Development of a High Anthraquinone-Producing Escherichia coli Strain Using Malonyl-CoA Supply Pathway Engineering

Abstract

Anthraquinones are valuable compounds that are traditionally used as natural pigments and have diverse pharmacological activities, including antimicrobial and anticancer effects. In this study, we aimed to enhance the production of 1,3,5-trihydroxyanthraquinone (AQ256) using Escherichia coli (E. coli) as a host. AQ256 is biosynthesized from eight malonyl-CoA molecules via the type II polyketide synthase pathway. However, previous studies have reported very low production levels of AQ256 in E. coli (approximately 2.5 mg/L), mainly because of limited malonyl-CoA availability. To address this, we introduced a heterologous malonate assimilation pathway and reinforced the endogenous malonyl-CoA biosynthesis pathway. An E. coli strain harboring AQ256 biosynthetic genes from Photorhabdus laumondii TTO1 produced only 1.3 mg/L AQ256. Upon introducing the malonate assimilation pathway and cultivating in malonate-supplemented Luria–Bertani medium, production increased to 3.8 mg/L. Further enhancement of the endogenous malonyl-CoA supply through the coexpression of pantothenate kinase and acetyl-CoA carboxylase resulted in strain AQ-04, which produced 12.3 mg/L AQ256. Optimization of cultivation conditions enabled AQ-04 to achieve 23.9 mg/L AQ256, a 9.6-fold increase compared to previous studies. Our results demonstrate that the combination of introducing a malonate assimilation pathway and enhancing native malonyl-CoA supply is a highly effective strategy for increasing malonyl-CoA availability. This approach is promising for the biosynthesis of a wide range of malonyl-CoA-derived compounds.

Introduction

Anthraquinones are a group of compounds with a basic 9,10-anthracenedione skeleton consisting of three tandemly fused hexacyclic ring systems with two carbonyl groups (Figure A). The backbone contains a π-conjugated system, allowing it to absorb visible light, and has therefore been historically used as a dye. , In addition, anthraquinones exhibit diverse pharmacological activities owing to their quinone structure, which can oxidize the substrate, leading to hydrogen (−H) substitution with various functional groups or sugars. − Consequently, anthraquinones exhibit bioactivity as laxatives, antibacterials, antifungals, and anticancer agents. ,

1.

(A) Biosynthetic pathway of AQ256 and (B) the plasmid for heterologous expression of the AQ256 gene cluster in E. coli AQ256 utilizes eight malonyl-CoA as a substrate, involving nine AQ256 biosynthetic enzymes (AntA–I) from P. laumondii TTO1 and endogenous E. coli enzymes functioning as MCAT. Since the original ant biosynthetic genes are organized into two operons (antABC and antDEFGHI), a plasmid was constructed to independently express each operon under separate T7 promoters. Abbreviations: CoA, coenzyme A; PAP, 3′-phosphoadenosine 5′-phosphate; AntA, ketoreductase; AntB, phosphopantetheinyl transferase; AntC, cyclase; AntDE, ketosynthase αβ heterodimer; AntF, acyl carrier protein; AntG, coenzyme A ligase; AntH, aromatase; AntI, hydrolase; MCAT, malonyl-CoA-acyl carrier protein transacylase.

Anthraquinones are secondary metabolites found in microorganisms, insects, and plants and are generally synthesized via the action of polyketide synthase (PKS). ,− Some anthraquinones in plants are biosynthesized from precursors derived from shikimate or terpenoid biosynthetic pathways. PKS enzymes are classified into three types based on their structural and mechanistic properties: I, II, and III. Anthraquinones are produced through complex reaction mechanisms mediated by type II PKS composed of monofunctional enzyme complexes. In the type II PKS reaction, polyketide chains of varying lengths are generated, depending on the number of starter and extender units, by the action of a ketosynthase (KS)/chain length factor (CLF), a minimal unit in type II PKS. Subsequently, aromatase/cyclase, dehydratases, and other dedicated tailoring enzymes generate aromatic scaffolds. ,

The large-scale production of various anthraquinones remains a challenge because of the high cost and time-consuming nature of plant and insect extraction. Microbial production is also complicated by the complexity of genetic modifications and lack of established large-scale fermentation techniques.

Recently, metabolic engineering and synthetic biology approaches have been explored to develop microbial strains with enhanced anthraquinone production. For example, the production of flavokermesic acid, dehydrorabelomycin, , and 1,3,5-trihydroxyanthraquinone (AQ256) has been reported in Escherichia coli (E. coli). However, the low production yields remain a major challenge for these anthraquinones.

This study aimed to achieve high-level heterologous production of anthraquinones using metabolic engineering, focusing on the development of an E. coli strain optimized for AQ256 production. E. coli is a well-characterized prokaryotic model organism with extensive genetic and metabolic information. Owing to its ease of genetic manipulation and rapid growth, E. coli has been widely used as a production host for valuable compounds, , including type II polyketide products, via the heterologous expression of biosynthetic gene clusters, making it a promising candidate for anthraquinone biosynthesis. AQ256 is an aromatic polyketide synthesized via type II PKS, and its biosynthetic pathway is well-characterized. Moreover, the culture medium takes on a color ranging from red to brown, which allows easy monitoring of production during the cultivation process.

AQ256 was biosynthesized using eight malonyl-CoA molecules as precursors by expressing nine ant genes (antA–I) derived from Photorhabdus laumondii (P. laumondii) TTO1 in E. coli (Figure A). Malonyl-CoA is essential for fatty acid biosynthesis; however, its accumulation is limited and the production capacity for nonessential metabolites is restricted. , In E. coli, a high malonyl-CoA supply can be achieved via two major strategies: strengthening endogenous biosynthetic pathways − and introducing heterologous metabolic pathways for malonyl-CoA synthesis from malonate. − Strengthening the endogenous malonyl-CoA biosynthetic pathway via acetyl-CoA carboxylase gene expression contributes to enhanced anthraquinone production. In E. coli, malonyl-CoA is synthesized from acetyl-CoA via a reaction catalyzed by acetyl-CoA carboxylase (Acc). On the other hand, heterologous malonyl-CoA supply pathways based on matB/matC from Rhizobium trifolii (MatB: malonyl-CoA synthetase; MatC: malonate transporter) have been widely investigated for enhancing the biosynthesis of value-added metabolites such as flavonoids and hydroxy acids. , However, the application of these pathways to anthraquinone biosynthesis remains limited, and their integration with endogenous malonyl-CoA biosynthetic pathway enhancement has not been thoroughly explored.

In this study, we first evaluated the effect of introducing an exogenous malonate assimilation pathway into E. coli on the production of AQ256. Additionally, strains with enhanced endogenous malonyl-CoA biosynthesis were constructed to investigate the combined effects of the two approaches. The AQ256 production levels of these engineered strains, along with the intracellular CoA-related metabolite pools, were evaluated. Finally, cultivation conditions were optimized by adjusting medium composition and initial cell density to maximize AQ256 production.

Results

Heterologous Production of AQ256 by E. coli BL21 (DE3)

AQ256 can be produced in E. coli by expressing nine ant genes (antA–I) derived from the P. laumondii TTO1 strain. In the AQ256 biosynthesis pathway, the apo-form acyl carrier protein (ACP; AntF), which lacks the 4′-phosphopantetheine prosthetic group and is catalytically inactive, is activated by phosphopantetheinyl transferase (AntB) and coenzyme A ligase (AntG). These enzymes consume coenzyme A (CoA) and convert apo-ACP into the holo-form, which carries the 4′-phosphopantetheine moiety and serves as the active carrier of acyl intermediates (Figure A). Subsequently, malonyl-CoA is loaded onto the ACP using malonyl-CoA-acyl carrier protein transacylase (MCAT). This catalytic reaction is presumably mediated by endogenous E. coli enzymes functioning as MCAT. The malonyl-ACP then undergoes polyketide chain elongation catalyzed by the ketosynthase αβ heterodimer (AntDE), ultimately leading to the decarboxylative Claisen condensation of eight malonyl-CoA molecules to form an octaketide chain. The resulting octaketide chain is cyclized by ketoreductase (AntA), aromatase (AntH), and cyclase (AntC). Furthermore, AntI, an unusual lyase, cleaves the acetyl unit while promoting cyclization to form a cyclized heptaketide scaffold and the consequent release of acetyl-ACP. The released heptaketide product is spontaneously oxidized to yield AQ256. ,

In this study, ant genes were divided into two operons (antA–C and antD–I) and ligated under the T7 promoter to produce the pACYC-AQ256 plasmid (Figure B). The E. coli BL21 (DE3) strain harboring pACYC-AQ256 was designated AQ-01. AQ-01 was cultivated in Luria–Bertani (LB) medium, and the AQ256 production level was evaluated. As a result, AQ256 production by AQ-01 reached 1.3 ± 0.1 mg/L at 48 h of cultivation (Figure A).

2.

Effect of the heterologous malonate assimilation pathway on AQ256 production in E. coli (A) AQ256 production by AQ-01 and AQ-02 with or without malonate supplementation. A “+” indicates the addition of 22 mM malonate in the medium. The AQ-01 strain contains the AQ256 biosynthetic genes, while the AQ-02 strain contains both the AQ256 biosynthetic genes and the malonate assimilation pathway. Time course of (B) malonate consumption and (C) growth of AQ-01 and AQ-02. Results are presented as the mean ± standard deviation of three biologically independent experiments.

Malonyl-CoA Supply Enhancement via Exogenous Malonate Assimilation Pathway Introduction

Since AQ256 biosynthesis requires eight molecules of malonyl-CoA (Figure A), we hypothesized that introducing the malonate assimilation pathway may enhance the malonyl-CoA supply and, consequently, AQ256 production. To convert malonate into malonyl-CoA, a Na⁺-dependent malonate transporter encoded by the malonate transporter gene (madLM) from Malonomonas rubra (M. rubra) and the malonyl-CoA synthetase gene (mcs) from Bradyrhizobium japonicum (B. japonicum) was expressed in E. coli. The expression of madLM and mcs in E. coli has been reported to increase the (2S)-naringenin production, which uses malonyl-CoA as a precursor. Here, plasmids harboring madLM and mcs with the T7 promoter were introduced into AQ-01 to construct strain AQ-02.

The effect of malonate supplementation (22 mM) in LB medium on AQ256 production was evaluated in the AQ-01 and AQ-02 strains. No significant increase in AQ256 production was observed upon malonate supplementation of AQ-01 (Figure A). In contrast, in AQ-02, although AQ256 production was 1.1 ± 0.1 mg/L without malonate supplementation, increased to 2.9 ± 0.1 mg/L with addition of malonate, representing a 2.7-fold increase. Moreover, AQ256 production in AQ-02 did not increase after 24 h in the absence of malonate, whereas it continued to increase in the presence of malonate, reaching 3.8 ± 0.1 mg/L at 72 h. AQ-02 consumed 3.6 mM malonate at 72 h, whereas no significant consumption was observed in AQ-01 (Figure B). Malonate supplementation showed no effect on cell growth (OD₆₀₀: 5.7 ± 0.1) in AQ-01 (Figure C); in AQ-02, OD₆₀₀ only reached to 4.3 ± 0.1 upon malonate supplementation at 72 h. These results indicate that although malonate supplementation reduced the cell density of AQ-02 cells, AQ256 production per cell increased. AQ256 production was enhanced by introducing the malonate assimilation pathway and malonate supplementation.

To examine whether the decrease in cell density caused by malonate supplementation could be alleviated, the AQ-02 strain was cultured under a reduced malonate concentration (7.3 mM). As a result, after 72 h of cultivation, cell growth remained similarly suppressed as under the 22 mM condition (OD₆₀₀: 4.3 ± 0.1) (Figure S1A), and AQ256 production decreased to 2.7 ± 0.1 mg/L (Figure S1B). Based on these results, subsequent experiments in this study were conducted using 22 mM malonate as the experimental condition.

Pp-coaA and Cg-acc Expression to Strengthen the Endogenous Malonyl-CoA Production Pathway

In this study, the effects of enhanced CoA synthesis and acetyl-CoA carboxylation on AQ256 production were investigated (Figure A). Pantothenate kinase (CoaA), which phosphorylates pantothenate, is the initial step of CoA biosynthesis to produce 4′-phosphopantothenate. The pantothenate kinase gene (Pp-coaA) from Pseudomonas putida (P. putida) was selected for overexpression because, unlike CoaA from E. coli, Pp-CoaA is not subject to feedback inhibition by CoA, acetyl-CoA, or malonyl-CoA. Pp-coaA overexpression enhances the supply of CoA and acetyl-CoA in E. coli. , Acetyl-CoA carboxylase catalyzes the ATP-dependent conversion of acetyl-CoA to malonyl-CoA. The acc gene from Corynebacterium glutamicum (C. glutamicum) (Cg-acc), which is evolutionarily distinct from the four-subunit Acc complex of E. coli, encodes a three-subunit enzyme complex that exhibits higher activity at low temperatures (approximately 23 °C) compared to Acc from E. coli. , Therefore, it is expected to efficiently convert acetyl-CoA to malonyl-CoA even under low-temperature conditions, which are generally required for the expression of polyketide synthase in E. coli.

3.

Effect of the enhanced endogenous malonyl-CoA biosynthesis pathway on AQ256 production in E. coli (A) Endogenous malonyl-CoA biosynthesis pathway of E. coli. Solid arrows indicate single reaction steps, and the dashed arrows indicate multiple enzymatic steps. The metabolic reactions enhanced in this study are indicated by colored arrows (red, blue, yellow, and green). Abbreviations: MadLM, malonate transporter from M. rubra; MCS, malonyl-CoA synthetase from B. japonicum; Acc, acetyl-CoA carboxylase from C. glutamicum; CoaA, pantothenate kinase from P. putida. Time course of (B) AQ256 production and (C) malonate consumption of AQ-02, AQ-03, and AQ-04 strains. Strains AQ-03 and AQ-04 were constructed by overexpressing Pp-coaA and both Pp-coaA and Cg-acc, respectively, in the AQ-02 host strain. Data represent the mean ± standard deviation from three independent biological experiments.

First, the AQ-03 strain was constructed by overexpressing Pp-coaA in the AQ-02 strain, and the AQ-04 strain was constructed by overexpressing both Pp-coaA and Cg-acc (accBC, accE, and dtsR1 genes). The strains were cultivated for 72 h in LB medium supplemented with malonate. As a result, AQ256 production in AQ-03 and AQ-04 reached 9.8 ± 0.3 and 12.3 ± 0.2 mg/L, respectively, representing 1.5-fold and 1.9-fold increases compared to AQ-02 (Figure B). Additionally, malonate consumption in the culture medium was 4.6 mM in AQ-02, 6.6 mM in AQ-03, and 7.5 mM in AQ-04 (Figure C). These results demonstrate that Pp-coaA and Cg-acc overexpression further enhanced malonate uptake and AQ256 production in E. coli strains harboring the malonate assimilation pathway.

To clarify the effects of enhancing only the endogenous pathway, the AQ-03 and AQ-04 strains were also cultivated in LB medium without malonate supplementation. After 72 h of cultivation, AQ256 production reached 1.5 ± 0.0 mg/L in AQ-03 and 3.5 ± 0.3 mg/L in AQ-04, both of which were lower than the corresponding values under malonate-supplemented conditions (Figure S2 and Figure B). These results clearly demonstrate that the improvement in AQ256 production was achieved through the synergistic effect of introducing a heterologous malonate assimilation pathway and enhancing the endogenous malonyl-CoA biosynthesis pathway.

Measurement of Intracellular CoA Compounds in Malonyl-CoA Supply-Enhanced Strains

To investigate the effect of gene overexpression on the intracellular pools of CoA-related compounds, we analyzed the accumulation of CoA-related compounds (CoA, acetyl-CoA, and malonyl-CoA) in the engineered cells. Strains AQ-01–AQ-04 were cultured in LB medium supplemented with 22 mM malonate. Intracellular metabolites were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to measure the concentrations of CoA-related compounds. Metabolic analysis revealed that the total concentration of CoA-related compounds in all strains was highest at 9 h of cultivation and then decreased over time (Figure ). As expected, at 9 h of cultivation, the malonyl-CoA concentration in strain AQ-02 (0.87 nmol/mg-DCW) was 6.2-fold higher than that in AQ-01 (0.14 nmol/mg-DCW). However, after 24 h of cultivation, no significant differences in malonyl-CoA accumulation were observed between the AQ-01 and AQ-02 cells. In strains AQ-03 and AQ-04, the endogenous malonyl-CoA supply pathway was enhanced in addition to the introduction of the malonate assimilation pathway. At 9 h of cultivation, the CoA accumulation levels in AQ-03 and AQ-04 were 1.4 and 1.1 nmol/mg-DCW and in acetyl-CoA were 1.5 and 2.0 nmol/mg-DCW, respectively. These values were higher than those observed in AQ-02 (CoA: 0.6 nmol/mg-DCW, acetyl-CoA: 1.0 nmol/mg-DCW). Despite the higher AQ256 production observed in AQ-03 and AQ-04 than in AQ-02 (Figure B), the accumulation of malonyl-CoA remained similar among these strains throughout the cultivation period. This result suggests that malonyl-CoA was rapidly consumed in strains AQ-02, AQ-03, and AQ-04. However, considering that the best-performing strain, AQ-04, consumed approximately 7.5 mM malonate while producing only 12.3 ± 0.2 mg/L AQ256 (Figure C), it is likely that a substantial portion of the generated malonyl-CoA was diverted to competing endogenous pathways, such as fatty acid biosynthesis.

4.

Intracellular concentration of CoA-related compounds in the engineered strains. The vertical axis represents the total concentration of CoA-related metabolites, including CoA, acetyl-CoA, and malonyl-CoA. Results are expressed as means ± standard deviation of three independent biological experiments.

Optimization of Medium Composition: Effects of Glucose and LB Medium Components

AQ256 production was conducted in LB medium without the addition of carbon sources other than malonate. Metabolite analysis revealed a rapid decrease in the levels of CoA, acetyl-CoA, and malonyl-CoA after 24 h of cultivation (Figure ). To further enhance the supply of CoA derivatives, we examined the effects of glucose supplementation or additional yeast extract (a mixture of vitamins and amino acids) to LB medium supplemented with malonate on the AQ256 production. The AQ-04 strain, which exhibited the highest AQ256 production, was used to evaluate these effects.

As shown in Figure A, the addition of glucose to the medium resulted in a 2.6-fold increase in OD₆₀₀ at 72 h of cultivation (OD₆₀₀ = 9.1 ± 0.2) compared to cultures without glucose. Notably, glucose supplementation promoted malonate consumption, leading to complete malonate depletion after 48 h of culture (Figure B). Glucose was almost completely consumed after 48 h (Figure S3). However, AQ256 production was only 5.6 ± 0.1 mg/L at 72 h, which was notably lower than the titer without adding glucose (Figure C). Next, the addition of 5 g/L yeast extract slightly increased OD₆₀₀ at 72 h (OD₆₀₀ = 4.5 ± 0.1) compared to control conditions (Figure A). Malonate consumption also improved marginally, with 10.1 mM being consumed within 72 h (Figure B). Furthermore, AQ256 production at 72 h reached 15.6 ± 0.2 mg/L, representing a 1.5-fold increase compared to cultures without additional yeast extract (Figure C). Although additional yeast extract enhanced AQ256 production, the increase from 24 h (12.4 ± 0.7 mg/L) to 72 h (15.6 ± 0.2 mg/L) was only 26%, indicating that AQ256 production did not significantly improve production in the stationary phase.

5.

Effect of glucose or yeast extract supplementation on AQ256 production by strain AQ-04 in LB medium containing 22 mM malonate. Time course of (A) growth, (B) malonate consumption, and (C) AQ256 production by AQ-04 in three different medium conditions. Data represent the mean ± standard deviation of three independent biological experiments.

To further investigate how medium composition affects AQ256 biosynthesis, we also evaluated AQ256 production in the AQ-04 strain using M9 medium containing 10 g/L glucose and 22 mM malonate as the carbon source. After 72 h of cultivation in M9 medium, AQ-04 exhibited an OD₆₀₀ of 1.2 ± 0.1 and produced 1.4 ± 0.0 mg/L AQ256 (Figure S4). These values were lower than those obtained when AQ-04 was cultivated in LB medium. Compared to minimal M9 medium conditions, the nutrient-rich LB medium provides a more favorable environment for both cellular growth and efficient AQ256 production.

Effect of Initial Cell Density on AQ256 Production

Our experiments demonstrated that most AQ256 production occurred within the first 24 h of cultivation. Analysis of CoA derivatives showed a rapid increase in the intracellular CoA concentration at the start of cultivation, followed by a sharp decline, which is consistent with a previous study on fatty acid biosynthesis. Therefore, to increase the number of cells involved in the production for AQ256 up to 24 h of incubation, the effects on the initial cell density (OD₆₀₀) were investigated.

The AQ-04 strain was cultured in LB medium supplemented with 5 g/L yeast extract, and the cells were harvested 6 h after IPTG induction. Harvested cells were inoculated into fresh medium (LB + 5 g/L yeast extract + 22 mM malonate) at OD₆₀₀ values of 0.5, 2.3, and 4.4. The results showed that after 72 h of cultivation, both OD₆₀₀ and malonate consumption increased proportionally with the initial cell density. At an initial OD₆₀₀ of 4.4, the final OD₆₀₀ reached 7.3 ± 0.1, and 12.8 mM malonate was consumed (Figure A,B). Along with the higher initial cell densities, AQ256 production increased, reaching 23.9 ± 0.9 mg/L at an initial OD₆₀₀ of 4.4, which was 2.2-fold higher than at an initial OD₆₀₀ of 0.5 and 9.6-fold higher than the previous study by Cummings et al. (2.5 mg/L) (Figure C).

6.

Effects of initial cell density on AQ256 production in the AQ-04 strain. Time course of (A) growth, (B) malonate consumption, and (C) AQ256 production by AQ-04 at different initial OD₆₀₀. (D) Ratio of intracellular and extracellular AQ256 levels after 72 h of cultivation. Results are presented as means ± standard deviation of three independent biological experiments.

During AQ-04 cultivation, both the cells and the culture medium exhibited a reddish-brown coloration. Therefore, we measured AQ256 concentration in both the intracellular and extracellular fractions after 72 h of cultivation (Figure D). The results indicated that the ratio of intracellular to extracellular AQ256 was approximately 46%:54%, regardless of the initial cell density, suggesting that nearly half of the AQ256 accumulated inside the cells. These findings indicated that increasing the initial cell density enhanced early cell growth and malonate uptake, thereby improving AQ256 production efficiency.

Finally, to investigate whether metabolic redirection in the AQ-04 strain through inhibition of fatty acid synthesis could enhance AQ256 production, we conducted cultivation under the condition that yielded the highest AQ256 production (initial OD₆₀₀ = 4.4) with the addition of 50 μM cerulenin, a known inhibitor of fatty acid synthase. However, after 72 h of cultivation, the AQ256 production decreased (4.6 ± 0.2 mg/L) compared to that without cerulenin supplementation (Figure S5). Although the reason for the decreased AQ256 production upon cerulenin addition remains unclear, Omura suggested that cerulenin may inhibit the reactions of PKS.

Discussion

Previous studies have primarily focused on enhancing the endogenous malonyl-CoA biosynthetic pathways to improve anthraquinone production. However, the effects of introducing an exogenous malonate assimilation pathway on anthraquinone production have not yet been investigated. Therefore, in this study, we first investigated the effect of strengthening the exogenous malonyl-CoA supply by introducing a malonate assimilation pathway on the production of AQ256. The results showed that the AQ-02 strain carrying the malonate assimilation pathway exhibited a 2.9-fold increase in AQ256 production compared to the AQ-01 strain, which lacked the malonate assimilation pathway (Figure A). In contrast, a previous study reported that overexpression of madLM and mcs in E. coli led to a 6.8-fold increase in (2S)-naringenin production. This difference in the rate of increase in production may be attributable to the different numbers of malonyl-CoA molecules required: the biosynthesis of (2S)-naringenin requires three molecules of malonyl-CoA, whereas AQ256 production requires eight. Therefore, in the present study, we developed the AQ-04 strain, in which the endogenous malonyl-CoA supply pathway was also enhanced, to further increase malonyl-CoA availability. The AQ-04 strain showed a 1.9-fold increase in AQ256 production compared to the AQ-02 strain (Figure B). These results indicate that enhancement of malonyl-CoA supply is effective in improving AQ256 production in E. coli. However, even in the AQ-04 strain, the intracellular level of CoA-related compounds, including malonyl-CoA, rapidly decreased after 24 h of cultivation (Figure ). In future studies, time course monitoring of the levels of overexpressed Pp-CoaA and intracellular Cg-Acc in the AQ-04 strain using proteomic analysis may help to clarify the underlying cause. To the best of our knowledge, this study is the first successful demonstration that the integration of exogenous malonate assimilation and endogenous malonyl-CoA biosynthetic pathway enhancement can synergistically increase the production of a malonyl-CoA-derived compound.

Despite the introduction of the malonate assimilation pathway, 14.5 mM malonate remained unconsumed in the culture medium of AQ-04 even after 72 h of cultivation in LB medium supplemented with 22 mM malonate (Figure C). The limited uptake of malonate under glucose-free conditions suggests that the conversion of malonate to malonyl-CoA is the rate-limiting step in the malonate assimilation pathway. This reaction is catalyzed by malonyl-CoA synthetase (MCS), an ATP-dependent enzyme. , Intracellular ATP levels were measured, which showed a gradual decrease after 9 h of cultivation (Figure S6), potentially limiting the activity of MCS and subsequently reducing the efficiency of malonate utilization. Consequently, the slow conversion of intracellular malonate to malonyl-CoA likely leads to malonate accumulation inside the cells, which in turn reduces the driving force for further malonate uptake from the medium. In contrast, when glucose was added to the medium to enhance CoA biosynthesis, the AQ-04 strain completely consumed all 22 mM malonate (Figure B). This suggests that glucose supplementation provided sufficient ATP to support MCS activity. Unexpectedly, the addition of glucose decreased AQ256 production (Figure C). Additionally, the total intracellular concentration of CoA-related compounds was lower after 9 h of cultivation under glucose-supplemented conditions than that under glucose-free conditions (Figure S7). These results suggest that glucose supplementation activated the TCA cycle, leading to the preferential utilization of acetyl-CoA for bacterial growth, whereas malonyl-CoA was diverted toward fatty acid biosynthesis required for cell proliferation. Indeed, under glucose-supplemented conditions, bacterial growth continued beyond 18 h of culture (Figure A). Given that glucose is an inexpensive carbon source, the development of engineering strategies to enhance AQ256 production from glucose remains an important challenge for future research.

An interesting phenomenon observed under glucose supplementation was the absence of reddish-brown coloration in the supernatant, which is typically associated with AQ256 production. A comparative analysis of intracellular and extracellular AQ256 levels revealed that under glucose-supplemented conditions, a larger proportion of AQ256 accumulated intracellularly (Figure S8). Although the exact reason for this remains unclear, the substrate specificity of various E. coli transporters can change depending on the environmental conditions. Furthermore, small organic acids such as acetate generated by glucose metabolism are preferentially exported over AQ256.

In addition to glucose supplementation, we also examined the effects of yeast extract supplementation. The addition of 5 g/L yeast extract to LB medium resulted in an increase in AQ256 production (Figure C). This improvement may be attributed to the presence of CoA biosynthesis precursors such as β-alanine and pantothenic acid in yeast extract. , These compounds may synergistically enhance the CoA supply in combination with the expression of Pp-coaA (Figure A).

To further enhance AQ256 production, improving the enzymatic activity of the AQ256 PKS is also critical. Yang et al. reported that the coexpression of polyketide chain-synthesizing enzymes (antD, antE, antF, antB, and antG; Figure A) and heterogeneous cyclases (zhuI and zhuJ from Streptomyces sp. R1128) in E. coli BAP1 resulted in the production of 88.0 mg/L flavokermesic acid and oktaketide. These findings suggest that in AQ256 biosynthesis, the enzymatic reactions for cyclization and subsequent acetyl-ACP cleavage (catalyzed by antA, antH, antC, and antI gene products; Figure A) might be the rate-limiting steps. Future studies should focus on optimizing the expression levels of these enzymes and/or enhancing their catalytic activities through protein engineering to improve AQ256 production.

In addition to enzymatic activity, limitations in the expression levels and stability of AntA–I proteins may also influence the efficiency of AQ256 biosynthesis, particularly during the stationary phase. Therefore, relative quantification of AntA–I protein expression at different cultivation time points using proteomic approaches may help clarify whether the reduced AQ256 production observed at later stages of cultivation is attributable to insufficient protein abundance.

Moreover, in the initially designed strain, the transfer of malonyl-CoA to ACP relied on the endogenous MCAT from E. coli. Since the compatibility between ACP and acyltransferase is known to affect the efficiency of polyketide biosynthesis, this step was considered a potential rate-limiting factor. Accordingly, we introduced the MCAT gene derived from P. laumondii TTO1 (Pl-mcat) into the AQ-04 strain. Indeed, introduction of Pl-mcat into the AQ-04 strain increased AQ256 production by 1.3-fold, reaching 16.3 ± 1.8 mg/L (Figure S9). This improvement may be attributed either to elevated MCAT expression or to the P. laumondii-derived MCAT being more suitable for transferring malonyl-CoA to ACP than the endogenous E. coli MCAT.

In this study, we established a method for high-yield production of AQ256 in E. coli. This system has the potential to be applied for the high-yield production of other valuable anthraquinone derivatives by incorporating other modification enzymes into the AQ256 biosynthetic system. The malonyl-CoA enhancement strategy established in this study may also be applicable to broader biosynthesis of polyketides, including other anthraquinones.

Methods

Strains and Media

The E. coli strains used in this study are listed in Table S1. The E. coli DH5α strain was used for plasmid construction and amplification. E. coli strains were routinely cultivated in LB medium (10 g/L tryptone, 5 g/L yeast extract, and 5 g/L NaCl; Nacalai Tesque, Kyoto, Japan) with appropriate antibiotics (50 μg/mL chloramphenicol, 100 μg/mL kanamycin, 50 μg/mL spectinomycin, and 100 μg/mL ampicillin) at 37 °C with shaking at 200 rpm.

Plasmid Construction

The plasmids and polymerase chain reaction (PCR) primers used in this study are listed in Tables S2 and S3. All synthetic genes were synthesized using GeneArt (Thermo Fisher Scientific, Waltham, MA, USA). All plasmids were constructed using an In-Fusion HD cloning kit (Takara Bio, Mountain View, CA, USA) according to the manufacturer’s instructions.

The pACYC-AQ256 plasmid was constructed as follows: AQ256 biosynthetic genes from the P. laumondii subsp. laumondii TTO1 strain (MIBiG no. BGC0000196) was synthesized in four fragments (Figure S10). The DNA fragment encoding antA–antC genes was amplified from the synthetic gene by PCR using AQ256_fragment4_Fw_NdeI and AQ256_fragment4_Rv_Xho primers. The antA–antC fragment was inserted into the pACYCDuet-1 vector, which was double-digested with NdeI and XhoI to generate the pACYC-AntA-C plasmid. The DNA fragment encoding antD–antI genes was amplified from the three synthetic gene fragments using AQ256_fragment1_Fw_NcoI, AQ256_fragment1_Rv, AQ256_fragment2_Fw, AQ256_fragment2_Rv, AQ256_fragment3_Fw, and AQ256_fragment3_Rv_PstI primers. The antD–antI fragments were inserted into pACYC-AntA-C, which was double-digested with NcoI and PstI, to generate the pACYC-AQ256 plasmid.

The pACYC-AQ256-MCAT plasmid was constructed as follows: The DNA fragment encoding the malonyl-CoA acyl carrier protein transacylase (MCAT) gene from P. laumondii TTO1 was amplified from the synthetic gene using the pACYC-MCAT-Fw and pACYC-MCAT Rv primers. The resulting MCAT fragment was inserted into pACYC-AQ256, which had been double-digested with XhoI and AvrII, to generate pACYC-AQ256-MCAT.

The pMW219-MadLM plasmid was constructed as follows: The DNA fragment encoding the madLM gene from M. rubra was amplified from the synthetic gene using madLM_Fw and madLM_Rv primers. The DNA fragments encoding the T7 promoter and T7 terminator were amplified from the pETDuet-1 vector using madLM_pro_Fw, madLM_pro_Rv, madLM_ter_Fw, and madLM_ter_Rv. The madLM, T7 promoter, and T7 terminator fragments were inserted into pMW219 DNA (Nippon Gene, Tokyo, Japan), which was double-digested with EcoRI and BamHI to generate the pMW219-MadLM plasmid.

The pET-mcs plasmid was constructed as follows: The mcs gene from B. japonicum was codon-optimized for E. coli and synthesized. The DNA fragment encoding the mcs gene was amplified from the synthetic gene using the pETDuet_MCS_Fw and pETDuet_MCS_Rv primers. The mcs fragment was inserted into pETDuet-1, which was double-digested with NcoI and BamHI to generate the pET-mcs plasmid.

The pCDF-CoaA plasmid was constructed as follows: The CoaA gene from P. putida was codon-optimized for E. coli and synthesized. The DNA fragment encoding coaA was amplified from the synthetic gene using the pCDF-coaA Fw and pCDF-coaA Rv primers. The coaA fragment was inserted into pCDFDuet-1, which was double-digested with NdeI and AvrII to generate pCDF-CoaA.

The plasmid pCDF-Acc-CoaA was constructed as follows: The DNA fragments encoding accBC, accE, and dtsR1 genes were amplified from the C. glutamicum ATCC 13032 genome using the pCDF-accBC Fw, pCDF-accBC Rv, pCDF-accE Fw, pCDF-accE Rv, pCDF-dtsR1 Fw, and pCDF-dtsR1 Rv primers. The DNA fragment encoding coaA was amplified from the synthetic gene using the pCDF-Acc-coaA Fw and pCDF-Acc-coaA Rv primers. The dtsR1 fragment was inserted into pCDFDuet-1, which was double-digested with NdeI and AvrII to generate the pCDF-dtsR1 plasmid. The accBC fragment was inserted into pCDF-dtsR1, which was digested with AvrII to generate the pCDF-dtsR1-AccBC plasmid. The accE fragment was inserted into pCDF-dtsR1-AccBC, which was digested with AvrII to generate pCDF-dtsR1-AccBCE. The coaA fragment was inserted into pCDF-dtsR1-AccBCE, which was digested with AvrII to generate pCDF-Acc-CoaA.

Transformation of E. coli

All plasmids were used to transform E. coli strains via electroporation.

Batch Fermentation

Precultivation of E. coli strains was conducted in 3 mL of LB medium containing the appropriate antibiotics in a shaker incubator at 37 °C and 200 rpm overnight. Precultured E. coli cells were inoculated (OD₆₀₀ = 0.05) in 20 or 35 mL LB medium containing 0.1 M MOPS (pH 7.0, adjusted with NaOH) with appropriate antibiotics in 100 mL baffled Erlenmeyer flasks. The cultures were initially incubated at 37 °C and 200 rpm in a shaking incubator (Bio Shaker BR-43FL; Taitec, Saitama, Japan). Once the culture reached the logarithmic growth phase (OD₆₀₀ = 0.6–0.8), 0.1 mM isopropyl β-d-thiogalactopyranoside (IPTG, Nacalai Tesque, Kyoto, Japan) and either 7.3 mM or 22 mM disodium malonate (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) were added, and the incubation temperature was changed to 20 °C with continued cultivation at 180 rpm.

To evaluate the effect of medium composition on AQ256 production, LB medium with 10 g/L glucose, LB medium with an additional 5 g/L yeast extract (10 g/L tryptone, 10 g/L yeast extract, and 5 g/L NaCl), or M9 minimal medium (6.78 g/L Na₂HPO₄, 3.0 g/L KH₂PO₄, 0.5 g/L NaCl, 1.0 g/L NH₄Cl, 2 mM MgSO₄, and 0.2 mM CaCl₂) supplemented with 10 g/L glucose were also used for batch cultivation. These media for batch cultivation were supplemented with 0.1 M MOPS (pH 7.0) and 22 mM disodium malonate.

To investigate the effect of initial OD₆₀₀ in batch culture, strain AQ-04 was cultivated under the conditions described above in LB medium supplemented with yeast extract and disodium malonate [10 g/L tryptone, 10 g/L yeast extract, 5 g/L NaCl, 22 mM disodium malonate, and 0.1 M MOPS (pH 7.0)]. After 6 h of induction with IPTG, the cells were harvested by centrifugation at 3500g for 5 min. Harvested cells were washed once with LB medium and resuspended in an appropriate volume of LB medium to prepare a concentrated cell suspension (OD₆₀₀ = 24). This suspension was then inoculated into fresh medium at designated initial OD₆₀₀ values of 0.5, 2.3, or 4.4. Cerulenin was added as needed to a final concentration of 50 μM.

AQ256 Extraction

A 200 μL sample of the culture medium containing E. coli cells was transferred to a 1.5 mL tube, frozen at – 30 °C, and subsequently thawed. Then, 200 μL of 1-butanol and 50 μL of 1 M HCl were added, and the mixture was vigorously shaken. The solution was mixed at room temperature using a microtube mixer at 2000 rpm for 10 min and then centrifuged at 10,000 rpm for 3 min. Under acidic conditions, the upper layer turned yellow, whereas the lower layer remained colorless. The upper layer was collected and mixed 1:1 (v/v) with methanol to prepare the AQ256 extract solution, which was used for analysis.

Intracellular CoA Compound Extraction

Extraction of intracellular metabolites from E. coli and sample preparation were performed as follows: Quenching was performed by adding an equal volume of ice-cold 40% ethanol solution containing 0.8% NaCl to the collected 2 mL culture. The mixture was centrifuged at 3500 rpm, – 16 °C, for 15 min, and the cell pellet was collected. For extraction, 0.375 mL of ethanol was added, followed by vigorous vortexing and incubation at 70 °C for 15 min. The sample was then cooled on ice for 2 min, and 0.375 mL of ultrapure water was added and mixed by inversion. Subsequently, chloroform (0.5 mL) was added, and the mixture was vortexed. The mixture was centrifuged at 4000g, 4 °C, for 5 min, and the supernatant was collected. The collected samples were vacuum-dried using a FreeZone 2.5 Plus system (Labconco, Kansas City, MO, USA), and the dried metabolites were stored at – 80 °C. Before analysis, the metabolites were redissolved in ultrapure water at an appropriate concentration.

Analytical Methods

The concentrations of glucose and malonate in the culture medium were analyzed using high-performance liquid chromatography (HPLC) (Shimadzu Corporation, Kyoto, Japan) equipped with an Aminex HPX-87H column (7.8 mm × 300 mm, particle size 9 μm; Bio-Rad). The details of this method have been reported previously.

The AQ256 standard was synthesized using ChemSpace (Riga, Latvia). The concentration of AQ256 was analyzed by HPLC using a Cosmosil 5C18-AR II column (2.0 mm × 150 mm) (Nacalai Tesque, Inc., Kyoto, Japan) with the column oven set at 40 °C. The sample injection volume was 10 μL. The mobile phase comprised acetonitrile (A) and 0.1% formic acid in water (B). The mobile phase flow rate was 0.3 mL/min. The gradient program started at 40% A for 2 min, increased to 98% A over 10 min, held for 2 min, returned to 40% A over 1 min, and maintained for 20 min. The detection was performed at 284 nm.

The concentrations of intracellular CoA compounds and ATP were analyzed by LC-MS/MS (Agilent 1260/6460; Agilent Technologies, Palo Alto, CA, USA). Separation was performed using an Atlantis T3 column (2.1 mm × 150 mm, 3 μm) (Waters, Milford, MA, USA) with 5% methanol/4 mM dibutylammonium acetate (DBAA) as mobile phase A and 75% acetonitrile as mobile phase B. The flow rate was set at 0.36 mL/min. The gradient program was as follows: an initial hold at 100% A for 1 min, a decrease to 20% A over 5 min, further reduction to 0% A over 3 min, maintained for 2.5 min, returned to 100% A, and a final hold for 2.5 min. MS analysis was performed in negative mode, with the following transition conditions (m/z_parent → m/z_daughter): CoA, 766.1 → 408; acetyl-CoA, 808.1 → 408; malonyl-CoA, 852.1 → 808.1; ATP, 506.0 → 158.8.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acssynbio.5c00354.

• Additional figures and lists of strains, plasmids, and primers used in this study; (Figure S1) effect of malonate concentration on AQ256 production in strain AQ-02; (Figure S2) comparison of AQ256 production in strains AQ-03 and AQ-04 with or without malonate supplementation; (Figure S3) time course of glucose concentration in the culture supernatant of strain AQ-04; (Figure S4) AQ256 production and cell growth of strain AQ-04 in M9 medium supplemented with glucose and malonate; (Figure S5) effect of cerulenin supplementation on AQ256 production in strain AQ-04; (Figure S6) time course of intracellular ATP concentration in strain AQ-04; (Figure S7) time course of CoA-related metabolites in strain AQ-04 with or without glucose supplementation; (Figure S8) ratio of intracellular to extracellular AQ256 in strain AQ-04 with or without glucose supplementation; (Figure S9) AQ256 production in strain AQ-05 overexpressing Pl-mcat; (Figure S10) organization and predicted functions of the ant biosynthetic gene cluster from Photorhabdus laumondii TTO1; (Table S1) list of AQ256-producing strains; (Table S2) list of plasmids; (Table S3) list of primers (PDF)

T.S., S.A., T.B., and T.H. designed the experiments. T.S., M.T., and I.T. performed the experiments. All authors discussed the results. T.S., S.A., T.B., and T.H. wrote the manuscript. S.A., T.B., and T.H. supervised all aspects of the study.

The authors declare no competing financial interest.