iGEM 2023 — Team hubu-china

# Notebook

## Overview

### 2022.12

#### Set up the 2023-HUBU-China team.

### 2022.12-2023.2

#### Experimental skills training and literature reading training.

### 2023.2-2023.4

#### Brainstorming.

#### Project discussion and literature research were carried out in groups.

#### The projects of each group are summarized and finally the topics to be explored by the team are determined.

### 2023.3.4-6

#### Summarizing the reading reports of the experimental group members, writing the first draft of the project report.

#### Designing the initial version of the technical roadmap, participating in adjustments to the presentation slides.

#### Conducting the first offline presentation.

### 2023.4.22

#### Introducing the experimental technology portion and answering some questions during the online exchange with four schools.

#### Some preliminary experiments have begun.

### 2023.5

#### The preliminary design of the complete project system and a large number of background research and literature research were completed.

#### The first new parts were designed and built for the project.

#### The experiment continued.

### 2023.6-2023.9

#### The second batch of new elements was constructed following suggestions from interviews with professionals and professors.

#### Feedback from experiments and [human practice](https://2023.igem.wiki/hubu-china/human-practices) is used to continuously improve our project (click here to jump to the Experiments and Human practice section).

#### The results of the vast majority of wet versus dry experiments covered by our project were obtained.

#### Complete the video and poster production and participate in the second synthetic biology competition.

#### Write the first draft of the wiki.

### 2023.10

#### Complete the final wiki version and upload it.

#### Complete the poster production.

#### All experiments were completed.

#### 2023 iGEM Giant Jamboree!

## Teamlog

## Brainstorming and meeting

## Experiment

## Isobutanol production: Group 1 and Group 2

## CO2 fixation: Group 2

# Engineering Success

## Overview

### In our project, we built an engineered Z.mobilis expression system for the production of isobutanol.In order to make them better perform the expected functions, we followed the DBTL principle to design and improve our engineering project.     We have designed Isobutanol Biosynthesis System and CO2 Concentration and Fixation System during the project. According to the test results,We added andreplaced the promoter of A4 plasmid to achieve update iteration, and at the same time added one copy of isobutanol production gene kdcA in the genome to achieve double copy to further improve isobutanol production. We selected the most common and deeply studied Calvin cycle pathway, and to improve carbon fixation efficiency, we decided to construct carboxysome in the strain for CO2 concentration. Therefore, this system is completed from two aspects.so that they finally achieved our expected results.

Figure 1 Engineering cycle ( DBTL )

### Learn about reagents and enzymes and other materials and methods can jump [Protocol](https://2023.igem.wiki/hubu-china/protocol) Protocol further browse.

## System Ⅰ Isobutanol Biosynthesis System

### Strains ZMQ1-ZMQ8 and plasmids A1-A4 were obtained from Zymomonas mobilis by introducing isobutanol synthesis pathway, inhibiting by-product synthesis, replacing promoter and a series of engineering methods.     On this basis, we follow the DBTL principle to learn and optimize, and improve the production process of isobutanol.     We have designed Isobutanol Biosynthesis System and CO2 Concentration and Fixation System during the project. According to the test results,We added andreplaced the promoter of A4 plasmid to achieve update iteration, and at the same time added one copy of isobutanol production gene kdcA in the genome to achieve double copy to further improve isobutanol production. We selected the most common and deeply studied Calvin cycle pathway, and to improve carbon fixation efficiency, we decided to construct carboxysome in the strain for CO2 concentration. Therefore, this system is completed from two aspects.so that they finally achieved our expected results.

Figure.2 A metabolic pathway constructed in Zymomonas mobilis

### Search for keto acid decarboxylase gene kdcA which is from LactococcusLactis,L.lactic.[(https://www.uniprot.org/uniprot/Q6QBS4)](https://www.uniprot.org/uniprot/Q6QBS4).     This gene was optimized and synthesized according to the codon preference of Zymomonas mobilis, and it was constructed into motomonas, completing the isobutanol synthesis pathway of motomonas.

Figure.3 Isobutanol synthesis pathway in Z.mobilis

## Genome Double Copy

### he inducible expression of kdcA gene was inserted into the genome of Zymomonas mobilis by homologous recombination to improve the stability of gene expression. At the same time, the kdcA gene was changed into a constitutive strong expression to improve the transcription level of kdcA gene. The effects of two constitutive strong promoters, Pgap and Peno, on isobutanol production were compared.To construct a stable isobutanol production strain, the codon-optimized kdcA gene driven by Ptet was integrated into the genome at either the chromosomal locus of ZMO1650 finally.     We designed a double copy substitution of the kdcA gene in the genome of the strain. By reviewing the strain sequence, we found that the 1650 and 1547 loci of the strain were non essential genes, and the substitution of these loci would not have a significant impact on the growth of the strain. Therefore, these two loci were edited as replacement loci.

### Firstly, construct the editing plasmids at the ZMO1650 and ZMO1547 loci.Synthesize the kdcA gene, then amplify the fragment and amplify the vector. Then, T5 exonuclease assisted cloning the vector and fragment, and transform the T5 system into Escherichia coli receptive cells; Complete editing plasmid construction     We detected the correct editing plasmid through colony PCR, then extracted the plasmid and electroporated it into the receptive cells of Actinomycetes to complete the final transformation. Afterwards, we also used monoclonal PCR to detect the recombinant strain and ultimately obtained our target strain 1650-kdcA, a single copy bacterium named ZMQ9. We then cultured and preserved the bacteria, completing the construction of the first copy of kdcA. We will make a single copy bacterium into a receptive state and use the same method to replace the second copy. The resulting double copy ZMO1650 and ZMO1547 were named ZMQ11.

### Comparison of isobutanol production between single copy and double copy strains through shaking table fermentation testing.     Fermentative strains: ZMQ9/ZMQ11     Under the same concentration of tetracycline induction, the double copy strain had higher isobutanol production, and the same strain had significant differences in isobutanol production under different concentrations of tetracycline induction.

Figure.4 Comparison of isobutanol production between single copy and double copy strains

### Because the isobutanol produced by Z.mobilis fermentation induced by different tetracycline concentrations varies greatly, it is necessary to be very careful and avoid errors in fermentation as much as possible，At the same time, consider reducing the gradient difference of tetracycline and reducing errors. It is believed that the yield of this strain is not high enough and can only be induced by tetracycline to produce isobutanol, which is too limited. We plan to improve the components to increase production

## A4 Plasmid Promoter Iteration Update

### We have constructed Plasmids A1-A4, but in actual experiments, the yield of the transformed strain is not high. Therefore, we have made modifications and optimizations on A4, mainly by making some changes to the promoter. We construct A5 plasmid with another conservative strong promoter Peno inserted to enhance the expression of the last two genes ilvC and ilvD.

### Afterwards, we identified the Ppdc and PBAD combined strong promoters, amplified the pyruvate decarboxylase gene promoter (Ppdc) from Zymomonas mobilis CP4. pBAD vector system is a reliable and controllable system for expressing recombinant proteins in bacteria, which controls the metabolism of L-arabinose in Escherichia coli through the araBAD operon. Relevant literature suggests that the expression of these promoters can increase yield.

### We obtained the promoter Peno, Ppdc, and PBAD templates and amplified the promoter protein fragment.Then we reverse expanded the vector and constructed the plasmid.Transformed them, finally verify with monoclonal PCR that we have successfully constructed the A5, A6, and A7 plasmids.     To increase the isobutanol production, two composite parts BBa\_K4666031 and BBa\_K4666032 were constructed improving upon BBa\_K2800020 with another constitutive strong promoter Peno or Pgap inserted to enhance the expression of the last two genes ilvC and ilvD . The new parts were cloned into the shuttle vector pEZ15Asp saparetely generating the plasmid A5 and A8, and then transformed into the recombinant ZMQ to generate the recombinant strains ZMQ-A5 and ZMQ-A8, respectively.

Figure.5 Features of the new composite parts of BBa\_K4666031 and the BBa\_K4666032.

### The results indicated that the improved composite part with another constitutive promoter inserted to drive the ilvC-ilvD genes was a practical strategy to enhance the isobutanol production in Z. mobilis, and especially, the promoter Peno (Peno-ilvC-ilvD ) was more effective than the promoter Pgap (Pgap-ilvC-ilvD ).     A5, A6, and A7 were transformed into ZMQ11 for cultivation, and the transformed strains were named ZMQ11-A5, ZMQ11-A6, and ZMQ11-A7. Tetracycline was added to induce fermentation, and their isobutanol production was tested. The results showed that ZMQ11-A6 and ZMQ11-A7 could express higher levels of isobutanol at lower tetracycline levels. Finally,ZMQ11-A6 has the highest production capacity.

Figure.6 Cell growth and isobutanol production of ZMQ11-A5,ZMQ11-A5 and ZMQ11-A5 with different tetracycline. concentrations as an inducer.

### From the above results, it can be seen that the yield of isobutanol in the optimized strain plasmid after transformation has significantly increased, indicating that it is necessary to replace the promoter, and the strain that replaces the strong combination promoter can undergo fermentation without too high tetracycline induction.

## Silencing of the pdc gene by using the CRISPRi system

### The clustered regularly interspaced short palindromic repeats (CRISPR)/dCas9 system has been widely applied in both transcriptional regulation and epigenetic studies. However, for multiple targets, independent expression of multiple single guide RNAs (sgRNAs) is needed, which is less convenient. To address the problem, we employed a DNase-dead Cpf1 mutant (ddCpf1) for multiplex gene regulation. We inserted the dcpf1 gene at the ZMO0038 site of the genome and transformed the pEZ39 plasmid at the same time of the replacement promoter optimization of the A6 plasmid. ddCpf1 can effectively repress both transcription elongation and initiation of target gene pdc.

### We designed primers and constructed recombinant strains.We used the PCR to amplify the dcpf1 gene,the spectinomycin resistance gene,the tetracycline-inducible promoter Ptet gene,the sequences of upsteam and downstream of the insertion site( the gRNA from Zymomonas mobilis) and the reverse-amplified of pUC57 vector sequence.The fragments are linked by overlap .The fagment and the vector are linked by T5 exonuclease assisted cloning.Transfer the recombinant plasmid into Zymomonas mobilis.The dcpf1 gene is integrated into the Zymomonas mobilis genomes of ZMO0038 by homologous recombination.

### The results of two repetitions showed that the isobutanol yield increased to about 6g/L after inhibiting the ethanol pathway.

Figure.7 Cell growth and isobutanol production of ZMQ11-A6-D1with different tetracycline. concentrations as an inducer.

### From the perspective of gene silencing effect, CRISPRi system can effectively inhibit pdc gene. However, A5 plasmid and dcpf1 gene are both driven by the tetracycline-inducible promoter Ptet, so A6 plasmid replaces Ptet promoter with Ppdc to improve stability.

## System Ⅱ CO2 Concentration and Fixation System

### In the metabolic pathway diagram, red arrows indicate enhanced isobutanol synthesis, gray arrows indicate inhibition of ethanol production, and blue indicates enhanced carbon fixation.An explanation of the illustrated enzyme is shown below.

Figure.8 A metabolic pathway constructed in Zymomonas mobilis.

## Construction of Calvin Benson Bassham (CCB) pathway

### There are six CO₂ fixation pathways in nature, which are Calvin cycle, 3-hydroxypropionic acid double cycle, reduced acetyl-CoA cycle, reducing tricarboxylic acid cycle, 4-hydroxypropionic acid cycle and 3-hydroxypropionic acid or 4-hydroxybutyric acid cycle. The Calvin cycle is the most important way of carbon sequestration in nature. We choose to use the Calvin cycle to achieve the conversion of xylose to ethanol.

Figure.9 Calvin Cycle.

### To construct the Calvin cycle, we followed the following steps: amplifying the fragment, constructing the plasmid.First amplifying the ribose-1,5-diphosphate carboxylase/oxygenase (Rubisco) gene rbc\_tb，and construct it on pEZ-Peno-rbc-Ptet-prk for the first conversion, then rbc \_tb and zphophoribokinase gene prkA were constructed onto plasmid pEZ-Ptet-prk and transformed into ZM8b. We verified their successful assembly by agarose gel electrophoresis and obtained two strains Experimental strain ZM8b（pEZ-Peno-rbc-Ptet-prk）and Intermediate ZM8b（pEZ-Ptet-prk）.

Figure.10 Coupling 5-carbon glucose metabolism and Calvin cycle

### We conducted fermentation tests on two different bacteria to detect their growth and metabolism. The results showed that:The above fermentation analysis showed that direct overexpression of rbc and prkA genes ZM8b（pEZ-Peno-rbc-Ptet-prk）would significantly inhibit the strain (nearly no bacterial growth). The effect of ZM8b（pEZ-Ptet-prk）is slightly better.

### We have reviewed relevant literature and learned that some organisms undergoing the Calvin cycle have CO2 concentration mechanisms. The rate - limiting enzyme of the Calvin cycle ，ribosugar-1,5-diphosphate carb oxylase/oxygenase (Rubisco) has bifunctional enzyme activity ，which can catalyze both carboxylation and oxygenation.Due to Rubisco being an inefficient enzyme with slow catalytic speed and poor ability to distinguish between competing substrates CO2 and O2, many Calvin cycle organisms require other auxiliary links to help improve carbon dioxide fixation efficiency. And our Z.mobilis8b also draws on learning and constructs its own CO2 concentration mechanism – carboxysome.Introduces carboxylation to assist carbon fixation, so as to solve the previous problems. In order to help improve its carbon sequestration efficiency.

## Construction of Carboxysome Shell Protein

### At the same time, the fusiform body is used to concentrate carbon dioxide. The fusiform body can integrate the CO₂ or bicarbonate ion on BuBP to convert it into two triphosphoglyceric acids, and improve the CO₂ fixation efficiency.

Figure.11 pUZ-Shell Plasmid Map (Fusion expression)

Figure.12 Carboxysome Structure

### Through consulting materials, we learned about the carboxysome of blue-green algae (Synechococus elongatus PCC 7942) and chose to introduce carboxysome genes from blue-green algae in order to improve carbon sequestration efficiency In NCBI[（ https://www.ncbi.nlm.nih.gov/ ）](https://www.ncbi.nlm.nih.gov/).In the database search for carboxyl matrix related gene sequences, carboxyl matrix protein related genes ccmK1, ccmK2, ccmO, ccmL, ccmM, and ccmN were obtained from Halotec sp. PCC 7418, and the carbonic anhydrase gene sequence was synthesized from Synechococus elongatus PCC 7942.     Transferred plasmids pUZ-Shell\_PrkA and pEZ39p\_Core into ZM8b to form a complete fusiosomal Calvin cycle strain. The plasmid pUZ-Shell\_PrkA included ccmK1, ccmK2, ccmO, ccmL, and mNeonGreen, a gene of carbonicanhydrase. Plasmid 39p-core contains carboxylsome connexin gene ccmM, recruitment protein gene ccmN and ribulose 1, 5-diphosphate carboxylase/oxygenase (Rubisco) gene rbc\_tb. At the same time, ZM8b (pEZ-gfp) was constructed as a control.pEZ-gfp was an empty plasmid with fluorescent protein gene. Flow cytometry was used to detect the successful transfer of the plasmid, the fluorescent protein was observed by microscope, and the fusifier was successfully constructed by transmission electron microscopy after purification.

### Overexpression of carboxylates at the same time (8b-Complete) could collect and concentrate the toxic intermediate metabolites in the carbon sequestration reaction and effectively alleviate the inhibition，and carbon fixation pathways can increase ethanol production to a certain extent.

Figure.13 Results of growth and metabolism analysis of carboxylate strain

### The 8b fermentation test only focuses on growth metabolism and ethanol production analysis. Our construction goal is to reduce emissions by utilizing CO2, but carbon utilization is difficult to monitor, which is a major challenge and an important part that needs improvement. To truly prove the improvement of carbon sequestration, some testing should be conducted in the field of carbon sequestration.In the future, the project can use non-grain raw materials to convert sugar, and then use Z.mobilis8b to produce isobutanol with zero carbon emission.

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# Result

### This year, we carried out experiments to optimize the isobutanol production in Zymomonas mobilis. Our results were divided into two parts: the first part was the isobutanol production pathway engineering, including the optimization of gene expression and the interference of ethanol production through CRISPRi system; the second part was associated with the carbon concentrating and fixation, including a synthetic carboxysome assembly and the Calvin cycle pathway transformation. The details of our results were displayed as follows.

## Part Ⅰ Isobutanol Biosynthesis

### Overview

### As shown in the Part Improvement, the kdcA gene inserted in the genome of Z. mobilis was better than in the plasmid, and the over-expression of the optimized synthetic operon Bsals2-ilvC-ilvD (A5 plasmid) was necessary to divert carbon flux to isobutanol production. In order to further increase the isobutanol production in Z. mobilis, we decided to increase the gene copy number of kdcA in the genome, as well as to further optimize the expression of the operon Bsals2-ilvC-ilvD. In addition, considering that the ethanol metabolism pathway can divert carbon flux, another strategy to delete or suppress the ethanol pathway is needed to redirect carbon flux from ethanol to isobutanol biosynthesis. Therefore, the dCpf1 assisted CRISPRi system was employed to inhibit the pdc expression, which is the key gene involved in the ethanol production. All the plasmids and recombinant strains constructed in this part were shown in Table 1-1 and Table 1-2.

Table 1-1 The plasmids constructed for the isobutanol biosynthesis

| Plasmids | Description |

| --- | --- |
| *A5* | pEZ15A-Ptet-Bsals2-Peno-ilvC-ilvD |
| *A6* | pEZ15A-Ppdc-Bsals2-Peno-ilvC-ilvD |
| *A7* | pEZ15A-PBAD-Bsals2-Peno-ilvC-ilvD |
| *D1* | pEZ39P-cpf1-gRNA-pdc |

Table 1-2 The strains constructed for the isobutanol biosynthesis

| Strains | Description |

| --- | --- |
| *ZMQ* | 0038::Ptet-dcpf1 |
| *ZMQ9* | 0038:Ptet-dcpf1,1650:Pgap-kdcA |
| *ZMQ11* | 0038:Ptet-dcpf1.1650:Pgap-kdcA,1547:Pgap-kdcA |
| *ZMQ9-A5* | ZMQ9 containing plasmid A5 |
| *ZMQ11-A5* | ZMQ11 containing plasmid A5 |
| *ZMQ11-A6* | ZMQ11 containing plasmid A6 |
 *ZMQ11-A7* | ZMQ11 containing plasmid A7 || *ZMQ11-A6-D1 ZMQ11* | ZMQ11 containing plasmid A6 and D1 |

### Results 1—Two copies of kdcA integrated in the genome

### The dcpf1 gene driven by tetracycline-inducible promoter Ptet was firstly integrated into the genome at the chromosomal locus of ZMO0038, and the recombinant strain ZMQ was used as the parent strain for subsequent isobutanol pathway engineering. With the codon-optimized kdcA gene driven by Pgap integrated into ZMO1650 and ZMO1547, two new recombinant strains ZMQ9 and ZMQ11 were obtained. Then the optimized A5 plasmid was transferred saperately in the two recombinants, resulting in the strain ZMQ9-A5 and ZMQ11-A5.

Fig.1-1 Cell growth and isobutanol production of ZMQ9-A5 and ZMQ11-A5 with different tetracycline concentrations.

### Fig.1-1 displayed the cell growth and isobutanol production of ZMQ9-A5 and ZMQ11-A5 with different concentrations of tetracycline. Under lower tetracycline induction, the isobutanol production of ZMQ9-A5 reached a maximum of 0.58 g/L, and the ZMQ11-A5 reached a maximum of 0.93 g/L. Under higher tetracycline induction, the isobutanol production of ZMQ9-A5 reached a maximum of 3.45 g/L. The results indicated that the double-copy kdcA in Z. mobilis was superior to single-copy kdcA. However, the A5 plasmid contained a tetracycline-inducible promoter Ptet, and the dcpf1 was driven by Ptet as well, which may mutually influence each other due to the tetracycline concentrations. Higher concentration of tetracycline was required for the higher isobutanol production.

### Results 2—Promoter Replacement

### To avoid the mutual influence of two Ptet promoters in ZMQ11-A5, the Ptet promoter in the A5 plasmid was replaced with constitutive promoter Ppdc or PBAD, constructing the plasmid A6 and A7. After transferred into ZMQ11, the recombinant strain ZMQ11-A6 and ZMQ11-A7 were obtained. The isobutanol production of ZMQ11-A6 and ZMQ11-A7 were evaluated with different concentrations of tetracycline and arabinose (Figs.1-2).

Fig.1-2 Cell growth and isobutanol production of ZMQ11-A6 and ZMQ11-A7.

### As expected, the isobutanol production in ZMQ11-A6 and ZMQ11-A7 were both signifcantly higher than in the ZMQ11-A5 with low concerntation of inducers induction. The maximum isobutanol titer in ZMQ11-A6 reached 4.12 g/L without tetracycline induction, and it in ZMQ11-A7 reached 2.58 g/L with 5 mg/mL arabinose induction. These results indicated that the isobutanol production increased without tetracycline competition. The isobutanol production in ZMQ11-A6 was higher than in ZMQ11-A7, suggesting that Ppdc promoter was more efficient than the PBAD promoter in isobutanol production. The ZMQ11-A6 with the highest isobutanol production was selected for pdc interference through CRISPRi strategy.

### Results 3—dCpf1 assisted CRISPRi system to inhibit the pdc expression

### The plasmid D1 containing gRNA targeting pdc was transferred into the recombinant strain ZMQ11-A6, and a new strain ZMQ11-A6-D1 was obtained. The isobutanol production in ZMQ11-A6-D1 was investigated with different tetracycline concentrations, the results were shown in Fig.1-3.

Fig.1-3 Isobutanol production and growth curve of ZMQ11-A6-D1 at different tetracycline concentrations

### Two repetitive experiments were performed. In the first experiment, the highest isobutanol production reached 5.7 g/L, and it was 5.58 g/L in the second experiment. The isobutanol production in ZMQ11-A6-D1 was higher than in ZMQ11-A6, which indicated that the pdc was silenced, and more carbon flux was red redirect iverted from ethanol production to isobutanol biosynthesis.

## Part Ⅱ CO2 Concentrating and Fixation

### Overview

### Considering the issue of carbon dioxide emissions in the isobutanol production pathway, our project also designed to introduce the heterologous Calvin cycle pathway coupled with a carboxysome for CO2 concentrating and CO2 fixation, thus, the CO2 released during the isobutanol fermentation can be absorbed and further transformed into isobutanol again, achieved an efficient zero-carbon isobutanol generation. All the plasmids and recombinant strains constructed in this part were shown in Table 2-1 and Table 2-2.

Table 2-1 The plasmids constructed for the CO2 concentrating and fixation

| Plasmids | Description |

| --- | --- |
| *pEZ-PrkA* | prkA from Synechococcus elongatus PCC 7942 driven by Ptet |
| *pEZ-Rbc-PrkA* | prkA from Synechococcus elongatus PCC 7942 driven by Ptet and rbc ( encoding Rubisco) from Thiobacillus denitrificans driven by Peno |
| *pEZ-GFP* | gene encoding mNeonGreen driven by Ptet |
| *pEZ-Shell* | genes encoding CcmK1, CcmK2, CcmO, CcmL from Synechococcus elongatus PCC 7942, and mNeonGreen driven by Ptet |
| *pEZ-Shell-PrkA* | genes encoding CcmK1, CcmK2, CcmO, CcmL from Synechococcus elongatus PCC 7942, and mNeonGreen, CA, PrkA driven by Ptet |
| *pEZ39p-Core* | genes encoding CcmM, CcmN from Synechococcus elongatus PCC 7942, and Rubisco driven by Ptet |

Table 2-2 The strains constructed for the CO2 concentrating and fixation

| Strains | Description |

| --- | --- |
| *8b-PrkA* | 8b derivative containing plasmid pEZ-PrkA |
| *8b-Rbc-PrkA* | 8b derivative containing plasmid pEZ-Rbc-PrkA |
| *8b-GFP* | 8b derivative containing plasmid pEZ-GFP |
| *8b-Shell* | 8b derivative containing plasmid pEZ-Shell |
| *8b-Shell-PrkA* | 8b derivative containing plasmid pEZ-Shell-PrkA |
| *8b-Complete* | 8b derivative containing plasmid pEZ-Shell-PrkA and pEZ39p-Core |

### Results 1—Carboxyl body characterization

### Carboxysome shell associated genes ccmK1, ccmK2, ccmO, and ccmL, as well as a fluorescent protein gene mNeonGreen fused to ccmK1 were cloned into the shuttle vector pEZ15Asp generating the plasmid pEZ-Shell. The shell protein CcmK1 fused to mGFP (mNeonGreen enconding protein) was used to evaluate the assembly of carboxysomal proteins in Z. mobilis. After transferred into Z. mobilis strain 8b, the xylose-engineered mutant strain, a new recombinant strain 8b-Shell was constructed. The fluorescence of the cell was detected by flow cytometry with different concentrations of tetracycline induction (0, 0.2, 0.8 µg/mL).      Fig.2-1A displayed that no fluorescence was detected in parental strain as a negative control. Fig.2-1B showed the fluorescence detection in 8b-GFP as a positive control. The fluorescence was high even with no tetracycline induction. Fig.2-1C showed the fluorescence in 8b-Shell expressing complete shell components. The fluorescence was significantly improved with the increase of tetracycline concentrations. The results in Fig.2-1 suggested that the recombinant carboxysome shell plasmid was successfully transformed in Z. mobilis.

Fig.2-1 Fluorescence intensity of 8b, 8b-GFP and 8b-Shell.

### Results 2—Intracellular localization of carboxysome

Fig.2-2 Fluorescence image of 8b-Complete

### 8b-Complete was further constructed to express the complete carboxysome as well as the Calvin cycle pathway with both pEZ-Shell-PrkA and pEZ39p-Core transformed in Z. mobilis. Expression of carboxysome components in 8b-Complete was induced by tetracycline. Through the fluorescence detection by fluorescence microscope, it was confirmed that the carboxysome was successfully assembled in the cytoplasm in Z. mobilis, since the fluorescence intensity in 8b-Complete was discerned as green puncta as shown in Fig.2-2.

### Results 3—Carboxysome structure characterization by TEM microscope

Fig.2-3 Thin-section TEM images of intracellular carboxysome in 8b, 8b-Shell and 8b-Complete.

### The formation of carboxysome structure was further verified by thin-section transmission electron microscopy (Fig.2-3). As expected, the carboxysome-like structures (∼400 nm in diameter) with a high internal protein density were observed in both recombinant 8b-Shell and 8b-Complete cells but are invisible in the parental 8b cells without carboxysome-expressing vectors.

Fig.2-4 Thin-section TEM images of extracellular carboxysomes isolated from 8b-Shell and 8b-Complete.

### The recombinant carboxysome was then purified by sucrose density gradient centrifugation, following the induction of tetracycline. The purified carboxysome structure was verified by thin-section transmission electron microscopy as well. The images in Fig.2-4 showed that both the purified carboxysomes from 8b-Shell and 8b-Complete were 50 nm in diameter. However, they did not exhibit manifestly a regular icosahedral shape and symmetry. All these results suggested that these carboxysome proteins were densely packed in the carboxysome-like structures in Z. mobilis.

### Results 4—Fermentation analysis of 8b-Complete

Fig.2-5 Cell growth, sugar utilization, and ethanol production of Z. mobilis recombinant strains.

### The capability of carbon fixation by recombinant strain 8b-Complete was evaluated under aerobic and anaerobic conditions, compared with 8b-PrkA and 8b-Rbc-PrkA with the Calvin Cycle pathway inserted but without carboxysome expression. As shown in Fig.2-5, the single expression of rbc and prkA (8b-Rbc-PrkA) significantly inhibited the cell growth of Z. mobilis under aerobic and anaerobic conditions (almost no bacterial growth), while the co-overexpression of carboxysome and Calvin Cycle pathway (8b-Complete) dramatically eliminated the inhibition. The result suggested that the Calvin Cycle pathway, essentially the toxic intermediate in the process of carbon fixation was encaptured in carboxysome. In addition, the ethanol production detection showed that higher ethanol titer was obtained by 8b-Complete compared with the control 8b-GFP, indicating that a certain amount of CO2 may be fixed for ethanol production in 8b-Complete.

## Summarize：

### So far, we have successfully increased the isobutanol production in Z. mobilis to 5.7 g/L, and we have demonstrated the successful assembly of synthetic carboxysome in the cell, which can be used for carbon dioxide fixation. We would further integrate the two parts above into a single strain, to achieve the zero-carbon isobutanol production.