Reversal of multidrug resistance in vitro and in vivo by 5-N-formylardeemin,a new ardeemin derivative

Because multidrug resistance (MDR) is a serious impediment to the use of chemotherapy in treating cancer patients, great efforts have been made to search for effective MDR-reversing agents. We have developed a brand new synthetic ardeemin derivative, 5-N-formylardeemin, and investigated the activity of which in reversing MDR in MDR cancer cell lines derived from human breast cancer (MCF-7-R) or lung cancer (A549-R). 5-N-formylardeemin strongly enhanced the anti-cancer efficacy of doxorubicin, vincristine through potentiation of apoptosis in both MCF-7-R and A549-R at relatively noncytotoxic concentrations in vitro. Mechanistic studies showed that 5-N-formylardeemin inhibited the expression of MDR-1 (P-gp) and increased the intracellular accumulation of cytotoxic drugs in the MDR cells, suggesting that 5-N-formylardeemin reverses MDR activities through inhibiting MDR-1 expression. Interestingly, 5-N-formylardeemin also sensitized the parent wild-type cancer cells toward these chemotherapeutic agents to various extents. Importantly, in vivo studies demonstrated that 5-N-formylardeemin significantly improved the therapeutic effects of doxorubicin in nude mice bearing A549-R xenografts, which was associated with reduced expression of MDR-1 protein level and increased apoptosis in tumor tissues. These results underscore 5-N-formylardeemin as a potential sensitizer for chemotherapy against multidrug resistant cancers.     

Cytotoxic Activities of Amino Acid‐Conjugate Derivatives of Abietane‐Type Diterpenoids against Human Cancer Cell Lines

Nine amino acid conjugate derivatives, each 2 – 10 and 12 – 20 , were prepared from abietic acid ( 1 ) and dehydroabietic acid ( 11 ), respectively, and they were evaluated for their cytotoxicities against four human cancer cell lines, i.e., leukemia (HL60), lung (A549), stomach (AZ521), and breast (SK‐BR‐3). All compounds showed cytotoxicities against HL60 with IC₅₀ values in the range of 2.3–37.3 μM . In addition, most of the derivatives exhibited moderate cytotoxicities against the other cancer cell lines. Among the derivatives, methyl N‐[18‐oxoabieta‐8,11,13‐trien‐18‐yl]‐L ‐tyrosinate ( 19 ) exhibited potent cytotoxic activities against four cancer cell lines with IC₅₀ values of 2.3 (HL60), 7.1 (A549), 3.9 (AZ521), and 8.1 μM (SK‐BR‐3). Furthermore, all derivatives were shown to possess high selective cytotoxic activities for leukemia cells, since they exhibited only weak cytotoxicities against normal lymphocyte cell line RPMI1788.     

Alteration of membrane lipid biophysical properties and resistance of human lung adenocarcinoma A549 cells to cisplatin

Alterations of membrane lipid biophysical properties of sensitive A₅₄₉ and resistant A₅₄₉/DDP cells to the Cis-dichlorodiammine platinum (Cisplatin) were performed by measurements of fluorescence and flow cytometry approaches using fluorescence dyes of DPH, N-AS and Merocyanine 540 (MC 540) respectively. Fatty acids of membrane lipid of the two cell lines were analyzed by gas chromatography. The results indicated clearly that fluorescence polarization (P) of the DPH probe is 0.169 for the sensitive A₅₄₉ cell and 0.194 for the resistant A₅₄₉/DDP cells. Statistical analysis showed significant difference between the two cell lines. The polarizations of 2-AS and 7-AS which reflect the fluidity of surface and middle of lipid bilayer are 0.134 and 0.144 for the sensitive A549 cells as well as 0.171 and 0.178 for the resistant A₅₄₉/DDP cells respectively, but there is no significant difference of the polarization of 12-AS between the two cell lines. This shows that alterations of the membrane fluidity of both cells were mainly located on the surface and middle of the lipid bilayer. In addition, the packing density of phospholipid molecules in the membrane of the two cell lines detected by MC540 probe indicated that lipid packing of A₅₄₉ cell membranes was looser than that of the A₅₄₉/DDP cells. And unsaturation degree of plasma membrane fatty acids of the A₅₄₉/DDP cells was also lower than that of A₅₄₉ cells. Taken together, it was proposed that the alteration of membrane lipid biophysical state may be involved in the resistance of A₅₄₉/DDP cells to cisplatin.     

Inhibition of P‐Glycoprotein‐Induced Multidrug Resistance by a Clerodane‐Type Diterpenoid from Sindora sumatrana

The aim of the present study was to investigate the effects of di‐ and sesquiterpenoids isolated from the pods of Sindora sumatrana Miq. (Leguminosae) on P‐glycoprotein (P‐gp) function in an adriamycin‐resistant human breast cancer cell line, MCF‐7/ADR. Over‐expression of P‐gp is known to be one of the mechanisms involved in multidrug resistance (MDR), which is a major obstacle in clinical cancer treatment. Among six di‐ and sesquiterpenoids extracted from S. sumatrana, (+)‐7β‐acetoxy‐15,16‐epoxycleroda‐3,13(16),14‐trien‐18‐oic acid ( 1 ) showed a strong P‐gp inhibitory effect, as great as that of verapamil, a representative P‐gp inhibitor. Compound 1 enhanced daunomycin accumulation more than fourfold and significantly decreased daunomycin efflux compared with control, resulting in a decrease in the IC₅₀ value for daunomycin. These results suggest that compound 1 inhibits the functioning of P‐gp and, therefore, can be developed as an MDR‐reversing agent.     

Effect of pharynx epithelial cells surface desialylation on receptor‐mediated adherence of Staphylococcus aureus

Aims: To characterize the interaction between cell surface carbohydrates and Staphylococcus aureus. Methods and Results: In the present study, in vitro adherence of S. aureus to Detroit 562 cells, amount of cell surface desialylation and effect of subterminal monosaccharides on desialylated glycoproteins on adherence was studied with colony counting, HPLC, fluorescence microscopy and fluorometric techniques. According to our findings, S. aureus adherence to pharynx cells was enhanced (40%) after neuraminidase treatment, and neuraminidase also cleave great amount of Detroit 562 cells surface sialic acid (39–60%). Adherence assay with various monosaccharides‐pretreated bacteria, and lectin competitive inhibition, showed that the residual subterminal galactose, fucose and N‐acetyl‐d ‐glucosamine remaining on desialylated Detroit 562 cell surface glycoproteins responsible for this binding. Conclusion: The results are the first to show that galactose, fucose and N‐acetyl‐d ‐glucosamine remaining on desialylated pharynx cell surface glycoproteins serve as the adhesine receptors for S. aureus. Significance and Impact of the Study: This study may explain the predisposition of severe S. aureus pneumonia complication in respiratory viral infections.     

Establishment and molecular characterization of decitabine‐resistant K562 cells

The clinical activity of decitabine (5‐aza‐2‐deoxycytidine, DAC), a hypomethylating agent, has been demonstrated in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) patients. However, secondary resistance to this agent often occurs during treatment and leads to treatment failure. It is important to clarify the mechanisms underlying the resistance for improving the efficacy. In this study, by gradually increasing concentration after a continuous induction of DAC, we established the DAC‐resistant K562 cell line (K562/DAC) from its parental cell line K562. The proliferation and survival rate of K562/DAC was significantly increased, whereas the apoptosis rate was remarkably decreased than that of K562 after DAC treatment. In K562/DAC, a total of 108 genes were upregulated and 118 genes were downregulated by RNA‐Seq. In addition, we also observed aberrant expression of DDX43/H19/miR‐186 axis (increased DDX43/H19 and decreased miR‐186) in K562/DAC cells. Ectopic expression of DDX43 in parental K562 cells rendered cells resistant to the DAC. Taken together, we successfully established DAC‐resistant K562 cell line which can serve as a good model for investigating DAC resistance mechanisms, and DDX43/H19/miR‐186 may be involved in DAC resistance in K562.     

Lignans from the Twigs of Euonymus alatus (Thunb.) Siebold and Their Biological Evaluation

A new sesquilignan, euonymolin A ( 1 ), and six known lignans, (?)‐de‐O‐methylmagnolin ( 2 ), (+)‐de‐O‐methylepimagnolin A ( 3 ), (+)‐syringaresinol ( 4 ), (+)‐pinoresinol ( 5 ), (+)‐medioresinol ( 6 ), and (+)‐lariciresinol 4′‐O‐β‐d ‐glucopyranoside ( 7 ), were isolated from the twigs of Euonymus alatus (Thunb .) Siebold (Celastraceae). The structures of the isolated compounds were elucidated based on spectroscopic analyses, including extensive 1D‐ and 2D‐NMR techniques, HR‐MS analysis and circular dichroism (CD) data, and the literature data. All of the isolated compounds were evaluated for antiproliferative activity against A549, SK‐OV‐3, SK‐MEL‐2, and HCT‐15 cell lines and inhibition of nitric oxide (NO) production in a lipopolysaccharide (LPS)‐activated BV2 cell line. All compounds showed cytotoxicity against the SK‐MEL‐2 cell line with IC₅₀ values of 23.24 – 48.14 μm and inhibited NO production in LPS‐activated BV‐2 cells with IC₅₀ values of 6.75 – 23.53 μm .     

Non‐invasive quantification of brain glycogen absolute concentration

The only currently available method to measure brain glycogen in vivo is ¹³C NMR spectroscopy. Incorporation of ¹³C‐labeled glucose (Glc) is necessary to allow glycogen measurement, but might be affected by turnover changes. Our aim was to measure glycogen absolute concentration in the rat brain by eliminating label turnover as variable. The approach is based on establishing an increased, constant ¹³C isotopic enrichment (IE). ¹³C‐Glc infusion is then performed at the IE of brain glycogen. As glycogen IE cannot be assessed in vivo, we validated that it can be inferred from that of N‐acetyl‐aspartate IE in vivo: After [1‐¹³C]‐Glc ingestion, glycogen IE was 2.2 ± 0.1 fold that of N‐acetyl‐aspartate (n = 11, R² = 0.77). After subsequent Glc infusion, glycogen IE equaled brain Glc IE (n = 6, paired t‐test, p = 0.37), implying isotopic steady‐state achievement and complete turnover of the glycogen molecule. Glycogen concentration measured in vivo by ¹³C NMR (mean ± SD: 5.8 ± 0.7 μmol/g) was in excellent agreement with that in vitro (6.4 ± 0.6 μmol/g, n = 5). When insulin was administered, the stability of glycogen concentration was analogous to previous biochemical measurements implying that glycogen turnover is activated by insulin. We conclude that the entire glycogen molecule is turned over and that insulin activates glycogen turnover.     

Synthesis of Lanostane‐Type Triterpenoid N‐Glycosides and Their Cytotoxicity against Human Cancer Cell Lines

Seventeen lanostane‐type triterpenoid derivatives ( 2 – 18 ), including 11N‐glycosides ( 8 – 18 ), were synthesized from the natural triterpenoid, lanosterol ( 1 ), and were evaluated for their cytotoxicity against the human cancer cell lines, HL‐60, A549, and MKN45, as well as the normal human lung cells, WI‐38. Among them, N‐β‐d ‐2‐acetamido‐2‐deoxyglucoside ( 10 ) showed cytotoxicity against HL‐60, A549, MKN45, and WI‐38 cells (IC₅₀ 0.0078 – 2.8 μm ). However, N‐β‐d ‐galactoside ( 12 ) showed cytotoxicity against HL‐60 and MKN45 cells (IC₅₀ 0.0021 – 4.0 μm ), but not the normal WI‐38 cells. Furthermore, Western blot analysis suggested that 12 induces apoptosis by activation of caspases‐3, 8, and 9. These results will be useful for the synthesis of other tetracyclic triterpenoids or steroid N‐glycosides to increase their cytotoxicity and apoptosis‐inducing activities.     

The Effect of TIGAR Knockdown on Apoptotic and Epithelial‐Mesenchymal Markers Expression in Doxorubicin‐Resistant Non‐Small Cell Lung Cancer A549 Cell Lines

Resistance to chemotherapeutic drugs is a critical problem in cancer therapy, but the underlying mechanism has not been fully elucidated. TP53‐induced glycolysis regulatory phosphatase (TIGAR), an important glycolysis and apoptosis regulator, plays a crucial role in cancer cell survival by protecting cells against oxidative stress‐induced apoptosis. In the present study, we investigated whether TIGAR is involved in epithelial‐mesenchymal transition (EMT) in doxorubicin (DOX)‐resistant human non‐small cell lung cancer (NSCLC), A549/DOX cells. We found that the expression of TIGAR was significantly higher in A549/DOX cells than in the parent A549 cell lines. siRNA‐mediated TIGAR knockdown reduced migration, viability and colony survival of doxorubicin‐resistant lung cancer cells. Also, TIGAR knockdown decreased pro‐survival protein Bcl‐2 and increased pro‐apoptotic Bax and cleaved poly (ADP‐ribose) polymerase (PARP). Moreover, TIGAR depletion significantly up‐regulated both caspase‐3 and caspase‐9 expression. Furthermore, TIGAR depletion up‐regulated the expression of E‐cadherin and down‐regulated the expression of vimentin. These results indicate that TIGAR knockdown may inhibit EMT in doxorubicin (DOX)‐resistant human NSCLC and may represent a therapeutic target for a non‐small lung cancer cells chemoresistance.     

P-糖蛋白在多药耐药的K562细胞转运苯妥英纳与卡马西平中的作用

研究证实,多药转运体与难治性癫痫耐药机制密切相关,P-糖蛋白在其中起重要作用．主要研究P-糖蛋白拮抗剂维拉帕米对P-糖蛋白过表达的K562细胞耐药性及细胞内苯妥英纳与卡马西平浓度的影响．首先建立了P-糖蛋白高表达的K562/Dox(阿霉素诱导)耐药细胞株,比较耐药细胞株和P-糖蛋白表达阴性的K562细胞株对苯妥英纳和卡马西平的耐药性,并观察给予维拉帕米后,耐药细胞内抗癫痫药物的浓度变化．结果发现,苯妥英纳和卡马西平对K562/Dox细胞株的半数抑制浓度(IC₅₀)明显高于K562细胞株,加入维拉帕米后,苯妥英纳和卡马西平对K562/Dox 细胞的IC₅₀明显下降,逆转倍数分别为2.5和1.5．进一步研究发现,K562/Dox细胞内苯妥英纳和卡马西平的浓度均显著少于其药敏K562细胞,仅分别为正常K562细胞的23.6%和32.2%．当加入维拉帕米后,K562/Dox细胞内抗癫痫药物浓度明显升高(P < 0.05)．由此证明,高表达的P-糖蛋白参与了细胞的药物转运,在难治性癫痫的耐药机制中扮演重要角色．     

Inducible character of β‐xylanase in a hyperproducing mutant of Thermomyces lanuginosus

Ten strains of Thermomyces lanuginosus from various culture collections were evaluated for extracellular endo‐β‐1,4‐xylanase production. The best xylanase producer (5771±173 nkat/mL) T. lanuginosus SK, was subjected to UV and N‐methyl‐N‐nitro‐N‐nitrosoguanidine mutagenesis. A mutant strain T. lanuginosus MC134, that showed on oatspelts xylan a 1.5 fold higher xylanase production than the parent strain SK, was subjected to a study of the regulation of xylanase synthesis during growth on various carbohydrates and during induction in glucose‐grown cells. In the growth experiments the highest production of xylanase was observed in the presence of xylans, however, an appreciable amount of the enzyme, about 10%, was also produced during growth on xylose. Xylobiose was found to be the most efficient xylanase inducer in the glucose‐grown cells. Its induction efficiency was followed by xylose, beechwood and birchwood xylan. Xylanase induction by polysaccharides started several hours later but proceeded for a longer time than that induced by the low molecular mass inducers, indicating that the polysaccharides serve as more sustainable source of inducers and that they have to be first hydrolyzed by the low level of constitutively synthesized xylanase. The repression of the induction of xylanase by glucose confirmed that the xylanase synthesis in the mutant strain is similar to the parent strain and exhibits an induction‐repression regulation mechanism.     

Limonoids and Flavonoids from the Flowers of Azadirachta indica var. siamensis,and Their Melanogenesis‐Inhibitory and Cytotoxic Activities

A new limonoid, 7‐O‐acetyl‐7‐O‐debenzoyl‐22‐hydroxy‐21‐methoxylimocinin ( 2 ), and two new flavonoids, 3′‐(3‐hydroxy‐3‐methylbutyl)naringenin ( 7 ) and 4′‐O‐methyllespedezaflavanone C ( 9 ), along with nine known compounds, including two limonoids, 1 and 3 , and seven flavonoids, 4 – 6, 8 , and 10 – 12 , were isolated from a MeOH extract of the flowers of Azadirachta indica A.Juss. var. siamensis Valeton (Siamese neem tree; Meliaceae). The structures of new compounds were elucidated on the basis of extensive spectroscopic analysis and comparison with literature data. All of these compounds were evaluated for their melanogenesis‐inhibitory activities in B16 melanoma cells induced with α‐melanocyte‐stimulating hormone (α‐MSH). Compound 2 (16.9% melanin content at 30 μM ), 6‐deacetylnimbin ( 3 ; 49.6% melanin content at 100 μM ), and kaempferide ( 10 ; 41.7% melanin content at 10 μM ) exhibited inhibitory effects with no, or almost no, toxicity to the cells (81.0–111.7% cell viability). In addition, evaluation of their cytotoxic activities against HL60, A549, AZ521, and SK‐BR‐3 human cancer cell lines, isoazadironolide ( 1 ), 4′‐O‐methyl‐8‐prenylnaringenin ( 5 ), euchrestaflavanone A ( 8 ), 9 , and 3‐methoxy‐3′‐prenylnaringenin ( 12 ) revealed potent cytotoxicities against one or more cell lines with IC₅₀ values in the range of 4.5–9.9 μM .     

Successful selection of an infection‐protective anti‐Staphylococcus aureus monoclonal antibody and its protective activity in murine infection models

Recent clinical trials to develop anti‐methicillin‐resistant Staphylococcus aureus (MRSA) therapeutic antibodies have met unsuccessful sequels. To develop more effective antibodies against MRSA infection, a panel of mAbs against S. aureus cell wall was generated and then screened for the most protective mAb in mouse infection models. Twenty‐two anti‐S. aureus IgG mAbs were obtained from mice that had been immunized with alkali‐processed, deacetylated cell walls of S. aureus. One of these mAbs, ZBIA5H, exhibited life‐saving effects in mouse models of sepsis caused by community‐acquired MRSA strain MW2 and vancomycin‐resistant S. aureus strain VRS1. It also had a curative effect in a MW2‐caused pneumonia model. Curiously, the target of ZBIA5H was considered to be a conformational epitope of either the 1,4‐β‐linkage between N‐acetylmuramic acid and N‐acetyl‐D‐glucosamine or the peptidoglycan per se. Reactivity of ZBIA5H to S. aureus whole cells or purified peptidoglycan was weaker than that of most of the other mAbs generated in this study. However, the latter mAbs did not have the protective activities against S. aureus that ZBIA5H did. These data indicate that the epitopes that trigger production of high‐yield and/or high‐affinity antibodies may not be the most suitable epitopes for developing anti‐infective antibodies. ZBIA5H or its humanized form may find a future clinical application, and its target epitope may be used for the production of vaccines against S. aureus infection.     

NMR studies of the relationship between the changes of membrane lipids and the cisplatin-resistance of A549/DDP cells

Changes of membrane lipids in cisplatin-sensitive A549 and cisplatin-resistant A549/DDP cells during the apoptotic process induced by a clinical dose of cisplatin (30 μM) were detected by ¹H and ³¹P-NMR spectroscopy and by membrane fluidity measurement. The apoptotic phenotypes of the two cell lines were monitored with flow cytometry. The assays of apoptosis showed that significant apoptotic characteristics of the A549 cells were induced when the cells were cultured for 24 hours after treatment with cisplatin, while no apoptotic characteristic could be detected for the resistant A549/DDP cells even after 48 hours. The results of ¹H-NMR spectroscopy demonstrated that the CH₂/CH₃ and Glu/Ct ratios of the membrane of A549 cells increased significantly, but those in A549/DDP cell membranes decreased. In addition, the Chol/CH₃ and Eth/Ct ratios decreased for the former but increased for the latter cells under the same conditions. ³¹P-NMR spectroscopy indicated levels of phosphomonoesters (PME) and ATP decreased in A549 but increased in A549/DDP cells after being treated with cisplatin. These results were supported with the data obtained from ¹H-NMR measurements. The results clearly indicated that components and properties of membrane phospholipids of the two cell lines were significantly different during the apoptotic process when they were treated with a clinical dose of cisplatin. Plasma membrane fluidity changes during cisplatin treatment as detected with the fluorescence probe TMA-DPH also indicate marked difference between the two cell lines. We provided evidence that there are significant differences in plasma membrane changes during treatment of cisplatin sensitive A549 and resistant A549/DDP cells.     

Reversal of p-glycoprotein-mediated multidrug resistance by CJX1, an amlodipine derivative, in doxorubicin-resistant human myelogenous leukemia (K562/DOX) cells

P-glycoprotein-mediated drug efflux can yield a multidrug resistance (MDR) phenotype that is associated with a poor response to cancer chemotherapy. Development of safe and effective MDR reversing agents is an important approach in the clinic. The aim of this study was to observe the effects of CJX1, an amlodipine derivative, on the inhibition of P-gp function and P-gp-mediated MDR in K562/DOX cells and parental K562 cells. Based on the flow cytometric technology, the uptake, accumulation and efflux of rhodamine123 (Rh123) were detected in these cells by measuring Rh123-associated mean fluorescence intensity (MFI). The effects of CJX1 on the doxorubicin cytotoxicity were evaluated by assaying for MTT (3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium bromide) reduction and the reversal fold (RF) values. The DNA content, percentage of apoptosis and cell cycle analysis were monitored with flow cytometry. Intracellular accumulation of doxorubicin was also assessed by the determination of doxorubicin-associated MFI. Verapamil was employed as a comparative agent. Incubation of K562/DOX cells with CJX1 caused a marked increase in uptake and a notable decrease in efflux of Rh123, No such results were found in parental K562 cells. The inhibitory effect of the agent of P-gp function was reversible, but it persisted at least for 90 min after removal of 2.5 microM CJX1 from incubation medium. The doxorubicin-induced cytotoxicity, apoptosis and cell cycle perturbations were significantly potentiated by CJX1. The intracellular accumulation of doxorubicin was enhanced in the presence of various concentrations of CJX1. The CJX1 exhibited potent effects in vitro in the reversal of P-gp-mediated MDR, suggesting that the compound may become a candidate of effective MDR reversing agent in cancer chemotherapy.     

Modular pathway engineering of key carbon‐precursor supply‐pathways for improved N‐acetylneuraminic acid production in Bacillus subtilis

N‐acetylneuraminic acid (NeuAc) is widely used as a nutraceutical for facilitating infant brain development, maintaining brain health, and enhancing immunity. Currently, NeuAc is mainly produced by extraction from egg yolk and milk, or via chemical synthesis. However, its low concentration in natural resources and its non‐ecofriendly chemical synthesis result in insufficient NeuAc production and environmental pollution, respectively. In this study, improved NeuAc production was attained via modular pathway engineering of the supply pathways of two key precursors—N‐acetylglucosamine (GlcNAc) and phosphoenolpyruvate (PEP)—and by balancing NeuAc biosynthesis and cell growth in engineered Bacillus subtilis. Specifically, we used a previously constructed GlcNAc‐producing B. subtilis as the initial host for NeuAc biosynthesis. First, we constructed a de novo NeuAc biosynthetic pathway utilizing glucose by coexpressing glucosamine‐6‐phosphate acetyl‐transferase (GNA1), N‐acetylglucosamine 2‐epimerase (AGE), and N‐acetylneuraminic acid synthase (NeuB), resulting in 0.33 g/l NeuAc production. Next, to balance the supply of the two key precursors for NeuAc biosynthesis, modular pathway engineering was performed. The optimal strategy for balancing the GlcNAc module and PEP supply module involved the use of an engineered, unique glucose and malate coutilization pathway in B. subtilis, supplied with both glucose (for the GlcNAc moiety) and malate (for the PEP moiety) at high strength. This led to 1.65 g/L NeuAc production, representing a 5.0‐fold improvement over the existing methods. Furthermore, to enhance the NeuAc yield on cell, glucose and malate coutilization pathways were engineered to balance NeuAc biosynthesis and cell growth via the blocking of glycolysis, the introduction of the Entner–Doudoroff pathway, and the overexpression of the malic enzyme YtsJ. NeuAc titer reached 2.18 g/L, with 0.38 g/g dry cell weight NeuAc yield on cell, which represented a 1.32‐fold and 2.64‐fold improvement over the existing methods, respectively. The strategy of modular pathway engineering of key carbon precursor supply pathways via engineering of the unique glucose‐malate coutilization pathway in B. subtilis should be generically applicable for engineering of B. subtilis for the production of other important biomolecules. Our study also provides a good starting point for further metabolic engineering to achieve industrial production of NeuAc by a Generally Regarded As Safe bacterial strain.