Biotechnological production of enantiomeric pure lactic acid from renewable resources: recent achievements, perspectives, and limits

Lactic acid (LA) is an important and versatile chemical that can be produced from renewable resources such as biomass. LA is used in the food, pharmaceutical, and polymers industries and is produced by microorganism fermentation; however, most microorganisms cannot directly utilize biomass such as starchy materials and cellulose. Here, we summarize LA production using several kinds of genetically modified microorganisms, such as LA bacteria, Escherichia coli, Corynebacterium glutamicum, and yeast. Using gene manipulation and metabolic engineering, the yield and optical purity of LA produced from biomass has been significantly improved. In this review, the drawbacks as well as improvements of LA production by fermentation is discussed.     

Quantitative trait loci regulating sugar moiety of acylsugars in tomato

Acylsugars are broad-spectrum insect resistance sugar esters produced at very high levels by some accessions of the wild tomato, Solanum pennellii. Transferring acylsugar production from S. pennellii LA716 to cultivated tomato through traditional breeding developed the benchmark acylsugar breeding line CU071026. The base moiety of acylsugars (sucrose vs. glucose) can vary among S. pennellii accessions. Additionally the accession S. pennellii LA716 produces almost exclusively acylglucoses, but the breeding line CU071026 derived from S. pennellii LA716 produces exclusively acylsucroses. This study uses a BC1F1 and a BC1F2 population derived from the cross CU071026 × (CU071026 × S. pennellii LA716) to identify and confirm the action of three quantitative trait loci (QTL) on chromosomes 3, 4, and 11. The QTL on chromosomes 3 and 11 are both required for acylglucose production, while addition of the chromosome 4 QTL affects the level of acylglucose produced in the presence of the QTL on chromosomes 3 and 11. A three-way interaction between these acylglucose QTL was confirmed with a post hoc ANOVA. Identification of these three QTL provides a blueprint for breeding to shift acylsucrose production to acylglucose production in tomato breeding lines. The implications of these QTL and two additional QTL affecting total acylsugar level in the BC1F2 are discussed.     

Kinetic modeling and scale up of lipoic acid (LA) production from Saccharomyces cerevisiae in a stirred tank bioreactor

Scale up studies for production of lipoic acid (LA) from Saccharomyces cerevisiae have been reported in this paper for the first time. LA production in batch mode was carried out in a stirred tank bioreactor at varying agitation and aeration with maximum LA production of 512 mg/L obtained at 350 rpm and 25 % dissolved oxygen in batch culture conditions. Thus, LA production increased from 352 mg/L in shake flask to 512 mg/L in batch mode in a 5 L stirred tank bioreactor. Biomass production under these conditions was mathematically explained using logistic equation and data obtained for LA production and substrate utilization were successfully fitted using Luedeking–Piret and Mercier’s models. The kinetic studies showed LA production to be growth associated. Further enhancement of LA production was carried out using fed-batch (variable volume) and semi-continuous modes of fermentation. Semi-continuous fermentation with three feeding cycles of sucrose effectively increased the production of LA from 512 to 725 mg/L.     

Lauric Acid Alleviates Neuroinflammatory Responses by Activated Microglia: Involvement of the GPR40-Dependent Pathway

In several neurodegenerative diseases such as Alzheimer’s disease (AD), microglia are hyperactivated and release nitric oxide (NO) and proinflammatory cytokines, resulting its neuropathology. Mounting evidence indicates that dietary supplementation with coconut oil (CNO) reduces the cognitive deficits associated with AD; however, the precise mechanism(s) underlying the beneficial effect of CNO are unknown. In the present study, we examined the effects of lauric acid (LA), a major constituent of CNO, on microglia activated experimentally by lipopolysaccharide (LPS), using primary cultured rat microglia and the mouse microglial cell line, BV-2. LA attenuated LPS-stimulated NO production and the expression of inducible NO synthase protein without affecting cell viability. In addition, LA suppressed LPS-induced reactive oxygen species and proinflammatory cytokine production, as well as phosphorylation of p38-mitogen activated protein kinase and c-Jun N-terminal kinase. LA-induced suppression of NO production was partially but significantly reversed in the presence of GW1100, an antagonist of G protein-coupled receptor (GPR) 40, which is an LA receptor on the plasma membrane. LA also decreased LPS-induced phagocytosis, which was completely reversed by co-treatment with GW1100. Moreover, LA alleviated amyloid-β-induced enhancement of phagocytosis. These results suggest that attenuation of microglial activation by LA may occur via the GPR40-dependent pathway. Such effects of LA may reduce glial activation and the subsequent neuronal damage in AD patients who consume CNO.     

Comprehensive utilization of waste hemicelluloses during ethanol production to increase lactic acid yield: from pretreatment to fermentation

Background

Reducing the cost of producing cellulosic ethanol is essential for the industrialization of biorefinery. Several processes are currently under investigation, but few of these techniques are entirely satisfactory in terms of competitive cost or environmental impact. In this study, a new ethanol and lactic acid (LA) coproduction is proposed. The technique involved addition of waste alkaline peroxide pretreated hydrolysate (mainly LA and hemicelluloses) to the reaction mixture after ethanol fermentation (mainly LA and xylose) to reduce the ethanol production cost.

Results

The following processes were investigated to optimize LA production: no addition of hemicelluloses or hydrolysate, addition of recycled hemicelluloses, and addition of concentrated hydrolysate. The addition of concentrated hydrolysate at 48 hours, which resulted in a maximum LA concentration of 22.3 g/L, was the most environment-friendly and cost-effective process. After the improved fermentation, 361 mg LA and 132 mg ethanol were produced from 1 g of raw poplar wood. That is, the production of one gallon of ethanol produced $9 worth of LA.

Conclusions

The amount of LA produced from the pretreated hydrolysate and reaction mixture after ethanol fermentation cannot be underestimated. The recovery of hydrolysate rich in LA and hemicelluloses (or xylose) significantly improved LA yield and further reduced the ethanol production cost.

     

alpha-Lipoic acid inhibits inflammatory bone resorption by suppressing prostaglandin E2 synthesis

alpha-Lipoic acid (LA) has been intensely investigated as a therapeutic agent for several pathological conditions, including diabetic polyneuropathy. In the present study, we examined the effects of LA on osteoclastic bone loss associated with inflammation. LA significantly inhibited IL-1-induced osteoclast formation in cocultures of mouse osteoblasts and bone marrow cells, but LA had only a marginal effect on osteoclastogenesis from bone marrow macrophages induced by receptor activator of NF-kappaB ligand (RANKL). LA inhibited both the sustained up-regulation of RANKL expression and the production of PGE2 induced by IL-1 in osteoblasts. In addition, treatment with either prostaglandin E2 (PGE2) or RANKL rescued IL-1-induced osteoclast formation inhibited by LA or NS398, a specific cyclooxygenase-2 (COX-2) inhibitor, in cocultures. LA blocked IL-1-induced PGE2 production even in the presence of arachidonic acid, without affecting the expression of COX-2 and membrane-bound PGE2 synthase. Dihydrolipoic acid (the reduced form of LA), but not LA, attenuated recombinant COX-2 activity in vitro. LA also inhibited osteoclast formation and bone loss induced by IL-1 and LPS in mice. Our results suggest that the reduced form of LA inhibits COX-2 activity, PGE2 production, and sustained RANKL expression, thereby inhibiting osteoclast formation and bone loss in inflammatory conditions.     

鸡尾酒δ整合策略构建表达三类纤维素酶的酿酒酵母工程菌株及初步评价

木质纤维素乙醇具有替代化石燃料的潜力,其生产过程包括生物质预处理、纤维素酶生产、水解和发酵等多个步骤。将纤维素酶生产、水解和发酵组合在一起的统合生物加工过程（consolidated bioprocessing,CBP）由于能降低水解和发酵成本而具有应用于纤维素乙醇生产的潜力,该技术的关键是构建能有效降解纤维素的工程菌株,而构建表达纤维素酶的酿酒酵母即是其中一种选择。采用鸡尾酒多拷贝δ整合的策略将7种纤维素酶基因（Trichoderma reesei cbh1、cbh2和egl2,Aspergillus aculeatus cbh1、egl1和bgl1）表达盒整合至酿酒酵母W303-1A染色体上,经4轮整合筛选得到菌株LA1、LA2、LA3和LA4。对这4个菌株进行纤维素酶活性测定,结果表明从LA1到LA3各种纤维素酶活性呈递增趋势,而LA4的酶活性与LA3的酶活水平相当。对菌株LA3进行酸碱预处理玉米芯料的发酵评价,结果表明：①在外加商品化纤维素酶的情况下,与对照菌株W303-1A和AADY相比,LA3能有效利用纤维素料发酵产醇;②与分步整合的菌株W3相比,发酵性能更优;③培养基中的营养成分影响菌株发酵性能。这些结果表明,鸡尾酒δ整合是一种有效的构建酿酒酵母CBP菌株的方法。     

Lipoic acid stimulates cAMP production via G protein-coupled receptor-dependent and -independent mechanisms

Lipoic acid (LA) is a naturally occurring fatty acid that exhibits anti-oxidant and anti-inflammatory properties and is being pursued as a therapeutic for many diseases including multiple sclerosis, diabetic polyneuropathy and Alzheimer's disease. We previously reported on the novel finding that racemic LA (50:50 mixture of R-LA and S-LA) stimulates cAMP production, activates prostanoid EP2 and EP4 receptors and adenylyl cyclases (AC), and suppresses activation and cytotoxicity in NK cells. In this study, we present evidence that furthers our understanding of the mechanisms of action of LA. Using various LA derivatives, such as dihydrolipoic acid (DHLA), S,S-dimethyl lipoic acid (DMLA) and lipoamide (LPM), we discovered that only LA is capable of stimulating cAMP production in NK cells. Furthermore, there is no difference in cAMP production after stimulation with either R-LA, S-LA or racemic LA. Competition and synergistic studies indicate that LA may also activate AC independent of the EP2 and EP4 receptors. Pretreatment of PBMCs with KH7 (a specific peptide inhibitor of soluble AC) and the calcium inhibitor (Bapta) prior to LA treatment resulted in reduced cAMP levels, suggesting that soluble AC and calcium signaling mediate LA stimulation of cAMP production. In addition, pharmacological inhibitor studies demonstrate that LA also activates other G protein-coupled receptors, including histamine and adenosine but not the β-adrenergic receptors. These novel findings provide information to better understand the mechanisms of action of LA, which can help facilitate the use of LA as a therapeutic for various diseases.     

Too much linoleic acid promotes inflammation-doesn't it?

Controversy exists over how much linoleic acid (LA) should be consumed in a healthy diet. Some claim that high LA intake promotes inflammation through accumulation of tissue arachidonic acid (AA) and subsequent production of pro-inflammatory lipid mediators. Here the author reviews the current available evidence from human studies that address this issue. The data indicate that high LA in the diet or circulation is not associated with higher in vivo or ex vivo pro-inflammatory responses. Surprisingly, several studies showed that those individuals consuming the highest level of LA had the lowest inflammatory status. Recent findings suggest that LA and AA are involved in both pro- and anti-inflammatory signaling pathways. Thus, within the ranges of intake that are achievable for most human populations, the evidence do not support reducing LA intake below current consumption levels.     

Lipoic Acid Attenuates Inflammation via cAMP and Protein Kinase A Signaling

Background

Abnormal regulation of the inflammatory response is an important component of diseases such as diabetes, Alzheimer''s disease and multiple sclerosis (MS). Lipoic acid (LA) has been shown to have antioxidant and anti-inflammatory properties and is being pursued as a therapy for these diseases. We first reported that LA stimulates cAMP production via activation of G-protein coupled receptors and adenylyl cyclases. LA also suppressed NK cell activation and cytotoxicity. In this study we present evidence supporting the hypothesis that the anti-inflammatory properties of LA are mediated by the cAMP/PKA signaling cascade. Additionally, we show that LA oral administration elevates cAMP levels in MS subjects.

Methodology/Principal Findings

We determined the effects of LA on IL-6, IL-17 and IL-10 secretion using ELISAs. Treatment with 50 µg/ml and 100 µg/ml LA significantly reduced IL-6 levels by 19 and 34%, respectively, in T cell enriched PBMCs. IL-17 levels were also reduced by 35 and 50%, respectively. Though not significant, LA appeared to have a biphasic effect on IL-10 production. Thymidine incorporation studies showed LA inhibited T cell proliferation by 90%. T-cell activation was reduced by 50% as measured by IL-2 secretion. Western blot analysis showed that LA treatment increased phosphorylation of Lck, a downstream effector of protein kinase A. Pretreatment with a peptide inhibitor of PKA, PKI, blocked LA inhibition of IL-2 and IFN gamma production, indicating that PKA mediates these responses. Oral administration of 1200 mg LA to MS subjects resulted in increased cAMP levels in PBMCs four hours after ingestion. Average cAMP levels in 20 subjects were 43% higher than baseline.

Conclusions/Significance

Oral administration of LA in vivo resulted in significant increases in cAMP concentration. The anti-inflammatory effects of LA are mediated in part by the cAMP/PKA signaling cascade. These novel findings enhance our understanding of the mechanisms of action of LA.     

α-Lipoic acid increases tolerance of cardiomyoblasts to glucose/glucose oxidase-induced injury via ROS-dependent ERK1/2 activation

α-Lipoic acid (LA) has been shown to improve the diabetic cardiac symptoms. However, the underlying mechanisms have not been elucidated precisely. We have reported recently that LA potentially protected neurons from substance-induced apoptosis. We hypothesized that LA could attenuate cardiac cells death induced by oxidative stress derived from high glucose. To test this possibility, we examined the effects of LA on d-glucose/glucose oxidase (DG/GO, 30mM/5mU)-induced injury in rat cardiomyoblast H9c2 cells. We observed that LA pretreatment significantly increased cell viability in DG/GO-challenged cells. LA pretreatment also attenuated DG/GO-induced apoptosis as evidenced by decreases in both nuclear condensation and loss of mitochondrial potential. In addition, LA activated ERK1/2 and moderately increased ROS production. Blockade of ERK1/2 activation by PD98059 completely abolished LA-induced protection against DG/GO challenge. Inhibition of ROS by N-acetylcysteine abrogated LA-induced ERK1/2 activation and cytoprotection. Furthermore, we observed that the ROS production induced by LA was significantly slower and milder than that by DG/GO. Our results suggest that pretreatment with LA moderately increased ROS production to induce a preconditioning-like effect by ERK1/2 activation thereby increased tolerance of H9c2 cells to DG/GO challenge.     

Production of levulinic acid from steam exploded rice straw via solid superacid, S2O8(2-)/ZrO2-SiO2-Sm2O3

In this study, solid superacid was employed to catalyze the decomposition of steam exploded rice straw (SERS) for the production of levulinic acid, a versatile platform chemical. The results revealed that solid superacid, S(2)O(8)(2-)/ZrO(2)-SiO(2)-Sm(2)O(3), could be used as a substitute for homogenous acid to catalyze the production of LA from SERS and LA yield increased with the addition of solid superacid. It was also found that steam explosion combined with superfine grinding of rice straw could effectively increase LA yield for reducing particle size of rice straw and enhancing the accessibility of cellulose. Under optimal conditions of 200°C, 10 min, 13.3% of solid superacid to pretreated rice straw, and 1:15 of solid-liquid ratio, LA yield of the superfine grinding SERS was 70% of the theoretical yield, which was equivalent with the homogeneous acid-catalyzed production of LA.     

Lactic acid production from submerged fermentation of broken rice using undefined mixed culture

The present work aimed to characterize and optimize the submerged fermentation of broken rice for lactic acid (LA) production using undefined mixed culture from dewatered activated sludge. A microorganism with amylolytic activity, which also produces LA, Lactobacillus amylovorus, was used as a control to assess the extent of mixed culture on LA yield. Three level full factorial designs were performed to optimize and define the influence of fermentation temperature (20–50?°C), gelatinization time (30–60 min) and broken rice concentration in culture medium (40–80 g L^?1) on LA production in pure and undefined mixed culture. LA production in mixed culture (9.76 g L^?1) increased in sixfold respect to pure culture in optimal assessed experimental conditions. The optimal conditions for maximizing LA yield in mixed culture bioprocess were 31?°C temperature, 45 min gelatinization time and 79 g L^?1 broken rice concentration in culture medium. This study demonstrated the positive effect of undefined mixed culture from dewatered activated sludge to produce LA from culture medium formulated with broken rice. In addition, this work establishes the basis for an efficient and low-cost bioprocess to manufacture LA from this booming agro-industrial by-product.     

Divergent regulation of terpenoid metabolism in the trichomes of wild and cultivated tomato species

The diversification of chemical production in glandular trichomes is important in the development of resistance against pathogens and pests in two species of tomato. We have used genetic and genomic approaches to uncover some of the biochemical and molecular mechanisms that underlie the divergence in trichome metabolism between the wild species Solanum habrochaites LA1777 and its cultivated relative, Solanum lycopersicum. LA1777 produces high amounts of insecticidal sesquiterpene carboxylic acids (SCAs), whereas cultivated tomatoes lack SCAs and are more susceptible to pests. We show that trichomes of the two species have nearly opposite terpenoid profiles, consisting mainly of monoterpenes and low levels of sesquiterpenes in S. lycopersicum and mainly of SCAs and very low monoterpene levels in LA1777. The accumulation patterns of these terpenoids are different during development, in contrast to the developmental expression profiles of terpenoid pathway genes, which are similar in the two species, but they do not correlate in either case with terpenoid accumulation. However, our data suggest that the accumulation of monoterpenes in S. lycopersicum and major sesquiterpenes in LA1777 are linked both genetically and biochemically. Metabolite analyses after targeted gene silencing, inhibitor treatments, and precursor feeding all show that sesquiterpene biosynthesis relies mainly on products from the plastidic 2-C-methyl-d-erythritol-4-phosphate pathway in LA1777 but less so in the cultivated species. Furthermore, two classes of sesquiterpenes produced by the wild species may be synthesized from distinct pools of precursors via cytosolic and plastidial cyclases. However, highly trichome-expressed sesquiterpene cyclase-like enzymes were ruled out as being involved in the production of major LA1777 sesquiterpenes.     

Regulation of follicular luteinization by a gonadotropin-releasing hormone agonist: Relationship between steroidogenesis and apoptosis

The purpose of this study was to evaluate the effects of GnRH-analog (Leuprolide acetate, LA) administration on follicular luteinization in equine chorionic gonadotropin plus human chorionic gonadotropin (eCG + hCG)-superovulated prepubertal treated rats. Results indicate that LA treatment decreases circulating levels of progesterone (P) and P accumulation in collagenase-dispersed ovarian cell cultures, though estradiol(E₂) production is increased. These data suggest that cells from the LA group may be less luteinized following gonadotropin treatment. Studies performed on histological ovarian sections after different times of eCG administration showed that LA injections produce lower amounts of corpora lutea and antral follicles, and a greater number of atretic and preantral follicles. The basal and LH-stimulated P and progestagen accumulations are decreased in incubations of corpora lutea isolated from the LA group. In addition, the mitochondrial cholesterol side-chain cleavage (P450_SCC) levels in corpora lutea from LA-treated rats are reduced, indicating that the decrease in P production observed is due in part to an alteration in the steroidogenic luteal capability. Immunocytochemical localization of nuclei exhibiting DNA fragmentation by the technique of terminal deoxynucleotidyl transferase end-labeling showed that LA treatment causes an increase in the number of apoptotic cells in preantral and antral follicles at all times studied (1, 2, 4, or 7 days of LA administration). A similar effect, though less pronounced, was observed in corpora lutea. It is concluded that LA treatment produces a failure in the steroidogenic luteal capability and an increase of apoptotic mechanisms in the ovary, producing as a consequence an interference in the follicular recruitment, growth, and luteinization induced by gonadotropins. Mol. Reprod. Dev. 51:287–294, 1998. © 1998 Wiley-Liss, Inc.     

Optimization of Biomass Conversion to Levulinic Acid in Acidic Ionic Liquid and Upgrading of Levulinic Acid to Ethyl Levulinate

Levulinic acid (LA) is a versatile platform chemical that can be derived from biomass as an alternative to fossil fuel resources. Herein, the optimization of LA production from glucose and oil palm fronds (OPF) catalyzed by an acidic ionic liquid; 1-sulfonic acid-3-methyl imidazolium tetrachloroferrate ([SMIM][FeCl₄]) have been investigated. Response surface methodology based on Box-Behnken design was employed to optimize the LA yield and to examine the effect and interaction of reaction parameters on the LA production. The reaction parameters include reaction temperature, reaction time, feedstock loading, and catalyst loading. From the optimization study, the predicted mathematical models for LA production from glucose and OPF covered more than 90 % of the variability in the experimental data. At optimum conditions, 69.2 % of LA yield was obtained from glucose, while 24.8 % of LA yield was attained from OPF and registered 77.3 % of process efficiency. The recycled [SMIM][FeCl₄] gave sufficient performance for five successive cycles. Furthermore, the optimum LA produced from glucose and OPF can be directly converted to ethyl levulinate through esterification over the [SMIM][FeCl₄] catalyst. This study highlights the potential of [SMIM][FeCl₄] for biorefinery processing of renewable feedstocks at mild process conditions.     

Factors influencing biohydrogenation and conjugated linoleic acid production by mixed rumen fungi

The objective of this study was to evaluate the effect of soluble carbohydrates (glucose, cellobiose), pH (6.0, 6.5, 7.0), and rumen microbial growth factors (VFA, vitamins) on biohydrogenation of linoleic acid (LA) by mixed rumen fungi. Addition of glucose or cellobiose to culture media slowed the rate of biohydrogenation;only 35-40% of LA was converted to conjugated linoleic acid (CLA) or vaccenic acid (VA) within 24 h of incubation, whereas in the control treatment, 100% of LA was converted within 24 h. Addition of VFA or vitamins did not affect biohydrogenation activity or CLA production. Culturing rumen fungi at pH 6.0 slowed biohydrogenation compared with pH 6.5 or 7.0. CLA production was reduced by pH 6.0 compared with control (pH 6.5), but was higher with pH 7.0. Biohydrogenation of LA to VA was complete within 72 h at pH 6.0, 24 h at pH 6.5, and 48 h at pH 7.0. It is concluded that optimum conditions for biohydrogenation of LA and for CLA production by rumen fungi were provided without addition of soluble carbohydrates, VFA or vitamins to the culture medium; optimum pH was 6.5 for biohydrogenation and 7.0 for CLA production.     

Comparative toxicity of oleic and linoleic acid on human lymphocytes

Commercially available lipid emulsions for parenteral nutrition are mainly composed by long chain triacylglycerol containing a high proportion of linoleic acid (LA) or oleic acid (OA). The immunological impact of such therapy is particularly important because parenteral diets are often administered to critically ill patients as a mechanism to supply adequate nutrition during catabolic stress conditions. The comparative toxicity of OA and LA on human lymphocytes and the type of cell death induced by these fatty acids were determined in vitro. Parameters of cell death were investigated by flow cytometry-cell viability, DNA fragmentation, phosphatidylserine externalization, mitochondrial depolarization, neutral lipid accumulation and production of reactive oxygen species-and by fluorescence microscopy-chromatin condensation. Additionally a spectrofluorometric assay was employed to determine the activities of caspase--3, 6 and 8. Evidence is presented herein that OA is less toxic to human lymphocytes than LA. However, both fatty acids promoted apoptosis and necrosis of these cells. The mechanism of cell death induced by OA involved activation of caspase 3 while the mechanism of death induced by LA involved mitochondrial depolarization and ROS production. Importantly, neutral lipid accumulation may be a mechanism to protect lymphocytes against the toxicity induced by OA. OA may offer an immunological less problematic alternative to LA with respect to fatty acid composition of parenteral nutritional emulsions.     

Chaperone-aided expression of LipA and LplA followed by the increase in α-lipoic acid production

α-Lipoic acid (LA), a naturally occurring cofactor reported to be present in a diverse group of microorganisms, plants, and animal tissues, has been widely and successfully used as a therapy for a variety of diseases, including diabetes and heart disease. However, to date, recombinant DNA technology has not been applied for higher LA production due mainly to difficulties in the functional expression of key enzymes involved in LA production. Here, we report a study for higher LA production with the aid of chaperone plasmids, DnaKJE and trigger factor (Tf). The lipA and lplA genes encoding lipoate synthase and lipoate protein ligase in Pseudomonas fluorescens, respectively, were cloned and transformed into Escherichia coli K12. When they were overexpressed in E. coli, both LipA and LplA were expressed as inclusion bodies leading to no increase in LA production. However, when chaperone plasmids DnaKJE and Tf were coexpressed with lipA and lplA, the resulting recombinant E. coli strains showed higher LA production than the wild-type E. coli by 32–111%, respectively.