High‐level conversion of L‐lysine into 5‐aminovalerate that can be used for nylon 6,5 synthesis

L ‐Lysine is a potential feedstock for the production of bio‐based precursors for engineering plastics. In this study, we developed a microbial process for high‐level conversion of L ‐lysine into 5‐aminovalerate (5AVA) that can be used as a monomer in nylon 6,5 synthesis. Recombinant Escherichia coli WL3110 strain expressing Pseudomonas putida delta‐aminovaleramidase (DavA) and lysine 2‐monooxygenase (DavB) was grown to high density in fed‐batch culture and used as a whole cell catalyst. High‐density E. coli WL3110 expressing DavAB, grown to an optical density at 600 nm (OD₆₀₀) of 30, yielded 36.51 g/L 5AVA from 60 g/L L ‐lysine in 24 h. Doubling the cell density of E. coli WL3110 improved the conversion yield to 47.96 g/L 5AVA from 60 g/L of L ‐lysine in 24 h. 5AVA production was further improved by doubling the L ‐lysine concentration from 60 to 120 g/L. The highest 5AVA titer (90.59 g/L; molar yield 0.942) was obtained from 120 g/L L ‐lysine by E. coli WL3110 cells grown to OD₆₀₀ of 60. Finally, nylon 6,5 was synthesized by bulk polymerization of ?‐caprolactam and δ‐valerolactam prepared from microbially synthesized 5AVA. The hybrid system demonstrated here has promising possibilities for application in the development of industrial bio‐nylon production processes.     

Asymmetrically simultaneous synthesis of L‐homophenylalanine and N6‐protected‐2‐oxo‐6‐amino‐hexanoic acid by engineered Escherichia coli aspartate aminotransferase

L ‐Homophenylalanine (L ‐HPA) and N⁶‐protected‐2‐oxo‐6‐amino‐hexanoic acid (N⁶‐protected‐OAHA) can be used as building blocks for the manufacture of angiotensin‐converting enzyme inhibitors. To synthesize L ‐HPA and N⁶‐protected‐OAHA simultaneously from 2‐oxo‐4‐phenylbutanoic acid (OPBA) and N⁶‐protected‐L ‐lysine, several variants of Escherichia coli aspartate aminotransferase (AAT) were developed by site‐directed mutagenesis and their catalytic activities were investigated. Three kinds of N⁶‐protected‐L ‐lysine were tested as potential amino donors for the bioconversion process. AAT variants of R292E/L18H and R292E/L18T exhibited specific activities of 0.70±0.01 U/mg protein and 0.67±0.02 U/mg protein to 2‐amino‐6‐tert‐butoxycarbonylamino‐hexanoic acid (BOC‐lysine) and 2‐amino‐6‐(2,2,2‐trifluoro‐acetylamino)‐hexanoic acid, respectively. E. coli cells expressing R292E/L18H variant were able to convert OPBA and BOC‐lysine to L ‐HPA and 2‐oxo‐6‐tert‐butoxycarbonylamino‐hexanoic acid (BOC‐OAHA) with 96.2% yield in 8 h. This is the first report demonstrating a process for the simultaneous production of two useful building blocks, L ‐HPA and BOC‐OAHA. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Production of the Inaccessible Sesquiterpene (−)‐5‐Epieremophilene by Metabolically Engineered Escherichia coli

(?)‐5‐Epieremophilene, an epimer of the versatile sesquiterpene (+)‐valencene, is an inaccessible natural product catalyzed by three sesquiterpene synthases (SmSTPSs1‐3) of the Chinese medicinal herb Salvia miltiorrhiza, and its biological activity remains less explored. In this study, three metabolically engineered Escherichia coli strains were constructed for (?)‐5‐epieremophilene production with yields of 42.4–76.0 mg/L in shake‐flask culture. Introducing an additional copy of farnesyl diphosphate synthase (FDPS) gene through fusion expression of SmSTPS1‐FDPS or dividing the FDP synthetic pathway into two modules resulted in significantly improved production, and ultimately 250 mg of (?)‐5‐epieremophilene were achieved. Biological assay indicated that (?)‐5‐epieremophilene showed significant antifeedant activity against Helicoverpa armigera (EC₅₀=1.25 μg/cm²), a common pest of S. miltiorrhiza, implying its potential defensive role in the plant. The results provided an ideal material supply for studying other potential biological activities of (?)‐5‐epieremophilene, and also a strategy for manipulating terpene production in engineered E. coli using synthetic biology.     

Metabolic engineering of Escherichia coli for the production of 5-aminovalerate and glutarate as C5 platform chemicals

5-Aminovalerate (5AVA) is the precursor of valerolactam, a potential building block for producing nylon 5, and is a C5 platform chemical for synthesizing 5-hydroxyvalerate, glutarate, and 1,5-pentanediol. Escherichia coli was metabolically engineered for the production of 5-aminovalerate (5AVA) and glutarate. When the recombinant E. coli WL3110 strain expressing the Pseudomonas putida davAB genes encoding delta-aminovaleramidase and lysine 2-monooxygenase, respectively, were cultured in a medium containing 20 g/L of glucose and 10 g/L of l-lysine, 3.6 g/L of 5AVA was produced by converting 7 g/L of l-lysine. When the davAB genes were introduced into recombinant E. coli strainXQ56allowing enhanced l-lysine synthesis, 0.27 and 0.5 g/L of 5AVA were produced directly from glucose by batch and fed-batch cultures, respectively. Further conversion of 5AVA into glutarate could be demonstrated by expression of the P. putida gabTD genes encoding 5AVA aminotransferase and glutarate semialdehyde dehydrogenase. When recombinant E. coli WL3110 strain expressing the davAB and gabTD genes was cultured in a medium containing 20 g/L glucose, 10 g/L l-lysine and 10 g/L α-ketoglutarate, 1.7 g/L of glutarate was produced.     

Metabolic precursors enhance the production of poly‐ε‐lysine by Streptomyces noursei NRRL 5126

Poly‐ε‐lysine produced by streptomyces species is a promising biopolymer owing to its antimicrobial activity and safety for humans. A number of nutritional factors influencing poly‐ε‐lysine production by Streptomyces noursei NRRL 5126 were studied. Various metabolic precursors such as amino acids, tricarboxylic acid cycle intermediates and cofactors were investigated for improved production of poly‐ε‐lysine. Results indicated L ‐aspartate (2 mM) and citric acid (5 mM) to substantially increase the poly‐ε‐lysine production from 97.08 to 409.94 mg/L. Addition of citric acid after 24 h and L ‐aspartate after 36 h of fermentation medium further enhanced poly‐ε‐lysine production to 497.67 mg/L after a total fermentation time of 108 h. However, the use of cofactors of enzymes involved in the biosynthesis of poly‐ε‐lysine inhibited its production which is believed to be due to diversion of the flux to other metabolites.     

Strain engineering for high‐level 5‐aminolevulinic acid production in Escherichia coli

Herein, we report the development of a microbial bioprocess for high‐level production of 5‐aminolevulinic acid (5‐ALA), a valuable non‐proteinogenic amino acid with multiple applications in medical, agricultural, and food industries, using Escherichia coli as a cell factory. We first implemented the Shemin (i.e., C4) pathway for heterologous 5‐ALA biosynthesis in E. coli. To reduce, but not to abolish, the carbon flux toward essential tetrapyrrole/porphyrin biosynthesis, we applied clustered regularly interspersed short palindromic repeats interference (CRISPRi) to repress hemB expression, leading to extracellular 5‐ALA accumulation. We then applied metabolic engineering strategies to direct more dissimilated carbon flux toward the key precursor of succinyl‐CoA for enhanced 5‐ALA biosynthesis. Using these engineered E. coli strains for bioreactor cultivation, we successfully demonstrated high‐level 5‐ALA biosynthesis from glycerol (~30 g L^?1) under both microaerobic and aerobic conditions, achieving up to 5.95 g L^?1 (36.9% of the theoretical maximum yield) and 6.93 g L^?1 (50.9% of the theoretical maximum yield) 5‐ALA, respectively. This study represents one of the most effective bio‐based production of 5‐ALA from a structurally unrelated carbon to date, highlighting the importance of integrated strain engineering and bioprocessing strategies to enhance bio‐based production.     

Production of 3‐Fucosyllactose in Engineered Escherichia coli with α‐1,3‐Fucosyltransferase from Helicobacter pylori

3‐Fucosyllactose (3‐FL), one of the major oligosaccharides in human breast milk, is produced in engineered Escherichia coli. In order to search for a good α‐1,3‐fucosyltransferase, three bacterial α‐1,3‐fucosyltransferases are expressed in engineered E. coli deficient in β‐galactosidase activity and expressing the essential enzymes for the production of guanosine 5′‐diphosphate‐l ‐fucose, the donor of fucose for 3‐FL biosynthesis. Among the three enzymes tested, the fucT gene from Helicobacter pylori National Collection of Type Cultures 11637 gives the best 3‐FL production in a simple batch fermentation process using glycerol as a carbon source and lactose as an acceptor. In order to use glucose as a carbon source, the chromosomal ptsG gene, considered the main regulator of the glucose repression mechanism, is disrupted. The resulting E. coli strain of ?LP‐YA+FT shows a much lower performance of 3‐FL production (4.50 g L^?1) than the ?L‐YA+FT strain grown in a glycerol medium (10.7 g L^?1), suggesting that glycerol is a better carbon source than glucose. Finally, the engineered E. coli ?LW‐YA+FT expressing the essential genes for 3‐FL production and blocking the colanic acid biosynthetic pathway (?wcaJ) exhibits the highest concentration (11.5 g L^?1), yield (0.39 mol mol^?1), and productivity (0.22 g L^?1 h) of 3‐FL in glycerol‐limited fed‐batch fermentation.     

An aminoacylase activity from Streptomyces ambofaciens catalyzes the acylation of lysine on α‐position and peptides on N‐terminal position

The presence of aminoacylase activities was investigated in a crude extract of Streptomyces ambofaciens ATCC23877. First activities catalyzing the hydrolysis of N‐α or ε‐acetyl‐L‐lysine were identified. Furthermore, the acylation of lysine and different peptides was studied and compared with results obtained with lipase B of Candida antarctica (CALB). Different regioselectivities were demonstrated for the two classes of enzymes. CALB was able to catalyze acylation only on the ε‐position whereas the crude extract from S. ambofaciens possessed the rare ability to catalyze the N‐acylation on the α‐position of the lysine or of the amino‐acid in N‐terminal position of peptides. Two genes, SAM23877_1485 and SAM23877_1734, were identified in the genome of Streptomyces ambofaciens ATCC23877 whose products show similarities with the previously identified aminoacylases from Streptomyces mobaraensis. The proteins encoded by these two genes were responsible for the major aminoacylase hydrolytic activities. Furthermore, we show that the hydrolysis of N‐α‐acetyl‐L‐lysine could be attributed to the product of SAM23877_1734 gene.     

High‐yield lipid production from lignocellulosic biomass using engineered xylose‐utilizing Yarrowia lipolytica

Lignocellulosic biomass shows high potential as a renewable feedstock for use in biodiesel production via microbial fermentation. Yarrowia lipolytica, an emerging oleaginous yeast, has been engineered to efficiently convert xylose, the second most abundant sugar in lignocellulosic biomass, into lipids for lignocellulosic biodiesel production. Yet, the lipid yield from xylose or lignocellulosic biomass remains far lower than that from glucose. Here we developed an efficient xylose‐utilizing Y. lipolytica strain, expressing an isomerase‐based pathway, to achieve high‐yield lipid production from lignocellulosic biomass. The newly developed xylose‐utilizing Y. lipolytica, YSXID, produced 12.01 g/L lipids with a maximum yield of 0.16 g/g, the highest ever reported, from lignocellulosic hydrolysates. Consequently, this study shows the potential of isomerase‐based xylose‐utilizing Y. lipolytica for economical and sustainable production of biodiesel and oleochemicals from lignocellulosic biomass.     

Production of 8,11‐dihydroxy and 8‐hydroxy unsaturated fatty acids from unsaturated fatty acids by recombinant Escherichia coli expressing 8,11‐linoleate diol synthase from Penicillium chrysogenum

Hydroxy unsaturated fatty acids can be used as antimicrobial surfactants. 8,11‐Linoleate diol synthase (8,11‐LDS) catalyzes the conversion of unsaturated fatty acid to 8‐hydroperoxy unsaturated fatty acid, and it is subsequently isomerized to 8,11‐dihydroxy unsaturated fatty acid by the enzyme. The optimal reaction conditions of recombinant Escherichia coli expressing Penicillium chrysogenum 8,11‐LDS for the production of 8,11‐dihydroxy‐9,12(Z,Z)‐octadecadienoic acid (8,11‐DiHODE), 8,11‐dihydroxy‐9,12,15(Z,Z,Z)‐octadecatrienoic acid (8,11‐DiHOTrE), 8‐hydroxy‐9(Z)‐hexadecenoic acid (8‐HHME), and 8‐hydroxy‐9(Z)‐octadecenoic acid (8‐HOME) were pH 7.0, 25°C, 10 g/L linoleic acid, and 20 g/L cells; pH 6.0, 25°C, 6 g/L α‐linolenic acid, and 60 g/L cells; pH 7.0, 25°C, 8 g/L palmitoleic acid, and 25 g/L cells; and pH 8.5, 30°C, 6 g/L oleic acid, and 25 g/L cells, respectively. Under these optimized conditions, the recombinant cells produced 6.0 g/L 8,11‐DiHODE for 60 min, with a conversion of 60% (w/w) and a productivity of 6.0 g/L/h; 4.3 g/L 8,11‐DiHOTrE for 60 min, with a conversion of 72% (w/w) and a productivity of 4.3 g/L/h; 4.3 g/L 8‐HHME acid for 60 min, with a conversion of 54% (w/w) and a productivity of 4.3 g/L/h; and 0.9 g/L 8‐HOME for 30 min, with a conversion of 15% (w/w) and a productivity of 1.8 g/L/h. To best of our knowledge, this is the first report on the biotechnological production of 8,11‐DiHODE, 8,11‐DiHOTrE, 8‐HHME, and 8‐HOME. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:390–396, 2017     

1,3‐propanediol production from glucose by mixed‐culture fermentation of Zygosacharomyces rouxii and Klebsiella pneumonia

1,3‐propanediol is an important chemical widely used in polymer production. In this study, two strains, Zygosacharomyces rouxii JL2011 and Klebsiella pneumoniae S6, were used as a mixed culture for 1,3‐propanediol production directly from glucose. Two important parameters including inoculation time of K. pneumoniae S6 at stage of mixed culture and initial cell ratio of Z. rouxii JL2011 to K. pneumoniae S6 in mixed fermentation were optimized in culture flasks. In those experiments, the best results were obtained with a yield of 6.8 g/L 1,3‐propanediol from glucose when K. pneumoniae S6 was inoculated after 48 h in the culture of Z. rouxii JL2011 by mixed culture of Z. rouxii JL2011 and K. pneumoniae S6 with initial cell ratio of 1:200. In a 7‐L bioreactor, the maximum 1,3‐propanediol production could reach up to 15.2 g/L. Thus, this study presents an effective process for 1,3‐propanediol microbial production from glucose by using mixed culture of Z. rouxii JL2011 and K. pneumoniae S6. This work does not only demonstrate a new way to produce 1,3‐propanediol from a low‐cost feedstock, but may also make a valuable contribution to the development of a cost‐effective fermentation based on renewable resources.     

Screening of bacterial strains capable of converting biodiesel‐derived raw glycerol into 1,3‐propanediol, 2,3‐butanediol and ethanol

The ability of bacterial strains to assimilate glycerol derived from biodiesel facilities to produce metabolic compounds of importance for the food, textile and chemical industry, such as 1,3‐propanediol (PD), 2,3‐butanediol (BD) and ethanol (EtOH), was assessed. The screening of 84 bacterial strains was performed using glycerol as carbon source. After initial trials, 12 strains were identified capable of consuming raw glycerol under anaerobic conditions, whereas 5 strains consumed glycerol under aerobiosis. A plethora of metabolic compounds was synthesized; in anaerobic batch‐bioreactor cultures PD in quantities up to 11.3 g/L was produced by Clostridium butyricum NRRL B‐23495, while the respective value was 10.1 g/L for a newly isolated Citrobacter freundii. Adaptation of Cl. butyricum at higher initial glycerol concentration resulted in a PD_max concentration of ～32 g/L. BD was produced by a new Enterobacter aerogenes isolate in shake‐flask experiments, under fully aerobic conditions, with a maximum concentration of ～22 g/L which was achieved at an initial glycerol quantity of 55 g/L. A new Klebsiella oxytoca isolate converted waste glycerol into mixtures of PD, BD and EtOH at various ratios. Finally, another new C. freundii isolate converted waste glycerol into EtOH in anaerobic batch‐bioreactor cultures with constant pH, achieving a final EtOH concentration of 14.5 g/L, a conversion yield of 0.45 g/g and a volumetric productivity of ～0.7 g/L/h. As a conclusion, the current study confirmed the utilization of biodiesel‐derived raw glycerol as an appropriate substrate for the production of PD, BD and EtOH by several newly isolated bacterial strains under different experimental conditions.     

Multicomponent cellulase production by Cellulomonas biazotea NCIM‐2550 and its applications for cellulosic biohydrogen production

Among four cellulolytic microorganisms examined, Cellulomonas biazotea NCIM‐2550 can grow on various cellulosic substrates and produce reducing sugar. The activity of cellulases (endoglucanase, exoglucanase, and cellobiase), xylanase, amylase, and lignin class of enzymes produced by C. biazotea was mainly present extracellularly and the enzyme production was dependent on cellulosic substrates (carboxymethyl cellulose [CMC], sugarcane bagasse [SCB], and xylan) used for growth. Effects of physicochemical conditions on cellulolytic enzyme production were systematically investigated. Using MnCl₂ as a metal additive significantly induces the cellulase enzyme system, resulting in more reducing sugar production. The efficiency of fermentative conversion of the hydrolyzed SCB and xylan into clean H₂ energy was examined with seven H₂‐producing pure bacterial isolates. Only Clostridiumbutyricum CGS5 exhibited efficient H₂ production performance with the hydrolysate of SCB and xylan. The cumulative H₂ production and H₂ yield from using bagasse hydrolysate (initial reducing sugar concentration = 1.545 g/L) were approximately 72.61 mL/L and 2.13 mmol H₂/g reducing sugar (or 1.91 mmol H₂/g cellulose), respectively. Using xylan hydrolysate (initial reducing sugar concentration = 0.345 g/L) as substrate could also attain a cumulative H₂ production and H₂ yield of 87.02 mL/L and 5.03 mmol H₂/g reducing sugar (or 4.01 mmol H₂/g cellulose), respectively. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Porting the synthetic D‐glucaric acid pathway from Escherichia coli to Saccharomyces cerevisiae

D‐Glucaric acid can be produced as a value‐added chemical from biomass through a de novo pathway in Escherichia coli. However, previous studies have identified pH‐mediated toxicity at product concentrations of 5 g/L and have also found the eukaryotic myo‐inositol oxygenase (MIOX) enzyme to be rate‐limiting. We ported this pathway to Saccaromyces cerevisiae, which is naturally acid‐tolerant and evaluate a codon‐optimized MIOX homologue. We constructed two engineered yeast strains that were distinguished solely by their MIOX gene – either the previous version from Mus musculus or a homologue from Arabidopsis thaliana codon‐optimized for expression in S. cerevisiae – in order to identify the rate‐limiting steps for D‐glucaric acid production both from a fermentative and non‐fermentative carbon source. myo‐Inositol availability was found to be rate‐limiting from glucose in both strains and demonstrated to be dependent on growth rate, whereas the previously used M. musculus MIOX activity was found to be rate‐limiting from glycerol. Maximum titers were 0.56 g/L from glucose in batch mode, 0.98 g/L from glucose in fed‐batch mode, and 1.6 g/L from glucose supplemented with myo‐inositol. Future work focusing on the MIOX enzyme, the interplay between growth and production modes, and promoting aerobic respiration should further improve this pathway.     

Rutin treats myocardial damage caused by pirarubicin via regulating miR‐22‐5p‐regulated RAP1/ERK signaling pathway

Our experiments have previously demonstrated that rutin (RUT) can improve myocardial damage caused by pirarubicin (THP). However, the underlying molecular mechanisms remain uncertain. In this study, we developed an microRNA (miRNA) chip by replicating the rat model of THP‐induced myocardial injury and identified miR‐22‐5p and the RAP1‐member of RAS oncogene family/extracellular regulated protein kinases (RAP1/ERK) signaling pathway as an object of study. Also, in vivo experiments demonstrated that THP caused abnormal changes in the electrocardiogram, cardiac function, and histomorphology in rats (P < .01). THP also reduces the expression of miR‐22‐5p (P < .01) and increases the levels of RAP1/ERK signaling pathway‐related proteins (P < .01, P < .05). RUT significantly improved THP‐induced myocardial damage (P < .01), increased the expression of miR‐22‐5p (P < .01), and decreased the levels of RAP1/ERK signaling pathway‐related proteins (P < .01, P < .05). In vitro studies confirmed that Rap1a is one of the target genes of miR‐22‐5p. miR‐22‐5p overexpression in cardiomyocytes can affect the RAP1/ERK pathway and reduce reactive oxygen species production and cardiomyocyte apoptosis caused by THP (P < .01), which is consistent with the effect of RUT. Our results indicate that RUT treats THP‐induced myocardial damage, which may be achieved by upregulating miR‐22‐5p, causing changes in its target gene Rap1a and the RAP1/ERK pathway.     

The movement protein (NSm) of Tomato spotted wilt virus is the avirulence determinant in the tomato Sw‐5 gene‐based resistance

The avirulence determinant triggering the resistance conferred by the tomato gene Sw‐5 against Tomato spotted wilt virus (TSWV) is still unresolved. Sequence comparison showed two substitutions (C118Y and T120N) in the movement protein NSm present only in TSWV resistance‐breaking (RB) isolates. In this work, transient expression of NSm of three TSWV isolates [RB1 (T120N), RB2 (C118Y) and non‐resistance‐breaking (NRB)] in Nicotiana benthamiana expressing Sw‐5 showed a hypersensitive response (HR) only with NRB. Exchange of the movement protein of Alfalfa mosaic virus (AMV) with NSm supported cell‐to‐cell and systemic transport of the chimeric AMV RNAs into N. tabacum with or without Sw‐5, except for the constructs with NBR when Sw‐5 was expressed, although RB2 showed reduced cell‐to‐cell transport. Mutational analysis revealed that N120 was sufficient to avoid the HR, but the substitution V130I was required for systemic transport. Finally, co‐inoculation of RB and NRB AMV chimeric constructs showed different prevalence of RB or NBR depending on the presence or absence of Sw‐5. These results indicate that NSm is the avirulence determinant for Sw‐5 resistance, and mutations C118Y and T120N are responsible for resistance breakdown and have a fitness penalty in the context of the heterologous AMV system.     

Optimization of 1,2,4‐butanetriol production from xylose in Saccharomyces cerevisiae by metabolic engineering of NADH/NADPH balance

1,2,4‐Butanetriol (BT) is used as a precursor for the synthesis of various pharmaceuticals and the energetic plasticizer 1,2,4‐butanetriol trinitrate. In Saccharomyces cerevisiae, BT is biosynthesized from xylose via heterologous four enzymatic reactions catalyzed by xylose dehydrogenase, xylonate dehydratase, 2‐ketoacid decarboxylase, and alcohol dehydrogenase. We here aimed to improve the BT yield in S. cerevisiae by genetic engineering. First, the amount of the key intermediate 2‐keto‐3‐deoxy‐xylonate as described previously was successfully reduced in 41% by multiple integrations of Lactococcus lactis 2‐ketoacid decarboxylase gene kdcA into the yeast genome. Since the heterologous BT synthetic pathway is independent of yeast native metabolism, this manipulation has led to NADH/NADPH imbalance and deficiency during BT production. Overexpression of the NADH kinase POS5Δ17 lacking the mitochondrial targeting sequence to relieve NADH/NADPH imbalance resulted in the BT titer of 2.2 g/L (31% molar yield). Feeding low concentrations of glucose and xylose to support the supply of NADH resulted in BT titer of 6.6 g/L with (57% molar yield). Collectively, improving the NADH/NADPH ratio and supply from glucose are essential for the construction of a xylose pathway, such as the BT synthetic pathway, independent of native yeast metabolism.     

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.