Anaerobic degradation of trans-cinnamate and ω-phenylalkane carboxylic acids by the photosynthetic bacterium Rhodopseudomonas palustris: evidence for a β-oxidation mechanism

The mechanism responsible for the initial steps in the anaerobic degradation of trans-cinnamate and -phenylalkane carboxylates by the purple non-sulphur photosynthetic bacterium Rhodopseudomonas palustris was investigated. Phenylacetate did not support growth and there was a marked CO₂ dependence for growth on acids with greater side-chain lengths. Here, CO₂ was presumably acting as a redox sink for the disposal of excess reducing equivalents. Growth on benzoate did not require the addition of exogenous CO₂. Aromatic acids with an odd number of side-chain carbon atoms (3-phenylpropionate, 5-phenylvalerate, 7-phenylheptanoate) gave greater apparent molar growth yields than those with an even number of side-chain carbon atoms (4-phenylbutyrate, 6-phenylhexanoate, 8-phenyloctanoate). HPLC analysis revealed that phenylacetate accumulated and persisted in the culture medium during growth on these latter compounds. Cinnamate and benzoate transiently accumulated in the culture medium during growth on 3-phenylpropionate, and benzoate alone accumulated transiently during the course of trans-cinnamate degradation. The transient accumulation of 4-phenyl-2-butenoic acid occurred during growth on 4-phenylbutyrate, and phenylacetate accumulated to a 1:1 molar stoichiometry with the initial 4-phenylbutyrate concentration. It is proposed that the initial steps in the anaerobic degradation of trans-cinnamate and the group of acids from 3-phenylpropionate to 8-phenyloctanoate involves -oxidation of the side-chain.Abbreviation 3-PP 3-phenylpropionic acid - 4-PB 4-phenylbutyric acid - 5-PV 5-phenylvaleric acid - 6-PH 6-phenylhexanoic acid - 7-PH 7-phenylheptanoic acid - 8-PO 8-phenyloctanoic acid - 4-P2B 4-phenyl-2-butenoic acid - GC/MS Gas chromatography/Mass spectrometry - HPLC High-pressure liquid chromatography     

Engineering Escherichia coli for the synthesis of short- and medium-chain α,β-unsaturated carboxylic acids

Concerns over sustained availability of fossil resources along with environmental impact of their use have stimulated the development of alternative methods for fuel and chemical production from renewable resources. In this work, we present a new approach to produce α,β-unsaturated carboxylic acids (α,β-UCAs) using an engineered reversal of the β-oxidation (r-BOX) cycle. To increase the availability of both acyl-CoAs and enoyl-CoAs for α,β-UCA production, we use an engineered Escherichia coli strain devoid of mixed-acid fermentation pathways and known thioesterases. Core genes for r-BOX such as thiolase, hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase, and enoyl-CoA reductase were chromosomally overexpressed under the control of a cumate inducible phage promoter. Native E. coli thioesterase YdiI was used as the cycle-terminating enzyme, as it was found to have not only the ability to convert trans-enoyl-CoAs to the corresponding α,β-UCAs, but also a very low catalytic efficiency on acetyl-CoA, the primer and extender unit for the r-BOX pathway. Coupling of r-BOX with YdiI led to crotonic acid production at titers reaching 1.5 g/L in flask cultures and 3.2 g/L in a controlled bioreactor. The engineered r-BOX pathway was also used to achieve for the first time the production of 2-hexenoic acid, 2-octenoic acid, and 2-decenoic acid at a final titer of 0.2 g/L. The superior nature of the engineered pathway was further validated through the use of in silico metabolic flux analysis, which showed the ability of r-BOX to support growth-coupled production of α,β-UCAs with a higher ATP efficiency than the widely used fatty acid biosynthesis pathway. Taken together, our findings suggest that r-BOX could be an ideal platform to implement the biological production of α,β-UCAs.     

Ammonia lyases and aminomutases as biocatalysts for the synthesis of α-amino and β-amino acids

Ammonia lyases catalyse the reversible addition of ammonia to cinnamic acid (1: R=H) and p-hydroxycinnamic (1: R=OH) to generate L-phenylalanine (2: R=H) and L-tyrosine (2: R=OH) respectively (Figure 1a). Both phenylalanine ammonia lyase (PAL) and tyrosine ammonia lyase (TAL) are widely distributed in plants, fungi and prokaryotes. Recently there has been interest in the use of these enzymes for the synthesis of a broader range of L-arylalanines. Aminomutases catalyse a related reaction, namely the interconversion of α-amino acids to β-amino acids (Figure 1b). In the case of L-phenylalanine, this reaction is catalysed by phenylalanine aminomutase (PAM) and proceeds stereospecifically via the intermediate cinnamic acid to generate β-Phe 3. Ammonia lyases and aminomutases are related in sequence and structure and share the same active site cofactor 4-methylideneimidazole-5-one (MIO). There is currently interest in the possibility of using these biocatalysts to prepare a wide range of enantiomerically pure l-configured α-amino and β-amino acids. Recent reviews have focused on the mechanism of these MIO containing enzymes. The aim of this review is to review recent progress in the application of ammonia lyase and aminomutase enzymes to prepare enantiomerically pure α-amino and β-amino acids.     

Contributions of the peroxisome and β-oxidation cycle to biotin synthesis in fungi

The first step in the synthesis of the bicyclic rings of D-biotin is mediated by 8-amino-7-oxononanoate (AON) synthase, which catalyzes the decarboxylative condensation of l-alanine and pimelate thioester. We found that the Aspergillus nidulans AON synthase, encoded by the bioF gene, is a peroxisomal enzyme with a type 1 peroxisomal targeting sequence (PTS1). Localization of AON to the peroxisome was essential for biotin synthesis because expression of a cytosolic AON variant or deletion of pexE, encoding the PTS1 receptor, rendered A. nidulans a biotin auxotroph. AON synthases with PTS1 are found throughout the fungal kingdom, in ascomycetes, basidiomycetes, and members of basal fungal lineages but not in representatives of the Saccharomyces species complex, including Saccharomyces cerevisiae. A. nidulans mutants defective in the peroxisomal acyl-CoA oxidase AoxA or the multifunctional protein FoxA showed a strong decrease in colonial growth rate in biotin-deficient medium, whereas partial growth recovery occurred with pimelic acid supplementation. These results indicate that pimeloyl-CoA is the in vivo substrate of AON synthase and that it is generated in the peroxisome via the β-oxidation cycle in A. nidulans and probably in a broad range of fungi. However, the β-oxidation cycle is not essential for biotin synthesis in S. cerevisiae or Escherichia coli. These results suggest that alternative pathways for synthesis of the pimelate intermediate exist in bacteria and eukaryotes and that Saccharomyces species use a pathway different from that used by the majority of fungi.     

A general synthesis of long chain ω- and (ω-1)-hydroxy fatty acids

A method for the synthesis of long chain fatty acids substituted at the ω and ω-1 positions has been developed. The key step is the isomerization of the triple bond of an alkyn-1-ol from an internal position in the chain to the free terminus with a new, convenient reagent, sodium aminopropylamide (NaAPA). Standard functional group manipulations i.e., Jones oxidation, esterification and hydroboration of the triple bond are used to prepare ω-hydroxy fatty esters. The generality of the method is illustrated with syntheses of ω-hydroxy fatty esters with 24, 26, 28 and 30 carbon chains.In the 24 carbon series, hydration of the terminal triple bond of alkynoic ester 4a followed by reduction gave the (ω-1)-hydroxy ester.     

Peroxisomal β-oxidation of fatty acids in bovine and rat liver

Hepatic peroxisomal β-oxidation rates were compared in liver homogenates from cows and rats during different nutritional and physiological states. Peroxisomal oxidation in liver homogenates from cows represented 50% and 77% of the total capacity for the initial cycle of β-oxidation of palmitate and octanoate, respectively, but only 26% and 65% for rats. Lactation or food deprivation did not alter rates of hepatic peroxisomal β-oxidation of palmitate or octanoate in cows. Fasting and clofibrate treatment increased rates of total and peroxisomal β-oxidation of palmitate and octanoate in rat liver.     

In silico assessment of the metabolic capabilities of an engineered functional reversal of the β-oxidation cycle for the synthesis of longer-chain (C≥4) products

The modularity and versatility of an engineered functional reversal of the β-oxidation cycle make it a promising platform for the synthesis of longer-chain (C≥4) products. While the pathway has recently been exploited for the production of n-alcohols and carboxylic acids, fully capitalizing on its potential for the synthesis of a diverse set of product families requires a system-level assessment of its biosynthetic capabilities. To this end, we utilized a genome scale model of Escherichia coli, in combination with Flux Balance Analysis and Flux Variability Analysis, to determine the key characteristics and constraints of this pathway for the production of a variety of product families under fermentative conditions. This analysis revealed that the production of n-alcohols, alkanes, and fatty acids of lengths C3–C18 could be coupled to cell growth in a strain lacking native fermentative pathways, a characteristic enabling product synthesis at maximum rates, titers, and yields. While energetic and redox constraints limit the production of target compounds from alternative platforms such as the fatty acid biosynthesis and α-ketoacid pathways, the metabolic efficiency of a β-oxidation reversal allows the production of a wide range of products of varying length and functionality. The versatility of this platform was investigated through the simulation of various termination pathways for product synthesis along with the use of different priming molecules, demonstrating its potential for the efficient synthesis of a wide variety of functionalized compounds. Overall, specific metabolic manipulations suggested by this systems-level analysis include deletion of native fermentation pathways, the choice of priming molecules and specific routes for their synthesis, proper choice of termination enzymes, control of flux partitioning at the pyruvate node and the pentose phosphate pathway, and the use of an NADH-dependent trans-enoyl-CoA reductase instead of a ferredoxin-dependent enzyme.     

Kinetic advantage of the interaction between the fatty acid β-oxidation enzymes and the complexes of the respiratory chain

Respiration-linked oxidation of 3-hydroxybutyryl-CoA, crotonyl-CoA and saturated fatty acyl (C₄, C₈ and C₁₄)-CoA esters was studied in different mitochondrial preparations. Oxidation of acyl-CoA esters was poor in intact mitochondria; however, it was significant, as well as, NAD⁺ and CoA-dependent in gently and in vigorously sonicated mitochondria. The respiration-linked oxidation of crotonyl-CoA and 3-hydroxybutyryl-CoA proceeded at much higher rates (over 700%) in gently disrupted mitochondria than in completely disrupted mitochondria. The redox dye-linked oxidation of crotonyl-CoA (with inhibited respiratory chain) was also higher in gently disrupted mitochondria (149%) than in disrupted ones. During the respiration-linked oxidation of 3-hydroxybutyryl-CoA the steady-state NADH concentrations in the reaction chamber were determined, and found to be 8 μM in gently sonicated and 15 μM in completely sonicated mitochondria in spite of the observation that the gently sonicated mitochondria oxidized the 3-hydroxybutyryl-CoA much faster than the completely sonicated mitochondria. The NAD⁺-dependence of 3-hydroxybutyryl-CoA oxidation showed that a much smaller NAD⁺ concentration was enough to half-saturate the reaction in gently disrupted mitochondria than in completely disrupted ones. Thus, these observations indicate the positive kinetic consequence of organization of β-oxidation enzyme in situ. Respiration-linked oxidation of bytyryl-, oxtanoyl- and palmitoyl-CoA was also studied and these CoA intermediates were oxidized at approx. 50% of the rate of crotonyl- and 3-hydroxybutyryl-CoA in the gently disrupted mitochondria. In vigorously disrupted mitochondria the oxidation rate of these saturated acyl-CoA intermediates was hardly detectable indicating that the connection between the acyl-CoA dehydrogenase and the respiratory chain had been disrupted.     

Chemical synthesis and biological evaluation of ω-hydroxy polyunsaturated fatty acids

ω-Hydroxy polyunsaturated fatty acids (PUFAs), natural metabolites from arachidonic acid (ARA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) were prepared via convergent synthesis approach using two key steps: Cu-mediated CC bond formation to construct methylene skipped poly-ynes and a partial alkyne hydrogenation where the presence of excess 2-methyl-2-butene as an additive that is proven to be critical for the success of partial reduction of the poly-ynes to the corresponding cis-alkenes without over-hydrogenation. The potential biological function of ω-hydroxy PUFAs in pain was evaluated in naive rats. Following intraplantar injection, 20-hydroxyeicosatetraenoic acid (20-HETE, ω-hydroxy ARA) generated an acute decrease in paw withdrawal thresholds in a mechanical nociceptive assay indicating pain, but no change was observed from rats which received either 20-hydroxyeicosapentaenoic acid (20-HEPE, ω-hydroxy EPA) or 22-hydroxydocosahexaenoic acid (22-HDoHE, ω-hydroxy DHA). We also found that both 20-HEPE and 22-HDoHE are more potent than 20-HETE to activate murine transient receptor potential vanilloid receptor1 (mTRPV1).     

n-alkane oxidation by apseudomonas formation and β-oxidation of intermediate fatty acids

Summary From a culture broth ofPseudomonas aeruginosa (KSLA strain 473) grown on heptane as the sole source of carbon, fatty acids could be isolated after a period of decreased oxygen supply. The corresponding methyl esters—obtained by treatment with diazomethane—were separated by gas-liquid chromatography and identified by mass spectrometry. Heptylic, valeric and propionic acids were shown to be present in the original culture broth. Using the same techniques the formation of caproic acid from hexane was shown to occur, whereas the amount of butyric acid formed was extremely small and inconsistent. These results show conclusively that this microbiological oxidation of heptane and hexane proceeds by way of the corresponding fatty acids, which are further degraded by β-oxidation. The absence of caproic and valeric acids in heptane and hexane oxidation, respectively, shows that decarboxylation of fatty acids does not occur.     

Metabolic engineering of β-oxidation to leverage thioesterases for production of 2-heptanone, 2-nonanone and 2-undecanone

Medium-chain length methyl ketones are potential blending fuels due to their cetane numbers and low melting temperatures. Biomanufacturing offers the potential to produce these molecules from renewable resources such as lignocellulosic biomass. In this work, we designed and tested metabolic pathways in Escherichia coli to specifically produce 2-heptanone, 2-nonanone and 2-undecanone. We achieved substantial production of each ketone by introducing chain-length specific acyl-ACP thioesterases, blocking the β-oxidation cycle at an advantageous reaction, and introducing active β-ketoacyl-CoA thioesterases. Using a bioprospecting approach, we identified fifteen homologs of E. coli β-ketoacyl-CoA thioesterase (FadM) and evaluated the in vivo activity of each against various chain length substrates. The FadM variant from Providencia sneebia produced the most 2-heptanone, 2-nonanone, and 2-undecanone, suggesting it has the highest activity on the corresponding β-ketoacyl-CoA substrates. We tested enzyme variants, including acyl-CoA oxidases, thiolases, and bi-functional 3-hydroxyacyl-CoA dehydratases to maximize conversion of fatty acids to β-keto acyl-CoAs for 2-heptanone, 2-nonanone, and 2-undecanone production. In order to address the issue of product loss during fermentation, we applied a 20% (v/v) dodecane layer in the bioreactor and built an external water cooling condenser connecting to the bioreactor heat-transferring condenser coupling to the condenser. Using these modifications, we were able to generate up to 4.4 g/L total medium-chain length methyl ketones.     

Substrate inhibition and affinities of the glyoxysomal β-oxidation of sunflower cotyledons

During the glyoxysomal β-oxidation of long-chain acyl-CoAs, short-chain intermediates accumulate transiently (Kleiter and Gerhardt 1998, Planta 206: 125–130). The studies reported here address the underlying factors. The studies concentrated upon the aspects of (i) chain length specificity and (ii) metabolic regulation of the glyoxysomal β-oxidation of sunflower (Helianthus annuus L.) cotyledons. (i) Concentration-rate curves of the β-oxidation of acyl-CoAs of various chain lengths showed that the β-oxidation activity towards long-chain acyl-CoAs was higher than that towards short-chain acyl-CoAs at substrate concentrations <20 μM. At substrate concentrations >20 μM, long-chain acyl-CoAs were β-oxidized more slowly than short-chain acyl-CoAs because the β-oxidation of long-chain acyl-CoAs is subject to substrate inhibition which had already started at 5–10 μM substrate concentration and results from an inhibition of the multifunctional protein (MFP) of the β-oxidation reaction sequence. However, low concentrations of free long-chain acyl-CoAs are rather likely to exist within the glyoxysomes due to the acyl-CoA-binding capacity of proteins. Consequently, the β-oxidation rate towards a parent long-chain acyl-CoA will prevail over that towards the short-chain intermediates. (ii) Low concentrations (≤5 μM) of a long-chain acyl-CoA exerted an inhibitory effect on the β-oxidation rate of butyryl-CoA. Reversibility of the inhibition was observed as well as metabolization of the inhibiting long-chain acyl-CoA. Regarding the activities of the individual β-oxidation enzymes towards their C₄ substrates in the presence of a long-chain acyl-CoA, the MFP activity exhibited strong inhibition. This inhibition appears not to be due to the detergent-like physical properties of long-chain acyl-CoAs. The results of the studies, which are consistent with the observation that short-chain intermediates accumulate transiently during complete degradation of a long-chain acyl-CoA, suggest that the substrate concentration-dependent chain-length specificity of the β-oxidation and a metabolic regulation at the level of MFP are factors determining this transient accumulation. Received: 2 February 1999 / Accepted: 14 April 1999     

Characterization of ELP-fused ω-Transaminase and Its Application for the Biosynthesis of β-Amino Acid

Optically pure amines, β-amino acids and γ-amino acids are the valuable precursors to produce biologically active compounds. The ω-TAs are the class of enzymes which are widely used to produce such compounds. In this work (S)-ω-transaminase from the thermophilic eubacterium Sphaerobacter thermophilus (St-TA) was fused with Elastin-like polypeptides (ELPs) through the cloning process and expressed in E. coli cells. The characterization of this fusion complex was performed with respect to thermostability and effect of DMSO. Where in case of St-TA-ELP-V₆₀, major difference in the transition temperature (T_t) was observed, wherein a T_t of 38 and 70°C was observed at the increasing concentration of DMSO from 5 to 25% (v/v). Interestingly, these fusion proteins the activity was preserved even after the aggregation of fusion complex at Tt. The substrate specificity and product inhibition analysis showed that ω-TA-ELPs had comparable results as that of wild type ω-TA. Moreover, the fused ω-TA could be efficiently reused for up to 20 batches of transamination reaction. Furthermore, the applicability of the fusion protein for the production of a sitagliptin precursor (R)-3-amino-4-(2,4,5-triflurophenyl) butanoic acid (3-ATfBA) was evaluated, wherein 3-ATfBA was synthesized with good conversion (65%).     

Reaction modelling and simulation to assess the integrated use of transketolase and ω-transaminase for the synthesis of an aminotriol

The most attractive, as well as challenging, multistep organic syntheses would preferably be carried out in a single reactor, as a one-pot synthesis. For biocatalytic syntheses, multistep reactions in one-pot mode bring a number of advantages, while at the same time raising unique challenges such as the compatibility of different biocatalysts. In this paper, we have developed a transketolase–transaminase (TK-TAm) two-step one-pot aminotriol synthesis reaction model, which integrates reaction kinetic models with process characterization (consisting of component degradation as a function of pH and concentration, aldehyde toxicity towards the enzyme, and ketol donor and acceptor side-reactions with TAm). Based on the analysis of the effect of the TAm/TK activity ratio on product yield, simulations provided guidance for further process and biocatalyst development.