Production of volatile organic compounds by mycobacteria

The need for improved rapid diagnostic tests for tuberculosis disease has prompted interest in the volatile organic compounds (VOCs) emitted by Mycobacterium tuberculosis complex bacteria. We have investigated VOCs emitted by Mycobacterium bovis BCG grown on Lowenstein-Jensen media using selected ion flow tube mass spectrometry and thermal desorption-gas chromatography-mass spectrometry. Compounds observed included dimethyl sulphide, 3-methyl-1-butanol, 2-methyl-1-propanol, butanone, 2-methyl-1-butanol, methyl 2-methylbutanoate, 2-phenylethanol and hydrogen sulphide. Changes in levels of acetaldehyde, methanol and ammonia were also observed. The compounds identified are not unique to M.?bovis BCG, and further studies are needed to validate their diagnostic value. Investigations using an ultra-rapid gas chromatograph with a surface acoustic wave sensor (zNose) demonstrated the presence of 2-phenylethanol (PEA) in the headspace of cultures of M.?bovis BCG and Mycobacterium smegmatis, when grown on Lowenstein-Jensen supplemented with glycerol. PEA is a reversible inhibitor of DNA synthesis. It is used during selective isolation of gram-positive bacteria and may also be used to inhibit mycobacterial growth. PEA production was observed to be dependent on growth of mycobacteria. Further study is required to elucidate the metabolic pathways involved and assess whether this compound is produced during in vivo growth of mycobacteria.     

Metabolic engineering of Escherichia coli for 1-butanol production

Compared to ethanol, butanol offers many advantages as a substitute for gasoline because of higher energy content and higher hydrophobicity. Typically, 1-butanol is produced by Clostridium in a mixed-product fermentation. To facilitate strain improvement for specificity and productivity, we engineered a synthetic pathway in Escherichia coli and demonstrated the production of 1-butanol from this non-native user-friendly host. Alternative genes and competing pathway deletions were evaluated for 1-butanol production. Results show promise for using E. coli for 1-butanol production.     

Volatile components from the mandibular glands of the turtle ants, Cephalotes alfaroi and Cephalotes cristatus

GC–MS analysis of whole head extracts from the turtle ants, Cephalotes alfaroi and Cephalotes cristatus, showed that 4-heptanone and 4-heptanol were the major volatile components in the mandibular glands. 4-Heptanone and 4-heptanol have rarely been identified in mandibular gland secretions from other ant genera. Thus, these compounds may be chemotaxonomic markers for the genus Cephalotes, since they have been identified in the mandibular glands from all members of this genus that have been investigated to date. Minor components identified in the whole head extracts of these ants were 3-methyl-1-butanol, 3-heptanone, 3-hexanol, 2- and 3-methylbutanoic acids, 2-methyl-4-heptanone, 2-phenylethanol and phenol. To our knowledge, this is the first time that 2-methyl-4-heptanone and phenol have been reported in the mandibular gland secretion from any Formicid.     

Yeast strain effect on the concentration of major volatile compounds and sensory profile of wines from Vitis vinifera var. Treixadura

The fermentative ability of five Saccharomyces cerevisiae strains and their influence on the aroma and sensory properties of wine from Treixadura were evaluated to determine the most suitable yeast that produces a high quality wine from this grapevine variety. The results indicated that all strains, except T2, were able to lead the vinification process and have good fermentative powers. The chemical composition of wines obtained with resident cellar yeasts consisted of a significant amount of glycerol, a compound that contributes to the structure and smoothness in taste of the wine. In addition, these strains were good producers of acetates, ethyl esters and fatty acids, compounds that positively influence wine aroma. Compounds with a direct contribution to the aroma of Treixadura wines (Odour Activity Value >1) included 2-methyl-1-butanol, 3-methyl-1-butanol, 2-phenylethanol, isoamyl and ethyl acetate and the ethyl esters, ethyl butyrate, ethyl hexanoate, ethyl octanoate and ethyl decanoate. Sensory analysis supported the fact that cellar strains produced more fruity and floral wines than commercial or spontaneous fermentations. We conclude that resident cellar yeasts enhanced sensory quality of wines from Treixadura.     

Enzymes catalyzed esterification of N-protected amino acids with secondary alcohols

Secondary alcohols were converted to their corresponding esters with N-Cbz-L-amino acids by Celite-immobilized protease or lipase in organic solvents at pH 7.5. The esterification of 2-butanol and 2-phenylethanol were achieved up to 71% yields. The optimal reaction condition for Aspergillus oryzae protease (AOP) catalyzed synthesis of N-Cbz-L-aspartyl- sec-butyl ester (97.6 de) was at pH 7.5 and a duration of 96 hours.     

Engineering <Emphasis Type="Italic">Bacillus subtilis</Emphasis> for isobutanol production by heterologous Ehrlich pathway construction and the biosynthetic 2-ketoisovalerate precursor pathway overexpression

In the present work, Bacillus subtilis was engineered as the cell factory for isobutanol production due to its high tolerance to isobutanol. Initially, an efficient heterologous Ehrlich pathway controlled by the promoter P₄₃ was introduced into B. subtilis for the isobutanol biosynthesis. Further, investigation of acetolactate synthase of B. subtilis, ketol-acid reductoisomerase, and dihydroxy-acid dehydratase of Corynebacterium glutamicum responsible for 2-ketoisovalerate precursor biosynthesis showed that acetolactate synthase played an important role in isobutanol biosynthesis. The overexpression of acetolactate synthase led to a 2.8-fold isobutanol production compared with the control. Apart from isobutanol, alcoholic profile analysis also confirmed the existence of 1.21 g/L ethanol, 1.06 g/L 2-phenylethanol, as well as traces of 2-methyl-1-butanol and 3-methyl-1-butanol in the fermentation broth. Under microaerobic condition, the engineered B. subtilis produced up to 2.62 g/L isobutanol in shake-flask fed-batch fermentation, which was 21.3% higher than that in batch fermentation.     

Stripping of nonionic surfactants from the coacervate phase of cloud point system for lipase separation by Winsor II microemulsion extraction with the direct addition of alcohols

Extractive microbial fermentation of lipase by Serratia marcescens ECU1010 in cloud point system was previously carried out in the cloud point system. The direct addition of different alcohols, including iso-butanol, 2-phenylethanol and 1-octanol, into the coacervate phase of the clear supernatant of the fermentation broth formed microemulsion, where the nonionic surfactants and lipase were unevenly partitioned between the different phases in the microemulsion system. The polarity of alcohols strongly affected the microemulsion type at room temperature condition. The results indicated that the Winsor II microemulsion, formed by the addition of iso-butanol or 2-phenylethanol as the organic solvent, favored the stripping of the nonionic surfactant into the O_m phase, whereas the lipase was left in the excess aqueous phase. However, the Winsor I microemulsion, formed by the addition of 1-octanol as the organic solvent, failed to separate the lipase from the nonionic surfactant in the coacervate phase of cloud point system, because the nonionic surfactant and lipase were partitioned into the W_m phase at the same time. Moreover, in the Winsor II microemulsion extraction with 2-phenylethanol as the organic solvent, in which case the protein–surfactant complexes were absent at the interface between the O_m phase and the excess aqueous phase, the high lipase recovery (above 80%) and good nonionic surfactant removal were achieved. The effect of nonionic surfactants on lipase activity was also presented.     

Production of Aromatic Compounds by Metabolically Engineered Escherichia coli with an Expanded Shikimate Pathway

Escherichia coli was metabolically engineered by expanding the shikimate pathway to generate strains capable of producing six kinds of aromatic compounds, phenyllactic acid, 4-hydroxyphenyllactic acid, phenylacetic acid, 4-hydroxyphenylacetic acid, 2-phenylethanol, and 2-(4-hydroxyphenyl)ethanol, which are used in several fields of industries including pharmaceutical, agrochemical, antibiotic, flavor industries, etc. To generate strains that produce phenyllactic acid and 4-hydroxyphenyllactic acid, the lactate dehydrogenase gene (ldhA) from Cupriavidus necator was introduced into the chromosomes of phenylalanine and tyrosine overproducers, respectively. Both the phenylpyruvate decarboxylase gene (ipdC) from Azospirillum brasilense and the phenylacetaldehyde dehydrogenase gene (feaB) from E. coli were introduced into the chromosomes of phenylalanine and tyrosine overproducers to generate phenylacetic acid and 4-hydroxyphenylacetic acid producers, respectively, whereas ipdC and the alcohol dehydrogenase gene (adhC) from Lactobacillus brevis were introduced to generate 2-phenylethanol and 2-(4-hydroxyphenyl)ethanol producers, respectively. Expression of the respective introduced genes was controlled by the T7 promoter. While generating the 2-phenylethanol and 2-(4-hydroxyphenyl)ethanol producers, we found that produced phenylacetaldehyde and 4-hydroxyphenylacetaldehyde were automatically reduced to 2-phenylethanol and 2-(4-hydroxyphenyl)ethanol by endogenous aldehyde reductases in E. coli encoded by the yqhD, yjgB, and yahK genes. Cointroduction and cooverexpression of each gene with ipdC in the phenylalanine and tyrosine overproducers enhanced the production of 2-phenylethanol and 2-(4-hydroxyphenyl)ethanol from glucose. Introduction of the yahK gene yielded the most efficient production of both aromatic alcohols. During the production of 2-phenylethanol, 2-(4-hydroxyphenyl)ethanol, phenylacetic acid, and 4-hydroxyphenylacetic acid, accumulation of some by-products were observed. Deletion of feaB, pheA, and/or tyrA genes from the chromosomes of the constructed strains resulted in increased desired aromatic compounds with decreased by-products. Finally, each of the six constructed strains was able to successfully produce a different aromatic compound as a major product. We show here that six aromatic compounds are able to be produced from renewable resources without supplementing with expensive precursors.     

Screening of yeasts for the production of the aroma compound 2-phenylethanol in a molasses-based medium

Fourteen yeast strains were screened for production of 2-phenylethanol from l-phenylalanine with molasses as carbon source. Up to 1 g 2-phenylethanol l^–1 was obtained. Using oleyl alcohol as a second phase for in situ product removal to enhance the production of 2-phenylethanol increased the yield to about 3 g 2-phenylethanol l^–1 at 35 °C. The most productive strains were Kluyveromyces marxianus CBS 600 and CBS 397.     

Metabolic engineering of Escherichia coli for 1-butanol and 1-propanol production via the keto-acid pathways

Production of higher alcohols via the keto-acid intermediates found in microorganism's native amino-acid pathways has recently shown promising results. In this work, an Escherichia coli strain that produces 1-butanol and 1-propanol from glucose was constructed. The strain first converts glucose to 2-ketobutyrate, a common keto-acid intermediate for isoleucine biosynthesis. Then, 2-ketobutyrate is converted to 1-propanol through reactions catalyzed by the heterologous decarboxylase and dehydrogenase, or to 1-butanol via the chemistry involved in the synthesis of the unnatural amino acid norvaline. We systematically improved the synthesis of 1-propanol and 1-butanol through deregulation of amino-acid biosynthesis and elimination of competing pathways. The final strain demonstrated a production titer of 2 g/L with nearly 1:1 ratio of butanol and propanol.     

Current knowledge on isobutanol production with Escherichia coli, Bacillus subtilis and Corynebacterium glutamicum

Due to steadily rising crude oil prices great efforts have been made to develop designer bugs for the fermentative production of higher alcohols, such as 2-methyl-1-butanol, 3-methyl-1-butanol and 2-Methyl-1-propanol (isobutanol), which all possess quality characteristics comparable to traditional oil based fuels. The common metabolic engineering approach uses the last two steps of the Ehrlich pathway, catalyzed by 2-ketoacid decarboxylase and an alcohol dehydrogenase converting the branched chain 2-ketoacids of L-isoleucine, L-leucine, and L-valine into the respective alcohols. This strategy was successfully used to engineer well suited and industrially employed bacteria, such as Escherichia coli, Bacillus subtilis and Corynebacterium glutamicum for the production of higher alcohols. Among these alcohols, isobutanol is currently the most promising one regarding final titer and yield. This article summarizes the current knowledge and achievements on isobutanol production with E. coli, B. subtilis and C. glutamicum regarding the metabolic engineering approaches and process conditions.     

Tuning the specificity of the recombinant multicomponent toluene o-xylene monooxygenase from Pseudomonas sp. strain OX1 for the biosynthesis of tyrosol from 2-phenylethanol

Biocatalysis is today a standard technology for the industrial production of several chemicals, and the number of biotransformation processes running on a commercial scale is constantly increasing. Among biocatalysts, bacterial multicomponent monooxygenases (BMMs), a diverse group of nonheme diiron enzymes that activate dioxygen, are of primary interest due to their ability to catalyze a variety of complex oxidations, including reactions of mono- and dihydroxylation of phenolic compounds. In recent years, both directed evolution and rational design have been successfully used to identify the molecular determinants responsible for BMM regioselectivity and to improve their activity toward natural and nonnatural substrates. Toluene o-xylene monooxygenase (ToMO) is a BMM isolated from Pseudomonas sp. strain OX1 which hydroxylates a wide spectrum of aromatic compounds. In this work we investigate the use of recombinant ToMO for the biosynthesis in recombinant cells of Escherichia coli strain JM109 of 4-hydroxyphenylethanol (tyrosol), an antioxidant present in olive oil, from 2-phenylethanol, a cheap and commercially available substrate. We initially found that wild-type ToMO is unable to convert 2-phenylethanol to tyrosol. This was explained by using a computational model which analyzed the interactions between ToMO active-site residues and the substrate. We found that residue F176 is the major steric hindrance for the correct positioning of the reaction intermediate leading to tyrosol production into the active site of the enzyme. Several mutants were designed and prepared, and we found that the combination of different mutations at position F176 with mutation E103G allows ToMO to convert up to 50% of 2-phenylethanol into tyrosol in 2 h.     

Enhancement of 2-phenylethanol productivity by Saccharomyces cerevisiae in two-phase fed-batch fermentations using solvent immobilization

The bioconversion of L-phenylalanine to 2-phenylethanol by Saccharomyces cerevisiae in fed-batch experiments has shown that concentrations of 2-phenylethanol of >2.9 g/L have a negative impact on the oxidative capacity of the yeast. Without tight control on ethanol production, and hence on the feed rate, ethanol rapidly accumulates in the culture media, resulting in complete inhibition of cell growth before the maximal 2-phenylethanol concentration of 3.8 g/L, obtained in the absence of ethanol production, could be achieved. This effect was attributed to a cumulative effect of ethanol and 2-phenylethanol, which reduced the tolerance of the cells for these two products. To enhance the productivity of the bioconversion, a novel in situ product recovery strategy, based on the entrapment of an organic solvent (dibutylsebacate) into a polymeric matrix of polyethylene to form a highly absorbent and chemically and mechanically stable composite resin, was developed. Immobilization of the organic solvent successfully prevented phase toxicity of the solvent and allowed for an efficient removal of 2-phenylethanol from the bioreactor without the need for prior cell separation. The use of the composite resin increased the volumetric productivity of 2-phenylethanol by a factor 2 and significantly facilitated downstream processing, because no stable emulsion was formed. The 2-phenylethanol could be backextracted from the composite resin, yielding a concentrated and almost cell-free solution. In comparison to two-phase extractive fermentations with cells immobilized in alginate-reinforced chitosan beads, the use of a composite resin was extremely inexpensive and simple. In addition, the composite resin was found to be insensitive to abrasion and chemically stable, such that sterilization with 2 M NaOH or heat was possible. Finally, the composite resin could be produced on a large scale using commercially available equipment.     

Engineering Corynebacterium glutamicum for isobutanol production

The production of isobutanol in microorganisms has recently been achieved by harnessing the highly active 2-keto acid pathways. Since these 2-keto acids are precursors of amino acids, we aimed to construct an isobutanol production platform in Corynebacterium glutamicum, a well-known amino-acid-producing microorganism. Analysis of this host’s sensitivity to isobutanol toxicity revealed that C. glutamicum shows an increased tolerance to isobutanol relative to Escherichia coli. Overexpression of alsS of Bacillus subtilis, ilvC and ilvD of C. glutamicum, kivd of Lactococcus lactis, and a native alcohol dehydrogenase, adhA, led to the production of 2.6 g/L isobutanol and 0.4 g/L 3-methyl-1-butanol in 48 h. In addition, other higher chain alcohols such as 1-propanol, 2-methyl-1-butanol, 1-butanol, and 2-phenylethanol were also detected as byproducts. Using longer-term batch cultures, isobutanol titers reached 4.0 g/L after 96 h with wild-type C. glutamicum as a host. Upon the inactivation of several genes to direct more carbon through the isobutanol pathway, we increased production by ∼25% to 4.9 g/L isobutanol in a ∆pyc∆ldh background. These results show promise in engineering C. glutamicum for higher chain alcohol production using the 2-keto acid pathways.     

Substrate entropy in enzyme enantioselectivity: an experimental and molecular modeling study of a lipase

The temperature dependence of the enantioselectivity of Candida antarctica lipase B for 3-hexanol, 2-butanol, 3-methyl-2-butanol, 3,3-dimethyl-2-butanol, and 1-bromo-2-butanol revealed that the differential activation entropy, deltaR-SdeltaS, was as significant as the differential activation enthalpy, DeltaR-SdeltaH, to the enantiomeric ratio, E. 1-Bromo-2-butanol, with isosteric substituents, displayed the largest deltaR-SdeltaS. 3-Hexanol displayed, contrary to other sec-alcohols, a positive deltaR-SdeltaS. In other words, for 3-hexanol the preferred R-enantiomer is not only favored by enthalpy but also by entropy. Molecular dynamics (MD) simulations and systematic search calculations of the substrate accessible volume within the active site revealed that the (R)-3-hexanol transition state (TS) accessed a larger volume within the active site than the (S)-3-hexanol TS. This correlates well with the higher TS entropy of (R)-3-hexanol. In addition, this enantiomer did also yield a higher number of allowed conformations, N, from the systematic search routines, than did the S-enantiomer. The substrate accessible volume was greater for the enantiomer preferred by entropy also for 2-butanol. For 3,3-dimethyl-2-butanol, however, neither MD-simulations nor systematic search calculations yielded substrate accessible volumes that correlate to TS entropy. Ambiguous results were achieved for 3-methyl-2-butanol.     

Amino Acid Sequences of Glycopeptides from Cucumisin

Oxidation of short-chain iso-alkanes (isobutane, isopentane, 2-methylpentane, and 3-methylpentane) was studied with propane-grown resting mycelia of Scedosporium sp. A-4. Isobutane was oxidized to terf-butanol, but both isobutane and tert-butanol were not used for growth. Isopentane was oxidized to 3-methyl-1-butanol, 2-methyl-2-butanol, and 3-methyl-2-butanol but not to 2-methyl-1-butanol. 2-Methylpentane was oxidized to 4-methyl-1-pentanol, 2-methyl-2-pentanol, and 4-methyl-2-pentanol but not to 2-methyl-1-pentanol or 2-methyl-3-pentanol. 3-Methylpentane was not oxidized. Oxidation of branched alcohols was also studied.     

Production of 2-methyl-1-butanol and 3-methyl-1-butanol in engineered Corynebacterium glutamicum

The pentanol isomers 2-methyl-1-butanol and 3-methyl-1-butanol represent commercially interesting alcohols due to their potential application as biofuels. For a sustainable microbial production of these compounds, Corynebacterium glutamicum was engineered for producing 2-methyl-1-butanol and 3-methyl-1-butanol via the Ehrlich pathway from 2-keto-3-methylvalerate and 2-ketoisocaproate, respectively. In addition to an already available 2-ketoisocaproate producer, a 2-keto-3-methylvalerate accumulating C. glutamicum strain was also constructed. For this purpose, we reduced the activity of the branched-chain amino acid transaminase in an available C. glutamicum l-isoleucine producer (K2P55) via a start codon exchange in the ilvE gene enabling accumulation of up to 3.67 g/l 2-keto-3-methylvalerate. Subsequently, nine strains expressing different gene combinations for three 2-keto acid decarboxylases and three alcohol dehydrogenases were constructed and characterized. The best strains accumulated 0.37 g/l 2-methyl-1-butanol and 2.76 g/l 3-methyl-1-butanol in defined medium within 48 h under oxygen deprivation conditions, making these strains ideal candidates for additional strain and process optimization.     

Sol–gels and cross-linked aggregates of lipase PS from Burkholderia cepacia and their application in dry organic solvents

Lipase PS from Burkholderia cepacia (formerly Pseudomonas cepacia) was successfully immobilized in sol–gels under low methanol conditions using lyophilization in order to dry the gel. The enzyme was also cross-linked with glutaraldehyde to CLEAs without any additives. These immobilized enzyme preparations were employed for the highly enantioselective acylations of 1-phenylethanol (1), 1-(2-furyl)ethanol (2) and N-acylated 1-amino-2-phenylethanol (3) with vinyl acetate in organic solvents. Enzymatic hydrolysis of the obtained ester product was observed as a side reaction of the acylation of 3 in the presence of lipase PS powder. Hydrolysis was suppressed when the immobilized preparations of lipase PS were used.