1. Designing and constructing minimalist prokaryotic polyploid Escherichia coli (PMEC)

Introduction

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Figure 1 Polyploid strain construction method, Photo credit: Wang Sumeng. E. coli to produce 3-hydroxypropionate and L-threonine using synthetic biology strategies. 2023. Shandong University, PhD dissertation.

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In the course of previous studies, we found that it was possible to design and construct polyploid E. coli by finely regulating the expression level of ftsZ gene. We insert a weakly expressing element of “KanR-terminator-promoter-RBS” in front of the start codon of the ftsZ gene of one of the replication forks of E. coli to construct polyploid E. coli. After completing the construction, we performed validation and phenotype analysis.

1.1 Comparing the expression intensities of different promoter-RBS in DGF298

Our method to regulate ftsZ gene expression comes from a paper by Su-Meng Wang[1], in which the authors selected the terminator B1006 from the iGEM website (http:/lparts.igem.org/Main Page), promoters with different strengths including J23100, J23110, J23116, J23109, J23113 and J23103 and constructed the characterization plasmids pCL-100RFP, pCL-110RFP, pCL-116RFP, pCL-109RFP, pCL-113RFP, and pCL-103RFP. These plasmids were tested by linking the reporter gene rfp to either the J23100/J23110/J23116-B0034 or J23103/J23113/J23109-B0033 seamlessly cloned onto plasmid pCL1920 was constructed.

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Figure 2 Characterisation results

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And in order to construct a minimalist polyploid E. coli strain, we need to re-characterize the standard strengths of these plasmids in DGF-298. From there, the next step is to determine which promoter to choose in combination with RBS.

1.2 Constructing minimalist prokaryotic polyploid Escherichia coli

First, we introduced a homologous recombination system by introducing the PTK-Red plasmid to the DGF298 strain by electroporation, and then constructed and transferred ftsZ-Kan fragments containing the weakly expressed element “KanR-terminator-promoter-RBS” into DGF298.

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Figure 3 Map of plasmid pTKRED

Figure 4 Map of fragment JAR3-LR-FtsZ-kan

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Later, we further verified the ploidy of the new DGF298-103Z cells: DAPI staining and flow cytometry analysis showed that some cells of the strain DGF298-103Z were long strips, different from the oval shape of DGF298, the chromosome fluorescence intensity was higher than that of DGF298, and some cells contained two chromosomes, engineered chromosomes and wild-type chromosomes(Fig.5,6)

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Figure 5 DGF298-103Z DAPI staining results

Figure 6 DGF298 DAPI staining results

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The results of continuous transfer and PCR verification showed that after six transfers, the strain DGF298-103Z still contained engineered chromosomes of 1925 bp and wild type chromosomes of 724 bp (Fig.7), suggesting the engineered chromosomes were highly stable.

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Figure 7 Results of verification of single colonies isolated

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1.3 Morphological analysis

The results of field emission scanning electron microscopy showed that some cells of DGF298-103Z were elongated in morphology, which was different from the oval shape of DGF298, and due to the slow down of cytokinesis caused by the weak expression of ftsZ gene, most cells of DGF298-103Z could be seen to have Z-loops. Through statistical analysis, we found that almost 59% of DGF298-103Z cells were longer than 2.0 μm, while 77% of DGF298 cells were shorter than 2.0 μm. DGF298-103Z had a larger average cell volume (p<0.05), but its specific surface area decreased.

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Figure 8 Scanning electron microscopy results of DGF298 (10000× left, 20000× right)

Figure 9 Scanning electron microscopy results of DGF298-103Z (10000× left, 20000× right)

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1.4 pH growth-dependent analysis

With the decrease of pH in LB medium, polyploid E. coli DGF298-103Z began to show a growth advantage, which was manifested by the fact that the stunt period was significantly shorter than DGF298 in pH = 6 medium, and the mass doubling time of DGF298-103Z was 231.8% longer than that under pH = 7 and under pH = 5, but the mass time of haploid E. coli DGF298 was extended by 1300.1%. In M9 medium, DGF298-103Z was poorly grown in DGF298 at pH = 7, but not much in pH = 5, and interestingly, in M9 medium, DGF-298 and DGF298-103Z had the shortest mass doubling time at pH = 6. The shortening of the mass doubling time of DGF298-103Z under the slightly acidic conditions of M9 medium may bring advantages to its fermentation production in M9 medium. These results indicate that polyploid E. coli DGF298-103Z has a stronger tolerance to pH.

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Figure 10 DGF298/DGF298-103Z grown in LB medium at different pH (n=3) with left-to-right pH of 5, 6, and 7, respectively

Figure 11 DGF298/DGF298-103Z grown in M9 medium at different pH (n=3) with left-to-right pH of 5, 6, and 7, respectively

Figure 12 Doubling time of DGF-298/DGF-298-103Z in M9 medium at different pH (LB medium on the left)

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1.5 Growth-dependent analysis of acetate

The growth curve and mass doubling time of DGF298 were basically the same when cultured in M9 medium containing 0-4 g/L acetate, while DGF298-103Z was cultured in M9 medium with 4 g/L acetate by 191.6% compared with the control group, which may be due to the fact that the effect of acetate on the mass doubling time of polyploid DGF298-103Z was higher than that of DGF298. These results indicated that polyploid E. coli DGF298-103Z grew worse than haploid E. coli DGF298 and had a longer mass doubling time than DGF-298 when acetate was added.

These analyses suggest that polyploid E. coli DGF298-103Z has poor acetate tolerance, possibly because the original DGF298 strain knocked out the gene ACS, which encodes acetyl-CoA synthetase, which catalyzes the conversion of acetic acid to acetyl-CoA.

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Figure 13 DGF-298/DGF-298-103Z growth in M9 medium with different acetate concentrations (n=3)

Figure 14 Doubling time of DGF-298/DGF-298-103Z when grown in M9 medium at different acetate concentrations

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1.6 Temperature-dependent analysis

The results showed that the change of temperature had a great effect on the generation time, and both haploid E. coli DGF298 and polyploid E. coli DGF298-103Z had the shortest at 37°C and could not grow at 45°. For polyploid E. coli DGF298-103Z, low temperature has a greater effect on its generation. DGF298 increased by 118.7% at 42°C and 515.6% at 30°C. However, DGF298-103Z showed better tolerance to higher temperature, and the difference between it and DGF298 at 30°C, 37°C, and 42°C was 3.87h, 0.337h, and 0.04h, respectively, and with the increase of temperature, it was closer to that of DGF298, and its delay period at 42°C was significantly shorter than that of DGF29. At 42°C, the polyploid generation increased by 118.8%, which was less than the increase of DGF298 by 171.1% (Fig.15).The above results indicated that the perturbation of polyploid E. coli DGF298-103Z was less affected by high temperature.

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Figure 15 Growth curves and mass doubling times of DGF-298/DGF-298-103Z in M9 medium at different temperatures (n=3)

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Summary

Thus, we proposed the concept of minimalist polyploid E. coli, which has both wild-type chromosomes and highly stable engineered chromosomes, and has morphological changes such as increased average cell length, basically the same width, increased average cell volume and surface area, and reduced specific surface area compared with haploid E. coli. Robustness analysis showed that compared with haploid chassis cells, polyploid E. coli showed better resistance to stress under high temperature and pH perturbation.

2. Transcriptomic Analysis and Metabolic Network Modeling

Introduction

In order to explore the changes of protein expression and carbon flux in the polyploidized strain DGF298-103Z, we performed 16s rRNA sequencing identification analysis and transcription library analysis of polyploid E. coli DGF298-103Z and haploid E. coli DGF298. By substituting the transcriptome sequencing results into the Cobrapy metabolic network model of dry lab, we predicted that the down-regulation of two genes (nuoF and pfkB) would have a greater impact on the growth of polyploid E. coli, and we engineered the polyploid E. coli by upregulating these two genes by combining promoters and RBS with different intensities.

2.1 16S rRNA sequencing identification analysis

In the sequencing analysis of a total of 6 samples of DGF-298 and DGF-298-103Z, the purity of the strains was verified by 16sRNA sequencing, which avoided the risk of losing strains due to infection in the middle of the experiment. At the same time, RNA is free of pigments, proteins, sugars and other impurities, RQN≥6.5, 28/23S brightness is greater than 18/16S, the total amount meets the needs of two standard library construction, and can be used for transcriptome analysis and library construction follow-up experiments. (Fig.16, 17; Table.1)

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Figure 16 Results for DGF298 and DGF298-103Z (DNA Marker: 100, 250, 500, 750, 1000, 2000, 3000, 5000bp)

Figure 17 Detection diagram of GF-298 / DGF-298-103Z (corresponding to samples No.1-6)

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Table 1 Results of the 16sRNA test for DGF298 and DGF298-103Z//caption

Sample number	Sample name	Concentration (NG / μ l)	OD260/OD280	OD260/OD230	RQN	NT contrast results
1	DGF298-1	538.74	2.04	2.33	10	Escherichia_coli
2	DGF298-2	519.11	2.03	2.03	10	Escherichia_coli
3	DGF298-3	400.04	2.21	2.21	10	Escherichia_coli
4	DGF298-4	292.5	2.25	2.35	10	Escherichia_coli
5	DGF298-5	237.02	2.13	2.13	10	Escherichia_coli
6	DGF298-6	430.98	2.32	2.32	10	Escherichia_coli

2.2 Transcription repositories

In transcriptomic analysis, we found that the expression levels of carboxylic acid synthesis pathways, amino acid synthesis pathways, and sulfur compound synthesis pathways were upregulated in polyploids (Fig.18).

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Figure 18 Results of the transcriptome analysis of DGF298 and DGF298-103Z

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2.3 Modular control

Sequencing showed that we successfully constructed and transferred the PACYC-Ptac-pfkA-nuoF plasmid containing different expression intensities combinations into DGF298-103Z (Fig.19). The qPCR results showed significant up-regulation of nuoF or pfkB genes in the experimental group with regulatory combinations of moderate intensity (M) and high intensity (U) (Fig.20). By characterizing the growth curves of strains with different intensity regulatory modules, we found that some experimental groups had earlier log phase compared with DGF298-103Z in the control group (Fig.21).

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Figure 19 Different combinations of modular control

Figure 20 Results of RT-qPCR for gene expression measurements

Figure 21 Characterization results of modularly modulation

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Summary

In this part of work, we learned about the expression level of carboxylic acid synthesis, amino acid synthesis, and sulfate compound synthesis pathway were upregulated in polyploid strain through 16sRNA sequencing and transcriptional assembly library of DGF298-103Z, which indicates that polyploidization may bring more excellent fermentation potential. After modeling the metabolic pathways of DGF298-103Z and DGF298, we surrogate transcriptome data and predicted that downregulation of the nuoF and pfkB genes severely affected strain growth. To improve the model, we tried up-regulatiing these two genes through a combination of promoter and RBS of different intensity, and the characterization results showed accelerated growth and metabolism of some experimental groups.

3. Fermentation potential analysis of novel chassis cells

Introduction

To analyze the production capacity of new chassis cells for different high value-added products, we selected appropriate carriers, constructed different heterologous gene plasmids and introduced them into cells for protein expression levels and PHB fermentation production analysis. After that, in an attempt to combine the possible sensitivity of the shuttle promoter of Corynebacterium glutamami to specific metabolic conditions with the rapid multiplication and ease of handling of E. coli, we introduced the shuttle promoters into E. coli and evaluated their expression levels. At the same time, we found that the TCA cycle and PHB synthesis reaction will compete for acetyl-CoA in the intracellular metabolic flow, and in order to try to achieve the efficient allocation of carbon flux in the initial growth and production metabolism stages, we introduced a dynamic temporal expression system to DGF298-103Z.

3.1 Protein expression levels

Sequencing showed that we successfully constructed and transferred the plasmid PACYC-Ptac-GFP-Amp into DGF298-103Z and DGF298. We used the expression intensity of the reporter gene GFP to characterize the protein expression levels of the strains. When observing single colonies with a blue light instrument, we found that the colony fluorescence intensity of polyploid DGF298-103Z was higher than that of haploid DGF298(Fig.26).

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Figure 22 Map of plasmid PACYC-Ptac-GFP-Amp

Figure 23 Sequencing results of PACYC-Ptac-GFP-Amp

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Characterization showed that the GFP expression levels of GFP (GFP/OD600) in both polyploid and haploid strains in LB medium were higher than in M9 medium(Fig.25), and that the protein expression intensity of polyploid DGF298-103Z was about 50% higher than DGF298 in both environments, both strains had significantly earlier logarithmic phase in LB medium (Fig.24). This indicates that polyploid E. coli has higher protein expression levels and that LB medium is more suitable for rapid bacterial growth than M9 medium.

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Figure 24 OD600 of DGF-298/ DGF-298-103z （M9 on the left, LB on the right, 3 replicates）

Figure 25 GFP/OD600 of DGF-298 /DGF-298-103z (LB medium on the left, M9 medium on the right, three replicates)

Figure 26 The blue light meter detects the green fluorescence of DFG298-103Z(right) and DGF298(left)

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3.2 Shake bottle simulation fermentation production of PHB

Sequencing showed that we successfully constructed and transferred plasmid pUC-Pcon-both into DGF298-103Z, DGF298 and W3110. In M9 medium supplemented with 40g / L glucose as the carbon source, DGF298 and DGF298-103Z showed more rapid and stable initial growth than wild-type strain W3110(Fig.28), possibly due to the synergistic action of DGF298 strain gcvA deletion and functional ilvG that resist the growth inhibitory feedback of valine accumulation. However, polyploid DGF298-103Z grew slower in middle and late stages than haploid DGF298 and consumed glucose slower in the initial stage, probably due to its metabolic burden and aberrant chromosome number. The total amount of glucose consumed by DGF298-103Z fermentation was 13.9% and 19% higher than W3110 and DGF298, respectively (Fig.29).

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Figure 27 Map of plasmid pUC-Pcon-PHB

Figure 28 OD600 of DGF-298 and DGF-298-103z during fermentation (three replicates）

Figure 29 Change in glucose concentration in medium (left) vs. total sugar consumption (right) (average of three replicates in each group)

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3.3 Amino acid determination

By analyzing the content of free amino acids in the fermentation broth, we found that the free phenylalanine Phe production of DGF298-103Z, DGF298 and W3110 was more prominent than other amino acids, and the Phe production of DGF298-103Z was 7.98 nmol, which was 19% and 211% higher than that of DGF298 and W3110, respectively (Fig.30).

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Figure 30 Amino acid fermentation yield of W3110, DGF-298，DGF-298-103Z

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3.4 Analysis of the PHB yield

We isolated single colonies of DGF298-103Z, DGF298 and W3110 in the late stage of fermentation, and after Nile red staining we found that the fluorescence intensity of DGF298-103Z was higher (Fig.31), implying a higher PHB yield. Further determination of intracellular PHB content by gas chromatography showed that the average total PHB production of DGF298-103Z, DGF298 and W3110 in the simulated fermentation was 0.025477g /L, and the total PHB production of 0.014595333g/L,0.013395333g/L and DGF298-103Z was 43.7% and 47.4% higher than that of DGF298 and W3110, respectively (Fig.32).

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Figure 31 Nile red staining results at the end of fermentation (DGF-298, W3110, DGF-298-103Z, from left to right)

Figure 32 Total amount of PHB in the bacteria after fermentation (three controls)

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3.5 Screening shuttle promoters

When designing metabolic pathways for downstream products, our team tried to expand the control of the expression strength of specific genes by introducing shuttle promoters. Therefore, we compared the transcriptome sequencing results of different Corynebacterium glutamicum strains, identified promoters and RBSs with different expression strengths, and applied them to E. coli chassis cells for expression (Transcriptome data provided by Dr. Xin Jin from Prof. Quanfeng Liang’s group）.

The PACYAC-RFP series of plasmids we constructed were transferred into E.coli DH5α after verifying. We used the expression intensity of the reporter gene RFP to characterize the expression intensity of different shuttle promoters and RBS. The results showed significant differences in the expression intensity of different shuttle promoters in E. coli chassis cells (Fig.33).

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Figure 33 Characterization results of the shuttle promoter

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3.6 Self-induced dynamic time-sequence modulated cascade lines

The starting plasmids for our dynamic cascade regulation; pCDF-QS-GFP and Plas-RFP were provided by Xiaomeng Li from Prof.

We tried to replace the GFP gene pre-promoter on the plasmid with different newly discovered shuttle promoters, obtaining the plasmid QS-P02-LasI-GFP, in which the primers LasI-P02-R and LasI-P02-F could be redesigned according to the different promoters. Finally, the constructed plasmids with a series of different promoters were co-transfected with plasmid Plas-RFP into DGF-298 chemoreceptor state, and the newly constructed strains were named L02 and L38, respectively.

After analyzing the experimental results, it was found that replacing different promoters with different strengths for the cascade of self-induced dynamic timing control would result in different response effects, which increased the diversity of the time difference of the cascade. From the result, we can see that the normalized difference in response time between GFP fluorescent proteins and RFP fluorescent proteins of strains L02 and L38 were about 193 min and 164 min, respectively (Fig. 34).

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Figure 34 Normalized curves of self-induced dynamic chronological regulatory cascade lines regulating the intensity of LasI synthase expression

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The normalized difference in response time between GFP fluorescent proteins and RFP fluorescent proteins was expanded after promoter replacement by LasI synthase, suggesting that the expression intensity of LasI synthase can be efficiently regulated using promoter engineering to generate small molecules of auto-inducer to induce dynamic, time-sequential expression of upstream and downstream genes at different time intervals.

Summary

Through the above study, we concluded that the polyploid strain DGF298-103Z in the genome streamlined DGF strain with rapid and stable fermentation initial growth and the advantages of intracellular carbon metabolism flow suitable for amino acid production, further improve the protein expression level and the synthesis of heterologous secondary metabolites PHB, with more excellent fermentation production potential. By introducing the shuttle promoter of Corynebacterium glutamicum into E. coli DH5α and characterizing it, we obtained the expression strengths of different shuttle promoters in E. coli and applied them to the construction of dynamic cascade regulatory lines, and successfully expanded the normalized differences of different temporal response times. In the future, we still hope to further utilize the newly discovered synthetic biology components to replace the promoters of different components in the QS system, such as LasI, LasR, TraR, etc., so as to achieve a finer temporal regulation of the TCA reaction and PHB production.

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