A Bifidobacterium animalis subsp. lactis strain that can suppress Helicobacter pylori: isolation, in vitro and in vivo validation.

The administration of probiotics is an effective approach for treatment of Helicobacter pylori, which is associated with human gastrointestinal diseases and cancers. To explore more effective probiotics for H. pylori infection elimination, bacteria from infant feces were screened in this study. We successfully isolated the Bifidobacterium animalis subsp. lactis strains and evaluated its efficacy to inhibit H. pylori growth in vitro and in vivo. The results showed that a B. animalis strain (named BB18) sustained a high survival rate after incubation in gastric juice. The rapid urease test suggested that B. animalis BB18 reduced pathogen loads in H. pylori-infected Mongolian gerbils. Alleviation of H. pylori infection-induced gastric mucosa damage and decreased levels inflammatory cytokines were observed after the B. animalis BB18 administration. These findings demonstrated that B. animalis BB18 can inhibit H. pylori infection both in vitro and in vivo, suggesting its potential application for the prevention and eradication therapy of H. pylori infection.

Knockout of a PLD gene in Schizochytrium limacinum SR21 enhances docosahexaenoic acid accumulation by modulation of the phospholipid profile.

BACKGROUND: The hydrolysis and transphosphatidylation of phospholipase D (PLD) play important roles in the interconversion of phospholipids (PLs), which has been shown to profoundly impact lipid metabolism in plants. In this study, the effect of the PLD1 gene of Schizochytrium limacinum SR21 (S. limacinum SR21) on lipid metabolism was investigated. RESULTS: PLD1 knockout had little impact on cell growth and lipid production, but it significantly improved the percentage of polyunsaturated fatty acids in lipids, of which docosahexaenoic acid (DHA) content increased by 13.3% compared to the wild-type strain. Phospholipomics and real-time quantitative PCR analysis revealed the knockout of PLD1 reduced the interexchange and increased de novo synthesis of PLs, which altered the composition of PLs, accompanied by a final decrease in phosphatidylcholine (PC) and an increase in phosphatidylinositol, lysophosphatidylcholine, and phosphatidic acid levels. PLD1 knockout also increased DHA content in triglycerides (TAGs) and decreased it in PLs. CONCLUSIONS: These results indicate that PLD1 mainly performs the transphosphatidylation activity in S. limacinum SR21, and its knockout promotes the migration of DHA from PLs to TAGs, which is conducive to DHA accumulation and storage in TAGs via an acyl CoA-independent pathway. This study provides a novel approach for identifying the mechanism of DHA accumulation and metabolic regulation strategies for DHA production in S. limacinum SR21.

Strategies for high cell density cultivation of Akkermansia muciniphila and its potential metabolism.

IMPORTANCE: Currently, there is significant interest in Akkermansia muciniphila as a promising next-generation probiotic, making it a hot topic in scientific research. However, to achieve efficient industrial production, there is an urgent need to develop an in vitro culture method to achieve high biomass using low-cost carbon sources such as glucose. This study aims to explore the high-density fermentation strategy of A. muciniphila by optimizing the culture process. This study also employs techniques such as LC-MS and RNA-Seq to explain the possible regulatory mechanism of high-density cell growth and increased cell surface hydrophobicity facilitating cell colonization of the gut in vitro culture. Overall, this research sheds light on the potential of A. muciniphila as a probiotic and provides valuable insights for future industrial production.

Transesterification of phosphatidylcholine with DHA-rich algal oil using immobilized Candida antarctica lipase B to produce DHA-phosphatidylcholine.

Docosahexaenoic acid (DHA) enriched with phospholipids (PLs) (DHA-PLs) is a type of structured PL with good physicochemical and nutritional properties. Compared to PLs and DHA, DHA-PLs has higher bioavailability and structural stability and many nutritional benefits. To improve the enzymatic synthesis of DHA-PLs, this study investigated the preparation of phosphatidylcholine (PC) enriched with DHA (DHA-PC) via enzymatic transesterification of algal oil, which is rich in DHA-triglycerides, using immobilized Candida antarctica lipase B (CALB). The optimized reaction system incorporated 31.2% DHA into the acyl chain of PC and converted 43.6% PC to DHA-PC within 72 h at 50 °C, 1:8 PC: algal oil mass ratio, 25% enzyme load (based on total substrate mass), and 0.02 g/mL molecular sieve concentration. Consequently, the side reactions of PC hydrolysis were effectively suppressed and products with high PC content (74.8%) were produced. Molecular structure analysis showed that exogenous DHA was specifically incorporated into the sn-1 site of the PC by immobilized CALB. Furthermore, the evaluation of reusability with eight cycles showed that the immobilized CALB had good operational stability in the present reaction system. Collectively, this study demonstrated the applicability of immobilized CALB as a biocatalyst for synthesizing DHA-PC and provided an improved enzyme-catalyzed method for future DHA-PL synthesis.

Metabolic mechanism of astaxanthin biosynthesis in Xanthophyllomyces dendrorhous in response to sodium citrate treatment.

Astaxanthin is an important ketocarotenoid widely used in industries. However, its application is limited because of its low yield. Sodium citrate (Na-citrate), one of the major carbon sources for microorganisms, can promote cell growth and product accumulation. The basidiomycetous red yeast Xanthophyllomyces dendrorhous was thus used to study the effect of Na-citrate on cell growth and astaxanthin synthesis. The highest biomass and astaxanthin yield (6.0 g/L and 22.5 mg/L) were obtained in shake-flask when 3 g/L Na-citrate was added at 24 h and were 1.8 and 2.0 times higher than those of the control group, respectively. Furthermore, metabolomics and real-time reverse transcription PCR (qRT-PCR) analysis were conducted to study the metabolic pathways of X. dendrorhous in response to Na-citrate. The qRT-PCR assay revealed that Na-citrate facilitated glucose consumption, promoted the metabolic flux from glycolysis, and regulated the tricarboxylic acid (TCA) cycle, providing more energy and substrates for the synthesis of astaxanthin. The gene analysis revealed that adding Na-citrate significantly upregulated the expression of six key genes (ICL, HMGS, crtE, crtYB, crtI, and crtS) involved in pathways related to astaxanthin biosynthesis. These results suggest that exogenous Na-citrate treatment is a potentially valuable strategy to stimulate astaxanthin production in X. dendrorhous.

A Novel Bioflocculant Produced by Cobetia marina MCCC1113: Optimization of Fermentation Conditions by Response Surface Methodology and Evaluation of Flocculation Performance when Harvesting Microalgae.

A preliminary study was carried out to optimize the culture medium conditions for producing a novel microbial flocculant from the marine bacterial species Cobetia marina. The optimal glucose, yeast extract, and glutamate contents were 30, 10, and 2 g/l, respectively, while the optimal initial pH of the culture medium was determined to be 8. Following response surface optimization, the maximum bioflocculant production level of 1.36 g/l was achieved, which was 43.40% higher than the original culture medium. Within 5 min, a 20.0% (v/v) dosage of the yielded bioflocculant applied to algal cultures resulted in the highest flocculating efficiency of 93.9% with Spirulina platensis. The bioflocculant from C. marina MCCC1113 may have promising application potential for highly productive microalgae collection, according to the findings of this study.

Genetic regulation and fermentation strategy for squalene production in Schizochytrium sp.

Squalene, as an important terpenoid, is extensively used in the medicine and health care fields owing to its functions of anti-oxidation, blood lipid regulation and cancer prevention. The marine microalgae, Schizochytrium sp., which acts as an excellent strain with potential of high squalene production was selected as the starting strain. The overexpressed strain with sqs gene got the reduced biomass and lipid, while the squalene titer was increased by 79.6% ± 4.7% to 12.8 ± 0.2 mg/L. In order to further increase squalene production, the recombinant strain (HS strain) with sqs and hmgr gene co-overexpression was further constructed. The biomass and squalene titer of the HS strain were increased by 13.6% ± 1.2% and 88.8% ± 5.3%, respectively, which indicated the carbon flux of the mevalonate pathway was enhanced for squalene accumulation. Regarding the squalene synthesis is completely coupled with cell growth, fermentation strategy to prolong the logarithmic growth phase was conducive to improve squalene production. Under the condition of optimal composition and concentrated medium, the squalene titer of HS strain was 27.0 ± 1.3 mg/L, which was 2.0 times that of the basal medium condition (13.5 ± 0.4 mg/L). This study which combined the metabolic engineering and fermentation strategy provides a new strategy for squalene production in Schizochytrium sp. KEY POINTS: •The overexpression of sqs and hmgr genes promoted carbon metabolism for squalene. •The optimal and concentrated media can increase squalene yield.

Study on the mechanism of efficient extracellular expression of toxic streptomyces phospholipase D in Brevibacillus choshinensis under Mg2+ stress.

BACKGROUND: Phospholipase D (PLD) has significant advantages in the food and medicine industries due to its unique transphosphatidylation. However, the high heterologous expression of PLD is limited by its cytotoxicity. The present study sought to develop an efficient and extracellular expression system of PLD in the non-pathogenic Brevibacillus choshinensis (B. choshinensis). RESULTS: The extracellular PLD was effectively expressed by the strong promoter (P2) under Mg2+ stress, with the highest activity of 10 U/mL. The inductively coupled plasma-mass spectrometry (ICP-MS) results elucidated that the over-expression of PLD by P2 promoter without Mg2+ stress induced the ionic homeostasis perturbation caused by the highly enhanced Ca2+ influx, leading to cell injury or death. Under Mg2+ stress, Ca2+ influx was significantly inhibited, and the strengths of P2 promoter and HWP gene expression were weakened. The study results revealed that the mechanism of Mg2+ induced cell growth protection and PLD expression might be related to the lowered strength of PLD expression by P2 promoter repression to meet with the secretion efficiency of B. choshinensis, and the redistribution of intracellular ions accompanied by decreased Ca2+ influx. CONCLUSIONS: The PLD production was highly improved under Mg2+ stress. By ICP-MS and qPCR analysis combined with other results, the mechanism of the efficient extracellular PLD expression under Mg2+ stress was demonstrated. The relatively low-speed PLD expression during cell growth alleviated cell growth inhibition and profoundly improved PLD production. These results provided a potential approach for the large-scale production of extracellular PLD and novel insights into PLD function.

Enhancing astaxanthin accumulation in Xanthophyllomyces dendrorhous by a phytohormone: metabolomic and gene expression profiles.

Xanthophyllomyces dendrorhous is a promising source of natural astaxanthin due to its ability to accumulate high amounts of astaxanthin. This study showed that 6-benzylaminopurine (6-BAP) is an effective substrate that enhances cell biomass and astaxanthin accumulation in X. dendrorhous. In the current study, the biomass and astaxanthin content in X. dendrorhous were determined to be improved by 21.98% and 24.20%, respectively, induced by 6-BAP treatments. To further understand the metabolic responses of X. dendrorhous to 6-BAP, time-course metabolomics and gene expression levels of X. dendrorhous cultures with and without 6-BAP feeding were investigated. Metabolome analysis revealed that 6-BAP facilitated glucose consumption, promoted the glycolysis, suppressed the TCA cycle, drove carbon flux of acetyl-CoA into fatty acid and mevalonate biosynthesis, and finally facilitated the formation of astaxanthin. ROS analysis suggested that the antioxidant mechanism in X. dendrorhous can be induced by 6-BAP. Additionally, the process of 6-BAP significantly upregulated the expression of six key genes involved in pathways related to astaxanthin biosynthesis. This research demonstrates the metabolomic mechanism of phytohormone stimulation of astaxanthin production iNn X. dendrorhous and presents a new strategy to improve astaxanthin production to prevent the dilemma of choosing between accumulation of astaxanthin and cell biomass.

Development of a fourth-order compact finite difference scheme for simulation of simulated-moving-bed process.

A fourth-order compact finite difference scheme was developed to solve the model equation of simulated moving bed, which has a boundary condition that is updated along the calculation process and cannot be described as an explicit function of time. Two different methods, direct method and pseudo grid point method, were proposed to deal with the boundary condition. The high accuracy of the two methods was confirmed by a case study of solving an advection-diffusion equation with exact solution. The developed compact finite difference scheme was then used to simulate the SMB processes for glucose-fructose separation and enantioseparation of 1,1'-bi-2-naphtol. It was found that the simulated results fit well with the experimental data. Furthermore, the developed method was further combined with the continuous prediction method to shorten the computational time and the results showed that, the computational time can be saved about 45%.

Functions of Enyolreductase (ER) Domains of PKS Cluster in Lipid Synthesis and Enhancement of PUFAs Accumulation in Schizochytrium limacinum SR21 Using Triclosan as a Regulator of ER.

The polyketide synthase (PKS) cluster genes are supposed to synthesize polyunsaturated fatty acids (PUFAs) in S. limacinum. In this study, two enyolreductase (ER) genes located on PKS cluster were knocked out through homologous recombination to explore their functions. The knock-out of OrfB-ER (located on OrfB subunit) decreased lipid content and had obvious decrease on PUFAs content, indicating OrfB-ER domain played a vital role on PUFAs synthesis; the knock-out of OrfC-ER (located on OrfC subunit) decreased SFAs content and increased total lipid content, indicating OrfC-ER domain was likely to be related with SFAs synthesis, and lipid production could be improved by down-regulating OrfC-ER domain expression. Therefore, the addition of triclosan as a reported regulator of ER domain induced the increase of PUFAs production by 51.74% and lipids yield by 47.63%. Metabolic analysis indicated triclosan played its role through inhibiting the expression of OrfC-ER to reduce the feedback inhibition of SFAs and further to enhance NADPH synthesis for lipid production, and by weakening mevalonate pathway and tricarboxylic acid (TCA) cycle to shift precursors for lipid and PUFAs synthesis. This research illuminates functions of two ER domains in S. limacinum and provides a potential targets for improving lipid production.

The role of fluconazole in the regulation of fatty acid and unsaponifiable matter biosynthesis in Schizochytrium sp. MYA 1381.

BACKGROUND: Schizochytrium has been widely used in industry for synthesizing polyunsaturated fatty acids (PUFAs), especially docosahexaenoic acid (DHA). However, unclear biosynthesis pathway of PUFAs inhibits further production of the Schizochytrium. Unsaponifiable matter (UM) from mevalonate pathway is crucial to cell growth and intracellular metabolism in all higher eukaryotes and microalgae. Therefore, regulation of UM biosynthesis in Schizochytrium may have important effects on fatty acids synthesis. Moreover, it is well known that UMs, such as squalene and ß-carotene, are of great commercial value. Thus, regulating UM biosynthesis may also allow for an increased valuation of Schizochytrium. RESULTS: To investigate the correlation of UM biosynthesis with fatty acids accumulation in Schizochytrium, fluconazole was used to block the sterols pathway. The addition of 60 mg/L fluconazole at 48 h increased the total lipids (TLs) at 96 h by 16% without affecting cell growth, which was accompanied by remarkable changes in UMs and NADPH. Cholesterol content was reduced by 8%, and the squalene content improved by 45% at 72 h, which demonstrated fluconazole's role in inhibiting squalene flow to cholesterol. As another typical UM with antioxidant capacity, the ß-carotene production was increased by 53% at 96 h. The increase of squalene and ß-carotene could boost intracellular oxidation resistance to protect fatty acids from oxidation. The NADPH was found to be 33% higher than that of the control at 96 h, which meant that the cells had more reducing power for fatty acid synthesis. Metabolic analysis further confirmed that regulation of sterols was closely related to glucose absorption, pigment biosynthesis and fatty acid production in Schizochytrium. CONCLUSION: This work first reported the effect of UM biosynthesis on fatty acid accumulation in Schizochytrium. The UM was found to affect fatty acid biosynthesis by changing cell membrane function, intracellular antioxidation and reducing power. We believe that this work provides valuable insights in improving PUFA and other valuable matters in microalgae.

Screening chemical modulators of benzoic acid derivatives to improve lipid accumulation in Schizochytrium limacinum SR21 with metabolomics analysis.

BACKGROUND: Schizochytrium sp. is a marine fungus with great potential as an alternative commercial source of lipids rich in polyunsaturated fatty acids (PUFAs). To further increase lipid accumulation in Schizochytrium sp., the effect of exogenous additives has become one of the hotspots of current research. Although benzoic acid derivatives showed positive effects on lipid accumulation in Schizochytrium, the biochemical mechanism needs further investigation. RESULTS: Four benzoic acid derivatives (sodium benzoate, p-aminobenzoic acid, p-methyl benzoic acid and folic acid) were screened and evaluated for their effect on lipid accumulation in Schizochytrium limacinum SR21. The lipid yield was increased by 56.84% with p-aminobenzoic acid (p-ABA) at a concentration of 200 mg/L among the four tested chemical modulators. The metabolomics analysis showed that 200 mg/L p-ABA was optimal for promoting glucose catabolism in glycolysis with an increase in the mevalonate pathway and a weakening of the tricarboxylic acid (TCA) cycle. Moreover, p-ABA increased NADPH generation by enhancing the pentose phosphate pathway (PPP), ultimately redirecting the metabolic flux to lipid synthesis. Fed-batch fermentation further proved that p-ABA could significantly increase the yield of lipid by 30.01%, reaching 99.67 g/L, and the lipid content was increased by 35.03%, reaching 71.12%. More importantly, the yields of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) were increased by 33.28% and 42.0%, respectively. CONCLUSION: The addition of p-ABA could promote the synthesis of tetrahydrofolate, enhancing NADPH, which ultimately promoted the flow of carbon flux to lipid synthesis. These findings provide a valuable strategy for improving the lipid accumulation in Schizochytrium by additives.

Strategy and regulatory mechanisms of glutamate feeding to enhance astaxanthin yield in Xanthophyllomyces dendrorhous.

Xanthophyllomyces dendrorhous is an excellent industrial source for production of natural astaxanthin, but the yield of astaxanthin is relative low due to the contradiction between biomass weight and astaxanthin accumulation. Glutamate, a metabolite connecting nitrogen and carbon metabolisms, is probably a promising entry point to interfere cellular metabolisms. Thus, the effect of glutamate on cell growth and astaxanthin accumulation in X. dendrorhous was investigated. Results showed that glutamate feeding facilitated glucose consumption and further led to the increment of astaxanthin content with little influence of cell growth. A comparative proteomics study was applied to decipher the regulatory mechanisms of enhanced astaxanthin biosynthesis in X. dendrorhous as a response to the glutamate feeding. The expressions of proteins with the highest degree of fold change were involved in carbohydrate, amino acids, and carotenogenesis metabolisms as well as redox and stress-associated metabolisms. In addition, a possible regulatory model of enhanced astaxanthin accumulation in response to glutamate feeding in X. dendrorhous is also proposed.

Changes to the tropomyosin structure alter the angiotensin-converting enzyme inhibitory activity and texture profiles of eel balls under high hydrostatic pressure.

Changes in the structure of tropomyosin (TM) altered the texture profiles of eel balls and the inhibitory activity of the angiotensin-converting enzyme (ACE). The secondary and tertiary structure of TM was determined after high hydrostatic pressure (HHP) treatment. The correlation between the spatial structure of TM and the texture profiles of eel balls was developed and discussed. The ß-sheet was converted to a ß-turn and a random coil when treated at HHP (200-400 MPa), meanwhile the α-helix unfolded and was converted into a ß-sheet, ß-turn and a random coil with treatment at 500 and 600 MPa. The surface hydrophobicity (H0) was increased and the sulfhydryl (SH) content decreased with an increase in the pressure. The results indicated that the texture profiles of eel balls showed a negative relationship with the α-helix, ß-sheet and SH content. The texture profiles of eel balls were greatly enhanced after treatment at 500 and 600 MPa, leading to the improved surface network of the eel ball products. The ACE inhibitory activity of TM after HHP treatment exhibited a positive relationship with the ß-sheet content in the protein. The ACE inhibitory activity was preserved under 600 MPa.

Using a trait-based approach to optimize mixotrophic growth of the red microalga Porphyridium purpureum towards fatty acid production.

BACKGROUND: Organic carbon sources have been reported to simultaneously increase the growth and lipid accumulation in microalgae. However, there have been no studies of the mixotrophic growth of Porphyridium purpureum in organic carbon media. In this study, three organic carbon sources, glucose, sodium acetate, and glycerol were used as substrates for the mixotrophic growth of P. purpureum. Moreover, a novel trait-based approach combined with Generalized Additive Modeling was conducted to determine the dosage of each organic carbon source that optimized the concentration of cell biomass or fatty acid. RESULTS: A 0.50% (w/v) dosage of glucose was optimum for the enhancement of the cell growth of P. purpureum, whereas sodium acetate performed well in enhancing cell growth, arachidonic acid (ARA) and eicosapentaenoic acid (EPA) content, and glycerol was characterized by its best performance in promoting both cell growth and ARA/EPA ratio. The optimum dosages of sodium acetate and glycerol for the ARA concentration were 0.25% (w/v) and 0.38% (v/v), respectively. An ARA concentration of 211.47 mg L-1 was obtained at the optimum dosage of glycerol, which is the highest ever reported. CONCLUSIONS: The results suggested that a comprehensive consider of several traits offers an effective strategy to select an optimum dosage for economic and safe microalgae cultivation. This study represents the first attempt of mixotrophic growth of P. purpureum and proved that both biomass and ARA accumulation could be enhanced under supplements of organic carbon sources, which brightens the commercial cultivation of microalgae for ARA production.

Functions of PKS Genes in Lipid Synthesis of Schizochytrium sp. by Gene Disruption and Metabolomics Analysis.

Schizochytrium sp. is a kind of marine microalgae with great potential as promising sustainable source of polyunsaturated fatty acids (PUFAs). Polyketide synthase-like (PKS synthase) is supposed to be one of the main ways to synthesize PUFAs in Schizochytrium sp. In order to study the exact relationship between PKS and PUFA biosynthesis, chain length factor (CLF) and dehydrogenase (DH) were cloned from the PKS gene cluster in Schizochytrium sp., then disrupted by homologous recombination. The results showed that DH- and CLF-disrupted strains had significant decreases (65.85 and 84.24%) in PUFA yield, while the saturated fatty acid (SFA) proportion in lipids was slightly increased. Meanwhile, the disruption of CLF decreased the C-22 PUFA proportion by 57.51% without effect on C-20 PUFA accumulation while DH-disrupted mutant decreased the production of each PUFA. Combined with analysis of protein prediction, it indicated that CLF gene exerted an enormous function on the carbon chain elongation in PUFA synthesis, especially for the final elongation from C-20 to C-22 PUFAs. Metabolomics analysis also suggested that the disruption of both genes resulted in the decrease of PUFAs but increase of SFAs, thus weakening glycolysis and tricarboxylic acid (TCA) cycle pathways. This study offers a broad new vision to research the mechanism of PUFA synthesis in Schizochytrium sp.

Overexpression of Malonyl-CoA: ACP Transacylase in Schizochytrium sp. to Improve Polyunsaturated Fatty Acid Production.

Polyunsaturated fatty acids (PUFAs) have been widely applied in the food and medical industry. In this study, malonyl-CoA: ACP transacylase (MAT) was overexpressed through homologous recombination to improve PUFA production in Schizochytrium. The results showed that the lipid and PUFA concentration were increased by 10.1 and 24.5% with MAT overexpression, respectively. Metabolomics analysis revealed that the intracellular tricarboxylic acid cycle was weakened and glucose absorption was accelerated in the engineered strain. In the mevalonate pathway, intracellular carotene content was decreased, and the carbon flux was then redirected toward PUFA synthesis. Furthermore, a glucose fed-batch fermentation was finally performed with the engineered Schizochytrium. The total lipid yield was further increased to 110.5 g/L, 39.6% higher than the wild strain. Docosahexaenoic acid and eicosapentaenoic acid yield were enhanced to 47.39 g/L and 1.65 g/L with an increase of 81.5 and 172.5%, respectively. This study provided an effective metabolic engineering strategy for industrial PUFA production.

Enhancement of cell growth and phycocyanin production in Arthrospira (Spirulina) platensis by metabolic stress and nitrate fed-batch.

Arthrospira (Spirulina) platensis is known to have high-quality proteins content and phycocyanin as one of the major pigment constituents of the cells, and the most challenging problem associated with phycocyanin production in Arthrospira is to optimize its intracellular accumulation. The present study evaluated the metabolic stress conditions (by nutrient enrichment) of Arthrospira platensis FACHB-314 for boosting biomass growth and high content phycocyanin accumulation. Experimental results showed that 5 mM sodium glutamate and 7.5 mM succinic acid could enhance biomass yield as well as phycocyanin accumulation compared with that of the control groups. The present study demonstrates that the biomass growth and phycocyanin accumulation were significantly enhanced in fed-batch cultivation of Arthrospira platensis by applying the substrates as metabolic stress agents combined with nitrate feeding strategy. cobA/hemD, hemG and ho genes presented the over-expression level with adding sodium glutamate and succinic acid in cultures, respectively, compared to the control groups.

Overproduction of L-tryptophan via simultaneous feed of glucose and anthranilic acid from recombinant Escherichia coli W3110: Kinetic modeling and process scale-up.

L-tryptophan is an essential amino acid widely used in food and pharmaceutical industries. However, its production via Escherichia coli fermentation suffers severely from both low glucose conversion efficiency and acetic acid inhibition, and to date effective process control methods have rarely been explored to facilitate its industrial scale production. To resolve these challenges, in the current research an engineered strain of E. coli was used to overproduce L-tryptophan. To achieve this, a novel dynamic control strategy which incorporates an optimized anthranilic acid feeding into a dissolved oxygen-stat (DO-stat) glucose feeding framework was proposed for the first time. Three original contributions were observed. Firstly, compared to previous DO control methods, the current strategy was able to inhibit completely the production of acetic acid, and its glucose to L-tryptophan yield reached 0.211 g/g, 62.3% higher than the previously reported. Secondly, a rigorous kinetic model was constructed to simulate the underlying biochemical process and identify the effect of anthranilic acid on both glucose conversion and L-tryptophan synthesis. Finally, a thorough investigation was conducted to testify the capability of both the kinetic model and the novel control strategy for process scale-up. It was found that the model possesses great predictive power, and the presented strategy achieved the highest glucose to L-tryptophan yield (0.224 g/g) ever reported in large scale processes, which approaches the theoretical maximum yield of 0.227 g/g. This research, therefore, paves the way to significantly enhance the profitability of the investigated bioprocess.