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 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).

    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/ ）.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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