Production Of L-alanine Research Articles

Systems engineering of Escherichia coli for high-level l-alanine production

l-alanine is a critical natural amino acid that is widely used in the food, pharmaceutical, and personal care industries. However, low titer and long fermentation period limits the industrial viability of l-alanine production. In this study, we engineered a microbial cell factory for efficient l-alanine synthesis using a variety of metabolic engineering strategies. Initially, l-alanine was biosynthesized through the heterologous introduction of alanine dehydrogenase from Geobacillus stearothermophilus. Thereafter, the l-alanine biosynthetic pathway and transport module were modified to improve its titers. Furthermore, a dynamic response system was constructed based on the l-alanine-driven PAla-7 promoter to regulate the tolerance target BetT expression and enhance the tolerance of Escherichia coli to high l-alanine concentrations. Finally, the resultant strain E. coli W-PAla-7-BetT could produce 134.5 g/L of l-alanine in 24 h in a 5-L bioreactor, with 5.6 g/L/h of productivity, which is the highest productivity reported to date. Our findings may help in constructing high-performance microbial cell factories for the production of l-alanine and other valuable chemicals.

Photo/biocatalytic system for visible-light driven L-alanine production from ammonia and pyruvate

The chemical structure of L-alanine is similar to that of L-lactic acid and its polymerisation, poly (L-alanine) (poly-L-Ala), is a nylon with the function of biodegradable. There is a need...

Optimizing Hexose Utilization Pathways of Cupriavidus necator for Improving Growth and L-Alanine Production under Heterotrophic and Autotrophic Conditions.

Cupriavidus necator is a versatile microbial chassis to produce high-value products. Blocking the poly-β-hydroxybutyrate synthesis pathway (encoded by the phaC1AB1 operon) can effectively enhance the production of C. necator, but usually decreases cell density in the stationary phase. To address this problem, we modified the hexose utilization pathways of C. necator in this study by implementing strategies such as blocking the Entner-Doudoroff pathway, completing the phosphopentose pathway by expressing the gnd gene (encoding 6-phosphogluconate dehydrogenase), and completing the Embden-Meyerhof-Parnas pathway by expressing the pfkA gene (encoding 6-phosphofructokinase). During heterotrophic fermentation, the OD600 of the phaC1AB1-knockout strain increased by 44.8% with pfkA gene expression alone, and by 93.1% with gnd and pfkA genes expressing simultaneously. During autotrophic fermentation, gnd and pfkA genes raised the OD600 of phaC1AB1-knockout strains by 19.4% and 12.0%, respectively. To explore the effect of the pfkA gene on the production of C. necator, an alanine-producing C. necator was constructed by expressing the NADPH-dependent L-alanine dehydrogenase, alanine exporter, and knocking out the phaC1AB1 operon. The alanine-producing strain had maximum alanine titer and yield of 784 mg/L and 11.0%, respectively. And these values were significantly improved to 998 mg/L and 13.4% by expressing the pfkA gene. The results indicate that completing the Embden-Meyerhof-Parnas pathway by expressing the pfkA gene is an effective method to improve the growth and production of C. necator.

Alanine dehydrogenases from four different microorganisms: characterization and their application in L-alanine production

BackgroundAlanine dehydrogenase (AlaDH) belongs to oxidoreductases, and it exists in several different bacteria species and plays a key role in microbial carbon and nitrogen metabolism, spore formation and photosynthesis. In addition, AlaDH can also be applied in biosynthesis of L-alanine from cheap carbon source, such as glucose.ResultsTo achieve a better performance of L-alanine accumulation, system evaluation and comparison of different AlaDH with potential application value are essential. In this study, enzymatic properties of AlaDH from Bacillus subtilis 168 (BsAlaDH), Bacillus cereus (BcAlaDH), Mycobacterium smegmatis MC2 155 (MsAlaDH) and Geobacillus stearothermophilus (GsAlaDH) were firstly carefully investigated. Four different AlaDHs have few similarities in optimum temperature and optimum pH, while they also exhibited significant differences in enzyme activity, substrate affinity and enzymatic reaction rate. The wild E. coli BL21 with these four AlaDHs could produce 7.19 g/L, 7.81 g/L, 6.39 g/L and 6.52 g/L of L-alanine from 20 g/L glucose, respectively. To further increase the L-alanine titer, competitive pathways for L-alanine synthesis were completely blocked in E. coli. The final strain M-6 could produce 80.46 g/L of L-alanine with a yield of 1.02 g/g glucose after 63 h fed-batch fermentation, representing the highest yield for microbial L-alanine production.ConclusionsEnzyme assay, biochemical characterization and structure analysis of BsAlaDH, BcAlaDH, MsAlaDH and GsAlaDH were carried out. In addition, application potential of these four AlaDHs in L-alanine productions were explored. The strategies here can be applied for developing L-alanine producing strains with high titers.

Activating a dormant metabolic pathway for high-temperature l-alanine production in Bacillus licheniformis

l-Alanine is an important amino acid widely used in food, medicine, materials, and other fields. Here, we develop Bacillus licheniformis as an efficient l-alanine microbial cell factory capable of realizing high-temperature fermentation. By enhancing the glycolytic pathway, knocking out the by-product pathways andoverexpressing the thermostable alanine dehydrogenase, the engineered B. licheniformis strain BLA3 produced 93.7g/L optically pure l-alanine at 50°C. Subsequently, d-alanine dependence of an alanine racemase-deficient strain is relieved by adaptive laboratory evolution, implying that a dormant alternative pathway for d-alanine synthesis is activated in the evolved strain. The d-amino acid aminotransferase Dat1 is shown to be a key enzyme in the dormant alternative pathway. Molecular mechanism of the d-alanine dependence is revealed via mutational analysis. This study demonstrates a novel technology for high-temperature l-alanine production and shows that activating dormant metabolic pathway(s) is an effective strategy of metabolic engineering.

Key technology for anaerobic fermentation of L-alanine and its commercialization

Traditionally, amino acids are produced mainly by chemical synthesis or aerobic fermentation. Compared to chemical synthesis, production of amino acids by microbial fermentation directly uses renewable resources as feedstock and this reduces the dependence on petroleum-based compounds and decreases pollutants generation and toxic substrates usage. Fermentation under aerobic conditions has been used widely for its fast growth and high titers. However, a large amount of carbon is used for cell growth and this results in high biomass but low yield of target chemicals. Unlike the long history of aerobic fermentation, the commercial production of amino acids by anaerobic fermentation is realized only in recent years. It has several advantages such as simpler operation, no need for oxygen supply, and high yield close to the theoretical maximum value. L-alanine is the first amino acid commercially produced by anaerobic fermentation. In this article, we summarize the key technology for anaerobic fermentative production of L-alanine and its commercialization. As it is shown to be low-cost, high-efficiency, and environmental-friendly, anaerobic fermentation is expected to be widely used in industrial process and brings greater economic values and social benefits in the future.

Optimization of L-alanine production in the recombinant Pediococcus acidilactici BD16 (alaD+)

L-alanine has an extensive array of applications in the food and pharmaceutical industries. However, its commercial utilization is still limited due to reasons like high production costs, poor substrate availability, slower fermentations, lower productivity and lesser purity. In the present study, a Generally Recognized As Safe (GRAS) Pedicoccus acidilactici BD16 (alaD+) strain was employed for the microbial synthesis of L-alanine. Primarily, a considerable enhancement of L-alanine production in the recombinant P. acidilactici BD16 (alaD+) was achieved through a batch fermentation process optimized using one variable approach and Response Surface Methodology. To further enhance L-alanine titers in the recombinant P. acidilactici BD16 (alaD+), a single phase fed-batch fermentation process was also employed and 261.3 g/L (2934 mM) L-alanine production was achieved within 48 h under optimized conditions. After extraction and crystallization, 94% crystal L-alanine (245.8 g/L; 2758 mM) was recovered from the culture broth with an enantiomeric purity of 97.3%. Owing to its high fermentative productivity, the recombinant P. acidilactici BD16 (alaD+) strain based L-alanine production bioprocess offers a great commercial potential and will eventually help in the global expansion of the microbial L-alanine market.

Metabolic Engineering of Pediococcus acidilactici BD16 for Heterologous Expression of Synthetic alaD Gene Cassette and L-Alanine Production in the Recombinant Strain Using Fed-Batch Fermentation

Metabolic engineering substantially aims at the development of more efficient, robust and industrially competitive microbial strains for the potential applications in food, fermentation and pharmaceutical industries. An efficient lab scale bioprocess was developed for high level fermentative production of L-alanine using metabolically engineered Pediococcus acidilactici BD16 (alaD+). Computational biology tools assisted the designing of a synthetic alaD gene cassette, which was further cloned in shuttle vector pLES003 and expressed using an auto-inducible P289 promoter. Further, L-alanine production in the recombinant P. acidilactici BD16 (alaD+) strain was carried out using fed-batch fermentation under oxygen depression conditions, which significantly enhanced L-alanine levels. The recombinant strain expressing the synthetic alaD gene produced 229.12 g/L of L-alanine after 42 h of fed-batch fermentation, which is the second highest microbial L-alanine titer reported so far. After extraction and crystallization, 95% crystal L-alanine (217.54 g/L) was recovered from the culture broth with an enantiomeric purity of 97%. The developed bioprocess using recombinant P. acidilactici BD16 (alaD+) is suggested as the best alternative to chemical-based commercial synthesis of L-alanine for potential industrial applications.

Metabolic engineering of microorganisms for L-alanine production.

L-alanine is extensively used in chemical, food, and medicine industries. Industrial production of L-alanine has been mainly based on the enzymatic process using petroleum-based L-aspartic acid as the substrate. L-alanine production from renewable biomass using microbial fermentation process is an alternative route. Many microorganisms can naturally produce L-alanine using aminotransferase or L-alanine dehydrogenase. However, production of L-alanine using the native strains has been limited due to their low yields and productivities. In this review, metabolic engineering of microorganisms for L-alanine production was summarized. Among them, the Escherichia coli strains developed by Dr. Lonnie Ingram's group which can produce L-alanine with anaerobic fermentation process had several advantages, especially having high L-alanine yield, and it was the first one that realized commercialization. L-alanine is also the first amino acid that could be industrially produced by anaerobic fermentation.

Characterization and rational modification of aspartate 4-decarboxylase from Acinetobacter radioresistens for the production of l-alanine.

Enzymatic synthesis of l-alanine has the advantages of less byproducts, strong stereoselectivity, and high catalytic efficiency. Aspartate 4-decarboxylase (ASD) is used industrially in DL-aspartic acid resolution and l-alanine production because it catalyzes the decarboxylation of l-aspartic acid. In this study, the ASD gene from Acinetobacter radioresistens (ArASD) was cloned, and its enzymatic properties were analyzed. ArASD is a dodecamer and has the highest enzyme activity ever reported to date. The optimal conditions for ArASD catalysis are 50°C and pH 4.5. Site-directed mutagenesis was used to improve ArASD stability under acidic conditions to compensate for its weak acid resistance, and the variant N35D with higher catalytic ability was obtained. The conversion by N35 recombinant cells of l-aspartic acid to l-alanine was 92.5% at pH 4.5% and 99.9% at pH 6.0, whereas that of the wild-type recombinant cells was 29.7% and 31.4%, respectively. Aspartase from Escherichia coli (AspA) was employed with ArASD to construct a dual-enzyme system that catalyzes fumaric acid to l-alanine, and the conversion reached 97.1% using recombinant cells harboring the dual-enzyme system. This study explored the enzymatic properties of ArASD and an effective strategy for the acidic resistance modification of ASD. Moreover, the strain expressing the ArASD variant and AspA engineered in this study has great potential application for the l-alanine production industry, especially in the case of high optical purity requirements.

Application of Pediococcus acidilactici BD16 (alaD +) expressing L-alanine dehydrogenase enzyme as a starter culture candidate for secondary wine fermentation

Secondary fermentation is generally recommended to improve the aroma, flavour, overall acceptability and stability of the wines. Pediococcus acidilactici BD16 expressing L-alanine dehydrogenase gene is hereby proposed as a starter culture bacterium for the value addition of ginger, kiwi, plum and rose wines through secondary fermentation. Secondary fermentation using Pediococcus acidilactici BD16 (alaD +) imparted a natural sweetness and several health-promoting attributes to the dry wines, thereby improving their sensory and quality attributes. GC-MS-based metabolic fingerprinting further revealed the presence of significantly higher levels of L-alanine and many bioactive secondary metabolites and flavour enhancers such as 3-butynol, acetaldehyde, alaninol, aminopentols, benzene methanol, octanoic acid, ribitol, etc. and therapeutic ingredients such as 8-azanonane, actinobolin, adamantanemethylamine, aminononadecane, amphetamine, benzeneethanamine, guanosine, heptadecanenitrile, isopropyluriedoacetate, octanoic acid, nortriptyline and rimantadine in the secondary fermented wines. Further, a probable metabolic pathway was constructed to indicate major metabolic biotransformations in the wines which accounted for the improved quality attributes and strongly anticipated their therapeutic potential. This study is probably the first of its kind to highlight the application of P. acidilactici BD16 (alaD +) for in situ L-alanine production during secondary wine fermentation for enhancing organoleptic and therapeutic attributes of interest to health-conscious consumers.

Biosynthesis of l-alanine from cis-butenedioic anhydride catalyzed by a triple-enzyme cascade via a genetically modified strain

We not only developed a strategy for the construction of multienzyme systems, which is of considerable value to the sustainable production and biocatalysis, but also provided an l-alanine production process that reduced wastewater and costs.

Development of a Cofactor Balanced, Multi Enzymatic Cascade Reaction for the Simultaneous Production of L-Alanine and L-Serine from 2-Keto-3-deoxy-gluconate

Enzymatic reaction cascades represent a powerful tool to convert biogenic resources into valuable chemicals for fuel and commodity markets. Sugars and their breakdown products constitute a significant group of possible substrates for such biocatalytic conversion strategies to value-added products. However, one major drawback of sugar cascades is the need for cofactor recycling without using additional enzymes and/or creating unwanted by-products. Here, we describe a novel, multi-enzymatic reaction cascade for the one-pot simultaneous synthesis of L-alanine and L-serine, using the sugar degradation product 2-keto-3-deoxygluconate and ammonium as precursors. To pursue this aim, we used four different, thermostable enzymes, while the necessary cofactor NADH is recycled entirely self-sufficiently. Buffer and pH optimisation in combination with an enzyme titration study yielded an optimised production of 21.3 +/− 1.0 mM L-alanine and 8.9 +/− 0.4 mM L-serine in one pot after 21 h.

Effect of Cold Acclimation on Selected Metabolic Enzymes During Diapause in The European Corn Borer Ostrinia nubilalis (Hbn.)

The European corn borer, Ostrinia nubilalis Hbn., is a pest Lepidopteran species whose larvae overwinter by entering diapause, gradually becoming cold-hardy. To investigate metabolic changes during cold hardening, activities of four metabolic enzymes – citrate synthase (CS), lactate dehydrogenase (LDH), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured in whole-body homogenates of pupae, non-diapausing and diapausing larvae acclimated to 5 °C, −3 °C and −16 °C. The highest CS activity was detected in non-diapausing larvae, reflecting active development, while the highest in vitro LDH activity was recorded in diapausing larvae at temperatures close to 0 °C, evidencing a metabolic switch towards anaerobic metabolism. However, in-gel LDH activity showed that production of pyruvate from lactate is triggered by sub-zero temperatures. The activities of both aminotransferases were highest in non-diapausing larvae. Our findings suggest that during diapause and cold hardening the aminotransferases catalyse production of L-alanine, an important cryoprotectant, and L-aspartate, which is closely tied to both transamination reactions and Krebs cycle. The results of this study indicate that, during diapause, the activity of metabolic enzymes is synchronized with exogenous factors, such as temperatures close to 0 °C. These findings support the notion that diapause is metabolically plastic and vibrant, rather than simply a passive, resting state.

Optimization of a reduced enzymatic reaction cascade for the production of L-alanine

Cell-free enzymatic reaction cascades combine the advantages of well-established in vitro biocatalysis with the power of multi-step in vivo pathways. The absence of a regulatory cell environment enables direct process control including methods for facile bottleneck identification and process optimization. Within this work, we developed a reduced, enzymatic reaction cascade for the direct production of L-alanine from D-glucose and ammonium sulfate. An efficient, activity based enzyme selection is demonstrated for the two branches of the cascade. The resulting redox neutral cascade is composed of a glucose dehydrogenase, two dihydroxyacid dehydratases, a keto-deoxy-aldolase, an aldehyde dehydrogenase and an L-alanine dehydrogenase. This artificial combination of purified biocatalysts eliminates the need for phosphorylation and only requires NAD as cofactor. We provide insight into in detail optimization of the process parameters applying a fluorescamine based L-alanine quantification assay. An optimized enzyme ratio and the necessary enzyme load were identified and together with the optimal concentrations of cofactor (NAD), ammonium and buffer yields of >95% for the main branch and of 8% for the side branch were achieved.

The Metabolic Responses to L-Glutamine of Livers from Rats with Diabetes Types 1 and 2.

There are several claims about the beneficial effects of supplementing L-glutamine to both type 1 and type 2 diabetes. The purpose of the present study was to provide detailed knowledge about the fate of this amino acid in the liver, the first organ that receives the compound when ingested orally. The study was done using the isolated perfused rat liver, an experimental system that preserves the microcirculation of the organ and that allows to measured several parameters during steady-state and pre steady-state conditions. L-Glutamine was infused in the portal vein (5 mM) and several parameters were monitored. Livers from type 1 diabetic rats showed an accelerated response to L-glutamine infusion. In consequence of this accelerated response livers from type 1 diabetic rats presented higher rates of ammonia, urea, glucose and lactate output during the first 25–30 minutes following L-glutamine infusion. As steady-state conditions approached, however, the difference between type 1 diabetes and control livers tended to disappear. Measurement of the glycogen content over a period of 100 minutes revealed that, excepting the initial phase of the L-glutamine infusion, the increased glucose output in livers from type 1 diabetic rats was mainly due to accelerated glycogenolysis. Livers from type 2 diabetic rats behaved similarly to control livers with no accelerated glucose output but with increased L-alanine production. L-Alanine is important for the pancreatic β-cells and from this point of view the oral intake of L-glutamine can be regarded as beneficial. Furthermore, the lack of increased glucose output in livers from type 2 diabetic rats is consistent with observations that even daily L-glutamine doses of 30 g do not increase the glycemic levels in well controlled type 2 diabetes patients.

Efficient L-Alanine Production by a Thermo-Regulated Switch in Escherichia coli.

L-Alanine has important applications in food, pharmaceutical and veterinary and is used as a substrate for production of engineered thermoplastics. Microbial fermentation could reduce the production cost and promote the application of L-alanine. However, the presence of L-alanine significantly inhibit cell growth rate and cause a decrease in the ultimate L-alanine productivity. For efficient L-alanine production, a thermo-regulated genetic switch was designed to dynamically control the expression of L-alanine dehydrogenase (alaD) from Geobacillus stearothermophilus on the Escherichia coli B0016-060BC chromosome. The optimal cultivation conditions for the genetically switched alanine production using B0016-060BC were the following: an aerobic growth phase at 33°C with a 1-h thermo-induction at 42°C followed by an oxygen-limited phase at 42°C. In a bioreactor experiment using the scaled-up conditions optimized in a shake flask, B0016-060BC accumulated 50.3g biomass/100g glucose during the aerobic growth phase and 96g alanine/100g glucose during the oxygen-limited phase, respectively. The L-alanine titer reached 120.8g/l with higher overall and oxygen-limited volumetric productivities of 3.09 and 4.18g/lh, respectively, using glucose as the sole carbon source. Efficient cell growth and L-alanine production were reached separately, by switching cultivation temperature. The results revealed the application of a thermo-regulated strategy for heterologous metabolic production and pointed to strategies for improving L-alanine production.

Reducing lactate secretion by ldhA Deletion in L-glutamate- producing strain Corynebacterium glutamicum GDK-9.

L-lactate is one of main byproducts excreted in to the fermentation medium. To improve L-glutamate production and reduce L-lactate accumulation, L-lactate dehydrogenase-encoding gene ldhA was knocked out from L-glutamate producing strain Corynebacterium glutamicum GDK-9, designated GDK-9ΔldhA. GDK-9ΔldhA produced approximately 10.1% more L-glutamate than the GDK-9, and yielded lower levels of such by-products as α-ketoglutarate, L-lactate and L-alanine. Since dissolved oxygen (DO) is one of main factors affecting L-lactate formation during L-glutamate fermentation, we investigated the effect of ldhA deletion from GDK-9 under different DO conditions. Under both oxygen-deficient and high oxygen conditions, L-glutamate production by GDK-9ΔldhA was not higher than that of the GDK-9. However, under micro-aerobic conditions, GDK-9ΔldhA exhibited 11.61% higher L-glutamate and 58.50% lower L-alanine production than GDK-9. Taken together, it is demonstrated that deletion of ldhA can enhance L-glutamate production and lower the unwanted by-products concentration, especially under micro-aerobic conditions.

Optimizing Cofactor Specificity of Oxidoreductase Enzymes for the Generation of Microbial Production Strains—OptSwap

Abstract Central oxidoreductase enzymes (eg, dehydrogenases, reductases) in microbial metabolism often have preferential binding specificity for one of the two major currency metabolites NAD(H) and NADP(H). These enzyme specificities result in a division of the metabolic functionality of the currency metabolites: enzymes reducing NAD+ to NADH drive oxidative phosphorylation, and enzymes reducing NADP+ to NADPH drive anabolic reactions. In this work, we introduce the computational method OptSwap, which predicts bioprocessing strain designs by identifying optimal modifications of the cofactor binding specificities of oxidoreductase enzyme and complementary reaction knockouts. Using the Escherichia coli genome-scale metabolic model iJO1366, OptSwap predicted eight growth-coupled production designs with significantly greater product yields or substrate-specific productivities than designs predicted with gene knockouts alone. These designs were identified for the production of L-alanine, succinate, acetate, an...

Inhibition of Alanine Aminotransferase in Silico and in Vivo Promotes Mitochondrial Metabolism to Impair Malignant Growth

Cancer cells commonly exhibit increased nonoxidative d-glucose metabolism whereas induction of mitochondrial metabolism may impair malignant growth. We have first used an in silico method called elementary mode analysis to identify inhibition of ALAT (l-alanine aminotransferase) as a putative target to promote mitochondrial metabolism. We then experimentally show that two competitive inhibitors of ALAT, l-cycloserine and β-chloro-l-alanine, inhibit l-alanine production and impair d-glucose uptake of LLC1 Lewis lung carcinoma cells. The latter inhibition is linked to an initial energy deficit, as quantified by decreased ATP content, which is then followed by an activation of AMP-activated protein kinase and subsequently increased respiration rates and mitochondrial production of reactive oxygen species, culminating in ATP replenishment in ALAT-inhibited LLC1 cells. Moreover, we observe altered phosphorylation of p38 MAPK (mitogen-activated protein kinase 14), ERK (extracellular signal-regulated kinase 1/2), and Rb1 (retinoblastoma 1) proteins, as well as decreased expression of Cdc25a (cell decision cycle 25 homolog A) and Cdk4 (cyclin-dependent kinase 4). Importantly, these sequelae of ALAT inhibition culminate in similarly reduced anchorage-dependent and anchorage-independent growth rates of LLC1 cells, together suggesting that inhibition of ALAT efficiently impairs cancer growth by counteracting the Warburg effect due to compensatory activation of mitochondrial metabolism.