Engineered escherichia coli for enhancing cell membrane stability and improving yield of caryophyllene and application

Abstract

The invention relates to a method for reinforcing crosslinking of an escherichia coli outer membrane and peptidoglycan to promote biosynthesis of beta-caryophyllene, in the biosynthesis process of terpenoid substances, the terpenoid compounds damage the integrity of cell membranes mainly by destroying the stability of the outer membrane, and in order to overcome the challenge, the biosynthesis of the beta-caryophyllene is promoted. A novel'cell membrane damage defense model 'is generated at the right time. By enhancing crosslinking between an outer membrane and peptidoglycan, the stability of the membrane is maintained under the stress of terpenoids, so that the metabolic activity of cells is remarkably improved. Through strategies of optimizing metabolic pathways, enhancing cell viability, refining fermentation process regulation and control and the like, the yield of the beta-caryophyllene reaches 15.6 g / L after 49 hours of fed-batch fermentation, and the highest record of producing the beta-caryophyllene by escherichia coli within the shortest fermentation period is created. The research provides a new method for effectively synthesizing other terpenoids to solve the challenge of cell membrane damage.

Description

Technical Field

[0001] This invention belongs to the field of synthetic biology, specifically relating to engineered Escherichia coli that enhances cell membrane stability and increases caryophyllene production, and its applications. Background Technology

[0002] Terpenes are a class of highly promising high-density fuels, among which β-caryophyllene is a bicyclic sesquiterpene compound. Its hydrogenation product exhibits high energy density (comparable to aviation fuel JP-10), low oxygen content, and excellent thermal stability, making it a highly promising bio-based aviation fuel alternative. Currently, β-caryophyllene is mainly obtained through three methods: traditional plant extraction, chemical synthesis, and novel microbial fermentation.

[0003] The main hosts for microbial fermentation synthesis of β-caryophyllene include Escherichia coli, yeast, and cyanobacteria. Among them, Escherichia coli has attracted much attention due to its advantages such as rapid growth, short fermentation cycle, and simple genetic editing. Currently, β-caryophyllene is synthesized through two pathways: methyl erythritol 4-phosphate (MEP) and mevalonic acid (MVA).

[0004] Many terpenoids exhibit cytotoxicity by disrupting cell membranes, which remains a key factor limiting terpene production. The hydrophobic alkyl chains of terpenes can insert into the phospholipid bilayer, leading to altered membrane fluidity, ion leakage, and impaired function of membrane-associated proteins. Changes in membrane permeability have a bidirectional regulatory effect on cell physiology, as permeability directly regulates metabolic homeostasis by affecting transmembrane transport efficiency. However, excessive increases in membrane permeability due to loss of resting membrane potential can disrupt ion balance and enzyme activity, ultimately leading to cell death. To date, research using membrane engineering techniques to increase the storage capacity of membrane alcohols in cell membranes to improve product accumulation is limited. The processes by which membrane stability networks sense terpene stress and coordinate feedback regulation remain largely unclear.

[0005] However, microbial methods have been used for β-caryophyllene synthesis. For example, reports indicate that a yeast microbial cell factory constructed using strategies such as enhancing precursor metabolic flux, optimizing terpene synthase activity, host chassis engineering, and fermentation process optimization achieved a β-caryophyllene yield of 21.4 g / L during fed-batch fermentation for approximately 250 h. However, this method has a long production cycle and high cost, limiting its application in β-caryophyllene synthesis.

[0006] Therefore, it is necessary to develop new strategies and systems to construct efficient cell factories for β-caryophyllene synthesis in Escherichia coli, which has a relatively fast reproduction rate. Summary of the Invention

[0007] To address the above problems, this invention provides an engineered Escherichia coli that produces high levels of β-caryophyllene, obtained by introducing an rpoS overexpression cassette into a starting Escherichia coli capable of synthesizing β-caryophyllene.

[0008] In one specific embodiment, the promoter sequence used for the rpoS overexpression cassette is selected from SEQ ID NO:3-5; or, the ribosome binding site sequence is selected from SEQ ID NO:6-8.

[0009] In one specific embodiment, the promoter sequence used for the overexpression cassette of the rpoS is shown in SEQ ID NO:5, and the ribosome binding site sequence is shown in SEQ ID NO:8.

[0010] In one specific embodiment, the amino acid sequence of the rpoS is shown in SEQ ID NO:1, or the nucleic acid sequence is shown in SEQ ID NO:2.

[0011] In one specific implementation, the engineered E. coli also incorporates one or more combinations of overexpression cassettes of lpp, ompA, and Pal.

[0012] In one specific embodiment, the amino acid sequence of the lpp is as shown in SEQ ID NO:9, or the nucleic acid sequence is as shown in SEQ ID NO:10;

[0013] The amino acid sequence of ompA is shown in SEQ ID NO:11, or the nucleic acid sequence is shown in SEQ ID NO:12; or

[0014] The Pal amino acid sequence is shown in SEQ ID NO:13, or the nucleic acid sequence is shown in SEQ ID NO:14.

[0015] In one specific implementation, the initiating Escherichia coli is an Escherichia coli that has been introduced into the MVA and MEP pathways.

[0016] The present invention also provides the application of the above-mentioned engineered Escherichia coli in the production of β-caryophyllene.

[0017] This study systematically investigated the effects of different terpenoid compounds on the cell membrane stability of *E. coli*. To mitigate membrane damage caused by the insertion of hydrophobic terpene chains into the phospholipid bilayer during terpene biosynthesis, a "cell membrane damage defense program" was developed to maintain membrane integrity and cellular metabolic activity. This defense program was further combined with metabolic pathway optimization, fine-tuning of rpoS expression to enhance cell robustness, and fermentation process optimization. In summary, these strategies yielded a β-caryophyllene titer of 15.6 g / L, the highest yield reported to date in *E. coli*. This work provides effective recommendations for the efficient biosynthesis of β-caryophyllene and offers valuable insights into improving microbial tolerance to high levels of terpenoid production. Attached Figure Description

[0018] Figure 1 The fermentation results of the initiating Escherichia coli strains WYQ03 and WYQ05, which can synthesize β-caryophyllene, are shown. In the figure, 'a' represents the β-caryophyllene yield of engineered strains WYQ01 and WYQ03 after 24 h of shake-flask fermentation, reaching the OD value. 600 a) Induction was initiated by adding 0.5 mM IPTG at a final concentration of 0.6; b) Cell growth curves during shake-flask fermentation of engineered strains WYQ01 and WYQ03; c) β-caryophyllene yield after 24 h of shake-flask fermentation of engineered strains WYQ03 and WYQ05, reaching OD. 600 When the concentration was 0.6, IPTG was added to a final concentration of 0.5 mM for induction; d represents the cell growth curves of engineered bacteria WYQ03 and WYQ05 during shake-flask fermentation.

[0019] Figure 2 The effects of terpenoid stimulation on cell membrane permeability in *Escherichia coli* were investigated. The effects of β-caryophyllene, α-pinene, and limonene on cell membrane permeability were observed using fluorescence microscopy and flow cytometry.

[0020] Figure 3 A cell membrane anti-damage strategy was designed. The effects of terpenoids on the transcriptional levels of genes related to membrane stability were investigated. Under terpenoid-induced stress, membrane damage defense mechanisms maintained cellular metabolic activity.

[0021] Figure 4 To finely regulate the effect of the regulatory factor rpoS on β-caryophyllene synthesis, the left figure shows the shake-flask fermentation results of β-caryophyllene with different promoter and RBS combinations regulating rpoS expression; the right figure shows the growth curves of the strains during β-caryophyllene fermentation with different promoter and RBS combinations regulating rpoS expression.

[0022] Figure 5The effects of a cell membrane damage defense model on β-caryophyllene production. The influence of regulating lpp gene expression intensity on β-caryophyllene synthesis. The effect of terpene stimulation on cell membrane structure after lpp gene overexpression.

[0023] Figure 6 The cells were fed in batches in a 5 L fermenter. When the cells grew to OD=12, 0.5 mM IPTG was added to induce the synthesis of β-caryophyllene. After 2 hours of induction, 10% n-dodecane was added for in-situ extraction. Detailed Implementation

[0024] The principles and features of the present invention are described below with reference to examples. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0025] Definition of technical terms

[0026] rpoS: RNA polymerase σS factor; Lpp: lipid phosphophosphatase; ompA: outer membrane protein A; Pal: peptidoglycan-associated lipoprotein; mvaE: acetyl-CoA thiolysis enzyme / HMG-CoA reductase; mvaS: 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase; ERG12: mevalonate kinase; ERG8: phosphate mevalonate kinase; ERG19: mevalonate pyrophosphate decarboxylase; IDI: isopentenyl pyrophosphate isomerase; IspA: farnesyl pyrophosphate synthase.

[0027] In this invention, overexpression refers to expression that exceeds the host's basal expression level.

[0028] 1. Construction of engineered bacteria for terpene compound production

[0029] The β-caryophyllene synthase QHS1 gene from Artemisia annua was strongly expressed in Escherichia coli BL21(DE3) using the T7 promoter, resulting in the MEP pathway β-caryophyllene-producing strain WYQ01.

[0030] To further increase the yield of β-caryophyllene, we introduced the heterologous MVA pathway into the WYQ01 engineered strain, loaded the mvaE and mvaS overexpression cassettes from Enterococcus faecalis into plasmid pWYQ03, and loaded the ERG12, ERG8, and ERG19 gene overexpression cassettes from Saccharomyces cerevisiae into plasmid pWYQ04, in order to increase the accumulation of IPP intermediate products.

[0031] To further enhance the downstream metabolic pathway from IPP to β-caryophyllene, we loaded overexpression cassettes of two key enzymes, IDI (IPP isomerase) and IspA (GPP synthase / FPP synthase), from E. coli, into plasmid pWYQ03.

[0032] pWYQ03 and pWYQ04 were co-transformed into BL21(DE3) to obtain the MEP and MVA dual-pathway engineered strain WYQ03. For example... Figure 1 As shown in a and b, the β-caryophyllene yield of the dual-pathway production strain WYQ03 was 1.549 mg / L, which was 3.35 times higher than that of the MEP single-pathway strain WYQ01 (0.356 mg / L), and the WYQ03 strain showed greater growth advantage.

[0033] To mitigate the cytotoxic effects of the accumulation of intermediate metabolites IPP, DMAPP, GPP, and FPP, we used IPP as a key node in the metabolic pathway and integrated overexpression cassettes of four single-copy genes (ERG12, ERG8, ERG19, and IDI) from the MVA pathway into the BL21(DE3) genome to weaken the metabolic flux from mevalonic acid to IPP and DMAPP, thus obtaining the engineered strain WYQ05. Figure 1 As shown in c and d, the β-caryophyllene yield of WYQ05 reached 273.05 mg / L, which is significantly increased by about 161 times, and this strain has a slight growth advantage during fermentation.

[0034] The results above indicate that integrating single-copy ERG12, ERG8, ERG19, and IDI genes into the genome balanced gene expression levels in the β-caryophyllene synthesis pathway, reduced the accumulation of toxic metabolites, and thus improved the synthesis efficiency of β-caryophyllene.

[0035] 2. Effects of terpenoid stimulation on Escherichia coli membrane permeability

[0036] Since the above experiments showed that the modification of E. coli significantly increased the content of terpenoids in the cells, this study further investigated the effects of three terpenoid compounds—α-pinene, limonene, and β-caryophyllene—on the membrane permeability of E. coli, particularly their effects on membrane disruption.

[0037] During the culture process, different concentrations of α-pinene, limonene, and β-caryophyllene were added to continuously expose *E. coli* for different time periods. Changes in membrane permeability were evaluated by propidium iodide (PI) staining, and analyzed by fluorescence microscopy and flow cytometry.

[0038] The results are as follows Figure 2As shown, 24 h of stimulation with caryophyllene significantly increased erythrocyte count and fluorescence intensity, indicating enhanced membrane permeability and cell membrane damage. Conversely, low concentrations of α-pinene (10 mM and 20 mM) significantly increased cell membrane permeability within 3 h of exposure, while simultaneously inhibiting cell growth. When *E. coli* was treated with limonene, even 1 mM stimulation for 1 h significantly increased membrane permeability, while 2 mM stimulation completely inhibited growth. These results indicate that the degree of cell membrane damage caused by terpenes is ranked in the order of β-caryophyllene > α-pinene > limonene, suggesting that the structure and concentration of terpenes have a significant impact on their cytotoxic effects on *E. coli* cell membranes.

[0039] 3. Design a model to defend against E. coli cell membrane damage.

[0040] In most Gram-negative bacteria, cell membrane stability is maintained primarily through various cross-linking mechanisms. These include covalent cross-linking of the outer membrane to peptidoglycan by the major lipoprotein LPP, non-covalent interactions between the outer membrane and peptidoglycan mediated by OmpA, and non-covalent interactions between peptidoglycan and the Tol-Pal complex.

[0041] To investigate the membrane stress response induced by terpenoids in Escherichia coli, we analyzed the transcriptional changes of genes related to maintaining cell membrane integrity after stimulation.

[0042] like Figure 3 As shown, the transcriptional level of the core anchoring protein LPP was significantly reduced, directly affecting the stability of the outer membrane and leading to increased membrane permeability. Simultaneously, the expression of the outer membrane structural protein OmpA was also significantly reduced, further impairing membrane integrity and triggering intrinsic repair and enhancement mechanisms. This synergistic defense response includes upregulating genes in the Tol-Pal system (tolQ, tolR, tola, tolB, PAL) to enhance membrane repair, activating lipopolysaccharide (LPS) biosynthesis by increasing lptD expression, and promoting peptidoglycan crosslinking by upregulating dacB and nlpI. Overall, this multilayered reinforcement strategy strengthens the entire membrane structure—from the outer membrane and its anchoring points to the cell wall. Simultaneously, it maintains the stability of the MLA system (mlaA, mlaC, mlaE) and MEPs to ensure effective phospholipid cycling and peptidoglycan remodeling, enabling the cell membrane to dynamically adapt and mitigate terpenoid-induced membrane stress.

[0043] Based on the analysis of terpene-induced cell membrane damage responses, this study designed three models to enhance and reinforce the cross-linking between the outer membrane and peptidoglycan. A multilayer anchoring network was established by strengthening LPP-mediated covalent cross-linking and the non-covalent interaction between OmpA and PAL. Increased expression levels of LPP, LPP-OmpA, and LPP-OmpA-PAL significantly reduced terpene-stimulated *E. coli* cell membrane permeability and increased metabolic activity, indicating a reduction in membrane damage. Notably, overexpression of lipoprotein LPP alone led to the most significant reduction in membrane permeability and the highest metabolic activity. These findings demonstrate that the outer membrane reinforcement strategy developed in this study effectively maintained the structural integrity of the *E. coli* cell membrane under terpene-induced stress.

[0044] 4. Enhancing the robustness of the strain improves the yield of β-caryophyllene.

[0045] Strains' robustness is crucial for industrial biomanufacturing, directly impacting production efficiency and costs. During fermentation, when cells are subjected to carbon and nitrogen starvation, increased osmotic pressure, low pH, elevated temperature, or low temperatures, the expression level of intracellular rpoS increases dramatically. At this point, RNA polymerase relies on rpoS to recognize promoters and activate the transcription of genes essential for survival under stress. Within bacteria, numerous environmental and intracellular signals participate in the regulation of rpoS, and these regulations can occur at the transcriptional and translational levels, degradation processes, and metabolic activity regulation.

[0046] Therefore, to improve the robustness of the strain during β-caryophyllene fermentation, the expression level of rpoS (amino acid sequence as shown in SEQ ID NO:1, nucleic acid sequence as shown in SEQ ID NO:2) in strain WYQ05 was finely regulated. The expression intensity of rpoS was adjusted by selecting promoters BBa_J23100, BBa_J23103, and BBa_J23116 (nucleic acid sequences as shown in SEQ ID NO:3-5, respectively) and ribosome binding sites BBa_B0034, BBa_B0064, and BBa_B0032 (nucleic acid sequences as shown in SEQ ID NO:6-8, respectively).

[0047] Analysis of reporter gene GFP expression intensity showed that the intensity of expression elements 100-34, 116-34, 103-34, 103-64, and 103-32 gradually decreased in the combination of promoter and RBS. Using combinations of these five expression element intensities to control rpoS expression, it was found that the lower the rpoS expression intensity, the higher the β-caryophyllene production. The strain with the lowest expression intensity, PJ23103-RBS0032, achieved a β-caryophyllene production of 410 mg / L, which was 46.4% higher than that of the engineered strain WYQ05. Furthermore, the ΔrpoS production of the rpoS knockout strain was 26.6% lower than that of the WYQ05 strain.

[0048] Real-time monitoring of the strain's growth during fermentation revealed that, compared to the control group WYQ05 where rpoS expression levels were not regulated, enhanced rpoS expression promoted cell growth, especially at moderate rpoS expression levels. However, rpoS knockout was detrimental to cell growth. This may be because moderate rpoS expression may balance stress adaptation and resource allocation, while excessive expression may inhibit genes related to rapid growth (such as ribosome synthesis genes). The rpoS-deficient mutant cannot induce key protective genes under stress, leading to decreased survival rates.

[0049] To verify whether regulating rpoS expression levels affects cellular metabolic activity, thereby promoting β-caryophyllene production, we performed MTT assays on the control strain WYQ05 (non-rpoS expression), the rpoS-overexpressing strain PJ23103-RBS0032, and the rpoS-knockout strain ΔrpoS. The results showed that the metabolic activity of the overexpressing strain PJ23103-RBS0032 was significantly higher than that of WYQ05, while the knockout strain was significantly lower.

[0050] These results indicate that regulating rpoS expression levels enhances cell robustness during fermentation, which is beneficial for cell growth and β-caryophyllene production. This is also the first time that rpoS has been regulated to increase β-caryophyllene production yield.

[0051] 5. Enhancing β-caryophyllene production using a cell membrane damage defense model.

[0052] Our research found that β-caryophyllene accumulation leads to increased cell membrane permeability and reduced cellular metabolic activity. This cellular metabolic disorder affects the efficient synthesis of β-caryophyllene.

[0053] To enhance β-caryophyllene production, a previously established cell membrane damage defense model was utilized. Three defense modules—LPP, LPP-OmpA, and LPP-OmpA-Pal—with different enhancement levels against terpene-induced membrane damage were introduced into the production strain PJ23103-RBS0032, constructing the engineered strain Lpp. E LPP-OmpA E and LPP-OmpA-PAI E Their defensive strength gradually weakens. For example... Figure 5 As shown, β-caryophyllene production increased proportionally with increasing membrane defense strength. Notably, Lpp expression alone resulted in a β-caryophyllene production of 589.5 mg / L, a 44% increase compared to strains without Lpp expression. PI staining revealed that Lpp... E Only a small fraction of the cells from the strain showed red fluorescence, while a large number of cells from the non-overexpressing strain PJ23103-RBS0032 showed strong red fluorescence, indicating extensive membrane damage.

[0054] Therefore, overexpression of lpp can reduce the permeability of the E. coli cell membrane and resist damage to the cell membrane caused by β-caryophyllene accumulation and osmotic pressure during fermentation. To systematically analyze the effects of enhancing and weakening lpp expression levels on β-caryophyllene production and cell growth, we inhibited lpp expression in the cell genome using CRISPRi technology to construct Lpp... I1 Lpp I2 Lpp I3 Strains. Compared with strain PJ23103-RBS0032, strain Lpp overexpressing lpp... E Both yield and growth were superior, with a yield reaching 589.5 mg / L, an increase of 44%. Inhibition of lpp was detrimental to β-caryophyllene production and growth, reducing yield by at least 73.2%. Furthermore, overexpression of lpp significantly reduced the accumulation of acetic acid, a major byproduct of E. coli fermentation, while increasing glucose consumption. Conversely, inhibition of lpp significantly increased acetic acid accumulation and decreased glucose consumption, while lactic acid levels remained largely unchanged. Cellular metabolic activity analysis revealed that overexpression of lpp significantly enhanced cellular metabolic activity, while inhibition of lpp significantly reduced it.

[0055] 6. Production condition optimization and batch-fed fermentation

[0056] During microbial fermentation, key factors such as dissolved oxygen (DO) level, available nitrogen source, vitamin B1 (thiamine) and biotin significantly affect the synthesis efficiency of target products and cell growth kinetics by regulating cell metabolic flux, energy supply and coenzyme activity.

[0057] Based on mechanisms such as carbon metabolic flux allocation, coenzyme-dependent enzymatic reactions, and redox balance, and considering the above factors, high-yield Lpp strains... E Fermentation validation was conducted. The experiment was first performed in shake flasks. To increase dissolved oxygen levels, we used custom-made 500 mL baffled shake flasks containing 30 mL of culture medium. 2.5 g / L yeast extract and 4 g / L tryptone, a readily available nitrogen source, were added to accelerate cell growth. Micronutrients of vitamin B1 (20 μg / L) and biotin (20 μg / L) were also added. β-caryophyllene yield and cell count were measured after 48 h of fermentation. The concentrations reached 1.104 g / L and 29 g / L, respectively, which are approximately 1.8 times the yield before optimization and the cell count. This is approximately four times the yield of β-caryophyllene produced using E. coli through shake-flask fermentation.

[0058] To verify the β-caryophyllene production performance of this strain during scale-up culture, this study conducted fed-batch fermentation in a 5-L fermenter. Real-time monitoring of β-caryophyllene yield, cell growth, and glucose consumption showed that after 49 h of induction, the β-caryophyllene yield and rate reached 15.6 g / L and 325 mg / L / h, respectively, representing the highest yield and rate of β-caryophyllene production reported by *E. coli* to date. Furthermore, it produced relatively little of the byproducts acetic acid and lactic acid. However, the cell density was lower during fed-batch fermentation, with a maximum... The yield was only about 75.4%, and future research could further improve the production and yield of β-caryophyllene by increasing cell density.

[0059] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An engineered Escherichia coli that produces high levels of β-caryophyllene, characterized in that, It was obtained by introducing an rpoS overexpression cassette into a starting Escherichia coli capable of synthesizing β-caryophyllene.

2. The engineered Escherichia coli according to claim 1, characterized in that, The promoter sequence used in the rpoS overexpression cassette is selected from SEQ ID NO:3-5; or the ribosome binding site sequence is selected from SEQ ID NO:6-8.

3. The engineered Escherichia coli according to claim 1, characterized in that, The promoter sequence used for the overexpression cassette of rpoS is shown in SEQ ID NO:5, and the ribosome binding site sequence is shown in SEQ ID NO:

8.

4. The engineered Escherichia coli according to claim 1, characterized in that, The amino acid sequence of the rpoS is shown in SEQ ID NO:1, or the nucleic acid sequence is shown in SEQ ID NO:

2.

5. The engineered Escherichia coli according to claim 1, characterized in that, It also introduced one or more combinations of overexpression boxes of lpp, ompA, and Pal.

6. The engineered Escherichia coli according to claim 5, characterized in that, The amino acid sequence of the lpp is shown in SEQ ID NO:9, or the nucleic acid sequence is shown in SEQ ID NO:10; The amino acid sequence of ompA is shown in SEQ ID NO:11, or the nucleic acid sequence is shown in SEQ ID NO:12; or The Pal amino acid sequence is shown in SEQ ID NO:13, or the nucleic acid sequence is shown in SEQ ID NO:

14.

7. The engineered Escherichia coli according to any one of claims 1-6, characterized in that, The initiating Escherichia coli is an Escherichia coli that has been introduced into the MVA and MEP pathways.

8. The use of the engineered Escherichia coli according to any one of claims 1-7 in the production of β-caryophyllene.

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