Production Of Shikimic Acid Research Articles

A chromosome-scale genome of Rhus chinensis Mill. provides new insights into plant-insect interaction and gallotannins biosynthesis.

Rhus chinensis Mill., an economically valuable Anacardiaceae species, is parasitized by the galling aphid Schlechtendalia chinensis, resulting in the formation of the Chinese gallnut (CG). Here, we report a chromosomal-level genome assembly of R. chinensis, with a total size of 389.40 Mb and scaffold N50 of 23.02 Mb. Comparative genomic and transcriptome analysis revealed that the enhanced structure of CG and nutritional metabolism contribute to improving the adaptability of R. chinensis to S. chinensis by supporting CG and galling aphid growth. CG was observed to be abundant in hydrolysable tannins (HT), particularly gallotannin and its isomers. Tandem repeat clusters of dehydroquinate dehydratase/shikimate dehydrogenase (DQD/SDH) and serine carboxypeptidase-like (SCPL) and their homologs involved in HT production were determined as specific to HT-rich species. The functional differentiation of DQD/SDH tandem duplicate genes and the significant contraction in the phenylalanine ammonia-lyase (PAL) gene family contributed to the accumulation of gallic acid and HT while minimizing the production of shikimic acid, flavonoids, and condensed tannins in CG. Furthermore, we identified one UDP glucosyltransferase (UGT84A), three carboxylesterase (CXE), and six SCPL genes from conserved tandem repeat clusters that are involved in gallotannin biosynthesis and hydrolysis in CG. We then constructed a regulatory network of these genes based on co-expression and transcription factor motif analysis. Our findings provide a genomic resource for the exploration of the underlying mechanisms of plant-galling insect interaction and highlight the importance of the functional divergence of tandem duplicate genes in the accumulation of secondary metabolites.

The preparation of a boronate affinity-based controlled oriented imprinting coating on a silica nanoparticle surface for the separation and purification of shikimic acid in herbal medicine.

Shikimic acid (SA) is one of the most effective drugs against the A (H1N1) virus and has high medicinal value. Additionally, it has the ability to generate non-toxic herbicides and antimicrobial medications. The extraction from plants has proven to be the main route of production of SA with economic benefits and environmental efficiency. Therefore, it is necessary to perform purification of SA from these herbal medicines before quantifying it. In this study, researchers employed a boronate affinity-based controlled oriented surface imprinting technique to produce molecularly imprinted polymers (MIPs) as highly effective solid phase extraction (SPE) adsorbents for the isolation and purification of SA. 3-Fluoro-4-formylphenylboronic acid functionalized silica nanoparticles were used as supporting materials for immobilizing SA. Poly(2-anilinoethanol) with a higher hydrophilic domain can be used as an effective imprinting coating. The prepared SA-imprinted silica nanoparticles exhibited several significant results, such as good specificity, high binding capacity (39.06 ± 2.24 mg g-1), moderate binding constant (6.61 × 10-4 M-1), fast kinetics (8 min) and low binding pH (pH 5.0) toward SA. The replication of SA-imprinted silica nanoparticles was deemed satisfactory. The SA-imprinted silica nanoparticles could be still reused after seven adsorption-desorption cycles, which indicated high chemical stability. In addition, the recoveries of the proposed method for SA at three spiked level analysis in star aniseed and meadow cranesbill were 96.2% to 109.0% and 91.6% to 103.5%, respectively. The SA-imprinted silica nanoparticles that have been prepared are capable of identifying the target SA in real herbal medicines. Our approach makes sample pre-preparation simple, fast, selective and efficient.

Metabolic Engineering of Shikimic Acid Biosynthesis Pathway for the Production of Shikimic Acid and Its Branched Products in Microorganisms: Advances and Prospects.

The shikimate pathway is a necessary pathway for the synthesis of aromatic compounds. The intermediate products of the shikimate pathway and its branching pathway have promising properties in many fields, especially in the pharmaceutical industry. Many important compounds, such as shikimic acid, quinic acid, chlorogenic acid, gallic acid, pyrogallol, catechol and so on, can be synthesized by the shikimate pathway. Among them, shikimic acid is the key raw material for the synthesis of GS4104 (Tamiflu®), an inhibitor of neuraminidase against avian influenza virus. Quininic acid is an important intermediate for synthesis of a variety of raw chemical materials and drugs. Gallic acid and catechol receive widespread attention as pharmaceutical intermediates. It is one of the hotspots to accumulate many kinds of target products by rationally modifying the shikimate pathway and its branches in recombinant strains by means of metabolic engineering. This review considers the effects of classical metabolic engineering methods, such as central carbon metabolism (CCM) pathway modification, key enzyme gene modification, blocking the downstream pathway on the shikimate pathway, as well as several expansion pathways and metabolic engineering strategies of the shikimate pathway, and expounds the synthetic biology in recent years in the application of the shikimate pathway and the future development direction.

An Antifungal Role of Hydrogen Sulfide on Botryosphaeria Dothidea and Amino Acid Metabolism Involved in Disease Resistance Induced in Postharvest Kiwifruit.

Botryosphaeria dothidea is a major pathogen responsible for postharvest kiwifruit soft rot. This study aimed to determine the influence of hydrogen sulfide (H2S) on postharvest resistance to kiwifruit soft rot and the antifungal role of H2S against B. dothidea. The results indicated that H2S (20 μl L−1) restricted the lesion area following inoculation with B. dothidea. H2S enhanced the production of shikimic acid, tyrosine, tryptophan, and phenylalanine while also increasing the total phenols, flavonoids, and lignin. H2S upregulated the expression of AcDHQS, AcSDH, AcSK, AcPAL, AcCAD, and AcCHS. Additionally, sodium hydrosulfide (NaHS)-released H2S inhibited mycelial growth. NaHS concentrations of 20 and 40 mmol L−1 significantly decreased the mycelial weight and malondialdehyde content (MDA) content while increasing cell membrane conductivity and membrane leakage. The results indicate that H2S induces resistance in kiwifruit via a microbicidal role and amino acid metabolism involved in postharvest kiwifruit disease resistance.

History-Driven Genetic Modification Design Technique Using a Domain-Specific Lexical Model for the Acceleration of DBTL Cycles for Microbial Cell Factories.

The development of microbes for conducting bioprocessing via synthetic biology involves design-build-test-learn (DBTL) cycles. To aid the designing step, we developed a computational technique that suggests next genetic modifications on the basis of relatedness to the user's design history of genetic modifications accumulated through former DBTL cycles conducted by the user. This technique, which comprehensively retrieves well-known designs related to the history, involves searching text for previous literature and then mining genes that frequently co-occur in the literature with those modified genes. We further developed a domain-specific lexical model that weights literature that is more related to the domain of metabolic engineering to emphasize genes modified for bioprocessing. Our technique made a suggestion by using a history of creating a Corynebacterium glutamicum strain producing shikimic acid that had 18 genetic modifications. Inspired by the suggestion, eight genes were considered by biologists for further modification, and modifying four of these genes proved experimentally efficient in increasing the production of shikimic acid. These results indicated that our proposed technique successfully utilized the former cycles to suggest relevant designs that biologists considered worth testing. Comprehensive retrieval of well-tested designs will help less-experienced researchers overcome the entry barrier as well as inspire experienced researchers to formulate design concepts that have been overlooked or suspended. This technique will aid DBTL cycles by feeding histories back to the next genetic design, thereby complementing the designing step.

Biosensor-Guided Atmospheric and Room-Temperature Plasma Mutagenesis and Shuffling for High-Level Production of Shikimic Acid from Sucrose in Escherichia coli.

Here, we first developed a combined strain improvement strategy of biosensor-guided atmospheric and room-temperature plasma mutagenesis and genome shuffling. Application of this strategy resulted in a 2.7-fold increase in the production of shikimic acid (SA) and a 2.0-fold increase in growth relative to those of the starting strain. Whole-cell resequencing of the shuffled strain and confirmation using CRISPRa/CRISPRi revealed that some membrane protein-related mutant genes are identified as being closely related to the higher SA titer. The engineered shuffling strain produced 18.58 ± 0.56 g/L SA from glucose with a yield of 68% (mol/mol) by fed-batch whole-cell biocatalysis, achieving 79% of the theoretical maximum. Sucrose-utilizing Escherichia coli was engineered for SA production by introducing Mannheimia succiniciproducens β-fructofuranosidase gene. The resulting sucrose-utilizing E. coli strain produced 24.64 ± 0.32 g/L SA from sucrose with a yield of 1.42 mol/mol by fed-batch whole-cell biocatalysis, achieving 83% of the theoretical maximum.

Metabolic Engineering of Shikimic Acid-Producing Corynebacterium glutamicum From Glucose and Cellobiose Retaining Its Phosphotransferase System Function and Pyruvate Kinase Activities

The production of aromatic compounds by microbial production is a promising and sustainable approach for producing biomolecules for various applications. We describe the metabolic engineering of Corynebacterium glutamicum to increase its production of shikimic acid. Shikimic acid and its precursor-consuming pathways were blocked by the deletion of the shikimate kinase, 3-dehydroshikimate dehydratase, shikimate dehydratase, and dihydroxyacetone phosphate phosphatase genes. Plasmid-based expression of shikimate pathway genes revealed that 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase, encoded by aroG, and DHQ synthase, encoded by aroB, are key enzymes for shikimic acid production in C. glutamicum. We constructed a C. glutamicum strain with aroG, aroB and aroE3 integrated. This strain produced 13.1 g/L of shikimic acid from 50 g/L of glucose, a yield of 0.26 g-shikimic acid/g-glucose, and retained both its phosphotransferase system and its pyruvate kinase activity. We also endowed β-glucosidase secreting ability to this strain. When cellobiose was used as a carbon source, the strain produced shikimic acid at 13.8 g/L with the yield of 0.25 g-shikimic acid/g-glucose (1 g of cellobiose corresponds to 1.1 g of glucose). These results demonstrate the feasibility of producing shikimic acid and its derivatives using an engineered C. glutamicum strain from cellobiose as well as glucose.

Dynamic Flux Balance Analysis to Evaluate the Strain Production Performance on Shikimic Acid Production in Escherichia coli.

Flux balance analysis (FBA) is used to improve the microbial production of useful compounds. However, a large gap often exists between the FBA solution and the experimental yield, because of growth and byproducts. FBA has been extended to dynamic FBA (dFBA), which is applicable to time-varying processes, such as batch or fed-batch cultures, and has significantly contributed to metabolic and cultural engineering applications. On the other hand, the performance of the experimental strains has not been fully evaluated. In this study, we applied dFBA to the production of shikimic acid from glucose in Escherichia coli, to evaluate the production performance of the strain as a case study. The experimental data of glucose consumption and cell growth were used as FBA constraints. Bi-level FBA optimization with maximized growth and shikimic acid production were the objective functions. Results suggest that the shikimic acid concentration in the high-shikimic-acid-producing strain constructed in the experiment reached up to 84% of the maximum value by simulation. Thus, this method can be used to evaluate the performance of strains and estimate the milestones of strain improvement.

Yeast gets a solar-powered boost

The simple brewer’s yeast Saccharomyces cerevisiae is one of the most well-characterized microorganisms on the planet. It is used to ferment alcohol, as well as synthesize drugs and feedstock chemicals. Now, researchers have created yeast that can perform another trick: harnessing sunlight. Neel Joshi’s group at Harvard University added semiconductor nanoparticles to the surface of genetically engineered yeast to generate an inorganic, biological, hybrid organism. The yeast grabs electrons, which are produced when light shines on the particles, to drive production of shikimic acid—used to make oseltamivir, sold under the brand name Tamiflu. Joshi tells C&EN that the project was conceived of by two postdocs, Junling Guo and Miguel Suástegui, who have backgrounds in materials science and metabolic engineering, respectively. “They came up with this idea,” Joshi explains, “which initially I didn’t think was going to work.” But he was proved wrong. “It worked beautifully.” The researchers fixed indium phosphide

Production and Synthetic Modifications of Shikimic Acid.

Shikimic acid is a natural product of industrial importance utilized as a precursor of the antiviral Tamiflu. It is nowadays produced in multihundred ton amounts from the extraction of star anise ( Illicium verum) or by fermentation processes. Apart from the production of Tamiflu, shikimic acid has gathered particular notoriety as its useful carbon backbone and inherent chirality provide extensive use as a versatile chiral precursor in organic synthesis. This review provides an overview of the main synthetic and microbial methods for production of shikimic acid and highlights selected methods for isolation from available plant sources. Furthermore, we have attempted to demonstrate the synthetic utility of shikimic acid by covering the most important synthetic modifications and related applications, namely, synthesis of Tamiflu and derivatives, synthetic manipulations of the main functional groups, and its use as biorenewable material and in total synthesis. Given its rich chemistry and availability, shikimic acid is undoubtedly a promising platform molecule for further exploration. Therefore, in the end, we outline some challenges and promising future directions.

Shikimic Acid Production by Fusarium decemcellulare, An Endophytic Fungus Isolated from Flacourtia inermis Fruits

Microorganisms have produced many of the most potent agents against bacterial and fungal infections, cancer and high cholesterol etc. Studies on secondary metabolites originating from endophytic fungi isolated in Sri Lanka are relatively less studied. Hence we studied the chemistry and bioactivity of an endophytic fungus Fusarium decemcellulare isolated from the fruits of Flacourtia inermis. A pure culture of fungus was fermented in potato dextrose broth (PDB) for 28 days. The fermentation medium and mycelium was extracted in to ethyl acetate (EtOAc) and methanol (MeOH). These extracts did not show good activity for bioassays against antioxidant, antifungal, α-amylase inhibitory, brine shrimp lethality and phytotoxicity. Chromatographic separation of the EtOAc extract furnished ergosterol while MeOH extract furnished shikimic acid. Isolation of endophytic fungus Fusarium decemcellulare from the fruits of F. inermis and the production of shikimic acid from F. decemcellulare are reported for the first time.

The Role of the ydiB Gene, Which Encodes Quinate/Shikimate Dehydrogenase, in the Production of Quinic, Dehydroshikimic and Shikimic Acids in a PTS- Strain of Escherichia coli

The culture of engineered Escherichia coli for shikimic acid (SA) production results in the synthesis of quinic acid (QA) and dehydroshikimic acid (DHS), reducing SA yield and impairing downstream processes. The synthesis of QA by quinate/shikimate dehydrogenase (YdiB, ydiB) has been previously proposed; however, the precise role for this enzyme in the production of QA in engineered strains of E. coli for SA production remains unclear. We report the effect of the inactivation or the overexpression of ydiB in E. coli strain PB12.SA22 on SA, QA, and DHS production in batch fermentor cultures. The results showed that the inactivation of ydiB resulted in a 75% decrease in the molar yield of QA and a 6.17% reduction in the yield of QA (mol/mol) relative to SA with respect to the parental strain. The overexpression of ydiB caused a 500% increase in the molar yield of QA and resulted in a 152% increase in QA (mol/mol) relative to SA, with a sharp decrease in SA production. Production of SA, QA, and DHS in parental and derivative ydiB strains suggests that the synthesis of QA results from the reduction of 3-dehydroquinate by YdiB before its conversion to DHS.

Studies on the production of shikimic acid using the aroK knockout strain of Bacillus megaterium.

Shikimic acid has various pharmaceutical and industrial applications. It is the sole chemical building block for the antiviral drug oseltamivir (Tamiflu(®)) and one of the potent pharmaceutical intermediates with three chiral centres. Here we report a modified strain of Bacillus megaterium with aroK (shikimate kinase) knock out to block the aromatic biosynthetic pathway downstream of shikimic acid. Homologous recombination based gene disruption approach was used for generating aroK knock out mutant of B. megaterium. Shake flask cultivation showed shikimic acid yield of 2.98g/L which is ~6 times more than the wild type (0.53g/L). Furthermore, the shikimate kinase activity was assayed and it was 32% of the wild type. Effect of various carbon sources on the production of shikimic acid was studied and fructose (4%, w/v) was found to yield maximum shikimic acid (4.94g/L). The kinetics of growth and shikimic acid production by aroK knockout mutant was studied in 10L bioreactor and the yield of shikimic acid had increased to 6g/L which is ~12 fold higher over the wild type. It is evident from the results that aroK gene disruption had an immense effect in enhancing the shikimic acid production.

Enhanced production of shikimic acid using a multi-gene co-expression system in Escherichia coli

Shikimic acid (SA) is the key synthetic material for the chemical synthesis of Oseltamivir, which is prescribed as the front-line treatment for serious cases of influenza. Multi-gene expression vector can be used for expressing the plurality of the genes in one plasmid, so it is widely applied to increase the yield of metabolites. In the present study, on the basis of a shikimate kinase genetic defect strain Escherichia coli BL21 (ΔaroL/aroK, DE3), the key enzyme genes aroG, aroB, tktA and aroE of SA pathway were co-expressed and compared systematically by constructing a series of multi-gene expression vectors. The results showed that different gene co-expression combinations (two, three or four genes) or gene orders had different effects on the production of SA. SA production of the recombinant BL21-GBAE reached to 886.38 mg·L−1, which was 17-fold (P < 0.05) of the parent strain BL21 (ΔaroL/aroK, DE3).

Editorial: Quantitative Systems Biology for Engineering Organisms and Pathways.

The Editorial on the Research Topic Quantitative Systems Biology for Engineering Organisms and Pathways The biological production of chemicals has gained interest due to its contribution to greener and sustainable processes. Discovering the metabolic capacities of microorganisms shows ascending promise. The understanding of the metabolism and its complex interactions within the process environment is crucial to successfully apply and design cell factories. With the advances of high throughput measurements of -omic levels in a cell, it is possible to decipher the knowledge at different biological processes as a whole. Combining the gained experimental data with computational methods, namely, employing systems biology, allows the development of new bio-products and new cell factories. This Research Topic of Frontiers in Bioengineering and Biotechnology includes six reviews, two mini-reviews, an opinion, and an original research article. Mainly, the importance of applying systems biology approaches for finding metabolic engineering targets is discussed. Valgepea et al. explained their opinion on the potential of proteome optimization in contribution to feasible development of bioprocesses. Delvigne et al. summarized the importance of using fluorescent reporter libraries for the optimization of microbial production under bioreactor environments. They focused on the current status of this technique in terms of methods and applications. Martinez et al. reported the rational design examples of Escherichia coli strains for the production of shikimic acid, a precursor aromatic compound in the synthesis of a drug, which is efficient against diverse viruses, including H5N1 and H1N1. In addition, they discussed the challenging tasks required for further improving the overproducing strains using global transcriptomic analyses. Vargas-Tah and Gosset explored the microbial production of two other aromatic intermediates: cinnamic and p-hydroxycinnamic acids. The approaches in metabolic engineering of various microorganisms to optimize the usage of raw material and increase the efficiencies of these products were explained. Licona-Cassani et al. provided recent efforts on the systems biology studies in actinomycetes, which are important pathogens and valuable sources of antibiotics, in relation to optimize the production of bioactive natural products. Ates evaluated another example on the application of omics technologies in her review, the production of microbial exopolysaccharides. In addition to these studies on the application of systems biology tools for industrial-scale production, Freudenau et al. presented a dynamic mathematical model constructed to understand the factors affecting the production of plasmid DNA as a pharmaceutical gene vector. Blombach and Takors reviewed the impacts of carbon dioxide/bicarbonate levels on the physiological, production, metabolism, and regulation processes in microbial and mammalian cultures, a relevant issue still poorly understood. Caspeta et al. focused on the lignocellulosic production of ethanol in Saccharomyces cerevisiae and reviewed the information available on the inhibitory conditions during such processes and related stress response mechanisms of the cells. Understanding the reprograming of cellular functions in response to stress environments is crucial, and Taymaz-Nikerel et al. reviewed the current knowledge gained mainly by transcriptomic studies carried out in S. cerevisiae. Furthermore, they addressed the requirements of construction of a quantitative whole cell model, which is the ultimate goal of systems biology. We expect that the developments in this field will continue to increase, eventually yielding quantitative predictive models, which will be useful in many areas.

HPLC法测定兴安落叶松针叶中莽草酸含量

本文采用高效液相色谱法(HPLC)对北方常见松科树种兴安落叶松针叶中莽草酸含量及其全年动态变化进行分析。从2015年5月份开始采摘新鲜松针叶片，采样时间为每月15日，选取5株树龄相近的兴安落叶松树，每次分别从同株树的上、中，下三个部位各采集新鲜叶片300.0 g，直至本年11月针叶脱落为止。所采集松针叶片按照采集月份分为7份，混合均匀，阴干。干燥叶片研磨粉碎后，采用超声辅助法，用甲醇提取针叶中的莽草酸，提取液经减压浓缩，甲醇复溶后，进行HPLC检测分析。HPLC检测结果表明：兴安落叶松针叶中莽草酸平均含量全年变化范围为0.04%~0.44%。在不同时期，兴安落叶松针叶中莽草酸的含量不同，在6月中旬含量达到最大值0.44%，而11月份达到最低值0.04%。本文对兴安落叶松针叶中莽草酸的含量进行初步测定，并为工业上生产莽草酸的原料找到良好的替代品。 In this paper, the dynamic change of shikimic acid contents in the needle of Larix gmelinii during a year was studied using the method of HPLC. The fresh pine needle from the upper, middle and lower parts of 5 Larix gmelinii trees with the same age was collected from middle of March to middle of November in 2015. The weight per sample was 300.0 g. The samples collected in the same month were homogeneous mixing and dried in the shade. Before using, the sample was grounded and sieved into homogeneous size. The dried sample powder was mixed with methonal for ultrasonic treatment to extract shikimic acid. The extract was concentrated and re-solved for analysis of HPLC. The results showed that the shikimic acid contents in the needle of Larix gmelinii ranged from 0.04% to 0.44%, while the contents of shikimic acid in Larix gmelinii changed during different months. The highest content reached 0.44% in the middle of June, with the time increasing; the lowest content reached 0.04% in November. The paper determines the contents of shikimic acid contents in the needle of Larix gmelinii, in order to find a good substitute of raw materials for industrial production of shikimic acid.

Generation of aroE overexpression mutant of Bacillus megaterium for the production of shikimic acid.

BackgroundShikimic acid, the sole chemical building block for the antiviral drug oseltamivir (Tamiflu®), is one of the potent pharmaceutical intermediates with three chiral centers. Here we report a metabolically engineered recombinant Bacillus megaterium strain with aroE (shikimate dehydrogenase) overexpression for the production of shikimic acid.ResultsIn a 7 L bioreactor, 4.2 g/L shikimic acid was obtained using the recombinant strain over 0.53 g/L with the wild type. The enhancement of total shikimate dehydrogenase activity was 2.13-fold higher than the wild type. Maximum yield of shikimic acid (12.54 g/L) was obtained with fructose as carbon source. It was isolated from the fermentation broth using amberlite IRA-400 resin and 89 % purity of the product was achieved.ConclusionThis will add up a new organism in the armory for the fermentation based production which is better over plant based extraction and chemical synthesis of shikimic acid.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0251-3) contains supplementary material, which is available to authorized users.

Shikimic acid, a base compound for the formulation of swine/avian flu drug: statistical optimization, fed-batch and scale up studies along with its application as an antibacterial agent

The sudden outbreak of swine flu has increased the global demand of shikimic acid which is an industrially interesting compound, as it is used as a key starting material for the synthesis of a neuraminidase inhibitor Tamiflu(®), for the treatment of antiviral infections such as swine flu. Statistical optimization and evaluation of medium components for the production of shikimic acid by Citrobacter freundii is addressed in the present investigation. Plackett-Burman design was applied for the screening of the most significant variables affecting shikimic acid production, where glucose, asparagine, KH2PO4, CaCO3 and agitation rate were the most significant factors. Response surface methodology was also employed to study the interaction among the most significant variables through which shikimic acid production increased to 12.76 g/L. Further, fed-batch studies resulted in the production of 22.32 g/L of shikimic acid. The scalability of the process was also confirmed by running 14 L bioreactor (7.5 L production medium) where 20.12 g/L of shikimic acid was produced. In addition the antibacterial activity of the shikimic acid produced was analysed against four Gram positive and four Gram negative bacteria and it was found to have a greater inhibition effect against the Gram negative bacteria.

Production of shikimic acid from Escherichia coli through chemically inducible chromosomal evolution and cofactor metabolic engineering

BackgroundShikimic acid (SA) produced from the seeds of Chinese star anise (Illicium verum) is a key intermediate for the synthesis of neuraminidase inhibitors such as oseltamivir (Tamiflu®), an anti-influenza drug. However, plants cannot deliver a stable supply of SA. To avoid the resulting shortages and price fluctuations, a stable source of affordable SA is required. Although recent achievements in metabolic engineering of Escherichia coli strains have significantly increased SA productivity, commonly-used plasmid-based expression systems are prone to genetic instability and require constant selective pressure to ensure plasmid maintenance. Cofactors also play an important role in the biosynthesis of different fermentation products. In this study, we first constructed an E. coli SA production strain that carries no plasmid or antibiotic marker. We then investigated the effect of endogenous NADPH availability on SA production.ResultsThe pps and csrB genes were first overexpressed by replacing their native promoter and integrating an additional copy of the genes in a double gene knockout (aroK and aroL) of E. coli. The aroG fbr , aroB, aroE and tktA gene cluster was integrated into the above E. coli chromosome by direct transformation. The gene copy number was then evolved to the desired value by triclosan induction. The resulting strain, E. coli SA110, produced 8.9-fold more SA than did the parental strain E. coli (ΔaroKΔaroL). Following qRT-PCR analysis, another copy of the tktA gene under the control of the 5Ptac promoter was inserted into the chromosome of E. coli SA110 to obtain the more productive strain E. coli SA110. Next, the NADPH availability was increased by overexpressing the pntAB or nadK genes, which further enhanced SA production. The final strain, E. coli SA116, produced 3.12 g/L of SA with a yield on glucose substrate of 0.33 mol/mol.ConclusionAn SA-producing E. coli strain that carries neither a plasmid nor an antibiotic marker was constructed by triclosan-induced chromosomal evolution. We present the first demonstration that increasing NADPH availability by overexpressing the pntAB or nadK genes significantly enhances SA production.

Metabolic flux responses to genetic modification for shikimic acid production by Bacillus subtilis strains

BackgroundShikimic acid (SA) is a key chiral starting molecule for the synthesis of the neuramidase inhibitor GS4104 against viral influenza. Microbial production of SA has been extensively investigated in Escherichia coli, and to a less extent in Bacillus subtilis. However, metabolic flux of the high SA-producing strains has not been explored. In this study, we constructed with genetic manipulation and further determined metabolic flux with 13C-labeling test of high SA-producing B. subtilis strains.ResultsB. subtilis 1A474 had a mutation in SA kinase gene (aroI) and accumulated 1.5 g/L of SA. Overexpression of plasmid-encoded aroA, aroB, aroC or aroD in B. subtilis revealed that aroD had the most significantly positive effects on SA production. Simultaneous overexpression of genes for 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase (aroA) and SA dehydrogenase (aroD) in B. subtilis BSSA/pSAAroA/pDGSAAroD resulted in SA production of 3.2 g/L. 13C-Metabolic flux assay (MFA) on the two strains BSSA/pHCMC04/pDG148-stu and BSSA/pSAAroA/pDGSAAroD indicated the carbon flux from glucose to SA increased to 4.6% in BSSA/pSAAroA/pDGSAAroD from 1.9% in strain BSSA/pHCMC04/pDG148-stu. The carbon flux through tricarboxylic acid cycle significantly reduced, while responses of the pentose phosphate pathway and the glycolysis to high SA production were rather weak, in the strain BSSA/pSAAroA/pDGSAAroD. Based on the results from MFA, two potential targets for further optimization of SA production were identified. Experiments on genetic deletion of phosphoenoylpyruvate kinase gene confirmed its positive influence on SA production, while the overexpression of the transketolase gene did not lead to increase in SA production.ConclusionOf the genes involved in shikimate pathway in B. subtilis, aroD exerted most significant influence on SA accumulation. Overexpression of plasmid-encoded aroA and aroD doubled SA production than its parent strain. MFA revealed metabolic flux redistribution among phosphate pentose pathway, glycolysis, TCA cycle in the low and high SA-producing B. subtilis strains. The high SA producing strain BSSA/pSAAroA/pDGSAAroD had increased carbon flux into shikimate pathway and reduced flux into TCA cycle.