK by In-fusion cloning method. The corrected colonies were verified by PCR, and sequencing. The plasmids of pRSFDuet-sfp-MmCARQ302E-YahK and pRSFDuet-sfp-MmCARQ302E-SpyTag-EutM-SpyCatcher-YahK were transferred into *E. coli* BL21 respectively to evaluate the effect of mutation on the activity of 1,5-PDO synthesis module. The plasmids of pRSFDuet-sfp-MmCARQ302E-SpyTag-EutM-SpyCatcher-YahK and pTrc99a-davB-davA-GabT were co-transformed into *E. coli* NT1003-ΔYcjQ to construct the engineered 1,5-PDO producing strain NT1003-P4-ΔYcjQ.

![](https://static.igem.wiki/teams/4800/wiki/main/
engineering-success/engineering-success-721.png)

Figure 22. (a) pRSFDuet-sfp-MmCARQ302E-SpyTag-EutM- SpyCatcher-YahK plasmid (b) MmCAR by the protein scaffold. (c) The colony PCR validation diagram.

Test

The effect of assembly on the activity of 1,5-PDO synthesis module was determined by detecting the level to convert 5-HV to 1,5-PDO. The 1,5-PDO production of the engineered strain NT1003-P4-ΔYcjQ was determined by the HPLC analysis. From the catalysis results, we found that the assembly of MmCAR into the scaffold of EutM could significantly increase the ability of the 1,5-PDO producing module, 1.8 fold-higher than that without the assembly. Through a fermentation experiment, the assembly of MmCAR could increase 1,5-PDO production to 23 mM (2.4 g/L) in the engineered *E. coli* NT1003-P4-ΔYcjQ.

![](https://static.igem.wiki/teams/4800/wiki/main/
engineering-success/engineering-success-731.png)

Figure 23. (a) Whole-cell catalytic results of scaffold 1,5-PDO sysnthesis module to convert 5-HV to 1,5-PDO. (b) The 1,5-pentanediol production by the engineered strain of *E. coli* NT1003-P4-ΔYcjQ.

Learn

In this cycle, we learned that the assembly of enzymes based on the protein scaffold is an efficient strategy for improving the titer of the targeted product. In comparison to the rational enzyme engineering strategy, the enzyme assembly was more convenient, especially aiming the enzymes that lack understanding of catalytic mechanisms. Through the literature study and communication with experts, we also learned that the assembly of multiple enzymes in a metabolic pathway is also an efficient strategy for the microbial metabolic engineering. In the future work, we will further optimize the assembly strategy to improve 1,5-PDO production.

In summary

The overall goal of the project was to develop a cell factory to produce 1,5-PDO from glucose through the synthetic biology. Firstly, an artificial 1,5-PDO biosynthetic pathway was designed. The screening of the functional enzymes successfully fulfill the pathway for 1,5-PDO production from glucose in a lysine-producing strain *Escherichia coli* NT1003. To further improve 1,5-PDO production, we optimized the gene expression in a high copy number plasmid. Subsequently, we knocked out the branched pathways involving in acetic acid accumulation and 1,5-PDO degradation. What is more, focusing on the limiting enzymes of MmCAR, we engineered the enzyme by a rational engineering strategy, and obtained the mutant enzymes with higher activity. Meanwhile, we employed an assembly strategy to further improve the activity of the limiting enzymes. Based on these engineering strategies, an 1,5-PDO producing strain was successfully developed and optimized. Figure 24 shows the results obtained after each attempt.

![](https://static.igem.wiki/teams/4800/wiki/main/
engineering-success/engineering-success-81.jpg)

Figure 24. Results involved 1,5-PDO content obtained at each step.

Reference

[1].Cen, X. et al. Metabolic Engineering of *Escherichia coli* for De Novo Production of 1,5-Pentanediol from Glucose. ACS. Synth. Biol. 10, 192-203. (2021).

[2].Chen, C. et al. Metabolic engineering of *Escherichia coli* for the efficient production of 5-hydroxyvaleric acid. Process. Biochem. 130, 625-633.(2023)

[3].Xin, W. et al. Efficient production of 5-aminovalerate from l-lysine by engineered *Escherichia coli* whole-cell biocatalysts. Journal of Molecular Catalysis B: Enzymatic. 134,115-121.(2016).

[4].Zhang, G. et al. Self-Assembling Protein Scaffold System for Easy in Vitro Coimmobilization of Biocatalytic Cascade Enzymes. ACS. Catal. 8(6), 5611-5620 (2018).

[5].Zhang, G. et al. Developing a Protein Scaffolding System for Rapid Enzyme Immobilization and Optimization of Enzyme Functions for Biocatalysis. ACS. Synth. Biol. 8(8), 1867-1876. (2019).

{% endblock %}

{% extends "layout2.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Make a useful contribution for future iGEM teams. Use this page to document that Conmmunication & Cooperation.{% endblock %}
{% block page\_content %}

1.Obtaining recombinant plasmids

Obtaining the target gene and skeleton

According to Restriction Endonuclease from Takara

| **Component** | **Volume** |
| --- | --- |
| Restriction Endonuclease | 1 μL |
| Buffer | 2 μL |
| DNA | 1 μg |
| ddH2O | up to 20 μL |

1. Prepare the system of Restriction Endonuclease cleaving DNA.

2. Restriction Endonucleases are incubated at 37°C for 1 h except for BamH I, which is incubated at 30°C. The mixture is further digested by reaction at 80°C for 5 min and stored at 4°C.

PCR system

| **Reagent** | **Volume** |
| --- | --- |
| Phanta Taq Master Mix(2x) | 25 μL |
| Primer F(10 μM) | 2 μL |
| Primer R(10 μM) | 2 μL |
| Template DNA(Plasmid) | ＜20 ng |
| ddH2O | Up to 50 μLg |

Thermocycling Conditions for a Routine PCR:

| **Temperature (℃)** | **Time** | **Cycles** |
| --- | --- | --- |
| 95 | 3 min |  |
| 95 | 15 sec | 25-35 |
| 55-72 | 15 sec |
| 72 | 30~60 sec/kb |
| 72 | 5 min |  |

DNA Purification

1. Column equilibrium: Add 500 μL of equilibrium solution BL to the adsorption column CB2, centrifuge at 12000 rpm for 1 min, pour out the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

2. Cut the single band of target DNA from the agarose gel, place it into a clean centrifuge tube, and weigh it.

3. Add moderate volume of solution PC to the glue block and place it in a water bath at 50°C for 10 min, during which the centrifuge tube is gently turned up and down to ensure that the glue block is fully dissolved.

4. Add the solution obtained in the previous step to an adsorption column CB2, centrifuge at 12000 rpm for 1 min, pour out the waste liquid in the collection tube, and put the adsorption column CB2 into the collection tube.

5. Add 600 μL of rinsing solution PW to the adsorption column CB2, centrifuge at 12000 rpm for 1 min, pour out the waste liquid in the collection tube, and put the adsorption column CB2 into the collection tube.

6. Repeat step 5.

7. The adsorption column CB2 is put into the collection tube and centrifuged at 12000 rpm for 2 min. The adsorption column is left at room temperature for several minutes and allowed to dry thoroughly.

8. Put the adsorption column CB2 into a clean centrifuge tube, add an appropriate amount of elution buffer EB to the middle position of the adsorption membrane by hanging drops, place it at room temperature for 2 min, and centrifuge at 12000 rpm for 2 min to collect the DNA solution.

One step cloning

1. Configuration of homologous recombination reaction system.

2. Use a pipettor to gently suck and mix (do not shake and mix violently), and briefly centrifugal the reaction solution to the bottom of the tube.

3. React at 37°C for 30 min, then drop to 4°C or immediately cool on ice.

One Step Cloning reaction system:

| **Component** | **Volume** |
| --- | --- |
| Linearized plasmid | 2 μL |
| The fragment to be inserted | 12 μL |
| 5 × CE II Buffer | 4 μL |
| Exnase II | 2 μL |

Verifying the recombinant plasmid

Agarose gel electrophoresis

1.Place the gel tray in the appropriate position in the gel cartridge and place the comb in the correct position.

2. Measure 0.5 g agarose, put it in a 250 mL Erlenmeyer flask, add 50 mL 1 × TAE buffer and mix, then put the Erlenmeyer flask in the oven and heat to boil until the agarose is completely dissolved.

3. Add Ultra GelRed (10,000 ×) to the solution.

4.Pour the solution into the gel casting tray.

5. After the gel has solidified, pull out the comb.

6. Place the gel in the electrophoresis chamber with enough 1x TAE buffer.

7. Transfer the mixture to the well on the gel with a pipette.

8. Power on, run at 130 V for 20 minutes.

2. Extract the plasmid

According to TIANprep MiNi Plasmid kit:

1. Column equilibrium: Add 500 μL of equilibrium solution BL to the adsorption column CP3, centrifuge at 12000 rpm for 1min, pour out the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

2. Take 1 to 5 mL of the bacterial solution cultivated overnight, add it to the centrifuge tube, centrifuge at 12000 rpm for 1 min, and remove the supernatant as much as possible.

3. Add 250 μL of solution P1 (with added RNaseA) to the centrifuge tube containing the bacterial precipitate to completely suspend the bacterial precipitate.

4. Add 250 μL solution P2 to the centrifuge tube and gently turn it up and down for 6 to 8 times to fully lyse the thallus.

5. Add 350 μL of solution P3 to the centrifuge tube and immediately turn it gently up and down 6 to 8 times, at which point a white flocculent precipitate appears. The samples are centrifuged at 12000 rpm for 10 min.

6. Transfer the supernatant collected in the previous step to the adsorption column CP3 using a pipetting gun. After centrifugation at 12000 rpm for 30~ 60 sec, the waste liquid in the collection tube is poured out and the adsorption column CP3 is placed into the collection tube.

7. Add 600 μL of rinsing solution PW to the adsorption column CP3 (absolute ethanol has been added), centrifuge at 12000rpm for 30~60 sec, pour out the waste liquid in the collection tube, and put the adsorption column CP3 into the collection tube.

8. Repeat steps.

9. The adsorption column CP3 is put into the collection tube and centrifuged at 12000 rpm for 2 min.

10. Place the adsorption column CP3 in a clean centrifuge tube, add 50 to 100 μL of elution buffer EB to the middle part of the adsorption membrane, place it at room temperature for 2 min, and then centrifuge at 12000 rpm for 2 min to collect the plasmid solution into the centrifuge tube.

3.Preparation of competent cells

### Preparation of chemically transformation competent cells

1. Add 1 mL of overnight culture seed medium to a shaker flask containing 100 mL of LB medium and culture in a shaker at 37°C to about OD600=0.5.

2. Centrifugal harvesting, 6000 rpm, 5 min.

3.Add 10 mL of pre-cooled 100 mM CaCl2 for resuspension.

4. Apply cold compress on ice for 20 min.

5. Centrifugal harvesting, 4000 rpm, 10 min.

6. Add 4 mL of pre-cooled 100 mM CaCl2 and resuspend again.

7. Apply cold compress on ice for 20 min.

8. Centrifugation, 4000 rpm, 10 min.

9. Add 1mL of pre-cooled 100 mM CaCl2 and 15% glycerol mixed solution.

10. Aliquot in pre-cooled 1.5 mL centrifuge tubes and store at -80°C.

### Preparation of electrocompetent cells

1. Single colony of plasmid-introduced strain is inoculated into 5mL LB and cultured for 12~14 h as seed medium.

2. Take 1~2 mL of seed medium into a shaker containing 100mL of LB medium and culture until the OD600 value reaches 0.4~0.5.

3. Transfer the solution to a 50mL centrifuge tube and apply cold compress on ice for 20 min.

4.Centrifuge at 4°C, 4000 rpm for 10 min.

5. Resuspend the precipitation by 40 mL of pre-cooled sterile water and centrifuge at 4°C, 4,000 rpm for 10 min.

6. Resuspend the precipitation by another 40 mL of pre-cooled sterile water and centrifuge at 4°C, 4000 rpm for 10 min.

7. Take 20 mL of pre-cooled glycerol with a volume fraction of 10% for resuspension and centrifuge at 4°C, 4000 rpm for 10 min.

8. Finally, resuspend with 300 μL of pre-cooled glycerol with a volume fraction of 10%, aliquot in 1.5 mL centrifuge tubes, and store at -80°C.

4. Transformation

### Chemically transformation

1.Take competent cells from -80°C refrigerator and put it on ice.

2. When the competent cells thaw, add 10 μL DNA ligation product or 2 μL plasmid per tube, place the mixture on ice for 30 min.

3. Heat shock at 42°C for exactly 45 seconds.

4.Put the 1.5 mL tubes back on ice for 3 min.

5. Add 800 μL LB fluid medium without antibiotics into the 1.5 mL tubes and then culture in the shaker incubator at 37°C for an hour.

6. Extract 100-200 μL bacteria liquid, transferred to LB solid medium with antibiotics and spread plates.

7.Place plates upside down and incubate at 37°C overnight.

### Electroformation

1. 100 μL of competent cells and 5μL of each plasmid are mixed and added to a precooled Electric rotor cup and placed on ice for 5 min.

2. Turn on the electrometer and set the program:

| **Condition** | **Numerical value** |
| --- | --- |
| Voltage | 2500 V |
| Capacitance | 25 μF |
| Resistance | 200 Ω |
| Cuvette | 2 mm |

3. Clean the water on the outer wall of the electric rotating cup and conduct electric rotating.

4. After electroporation, 800 μL LB medium is added Immediately, transfer to a 1.5 mL centrifuge tube, and incubate in a shaker at 37°C for 1 h.

5. Centrifuge at 4000 rpm for 3 min, 650 μL of supernatant is aspirated, and the remainder is resuspended on LB solid medium with corresponding antibiotics and incubated for 20 h.

5. Preparation of protein glue

According to SDS-PAGE Gel Rapid Preparation Kit

| **Component** | The volume (mL) of each component required to prepare different volumes of SDS-PAGE separation gels | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
| 8% glue | 5 | 10 | 15 | 20 | 30 | 50 |
| ddH2O | 2.4 | 4.7 | 7.1 | 9.5 | 14.2 | 23.7 |
| 30%Acr-bis (29:1) | 1.3 | 2.7 | 4.0 | 5.3 | 8.0 | 13.3 |
| Lower glue buffer (4X) | 1.25 | 2.5 | 3.75 | 5.0 | 7.5 | 12.5 |
| 10% gel polymerization catalyst | 0.05 | 0.1 | 0.15 | 0.2 | 0.3 | 0.5 |
| TEMED | 0.003 | 0.006 | 0.006 | 0.012 | 0.018 | 0.03 |

6. Protein purification

1. After the bacteria are collected and crushed, the ultrasonic crushing machine has a power of 30%, and the working time is 2 sec with an interval of 3 sec for a total of 10 min.

2. After crushing, the supernatant is centrifuged at 6000 rpm for 10 min to obtain the crude enzyme solution.

3. Add the corresponding proportion of gravity nickel column to the obtained crude enzyme solution, place it on ice, shake it thoroughly at 200 rpm for 20~30 min, so that the target enzyme can combine with the nickel column.

4. Gravity elution: Prepare centrifuge tubes filled with 700 μl Coomassie Brilliant Blue staining solution.The gradient concentration of imidazole (15 ~30 mM) is added to the purification column.The miscellaneous proteins are eluted. The protein elution will be completed until the Coomassie brilliant blue staining solution do not change color after adding the protein eluent to the Coomassie brilliant blue staining solution.

5. Purification: The gravity column is rinsed with a high concentration of imidazole (100~300 mM), and the eluate is collected as the target enzyme.

6. The collected and purified enzyme is transferred to an ultrafiltration tube for desalting and centrifuged at 4500 rpm for 30 min.

7. Add PBS to the ultrafiltration tube, wash, centrifuge at 4500 rpm for 30 min, and repeat twice.

8. Concentrate the pure enzyme to less than 500 μL and collect the enzyme solution.

7. Whole-cell catalysis

The colony was inoculated and cultured in the LB medium at 37℃ and 200 rpm. 0.1 mM of IPTG was added into the culture to induce protein expression when cells grew into an OD600=0.6~0.8. After overnight induction and cultivation, the cells were harvested by centrifugation and resuspended in 50 mM Phosphate Buffered Saline (pH 8.0) containing 300 mM Na2HPO4 and 100 mM NaH2PO4.

React the prepared system at 30℃ and 200 rpm.

System for whole-cell catalytic production of 5-aminovaleramide

| **Component** | **Content** |
| --- | --- |
| Cell concentration | OD600=5 |
| L-Lysine (100 g/L) | 1 mL |
| PBS | to 10 mL |

System for whole-cell catalytic production of 5-aminovalerate

| **Component** | **Content** |
| --- | --- |
| Cell concentration | OD600=5 |
| L-Lysine (100 g/L) | 1 mL |
| PBS | to 10 mL |

System for whole-cell catalytic production of 5-hydroxyvalerate

| **Component** | **Content** |
| --- | --- |
| Cell concentration | OD600=40 |
| 5-aminovalreate (100 g/L) | 250 μL |
| glucose (500 g/L) | 20 μL |
| CaCO3 | 0.1 g |
| α-ketoglutarate (100g/L) | 250 μL |
| PBS | to 5 mL |

System for whole-cell catalytic production of 1,5-pentanediol

| **Component** | **Content** |
| --- | --- |
| Cell concentration | OD600=20 |
| 5-Hydroxyvaleric (100 g/L) | 250 μL |
| glucose (560 g/L) | 90 μL |
| MnSO4·H2O (50 g/L) | 75 μL |
 PBS | to 5 mL |

8. Media and culture methods

The 5 mL seed medium NE of NT1003 contained 10 g/L of (NH4)2SO4 and 5 g/L of DL-monosodium glutamate, 10 g/L Yeast Extract, 0.655 g/L K2HPO4·3H2O, 0.55 g/L C3H3NaO3, 5 g/L Cane sugar, 0.3 g/L Methionine, 0.3 g/L Threonine, 1 g/L MgSO4·7H2O, 0.032 g/L FeSO4·7H2O and 0.077 g/L MnSO4·H2O. We used the final concentration of 100 μg/mL Amp, 50 μg/mL Kan, 25 μg/mL Cm in our experiments. Shaker cultures were performed in 50 mL shaker tubes containing 5 mL NE medium.

The fermentation medium NR of NT1003 contained 10 g/L of (NH4)2SO4 and 5 g/L of Tryptone, 2 g/L Yeast Extract, 0.5 g/L KCl, 1.6 g/L MgSO4·7H2O, 23.125 g/L MoPO5Na,10 g/L CaCO3, 0.086 g/L ZnSO4·7H2O, 0.077 g/L CuSO4, 0.032 g/L MnSO4·H2O, 20 g/L glucose, 0.036 g/L Methionine, 0.036 g/L Threonine, 0.036 g/L VB1, 0.006 g/L NAM and 0.036 mg/L biotin. Shake flask fermentation was carried out in 500mL conical flasks containing 30mL NR medium. Main cultures were inoculated at an initial OD600=0.2 from overnight precultures in NE medium. 1ml of the bacterial solution was added to the shaker. The bacteria were grown at 37°C and 200 rpm. Expression of pathway enzymes was induced by adding 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) when the cells density reached OD600=1.0. At the same time, the shaker temperature was changed to 30°C, and the bacteria were still grown at 200 rpm.

The culture medium of the fermenter was basically the same as the NR medium, except that the initial concentration of glucose was changed to 50 g/L, and other conditions were unchanged. Primary seed cultures were still performed using 50 mL shaker tubes containing 10 mL NE medium. Secondary seed cultures were still grown using 2000 mL conical flasks containing 250 mL. When the cells density reached OD600=2.0, all of them were transferred to the fermenter. The initial cells density in fermenter was OD600=0.2. The initial ventilation volume was 0.6 vvm. The temperature was controlled to 30°C by the instrument. The pH was controlled at 7.0 and regulated with ammonia. The supplementary culture medium contained 400 g/L ammonium sulfate and 600 g/L glucose, and the fermenter was controled the dissolved oxygen level at 20% to 30%. Samples were taken every 3 hours to detect the cells density, glucose content, lysine content and ammonia nitrogen content.

9. Analytical Method

The cells density was detected by the absorbance value at 600 nm by UV spectrophotometer.

SBA was used to detect the content of glucose and lysine. After calibration with 100 g/L glucose and lysine standard solution, the glucose and lysine content of the sample was detected.

Quantification of acetic acid, 5-hydroxyvalerate, 1,5-pentanediol and other organic acids were carried out by using High performance liquid chromatography (HPLC) equipped with an Aminex HPX-87H Column (300\*7.8 mm). HPLC used 8 mM sulfuric acid as the mobile with a flow rate of 0.6 mL/min, and detection via RID.

Amino acid derivatization of the samples was first performed using the GPC method. The samples were quantitatively analyzed by high-performance liquid chromatography (HPLC). The Column was EF-C18 5 μL 120Å 4.6\*250nm, the column temperature was 40℃, the mobile phase was 80% acetonitrile and 108 g/L sodium acetate dissolved in acetonitrile, and gradient washing was used. The flow rate was 1.0 mL/min, and VWD absorption detection was performed at 254 nm.

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{% block lead %}Make a useful contribution for future iGEM teams. Use this page to document that Conmmunication & Cooperation.{% endblock %}
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PART 1

### 1. Construction of a cell factory for the production of 1, 5-pentanediol

There is no natural pathway for 1, 5-pentanediol (1,5-PDO) synthesis in wild-type Escherichia coli. Nowadays, the potential 1,5-PDO biosynthetic pathways have been proposed by other researchers. Here we designed a novel artificial 1,5-PDO biosynthetic pathway and assembled it in *E. coli* to obtain the cell factory for 1,5-PDO bioproduction. In the designed pathway, lysine is used as a director precursor, which is then converted into 5-hydroxyvalerate (5-HV) and 1,5-PDO via two synthetic modules (5-HV synthesis module and 1,5-PDO synthesis module). The 5-HV synthesis module converts lysine into 5-HV via the oxidase, hydrolase, transaminase, and aldehyde reductase, while 1,5-PDO synthesis module converts 5-HV into 1,5-PDO via carboxylate reductase and aldehyde reductase. As some of these enzymes are not present in *E. coli*, we added several heterologous enzymes into the metabolic network model of *E. coli* iML1515, and successfully established a 1,5-PDO metabolic network model (Figure 1). Based on a flux balance analysis (FBA), the maximum theoretical yield of 1,5-PDO from glucose based on our designed pathway was 0.847 mol/molGlucose, further identifying the theoretically feasibility of the artificial 1,5-PDO biosynthetic pathway.

![](https://static.igem.wiki/teams/4800/wiki/main/
results/results-1-1.jpg		)

Figure 1 The establishment of 1,5-PDO metabolic network model in *E. coli*

Next, we choose a lysine-producing strain of *E. coli* NT1003 as our chassis for the construction of 1,5-PDO pathway, which could avoid excessive modification of the front-end genes. In the designed pathway, a total of six enzymes are required for the bioconversion of lysine to the final product 1,5-PDO. The 5-hydroxyvalerate module of L-lysine monooxygenase (DavB) from Pseudomonas putida KT2440, 5-aminopentanamidase (DavA) from Pseudomonas putida KT2440 and 4-aminobutyrate aminotransferase (GabT) from *E. coli* were inserted into the vector pTrc99a (Figure 2), and the 1,5-PDO module of aldehyde reductase (YahK) from *E. coli*, phosphopantetheinyl transferase (sfp) from Bacillus subtilis, carboxylic acid reductase (MmCAR) from Mycobacterium marinum was inserted into the vector pACYCDuet-1 under the control of Ptrc (Figure 2). The two plasmids were co-transferred into *E. coli* NT1003 to assembly the 1,5-PDO biosynthetic pathways, and obtained the engineered strain of NT1003-P1, which was further validated by fermentation in shake flask.

![](https://static.igem.wiki/teams/4800/wiki/main/
results/results-1-2.png	)

Figure 2 The assembly of 1,5-PDO metabolic pathway in *E. coli* NT1003

We cultivated the engineered strain NT1003-P1 in a fermentation medium supplied with 20 g/L glucose. During the whole fermentation, the cell growth and 1,5-PDO production were measured. As shown in Figure 3, after fermentation of 80 h, cells could grow into an OD600 of 10, and 2.82 mM (0.293 g/L) of 1,5-PDO was produced from glucose. The results further indicated the feasibility of our designed pathway experimentally.

![](https://static.igem.wiki/teams/4800/wiki/main/
results/results-1-3.png	)

Figure 3 Production of 1, 5-pentanediol by fermentation with engineered strain NT1003-P1

PART 2

### 2. The optimization of fermentation conditions

For the fermentation by the engineered strain NT1003-P1, we investigated the effect of IPTG addition time on the 1,5-PDO production. We divided the induction period into five periods according to the value of OD600, which are OD600 of 0-1,OD600 of 1-2, OD600 of 3-4, OD600 of 5-6, and OD600 of 7-8 respectively. When the fermentation was end, we analyzed the samples by HPLC. As the results shown in Figure 4, we determined that the production of 1,5-PDO exhibited the highest level when IPTG was added at the growth of OD600 1~2.

![](https://static.igem.wiki/teams/4800/wiki/main/
results/results-2-1.png	)

Figure 4 Comparison of 1,5-PDO fermentation yields at different induction time in strain NT1003-P1

PART 3

### 3. Comparison of catalytic activities of carboxylate reductase, aldehyde reductase and transaminase

In the process of metabolic pathway construction, we also constructed expression plasmids containing carboxylic acid reductase (Figure 5a), aldehyde reductase (Figure 6a) and transaminase (Figure 7a) from different organism sources, aiming to compare their catalytic efficiency and obtain the desired enzyme with higher activity.

By using a whole-cell catalysis process, the enzyme activity of carboxylic acid reductase, and aldehyde reductase was comparatively analyzed by measuring the 5-HV consumption and 1,5-PDO production with HPLC. By comparing the 5-HV consumption, MmCAR and MpCAR displayed the higher substrate consumption activity. The further detection of 1,5-PDO production showed that MmCAR from Mycobacterium marinum resulted in the highest activity for 1,5-PDO production (11.5 mM) (Figure 5b). Therefore, MmCAR was selected as the carboxylate reductase in the following strain construction.

![](https://static.igem.wiki/teams/4800/wiki/main/
results/results-3-1.png	)

Figure 5 (a) The screening of carboxylate reductase for the establishment of functional modules for 1,5-PDO bioproduction. (b) Comparing the activity of carboxylate reductase from different organisms

After identifying the efficient carboxylate reductase for 1,5-PDO synthesis module, we performed the synthesis of 1,5-PDO by a whole-cell catalysis process to compare the activities of the four aldehyde reductases by detecting 1,5-PDO production with HPLC. The results confirmed that YahK from *E. coli* exhibited the highest activity for 1,5-PDO production (Figure 6b). Thus, Yahk was selected to assembly the 1,5-PDO synthesis module in the following strain construction.

![](https://static.igem.wiki/teams/4800/wiki/main/
results/results-3-2.png	)

Figure 6 (a) The screening of aldehyde reductase for the establishment of functional modules for 1,5-PDO bioproduction (b) Comparing the activity of aldehyde reductases from different organisms

For the 5-HV synthesis module, the high activity of DavB and DavA to convert lysine to 5-aminovalerate (5-AVA) has been demonstrated. The identification of the aminotransferase that could catalyze 5-aminovalerate efficiently was critical for this module. Here, we determined the activity of aminotransferases by detecting the 5-HV production from 5-AVA with HPLC. As the results shown in Figure 7b, all six aminotransferases exhibit ammonia-transamination activity towards 5-AVA. Among them, GabT from *E. coli* exhibited the highest ammonia-transamination activity and achieved the highest 5-HV production (Figure 7b). Thus, GabT was selected to assembly the 5-HV synthesis module in the following strain construction.

![](https://static.igem.wiki/teams/4800/wiki/main/
results/results-3-3.png	)

Figure 7 (a) The screening of transaminase for the establishment of functional modules for 1,5-PDO bioproduction (b) Comparing the activity of aminotransferases from different organisms

PART 4

### 4. The predicted gene optimization targets by metabolic network model simulation

After obtaining the functional enzymes, we further assembly the pathway in *E. coli* NT1003, and obtained the engineering strain of NT1003-P1 that could produce 1,5-PDO shown in Figure 3. Based on a metabolic network model simulation by FSEOF algorithm, the overexpression of genes in 1,5-PDO synthetic pathway was predicted to be benefit for increasing 1,5-PDO production (Table 1). During the fermentation, we found that the intermediate of 5-HV was largely accumulated. In conjunction with the model simulation and experimental results, we supposed that the activity of 1,5-PDO synthesis module is not matched with the activity of 5-HV synthesis module. Increasing activity of 1,5-PDO synthesis module should be done. At the meantime, based on a metabolic network model simulation by OptKncok algorithm, several gene deletion targets were also proposed (Table 1).

Table 1 Predicted Gene optimization TARGETS based on the metabolic network simulation

| **Reaction ID** | **Reaction** | **Enzyme or gene** | **Optimization strategy** |
| --- | --- | --- | --- |
| ARND2 | 5-Hydroxypentanal + Nicotinamide adenine dinucleotide phosphate --> 1,5-Pentanediol + H+ + Nicotinamide adenine dinucleotide phosphate - reduced | 1.1.1.2 | Overexpression |
| APENTAMAH2 | 5-Aminopentanamide + H2O + H+ --> 5-Aminopentanoate + Ammonium | 3.5.1.30 | Overexpression |
| CARs | 5-hydroxypentanoate + ATP C10H12N5O13P3 + H+ + Nicotinamide adenine dinucleotide phosphate - reduced --> 5-Hydroxypentanal + AMP C10H12N5O7P + Nicotinamide adenine dinucleotide phosphate + Diphosphate | 1.2.1.30 | Overexpression |
| ASAD | L-Aspartate 4-semialdehyde + Nicotinamide adenine dinucleotide phosphate + Phosphate <=> 4-Phospho-L-aspartate + H+ + Nicotinamide adenine dinucleotide phosphate - reduced | 1.2.1.11 | Knockout |
| ACKr | Acetate +ATP <=>Acetyl phosphate+ ADP | 2.7.2.15, 2.7.2.1 | Knockout |
| PTAr | Acetyl-CoA + Phosphate <=> Acetyl phosphate + Coenzyme A | 2.3.1.8 | Knockout |
| PDH | Coenzyme A + Nicotinamide adenine dinucleotide + Pyruvate --> Acetyl-CoA + CO2 + Nicotinamide adenine dinucleotide - reduced | b0115 and b0116 and b0114 | Knockout |

PART 5

### 5. The optimization of 1,5-PDO cell factory by multiple engineering strategies

Based on experimental result analysis in conjunction with the model simulation guidance as descried above, we employed several engineering strategies to optimize strain performance, including optimizing gene expression by the high copy number plasmid, the engineering limiting enzyme as well as the assembly of limiting enzyme. What was more, the deletion of several genes in the branched pathway was also performed.

5.1 The optimization of gene expression in high copy number plasmid

Due to the low copy number of the pACYCDuet plasmid, we supposed that the expression of MmCAR was insufficient, and thus affected the synthesis of 1,5-PDO. We replaced pACYCDuet in engineered bacteria with a high copy number plasmid of pRSFDuet to express genes in 1,5-PDO synthesis module, and obtained engineered strain NT1003-P2. The yield of 1,5-PDO produced by the engineered strain NT1003-P2 was determined by the HPLC. After a total fermentation period of 84 h, 8.09 mM (0.843 g/L)1,5-PDO was produced by the engineered strain *E. coli* NT1003-P2, which is 3.5-fold higher than in *E. coli* NT1003-P1 (Figure 8), indicating the optimization of gene expression by changing the high copy number plasmid was benefit for 1,5-PDO production in our work. This result also confirmed that the optimization of strain in conjunction with the metabolic network model simulation is an efficient approach.

![](https://static.igem.wiki/teams/4800/wiki/main/
results/results-5-1-1.png	)

Figure 8 Comparison of fermentation yields between NT1003-P1 and NT1003-P2

5.2 The deletion of genes in branched pathways

By using the OptKncok algorithm, multiple bypass genes that might affect the synthesis of 1,5-PDO were predicted (Table 1). In combination with the knockout results without affecting the normal growth of *E. coli* and the occurrence of the exchange reaction, we decided to knock out the two genes of ackA and pta, which encode the enzymes (acetate kinase and phosphate acetyltransferase) that convert pyruvate to acetate, the main by-product for *E. coli* fermentation. In addition, gene involving in 1,5-PDO degradation was also evaluated.

The results in Figure 9 exhibited the effect of deleting genes of ackA and pta on the final 1,5-PDO production. In comparison to engineered strain NT1003-P2, the knockout of ackA-pta increased the 1,5-PDO titer by 16%, and the titer in strain NT1003-P2-Δacka-pta reached 9.3 mM (0.97 g/L) (Figure 9), indicating the positive effect on 1,5-PDO production by deleting branched pathway of acetic acid synthesis. This result is also another success of strain optimization guided by the metabolic network model simulation.

![](https://static.igem.wiki/teams/4800/wiki/main/
results/results-5-2-1.png	)

Figure 9 Comparison of production between NT1003-P2 and NT1003-P2-ΔackAa-pta.

During the fermentation, we also evaluated the 1,5-PDO degradation ability of *E. coli*. Within 24 h, *E. coli* NT1003-P1 could consume (100.77 mM) 10.5 g/L 1,5-PDO (Figure 10a). From the previous study in the lab, the gene of YcjQ was identified to be responsible for 1,5-PDO degradation. The deletion of YcjQ significantly decreased the 1,5-PDO degradation (Figure 10a). Subsequently, we evaluated the 1,5-PDO production in YcjQ-deleting strain. We found that the deletion of YcjQ gene markedly improved the final 1,5-PDO titer, 1.75-fold higher than the strain NT1003-P2 (Figure 10-b). These results demonstrates that NT1003-P2-ΔYcjQ performed better, and we decided to adopt this strain in the following experiments.

![](https://static.igem.wiki/teams/4800/wiki/main/
results/results-5-2-2.png	)

Figure 10 (a) Comparison of residual 1,5-PDO catalyzed by NT1003-P1 and NT1003-P1-∆YcjQ. (b) Comparison of fermentation yields between NT1003-P2 and NT1003-P2-∆YcjQ

5.3 The rational engineering MmCAR

In the 1,5-PDO module, MmCAR was a critical limited enzyme. It has been reported that MmCAR has low catalytic efficiency for medium and long chain carboxylic acids. To improve the activity of MmCAR, we employed a rational engineering strategy to design the mutant MmCAR with higher activity. In combination with protein structure simulation, and molecular autodock, we focused on the amino site of Q302, and mutant it to E302 to change the polarity of the residues. After obtaining the mutated MmCARQ302E, we replaced the original MmCAR in the engineering bacteria, obtained NT1003-P3-∆YcjQ. As the results shown in Figure 11, the mutation of MmCARQ302E could moderately increase the final 1,5-PDO production, and 14 mM (1.45g/L) of 1,5-PDO in the engineered *E. coli* NT1003-P3-ΔYcjQ was finally obtained from glucose (Figure 11).

![](https://static.igem.wiki/teams/4800/wiki/main/
results/results-5-3-1.png	)

Figure 11 Comparison of fermentation yields between NT1003-P2-∆YcjQ and NT1003-P3-∆YcjQ

5.4 The assembly of enzyme by EutM scaffold protein

In the reported study, enzyme assembly based on protein scaffolds has been widely used to enhance the catalytic efficiency of enzymes. After reviewing the literature and discussing within the group, we focused on the EutM from Salmonella enterica. EutM is known to self-assemble as filament-like structure in vivo, and has been developed as a powerful protein scaffolding system to rapidly co-immobilize enzymes for improving the biocatalysis efficiency. We added a peptide of SpyTag on the N-terminus of MmCARQ302E, and also ligated a gene encoding EutM-SpyCatcher in the plasmid for enzyme assembly in vivo . The engineered strain containing the assembled enzymes was named as NT1003-P4-ΔYcjQ. As shown in Figure 12, the 1,5-PDO production of the engineered strain NT1003-P4-ΔYcjQ was determined by the HPLC analysis. After fermentation of 96 h, the assembly of MmCAR could increase 1,5-PDO production by 60%, and 23 mM (2.4 g/L) of 1,5-PDO was finally obtained in the engineered *E. coli* NT1003-P4-ΔYcjQ .

![](https://static.igem.wiki/teams/4800/wiki/main/
results/results-5-4-1.png	)

Figure 12 Comparison of fermentation yields between NT1003-P3-∆YcjQ and NT1003-P4-∆YcjQ

Reference

[1].Xuecong Cen. et al. Metabolic Engineering of Escherichia coli for De Novo Production of 1,5-Pentanediol from Glucose. ACS Synthetic Biology 2021 10 (1), 192-203

[2].Cheng, J. et al. Efficient whole-cell catalysis for 5-aminovalerate production from L-lysine by using engineered Escherichia coli with ethanol pretreatment. Sci Rep 10, 990 (2020)

[3].Chen Chen. et al. Metabolic engineering of Escherichia coli for the efficient production of 5-hydroxyvaleric acid, Process Biochemistry, Volume 130, Pages 625-633 (2023).

[4].Xuecong Cen. et al. Metabolic engineering of Escherichia coli for high production of 1,5-pentanediol via a cadaverine-derived pathway. Metabolic Engineering, Volume 74, Pages 168-177 (2022).

[5].Xin Wang. Et al. Efficient production of 5-aminovalerate from l-lysine by engineered Escherichia coli whole-cell biocatalysts. Journal of Molecular Catalysis B: Enzymatic, Volume 134, Part A, Pages 115-121 (2016)

[6].Tayyab Islam. et al. Metabolic engineering of Escherichia coli for biological production of 1, 3-Butanediol, Bioresource Technology, Volume 376, 128911 (2023)

[7].Zhang, G. et al. Self-Assembling Protein Scaffold System for Easy in Vitro Coimmobilization of Biocatalytic Cascade Enzymes. ACS Catalysis, 8(6), 5611-5620.

[8].Guoqiang Zhang. et al. Chapter Thirteen-Protein-based scaffolds for enzyme immobilization. Claudia Schmidt-Dannert, Maureen B. Quin, Methods in Enzymology, Academic Press, Volume 617, Pages 323-362 (2019)

[9].Xiao-Bao Sun. et al. SpyTag/SpyCatcher molecular cyclization confers protein stability and resilience to aggregation, New Biotechnology, Volume 49, Pages 28-36 (2019)

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