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Glutarate is a five carbon platform chemical produced during the catabolism of L-lysine. It is known that it can be catabolized through the glutaryl-CoA dehydrogenation pathway. Here, we discover that Pseudomonas putida KT2440 has an additional glutarate catabolic pathway involving L-2-hydroxyglutarate (L-2-HG), an abnormal metabolite produced from 2-ketoglutarate (2-KG). In this pathway, CsiD, a Fe2+/2-KG-dependent glutarate hydroxylase, is capable of converting glutarate into L-2-HG, and LhgO, an L-2-HG oxidase, can catalyze L-2-HG into 2-KG. We construct a recombinant strain that lacks both glutarate catabolic pathways. It can produce glutarate from L-lysine with a yield of 0.85 mol glutarate/mol L-lysine. Thus, L-2-HG anabolism and catabolism is a metabolic alternative to the glutaryl-CoA dehydrogenation pathway in P. putida KT2440; L-lysine can be both ketogenic and glucogenic. [ABSTRACT FROM AUTHOR]

As an important method for building blocks synthesis, whole cell biocatalysis is hindered by some shortcomings such as unpredictability of reactions, utilization of opportunistic pathogen, and side reactions. Due to its biological and extensively studied genetic background, Pseudomonas putida KT2440 is viewed as a promising host for construction of efficient biocatalysts. After analysis and reconstruction of the lactate utilization system in the P. putida strain, a novel biocatalyst that only exhibited NAD-independent D-lactate dehydrogenase activity was prepared and used in L-2-hydroxy-carboxylates production. Since the side reaction catalyzed by the NAD-independent L-lactate dehydrogenase was eliminated in whole cells of recombinant P. putida KT2440, two important L-2-hydroxy-carboxylates (L-lactate and L-2-hydroxybutyrate) were produced in high yield and high optical purity by kinetic resolution of racemic 2-hydroxy carboxylic acids. The results highlight the promise in biocatalysis by the biotechnologically important organism P. putida KT2440 through genomic analysis and recombination. [ABSTRACT FROM AUTHOR]

5-Aminovalerate is a potential C5 platform chemical for synthesis of valerolactam, 5-hydroxyvalerate, glutarate, and 1,5-pentanediol. It is a metabolite of L-lysine catabolism through the aminovalerate pathway in Pseudomonas putida. L-Lysine monooxygenase (DavB) and 5-aminovaleramide amidohydrolase (DavA) play key roles in the biotransformation of L-lysine into 5-aminovalerate. Here, DavB and DavA of P. putida KT2440 were expressed, purified, and coupled for the production of 5-aminovalerate from L-lysine. Under optimal conditions, 20.8 g/L 5-aminovalerate was produced from 30 g/L L-lysine in 12 h. Because L-lysine is an industrial fermentation product, the two-enzyme coupled system presents a promising alternative for the production of 5-aminovalerate. [ABSTRACT FROM AUTHOR]

An NAD-dependent D-lactate dehydrogenase (D-nLDH) of Lactobacillus bulgaricus ATCC 11842 was rationally re-designed for asymmetric reduction of a homologous series of α-keto carboxylic acids such as phenylpyruvic acid (PPA), α-ketobutyric acid, α-ketovaleric acid, β-hydroxypyruvate. Compared with wild-type D-nLDH, the Y52L mutant D-nLDH showed elevated activities toward unnatural substrates especially with large substitutes at C-3. By the biocatalysis combined with a formate dehydrogenase for in situ generation of NADH, the corresponding (R)-α-hydroxy carboxylic acids could be produced at high yields and highly optical purities. Taking the production of chiral (R)-phenyllactic acid (PLA) from PPA for example, 50 mMPPA was completely reduced to (R)-PLA in 90 min with a high yield of 99.0% and a highly optical purity (.99.9% e.e.) by the coupling system. The results presented in this work suggest a promising alternative for the production of chiral α-hydroxy carboxylic acids. [ABSTRACT FROM AUTHOR]

Escherichia coli NusA, an essential component of the RNA polymerase elongation complex, is involved in transcriptional elongation, termination, anti-termination, cold shock and stress-induced mutagenesis. In this study, we demonstrated that NusA can self-assemble into oligomers under heat shock conditions and that this property is largely determined by the C-terminal domain. In parallel with the self-assembly process, NusA also acquires chaperone activity. Furthermore, NusA overexpression results in the enhanced heat shock resistance of host cells, which may be due to the chaperone activity of NusA. Our results suggest that E. coli NusA can act as a protector to prevent protein aggregation under heat stress conditions in vitro and in the NusA-overexpressing strain. We propose a new hypothesis that NusA could serve as a molecular chaperone in addition to its functions as a transcription factor. However, it remains to be further investigated whether NusA has the same function under normal physiological conditions. [ABSTRACT FROM AUTHOR]

Highly efficient d-lactate production by Sporolactobacillus sp. strain CASD was demonstrated in this study. Peanut meal was found to be a better nutrient than yeast extract, soybean meal, soybean peptone, corn steep, liquor beef extract, and ammonium sulfate in the production of d-lactate. To improve the utilization of peanut meal, the material was enzymatically hydrolyzed and simultaneously utilized as the nitrogen source in d-lactate fermentation. Very high d-lactate production (207 g/L) was obtained using 40 g/L of peanut meal in 30-L fed-batch fermentation, with the average productivity of 3.8 g/(L·h) and optical purity of 99.3%. The production of such a high concentration of optically pure d-lactate by strain CASD, with the simultaneous enzymatic hydrolysis of peanut meal and fermentation, represents a new cost-efficient and integrated method for d-lactate production using agricultural by-products. [ABSTRACT FROM AUTHOR]

Two proteins that might be responsible for D-lactic acid (D-LA) formation were screened from the genome database of Lactobacillus rhamnosus GG. The coding genes of the two proteins in L. rhamnosus CASL, ldhD1 and ldhD2, were cloned and expressed in Escherichia coli Rosetta with an inducible expression vector pETDuet™-1 (Novagen, Darmstadt, Germany), respectively. The two purified proteins, LdhD-1 and LdhD-2, migrated as a single protein band separately, both corresponding to an apparent molecular mass between 35 kDa and 45 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The specific activities of LdhD-1 and LdhD-2 catalyzing pyruvate to LA were 0.02 U/mg and 0.21 U/mg, respectively. The configuration of LA converted from pyruvate was determined using high-performance liquid chromatography equipped with a chiral column. Only D-LA was detected when LdhD-1 and LdhD-2 were tested. In summary, the two proteins cloned and expressed in this study were most probably responsible for D-LA formation during fermentation of L. rhamnosus CASL. [ABSTRACT FROM AUTHOR]

N-Acetyl- d-neuraminic acid (Neu5Ac) and its derivates are a very important group of biomolecules because these sugars occupy the terminal positions in numerous macromolecules, such as the glycans of glycoproteins, and are involved in many biological and pathological phenomena. The synthesis and applications of Neu5Ac are attracting much interest due to the potential applications of this compound in the pharmaceutical industry, such as in the synthesis of the anti-flu drug zanamivir. In this review article, we discuss existing knowledge on the biotechnological production and applications of Neu5Ac and also propose some guidelines for future studies. [ABSTRACT FROM AUTHOR]

N-Acetyl- d-neuraminic acid (Neu5Ac) is a precursor for producing many pharmaceutical drugs such as zanamivir which have been used in clinical trials to treat and prevent the infection with influenza virus, such as the avian influenza virus H5N1 and the current 2009 H1N1. Two recombinant Escherichia coli strains capable of expressing N-acetyl- d-glucosamine 2-epimerase and N-acetyl- d-neuraminic acid aldolase were constructed based on a highly efficient temperature-responsive expression system which is safe compared to chemical-induced systems and coupled in Neu5Ac production. Carbon sources were optimized for Neu5Ac production, and the concentration effects of carbon sources on the production were investigated. With 2,200 mM pyruvate as carbon source and substrate, 61.9 mM (19.1 g l−1) Neu5Ac was produced from 200 mM N-acetyl- d-glucosamine (GlcNAc) in 36 h by the coupled cells. Our Neu5Ac biosynthetic process is favorable compared with natural product extraction, chemical synthesis, or even many other biocatalysis processes. [ABSTRACT FROM AUTHOR]

During 2,3,5,6-tetramethylpyrazine production from glucose by Bacillus strains, a novel product was detected and identified as 2,4,5-trimethylimidazole (TMI) by GC/MS. TMI appeared in the culture medium only after glucose had been depleted and then increased to 0.25–0.31 g l−1 in 90–120 h. When the ammonium source was changed from (NH4)2SO4 to (NH4)2HPO4, only about one tenth of TMI was detected. Although the mechanistic events largely remain unclear, both microbial strains tested demonstrated similar dynamic processes of TMI production, suggesting that TMI formation is a genuine feature of Bacillus species. [ABSTRACT FROM AUTHOR]

Attempts were made with success to develop a two-step biocatalytic process for uridine 5′-monophosphate (UMP) production from orotic acid by Corynebacterium ammoniagenes ATCC 6872: the strain was first cultivated in a high salt mineral medium, and then cells were harvested and used as the catalyst in the UMP production reaction. Effects of cultivation and reaction conditions on UMP production were investigated. The cells exhibited the highest biocatalytic ability when cultivated in a medium containing corn steep liquor at pH 7.0 for 15 h in the exponential phase of growth. To optimize the reaction, both “one-factor-at-a-time” method and statistical method were performed. By “one-factor-at-a-time” optimization, orotic acid, glucose, phosphate ion (equimolar KH2PO4 and K2HPO4), MgCl2, Triton X-100 were shown to be the optimum components for the biocatalytic reaction. Phosphate ion and C. ammoniagenes cell were furthermore demonstrated as the most important main effects on UMP production by Plackett–Burman design, indicating that 5-phosphoribosyl-1-pyrophosphate (PRPP) synthesis was the rate-limiting step for pyrimidine nucleotides production. Optimization by a central composition design (CCD) was then performed, and up to 32 mM (10.4 g l−1) UMP was accumulated in 24 h from 38.5 mM (6 g l−1) orotic acid. The yield was threefold higher than the original UMP yield before optimization. [ABSTRACT FROM AUTHOR]

High vanillin productivity was achieved in the batch biotransformation of ferulic acid by Streptomyces sp. strain V-1. Due to the toxicity of vanillin and the product inhibition, fed-batch biotransformation with high concentration of ferulic acid was unsuccessful. To solve this problem and improve the vanillin yield, a biotransformation strategy using adsorbent resin was investigated. Several macroporous adsorbent resins were chosen to adsorb vanillin in situ during the bioconversion. Resin DM11 was found to be the best, which adsorbed the most vanillin and the least ferulic acid. When 8% resin DM11 (wet w/ v) was added to the biotransformation system, 45 g l−1 ferulic acid could be added continually and 19.2 g l−1 vanillin was obtained within 55 h, which was the highest vanillin yield by bioconversion until now. This yield was remarkable for exceeding the crystallization concentration of vanillin and therefore had far-reaching consequence in its downstream processing. [ABSTRACT FROM AUTHOR]

An optimization strategy based on desirability function approach (DFA) together with response surface methodology (RSM) has been used to optimize production medium in L-glutamine fermentation. Fermentation problems often force to reach a compromise between different experimental variables in order to achieve the most suitable strategy applying in industrial production. The importance of the use of multi-objective optimization methods lies in the ability to cope with this kind of problems. A sequential RSM with different combinations of glucose and (NH4)2SO4 was performed to attain the optimal medium (OM-1) in glutamine production. Based on the result of RSM and the evaluation of production cost, a more economical optimal medium (OM-2) was obtained with the aid of DFA. In DFA study, glutamate, the main by-product in glutamine fermentation as another response was considered. Compared with OM-1 in validated experiment, similar amounts of glutamine were obtained in OM-2 while the concentration of glutamate and the production cost decreased by 53.6 and 7.1%, respectively. [ABSTRACT FROM AUTHOR]

A bacterium designated as HS8 was newly isolated from soil based on its ability to degrade isoeugenol. The strain was identified as Bacillus subtilis according to its 16S rDNA sequence analysis and biochemical characteristics. The metabolic pathway for the degradation of isoeugenol was examined. Isoeugenol-diol, for the first time, was detected as an intermediate from isoeugenol to vanillin by a bacterial strain. Isoeugenol was converted to vanillin via isoeugenol-diol, and vanillin was then metabolized via vanillic acid to guaiacol by strain HS8. These metabolites, vanillin, vanillic acid, and guaiacol, are all valuable aromatic compounds in flavor production. At the same time, the bipolymerization of isoeugenol was observed, which produced dehydrodiisoeugenol and decreased the vanillin yield. High level of vanillic acid decarboxylase activity was detected in cell-free extract. These findings provided a detailed profile of isoeugenol metabolism by a B. subtilis strain for the first time, which would improve the production of valuable aromatic compounds by biotechnology. [ABSTRACT FROM AUTHOR]