Production of optically pure d-lactic acid from brown rice using metabolically engineered Lactobacillus plantarum

Simultaneous saccharification and fermentation (SSF) of d-lactic acid was performed using brown rice as both a substrate and a nutrient source. An engineered Lactobacillus plantarum NCIMB 8826 strain, in which the ʟ-lactate dehydrogenase gene was disrupted, produced 97.7 g/L d-lactic acid from 20% (w/v) brown rice without any nutrient supplementation. However, a significant amount of glucose remained unconsumed and the yield of lactic acid was as low as 0.75 (g/g-glucose contained in brown rice). Interestingly, the glucose consumption was significantly improved by adapting L. plantarum cells to the low-pH condition during the early stage of SSF (8–17 h). As a result, 117.1 g/L d-lactic acid was produced with a high yield of 0.93 and an optical purity of 99.6% after 144 h of fermentation. SSF experiments were repeatedly performed for ten times and d-lactic acid was stably produced using recycled cells (118.4–129.8 g/L). On average, d-lactic acid was produced with a volumetric productivity of 2.18 g/L/h over 48 h.

     

Sodium ions activated phosphofructokinase leading to enhanced d-lactic acid production by Sporolactobacillus inulinus using sodium hydroxide as a neutralizing agent

Sporolactobacillus inulinus is a superior d-lactic acid-producing bacterium and proposed species for industrial production. The major pathway for d-lactic acid biosynthesis, glycolysis, is mainly regulated via the two irreversible steps catalyzed by the allosteric enzymes, phosphofructokinase (PFK) and pyruvate kinase. The activity level of PFK was significantly consistent with the cell growth and d-lactic acid production, indicating its vital role in control and regulation of glycolysis. In this study, the ATP-dependent PFK from S. inulinus was expressed in Escherichia coli and purified to homogeneity. The PFK was allosterically activated by both GDP and ADP and inhibited by phosphoenolpyruvate; the addition of activators could partly relieve the inhibition by phosphoenolpyruvate. Furthermore, monovalent cations could enhance the activity, and Na⁺ was the most efficient one. Considering this kind activation, NaOH was investigated as the neutralizer instead of the traditional neutralizer CaCO₃. In the early growth stage, the significant accelerated glucose consumption was achieved in the NaOH case probably for the enhanced activity of Na⁺-activated PFK. Using NaOH as the neutralizer at pH 6.5, the fermentation time was greatly shortened about 22 h; simultaneously, the glucose consumption rate and the d-lactic acid productivity were increased by 34 and 17%, respectively. This probably contributed to the increased pH and Na⁺-promoted activity of PFK. Thus, fermentations by S. inulinus using the NaOH neutralizer provide a green and highly efficient d-lactic acid production with easy subsequent purification.

     

d-Lactic acid biosynthesis from biomass-derived sugars via Lactobacillus delbrueckii fermentation

Poly-lactic acid (PLA) derived from renewable resources is considered to be a good substitute for petroleum-based plastics. The number of poly l-lactic acid applications is increased by the introduction of a stereocomplex PLA, which consists of both poly-l and d-lactic acid and has a higher melting temperature. To date, several studies have explored the production of l-lactic acid, but information on biosynthesis of d-lactic acid is limited. Pulp and corn stover are abundant, renewable lignocellulosic materials that can be hydrolyzed to sugars and used in biosynthesis of d-lactic acid. In our study, saccharification of pulp and corn stover was done by cellulase CTec2 and sugars generated from hydrolysis were converted to d-lactic acid by a homofermentative strain, L. delbrueckii, through a sequential hydrolysis and fermentation process (SHF) and a simultaneous saccharification and fermentation process (SSF). 36.3 g L^?1 of d-lactic acid with 99.8 % optical purity was obtained in the batch fermentation of pulp and attained highest yield and productivity of 0.83 g g^?1 and 1.01 g L^?1 h^?1, respectively. Luedeking–Piret model described the mixed growth-associated production of d-lactic acid with a maximum specific growth rate 0.2 h^?1 and product formation rate 0.026 h^?1, obtained for this strain. The efficient synthesis of d-lactic acid having high optical purity and melting point will lead to unique stereocomplex PLA with innovative applications in polymer industry.     

An aldonolactonase AltA from Penicillium oxalicum mitigates the inhibition of β-glucosidase during lignocellulose biodegradation

Efficient deconstruction of lignocellulose is achieved by the synergistic action of various hydrolytic and oxidative enzymes. However, the aldonolactones generated by oxidative enzymes have inhibitory effects on some cellulolytic enzymes. In this work, d-glucono-1,5-lactone was shown to have a much stronger inhibitory effect than d-glucose and d-gluconate on β-glucosidase, a vital enzyme during cellulose degradation. AltA, a secreted enzyme from Penicillium oxalicum, was identified as an aldonolactonase which can catalyze the hydrolysis of d-glucono-1,5-lactone to d-gluconic acid. In the course of lignocellulose saccharification conducted by cellulases from P. oxalicum or Trichoderma reesei, supplementation of AltA was able to relieve the decrease of β-glucosidase activity obviously with a stimulation of glucose yield. This boosting effect disappeared when sodium azide and ethylenediaminetetraacetic acid (EDTA) were added to the saccharification system to inhibit the activities of oxidative enzymes. In summary, we describe the first heterologous expression of a fungal secreted aldonolactonase and its application as an efficient supplement of cellulolytic enzyme system for lignocellulose biodegradation.

     

Biosynthesis of <Emphasis Type="SmallCaps">d</Emphasis>-lactic acid from lignocellulosic biomass

d-lactic acid is a versatile and important industrial chemical that can be applied in the synthesis of thermal-resistant poly-lactic acid. Biosynthesis of d-lactic acid can be achieved by a variety of microorganisms, including lactic acid bacteria, yeast, and fungi; however, the final product yield, optical purity, and the utilization of both glucose and xylose are restricted. Consequently, engineered microbial systems are essential to attain high titer, productivity, and complete utilization of sugars. Herein, we critically evaluate the promising wild-type microorganisms, as well as genetically modified microorganisms to produce enantiomerically pure d-lactic acid, particularly from renewable lignocellulosic biomass. In addition, innovative bioreactor operation, metabolic flux analysis, and recent genetic engineering methods for targeted microbial d-lactic acid synthesis will be discussed.     

Enhancing fungal production of galactaric acid

Galactaric (mucic) acid is a symmetrical six carbon diacid which can be produced by oxidation of galactose with nitric acid, electrolytic oxidation of d-galacturonate or microbial conversion of d-galacturonate. Both salts and the free acid of galactarate have relatively low solubility, which may create challenges for a microbial host. Galactaric acid was most soluble at pH values around 4.7 in the presence of ammonium or sodium ions and less soluble in the presence of potassium ions. Solubility increased with increasing temperature. Production of galactaric acid by Trichoderma reesei D-161646 was dependent on temperature, pH and medium composition, being best at pH 4 and 35 °C. Up to 20 g L^?1 galactaric acid were produced from d-galacturonate using a fed-batch strategy with lactose as co-substrate and both ammonium and yeast extract as nitrogen sources. Crystals of galactaric acid were observed to form in the broth of some fermentations.

     

Membrane-Integrated Fermentation System for Improving the Optical Purity of D-Lactic Acid Produced during Continuous Fermentation

This report describes the production of highly optically pure D-lactic acid by the continuous fermentation of Sporolactobacillus laevolacticus and S. inulinus, using a membrane-integrated fermentation (MFR) system. The optical purity of D-lactic acid produced by the continuous fermentation system was greater than that produced by batch fermentation; the maximum value for the optical purity of D-lactic acid reached 99.8% enantiomeric excess by continuous fermentation when S. leavolacticus was used. The volumetric productivity of the optically pure D-lactic acid was about 12 g/L/h, this being approximately 11-fold higher than that obtained by batch fermentation. An enzymatic analysis indicated that both S. laevolacticus and S. inulinus could convert L-lactic acid to D-lactic acid by isomerization after the late-log phase. These results provide evidence for an effective bio-process to produce D-lactic acid of greater optical purity than has conventionally been achieved to date.     

Efficient Production of Optically Pure d-Lactic Acid from Raw Corn Starch by Using a Genetically Modified l-Lactate Dehydrogenase Gene-Deficient and α-Amylase-Secreting Lactobacillus plantarum Strain

In order to achieve direct and efficient fermentation of optically pure d-lactic acid from raw corn starch, we constructed l-lactate dehydrogenase gene (ldhL1)-deficient Lactobacillus plantarum and introduced a plasmid encoding Streptococcus bovis 148 α-amylase (AmyA). The resulting strain produced only d-lactic acid from glucose and successfully expressed amyA. With the aid of secreting AmyA, direct d-lactic acid fermentation from raw corn starch was accomplished. After 48 h of fermentation, 73.2 g/liter of lactic acid was produced with a high yield (0.85 g per g of consumed sugar) and an optical purity of 99.6%. Moreover, a strain replacing the ldhL1 gene with an amyA-secreting expression cassette was constructed. Using this strain, direct d-lactic acid fermentation from raw corn starch was accomplished in the absence of selective pressure by antibiotics. This is the first report of direct d-lactic acid fermentation from raw starch.Poly-lactic acid (PLA) is an important agro-based plastic that can be produced from inexpensive, renewable, and abundantly available biomass resources, including starchy materials. These resources have advantages over limited oil- and fossil-based sources, as they do not result in any net carbon dioxide release to the atmosphere (7). Recently, stereocomplex PLA, which is composed of both poly-l- and -d-lactic acid, has been attracting much attention due to its high thermostability. Stereocomplex-type polymers show a melting point (ca. 230°C) that is approximately 50°C higher than that of the respective single polymers (8). Therefore, d-lactic acid, in addition to l-lactic acid, which has been the focus of production to date, is of significant importance.Lactic acid bacteria (LAB) are promising microorganisms for the efficient production of lactic acid from various sugars, such as glucose, sucrose, and lactose. However, when starchy materials are used as a carbon source, they must be saccharified by physicochemical and enzymatic treatment because most LAB cannot utilize starchy materials directly (13). This makes the whole process less economically viable. Therefore, many researchers have examined the direct production of lactic acid from starchy materials by using wild amylolytic LAB (ALAB) (6, 24, 25) or genetically modified amylase-producing LAB (15, 16). Although d-lactic acid has been produced by fermentation from pretreated substrates such as rice starch (5) and by simultaneous saccharification and fermentation from cellulose (23), there have been no reports on the direct production of d-lactic acid from starchy materials. This is due to a lack of d-lactic acid-producing ALAB and difficulties in gene manipulation of d-lactic acid-producing LAB, such as Lactobacillus delbrueckii (22).We focused on Lactobacillus plantarum, which is an industrially important strain due to its environmental flexibility and its ability to assimilate a wide range of carbohydrates (9). In recent years, several gene manipulation methods for Lactobacillus plantarum have been established (18, 19). Moreover, the complete genome sequence has been decoded for L. plantarum NCIMB 8826 (9). Based on whole-genome analysis, L. plantarum possesses two types of lactate dehydrogenase (LDH), l-LDH and d-LDH, which convert pyruvate into l- and d-lactic acid, respectively. Ferain et al. (4) reported that chromosomal deletion in the ldhL1 gene of L. plantarum NCIMB 8826 provoked an absence of l-LDH activity and produced d-lactic acid from glucose.In the present study, to produce d-lactic acid directly from starch, we constructed an l-LDH-deficient, α-amylase-secreting L. plantarum strain. The engineered strain expressed α-amylase from Streptococcus bovis 148 (AmyA) (20) and efficiently degraded raw starch with the aid of a C-terminal starch-binding domain (11). Using this strain, we achieved the direct and efficient fermentation of optically pure d-lactic acid from raw corn starch.     

Highly efficient production of <Emphasis Type="SmallCaps">d</Emphasis>-lactic acid from chicory-derived inulin by <Emphasis Type="Italic">Lactobacillus bulgaricus</Emphasis>

Inulin is a readily available feedstock for cost-effective production of biochemicals. To date, several studies have explored the production of bioethanol, high-fructose syrup and fructooligosaccharide, but there are no studies regarding the production of d-lactic acid using inulin as a carbon source. In the present study, chicory-derived inulin was used for d-lactic acid biosynthesis by Lactobacillus bulgaricus CGMCC 1.6970. Compared with separate hydrolysis and fermentation processes, simultaneous saccharification and fermentation (SSF) has demonstrated the best performance of d-lactic acid production. Because it prevents fructose inhibition and promotes the complete hydrolysis of inulin, the highest d-lactic acid concentration (123.6 ± 0.9 g/L) with a yield of 97.9 % was obtained from 120 g/L inulin by SSF. Moreover, SSF by L. bulgaricus CGMCC 1.6970 offered another distinct advantage with respect to the higher optical purity of d-lactic acid (>99.9 %) and reduced number of residual sugars. The excellent performance of d-lactic acid production from inulin by SSF represents a high-yield method for d-lactic acid production from non-food grains.     

Purification,characterization, gene cloning and nucleotide sequencing of D-stereospecific amino acid amidase from soil bacterium: Delftia acidovorans

The D-amino acid amidase-producing bacterium was isolated from soil samples using an enrichment culture technique in medium broth containing D-phenylalanine amide as a sole source of nitrogen. The strain exhibiting the strongest activity was identified as Delftia acidovorans strain 16. This strain produced intracellular D-amino acid amidase constitutively. The enzyme was purified about 380-fold to homogeneity and its molecular mass was estimated to be about 50 kDa, on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme was active preferentially toward D-amino acid amides rather than their L-counterparts. It exhibited strong amino acid amidase activity toward aromatic amino acid amides including D-phenylalanine amide, D-tryptophan amide and D-tyrosine amide, yet it was not specifically active toward low-molecular-weight D-amino acid amides such as D-alanine amide, L-alanine amide and L-serine amide. Moreover, it was not specifically active toward oligopeptides. The enzyme showed maximum activity at 40°C and pH 8.5 and appeared to be very stable, with 92.5% remaining activity after the reaction was performed at 45°C for 30 min. However, it was mostly inactivated in the presence of phenylmethanesulfonyl fluoride or Cd²⁺, Ag⁺, Zn²⁺, Hg²⁺ and As³⁺ . The NH₂ terminal and internal amino acid sequences of the enzyme were determined; and the gene was cloned and sequenced. The enzyme gene damA encodes a 466-amino-acid protein (molecular mass 49,860.46 Da); and the deduced amino acid sequence exhibits homology to the D-amino acid amidase from Variovorax paradoxus (67.9% identity), the amidotransferase A subunit from Burkholderia fungorum (50% identity) and other enantioselective amidases.

     

Comprehensive assessment of the l-lysine production process from fermentation of sugarcane molasses

l-Lysine is an essential amino acid that can be produced by chemical processes from fossil raw materials, as well as by microbial fermentation, the latter being a more efficient and environmentally friendly procedure. In this work, the production process of l-lysine-HCl is studied using a systematic approach based on modeling and simulation, which supports decision making in the early stage of process design. The study considers two analysis stages: first, the dynamic analysis of the fermentation reactor, where the conversion of sugars from sugarcane molasses to l-lysine with a strain of Corynebacterium glutamicum is carried out. In this stage, the operation mode (either batch or fed batch) and operating conditions of the fermentation reactor are defined to reach the maximum technical criteria. Afterwards, the second analysis stage relates to the industrial production process of l-lysine-HCl, where the fermentation reactor, upstream processing, and downstream processing are included. In this stage, the influence of key parameters on the overall process performance is scrutinized through the evaluation of several technical, economic, and environmental criteria, to determine a profitable and sustainable design of the l-lysine production process. The main results show how the operating conditions, process design, and selection of evaluation criteria can influence in the conceptual design. The best plant design shows maximum product yield (0.31 g l-lysine/g glucose) and productivity (1.99 g/L/h), achieving 26.5% return on investment (ROI) with a payback period (PBP) of 3.8 years, decreasing water and energy consumption, and with a low potential environmental impact (PEI) index.

     

Semi-industrial scale (30 m<Superscript>3</Superscript>) fed-batch fermentation for the production of <Emphasis Type="SmallCaps">d</Emphasis>-lactate by <Emphasis Type="Italic">Escherichia coli</Emphasis> strain HBUT-D15

d(?)-lactic acid is needed for manufacturing of stereo-complex poly-lactic acid polymer. Large scale d-lactic acid fermentation, however, has yet to be demonstrated. A genetically engineered Escherichia coli strain, HBUT-D, was adaptively evolved in a 15% calcium lactate medium for improved lactate tolerance. The resulting strain, HBUT-D15, was tested at a lab scale (7 L) by fed-batch fermentation with up to 200 g L^?1 of glucose, producing 184–191 g L^?1 of d-lactic acid, with a volumetric productivity of 4.38 g L^?1 h^?1, a yield of 92%, and an optical purity of 99.9%. The HBUT-D15 was then evaluated at a semi-industrial scale (30 m³) via fed-batch fermentation with up to 160 g L^?1 of glucose, producing 146–150 g L^?1 of d-lactic acid, with a volumetric productivity of 3.95–4.29 g L^?1 h^?1, a yield of 91–94%, and an optical purity of 99.8%. These results are comparable to that of current industrial scale l(+)-lactic acid fermentation.     

Chirality Matters: Synthesis and Consumption of the d-Enantiomer of Lactic Acid by Synechocystis sp. Strain PCC6803

Both enantiomers of lactic acid, l-lactic acid and d-lactic acid, can be produced in a sustainable way by a photosynthetic microbial cell factory and thus from CO₂, sunlight, and water. Several properties of polylactic acid (a polyester of polymerized lactic acid) depend on the controlled blend of these two enantiomers. Recently, cyanobacterium Synechocystis sp. strain PCC6803 was genetically modified to allow formation of either of these two enantiomers. This report elaborates on the d-lactic acid production achieved by the introduction of a d-specific lactate dehydrogenase from the lactic acid bacterium Leuconostoc mesenteroides into Synechocystis. A typical batch culture of this recombinant strain initially shows lactic acid production, followed by a phase of lactic acid consumption, until production “outcompetes” consumption at later growth stages. We show that Synechocystis is able to use d-lactic acid, but not l-lactic acid, as a carbon source for growth. Deletion of the organism''s putative d-lactate dehydrogenase (encoded by slr1556), however, does not eliminate this ability with respect to d-lactic acid consumption. In contrast, d-lactic acid consumption does depend on the presence of glycolate dehydrogenase GlcD1 (encoded by sll0404). Accordingly, this report highlights the need to match a product of interest of a cyanobacterial cell factory with the metabolic network present in the host used for its synthesis and emphasizes the need to understand the physiology of the production host in detail.     

Non-Sterilized Fermentative Production of Polymer-Grade L-Lactic Acid by a Newly Isolated Thermophilic Strain Bacillus sp. 2–6

Background

The demand for lactic acid has been increasing considerably because of its use as a monomer for the synthesis of polylactic acid (PLA), which is a promising and environment-friendly alternative to plastics derived from petrochemicals. Optically pure l-lactic acid is essential for polymerization of PLA. The high fermentation cost of l-lactic acid is another limitation for PLA polymers to compete with conventional plastics.

Methodology/Principal Findings

A Bacillus sp. strain 2–6 for production of l-lactic acid was isolated at 55°C from soil samples. Its thermophilic characteristic made it a good lactic acid producer because optically pure l-lactic acid could be produced by this strain under open condition without sterilization. In 5-liter batch fermentation of Bacillus sp. 2–6, 118.0 g/liter of l-lactic acid with an optical purity of 99.4% was obtained from 121.3 g/liter of glucose. The yield was 97.3% and the average productivity was 4.37 g/liter/h. The maximum l-lactic acid concentration of 182.0 g/liter was obtained from 30-liter fed-batch fermentation with an average productivity of 3.03 g/liter/h and product optical purity of 99.4%.

Conclusions/Significance

With the newly isolated Bacillus sp. strain 2–6, high concentration of optically pure l-lactic acid could be produced efficiently in open fermentation without sterilization, which would lead to a new cost-effective method for polymer-grade l-lactic acid production from renewable resources.     

Enhancing l-malate production of Aspergillus oryzae FMME218-37 by improving inorganic nitrogen utilization

Microbial l-malate production from renewable feedstock is a promising alternative to petroleum-based chemical synthesis. However, high l-malate production of Aspergillus oryzae was achieved to date using organic nitrogen, with inorganic nitrogen still unable to meet industrial applications. In the current study, we constructed a screening system and nitrogen supply strategy to improve l-malate production with ammonium sulphate [(NH₄)₂SO₄] as the sole nitrogen source. First, we generated and identified a high-producing mutant FMME218-37, which stably boosted l-malate production from 30.73 to 78.12 g/L, using a combined screening system with morphological characteristics. Then, by analyzing the fermentation parameters and physiological characteristics, we further speculated the key factor was the unbalance of carbon and nitrogen absorption. Finally, the titer and productivity of l-malate was increased to 95.2 g/L and 0.57 g/(L h) by regulating the nitrogen supply module to balance carbon and nitrogen absorption, which represented the highest level in A. oryzae with (NH₄)₂SO₄ as nitrogen source achieved to date. Moreover, our findings using a low-cost substrate may lead to building an economical cell factory of A. oryzae for l-malate production.

     

Homofermentative production of d-lactic acid from sucrose by a metabolically engineered Escherichia coli

Escherichia coli W, a sucrose-positive strain, was engineered for the homofermentative production of d-lactic acid through chromosomal deletion of the competing fermentative pathway genes (adhE, frdABCD, pta, pflB, aldA) and the repressor gene (cscR) of the sucrose operon, and metabolic evolution for improved anaerobic cell growth. The resulting strain, HBUT-D, efficiently fermented 100?g?sucrose?l^?1 into 85?g?d-lactic acid?l^?1 in 72–84?h in mineral salts medium with a volumetric productivity of ~1?g?l^?1?h^?1, a product yield of 85?% and d-lactic acid optical purity of 98.3?%, and with a minor by-product of 4?g?acetate?l^?1. HBUT-D thus has great potential for production of d-lactic acid using an inexpensive substrate, such as sugar cane and/or beet molasses, which are primarily composed of sucrose.     

Effect of Light on the Rate of Glycolysis in Scenedesmus obliquus

The production of d-lactic acid, a major fermentation product of Scenedesmus obliquus strain D₃, is inhibited by light. This inhibition has been observed in the wild type as well as in Bishop's mutant 11. However, in mutant 8, which lacks photophosphorylation, light stimulated the formation of d-lactic acid.     

<Emphasis Type="SmallCaps">l</Emphasis>-Serine overproduction with minimization of by-product synthesis by engineered <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

The direct fermentative production of l-serine by Corynebacterium glutamicum from sugars is attractive. However, superfluous by-product accumulation and low l-serine productivity limit its industrial production on large scale. This study aimed to investigate metabolic and bioprocess engineering strategies towards eliminating by-products as well as increasing l-serine productivity. Deletion of alaT and avtA encoding the transaminases and introduction of an attenuated mutant of acetohydroxyacid synthase (AHAS) increased both l-serine production level (26.23 g/L) and its productivity (0.27 g/L/h). Compared to the parent strain, the by-products l-alanine and l-valine accumulation in the resulting strain were reduced by 87 % (from 9.80 to 1.23 g/L) and 60 % (from 6.54 to 2.63 g/L), respectively. The modification decreased the metabolic flow towards the branched-chain amino acids (BCAAs) and induced to shift it towards l-serine production. Meanwhile, it was found that corn steep liquor (CSL) could stimulate cell growth and increase sucrose consumption rate as well as l-serine productivity. With addition of 2 g/L CSL, the resulting strain showed a significant improvement in the sucrose consumption rate (72 %) and the l-serine productivity (67 %). In fed-batch fermentation, 42.62 g/L of l-serine accumulation was achieved with a productivity of 0.44 g/L/h and yield of 0.21 g/g sucrose, which was the highest production of l-serine from sugars to date. The results demonstrated that combined metabolic and bioprocess engineering strategies could minimize by-product accumulation and improve l-serine productivity.     

l-3-n-Butylphthalide Activates Akt/mTOR Signaling,Inhibits Neuronal Apoptosis and Autophagy and Improves Cognitive Impairment in Mice with Repeated Cerebral Ischemia–Reperfusion Injury

l-3-n-Butylphthalide (l-NBP) exerts neuroprotective effects in animal models of cerebral ischemia, but its potential benefits in repeated cerebral ischemia–reperfusion (RCIR) injury remain unknown. We investigated the effect of l-NBP on cognitive impairment induced by RCIR in mice. Male C57Bl/6 mice received sham surgery or bilateral common carotid artery occlusion (3 times, 20 min each) and were orally administered preoperative l-NBP (30 mg/kg/day, 7 days), postoperative l-NBP (30 or 60 mg/kg/day, 28 days) or postoperative vehicle (28 days). Learning and memory were assessed by the Morris water maze task and step-down passive avoidance test. Nissl staining was used to identify pathologic changes in the hippocampal CA1 region. The expressions of proteins associated with signaling, apoptosis and autophagy were assessed by quantitative PCR and western blot. RCIR induced deficits in learning and memory that were alleviated by preoperative or postoperative l-NBP administration. Pathologic lesions in the hippocampal CA1 region induced by RCIR were less severe in mice treated with l-NBP. Preoperative or postoperative l-NBP administration in mice receiving RCIR promoted hippocampal expression of phospho-Akt and phospho-mTOR (suggesting activation of Akt/mTOR signaling), increased the Bcl-2/Bax ratio (indicating suppression of apoptosis) and reduced the LC3-II/LC3-I ratio (implying inhibition of autophagy). Preoperative or postoperative l-NBP administration also depressed hippocampal levels of beclin-1 mRNA (indicating suppression of autophagy). These findings suggest that the effect of l-NBP to alleviate learning and memory deficits in mice following RCIR may involve activation of Akt/mTOR signaling and regulation of the expressions of proteins related to apoptosis and autophagy.