Valorization of single-carbon chemicals by using carboligases as key enzymes.

Single-carbon (C1) biorefinery plays a key role in the consumption of global greenhouse gases and a circular carbon economy. Thereby, we have focused on the valorization of C1 compounds (e.g. methanol, formaldehyde, and formate) into multicarbon products, including bioplastic monomers, glycolate, and ethylene glycol. For instance, methanol, derived from the oxidation of CH4, can be converted into glycolate, ethylene glycol, or erythrulose via formaldehyde and glycolaldehyde, employing C1 and/or C2 carboligases as essential enzymes. Escherichia coli was engineered to convert formate, produced from CO via CO2 or from CO2 directly, into glycolate. Recent progress in the design of biotransformation pathways, enzyme discovery, and engineering, as well as whole-cell biocatalyst engineering for C1 biorefinery, was addressed in this review.

Engineering of two thiamine diphosphate-dependent enzymes for the regioselective condensation of C1-formaldehyde into C4-erythrulose.

A number of carboligases, which catalyze condensation of C1- and/or C2-aldehydes into multi-carbon products, have been reported. However, their catalytic activities and/or regioselectivities remained rather low. Thereby, this study has focused on engineering of C1 and C2 carboligases for the regioselective condensation of C1-formaldehyde into C4-erythrulose via C2-glycolaldehyde. The crystal structure of the glyoxylate carboligase from Escherichia coli (EcGCL) was elucidated in complex with glycolaldehyde. A structure-guided rationale generated several mutants, one of whose catalytic activity reached 15.6 M-1·s-1, almost 10 times greater than the wild-type enzyme. Another variant (i.e., EcGCL_R484M/N283Q/L478M/M488L/R284K) has shown significantly increased stability to the glycolaldehyde toxicity, enabling production of glycolaldehyde to 31 mM from 75 mM formaldehyde (conversion: 83 %). Besides, the E1 subunit of α-ketoglutarate dehydrogenase complex from Vibrio vulnificus (VvSucA) was engineered as a regiospecific C2 carboligase for condensation of glycolaldehyde into erythrulose. The combination of EcGCL_R484M/N283Q/L478M/M488L/R284K and VvSucA_K228L led to the cascade production of erythrulose to 8 mM from 90 mM formaldehyde via glycolaldehyde without byproduct formation. This study will contribute to valorization of C1 gases into industrially relevant multi-carbon products in an environment-friendly way.

Efficient biotransformation of docosahexaenoic acid-rich oils into the lipid mediator resolvin D5 by cells expressing 15S-lipoxygenase using a bioreactor.

Resolvin D5 (RvD5), 7S,17S-dihydroxy-4Z,8E,10Z,13Z,15E,19Z-docosahexaenoic acid (DHA) is a specialized pro-resolving mediator (SPM) generated in human macrophages. It is implicated in the resolution of inflammation and synthesized using an inefficient chemical process. Here, DHA-enriched oil hydrolysate was prepared from oils by lipase with resin treatment and solvent extraction. The reaction factors on the biotransformation of oil hydrolysate into RvD5 were optimized using Escherichia coli expressing arachidonate double-oxygenating 15S-lipoxygenase. After optimization, the cells converted 5.0 mM (1.64 g/L) DHA in oil hydrolysate into 4.0 mM (1.44 g/L) RvD5 in a bioreactor for 3.0 h, which was 15-fold higher than that in a flask before optimization, and RvD5 with a purity of > 97% was prepared from reaction solution by treatments of resins. This is the first trial for the production of C22-dihydroxy fatty acid using a bioreactor. This study will contribute to the large-scale production of SPMs from oils.

Variability of oral/taste sensitivity to fat: An investigation of attribution from detection threshold methods with repeated measurements.

Accumulating psychophysical evidence suggests substantial individual variability in oral/taste sensitivity to non-esterified, long-chain fatty acids (NEFA), which is commonly referred to as fat taste or oleogustus. Recent studies have sought to determine its associations with human factors such as body mass index (BMI) and food preferences, as it has been claimed that excessive fat consumption is related to several health conditions, including obesity. Yet, the findings are controversial. On the other hand, it has been noted that considerable variability also occurs based on the methodology used to measure the fatty acid taste. Specifically, learning effects have been observed over repeated measurements of the detection threshold of NEFA, yet there has been no methodology available to take into account these learning effects. Accordingly, in the present study, a novel methodology using a descending-block dual reminder A-Not A (DR A-Not A) method with a warm-up has been proposed to measure the NEFA detection threshold based on the signal detection theory and considering NEFA taste learning effects over repeated sessions. Homogeneous subjects (young adult Korean females within the normal BMI range, non-vegetarians) were randomized to either the novel descending-block DR A-Not A method or ascending triangle method that is commonly used for fat perception studies. Pure oleic acid emulsions were used as fat taste stimuli to be discriminated from pure mineral water. Each subject completed 14 repeated visits. For the ascending triangle method, 14 thresholds were determined using a stopping rule, while for the novel method, 7 thresholds were determined each per two consecutive days, using a criterion of a lower limit of 50% confidence interval of d' = 0.5, considering the practical aspects of taste studies in food sensory science. Based on the group median results of the last two visits, the variability of the detection thresholds was reduced using the novel descending-block DR A-Not A method due to better learning effects over repeated sessions. This shows the potential of the descending-block DR A-Not A threshold method for further studies on oral/taste sensitivity to fat.

Structural and functional characterization of a thermostable secretory phospholipase A2 from Sciscionella marina and its application in liposome biotransformation.

Secretory phospholipase A2 (sPLA2), which hydrolyzes the sn-2 acyl bond of lecithin in a Ca2+-dependent manner, is an important enzyme in the oil and oleochemical industries. However, most sPLA2s are not stable under process conditions. Therefore, a thermostable sPLA2 was investigated in this study. A marine bacterial sPLA2 isolated from Sciscionella marina (Sm-sPLA2) was catalytically active even after 5 h of incubation at high temperatures of up to 50°C, which is outstanding compared with a representative bacterial sPLA2 (i.e. sPLA2 from Streptomyces violaceoruber; Sv-sPLA2). Consistent with this, the melting temperature of Sm-sPLA2 was measured to be 7.7°C higher than that of Sv-sPLA2. Furthermore, Sm-sPLA2 exhibited an improved biotransformation performance compared with Sv-sPLA2 in the hydrolysis of soy lecithin to lysolecithin and free fatty acids at 50°C. Structural and mutagenesis studies revealed that the Trp41-mediated anchoring of a Ca2+-binding loop into the rest of the protein body is directly linked to the thermal stability of Sm-sPLA2. This finding provides a novel structural insight into the thermostability of sPLA2 and could be applied to create mutant proteins with enhanced industrial potential.

Valorization of Soy Lecithin by Enzyme Cascade Reactions Including a Phospholipase A2, a Fatty Acid Double-Bond Hydratase, and/or a Photoactivated Decarboxylase.

A huge amount of phospholipids or lecithin is produced as a byproduct in the vegetable oil industry. However, most are just used as a feed additive. This study has focused on enzymatic valorization of lecithin. This was exploited by enzymatic transformation of soy lecithin into lysolecithin liposomes, including functional free fatty acids, hydroxy fatty acids, hydrocarbons, or secondary fatty alcohols. One of the representative examples was the preparation of lysolecithin liposomes containing secondary fatty alcohols [e.g., 9-Hydroxyheptadec-11-ene (9) and 9-heptadecanol (10)] by using a phospholipase A2 from Streptomyces violaceoruber, a fatty acid double-bond hydratase from Stenotrophomonas maltophilia, and a photoactivated decarboxylase from Chlorella variabilis NC64A. The engineered liposomes turned out to range ca. 144 nm in diameter by dynamic light scattering analysis. Thereby, this study will contribute to application of functional fatty acids and their derivatives as well as valorization of lecithin for the food and cosmetic industries.

Engineering of a bacterial outer membrane vesicle to a nano-scale reactor for the biodegradation of ß-lactam antibiotics.

Bacterial outer membrane vesicles (OMVs) are small unilamellar proteoliposomes, which are involved in various functions including cell to cell signaling and protein excretion. Here, we have engineered the OMVs of Escherichia coli to nano-scaled bioreactors for the degradation of ß-lactam antibiotics. This was exploited by targeting a ß-lactamase (i.e., CMY-10) into the OMVs of a hyper-vesiculating E. coli BL21(DE3) mutant. The CMY-10-containing OMVs, prepared from the E. coli mutant cultures, were able to hydrolyze ß-lactam ring of nitrocefin and meropenem to a specific rate of 6.6 × 10-8 and 3.9 × 10-12 µmol/min/µm3 of OMV, which is approximately 100 and 600-fold greater than those of E. coli-based whole-cell biocatalsyts. Furthermore, CMY-10, which was encapsulated in the engineered OMVs, was much more stable against temperature and acid stresses, as compared to free enzymes in aqueous phase. The OMV-based nano-scaled reaction system would be useful for the remediation of a variety of antibiotics pollution for food and agricultural industry.

Triplet-triplet annihilation-based photon-upconversion to broaden the wavelength spectrum for photobiocatalysis.

Photobiocatalysis is a growing field of biocatalysis. Especially light-driven enzyme catalysis has contributed significantly to expanding the scope of synthetic organic chemistry. However, photoenzymes usually utilise a rather narrow wavelength range of visible (sun)light. Triplet-triplet annihilation-based upconversion (TTA-UC) of long wavelength light to shorter wavelength light may broaden the wavelength range. To demonstrate the feasibility of light upconversion we prepared TTA-UC poly(styrene) (PS) nanoparticles doped with platinum(II) octaethylporphyrin (PtOEP) photosensitizer and 9,10-diphenylanthracene (DPA) annihilator (PtOEP:DPA@PS) for application in aqueous solutions. Photoexcitation of PtOEP:DPA@PS nanoparticles with 550 nm light led to upconverted emission of DPA 418 nm. The TTA-UC emission could photoactivate flavin-dependent photodecarboxylases with a high energy transfer efficiency. This allowed the photodecarboxylase from Chlorella variabilis NC64A to catalyse the decarboxylation of fatty acids into long chain secondary alcohols under green light (λ = 550 nm).

Enhancing acid tolerance of Escherichia coli via viroporin-mediated export of protons and its application for efficient whole-cell biotransformation.

Escherichia coli-based whole-cell biocatalysts are widely used for the sustainable production of value-added chemicals. However, weak acids present as substrates and/or products obstruct the growth and fermentation capability of E. coli. Here, we show that a viroporin consisting of the influenza A matrix-2 (M2) protein, is activated by low pH and has proton channel activity in E. coli. The heterologous expression of the M2 protein in E. coli resulted in a significant increase in the intracellular pH and cell viability in the presence of various weak acids with different lengths of carbon chains. In addition, the feasibility of developing a robust and efficient E. coli-based whole-cell biocatalyst via introduction of the proton-selective viroporin was explored by employing (Z)-11-(heptanolyoxy)undec-9-enoic acid (ester) and 2-fucosyllactose (2'-FL) as model products, whose production is hampered by cytosolic acidification. The engineered E. coli strains containing the proton-selective viroporin exhibited approximately 80% and 230% higher concentrations of the ester and 2'-FL, respectively, than the control strains without the M2 protein. The simple and powerful strategy developed in this study can be applied to produce other valuable chemicals whose production involves substrates and/or products that cause cytosolic acidification.

Increased Production of ω-Hydroxynonanoic Acid and α,ω-Nonanedioic Acid from Olive Oil by a Constructed Biocatalytic System.

ω-Hydroxynonanoic acid and α,ω-nonanedioic acid are used for synthesizing diverse chemicals. Although biological methods are developed, their concentrations are low due to the toxicity of high concentrations of the hydrophobic chemicals toward biocatalysts. Here, we constructed a biocatalytic system with high productivity by adding an adsorbent resin and a strong base anion-exchange resin, reducing the solubility of ω-hydroxynonanoic acid and α,ω-nonanedioic acid, feeding ω-hydroxynonanoic acid, and introducing a cofactor regeneration system. The constructed biocatalytic system converted 300 mM (83.9 g L-1) and 154 mM (43.5 g L-1) oleic acid in the olive oil hydrolysate obtained after resin extraction, which were derived from 110 and 54 g L-1 olive oil, respectively, into 202 mM (35.2 g L-1) ω-hydroxynonanoic acid and 103 mM (19.4 g L-1) α,ω-nonanedioic acid, which are 21- and 24-fold higher values than the previously reported results, respectively. This study may contribute to the industrial biosynthesis of ω-hydroxynonanoic acid and α,ω-nonanedioic acid from olive oil.

Construction of an engineered biocatalyst system for the production of medium-chain α,ω-dicarboxylic acids from medium-chain ω-hydroxycarboxylic acids.

Medium-chain α,ω-dicarboxylic acids produced from renewable long-chain fatty acids are valuable as precursors in the chemical industry. However, they are difficult to produce biologically at high concentrations. Although improved biocatalyst systems consisting of engineering of Baeyer-Villiger monooxygenases are used in the production of ω-hydroxycarboxylic acids from long-chain fatty acids, the engineering of biocatalysts involved in the production of α,ω-dicarboxylic acids from ω-hydroxycarboxylic acids has been rarely attempted. Here, we used highly active bacterial enzymes, Micrococcus luteus alcohol dehydrogenase and Archangium violaceum aldehyde dehydrogenase, for the efficient production of α,ω-dicarboxylic acids from ω-hydroxycarboxylic acids and constructed a biocatalyst with cofactor regeneration system by introducing NAD(P)H flavin oxidoreductase as the NAD(P)H oxidase. The inhibition of the biocatalyst by hydrophobic substrates was attenuated by engineering a biocatalyst system with an adsorbent resin, which allowed us to obtain 196 mM decanedioic, 145 mM undecanedioic, and 114 mM dodecanedioic acid from 200 mM of C10, C11, and C12 hydroxyl saturated carboxylic acids, respectively, and 141 mM undecanedioic acid from 150 mM C11 unsaturated carboxylic acids, with molar conversions of 98%, 97%, 95%, and 94%, respectively. The concentration of undecanedioic acid obtained was approximately 40-fold higher than that in the previously highest results. Our results from this study can be applied for the industrial production of medium-chain α,ω-dicarboxylic acids from renewable long-chain fatty acids.

Photobiocatalytic synthesis of chiral secondary fatty alcohols from renewable unsaturated fatty acids.

En route to a bio-based chemical industry, the conversion of fatty acids into building blocks is of particular interest. Enzymatic routes, occurring under mild conditions and excelling by intrinsic selectivity, are particularly attractive. Here we report photoenzymatic cascade reactions to transform unsaturated fatty acids into enantiomerically pure secondary fatty alcohols. In a first step the C=C-double bond is stereoselectively hydrated using oleate hydratases from Lactobacillus reuteri or Stenotrophomonas maltophilia. Also, dihydroxylation mediated by the 5,8-diol synthase from Aspergillus nidulans is demonstrated. The second step comprises decarboxylation of the intermediate hydroxy acids by the photoactivated decarboxylase from Chlorella variabilis NC64A. A broad range of (poly)unsaturated fatty acids can be transformed into enantiomerically pure fatty alcohols in a simple one-pot approach.

Genome-Scale Metabolic Network Reconstruction and In Silico Analysis of Hexanoic acid Producing Megasphaera elsdenii.

Hexanoic acid and its derivatives have been recently recognized as value-added materials and can be synthesized by several microbes. Of them, Megasphaera elsdenii has been considered as an interesting hexanoic acid producer because of its capability to utilize a variety of carbons sources. However, the cellular metabolism and physiology of M. elsdenii still remain uncharacterized. Therefore, in order to better understand hexanoic acid synthetic metabolism in M. elsdenii, we newly reconstructed its genome-scale metabolic model, iME375, which accounts for 375 genes, 521 reactions, and 443 metabolites. A constraint-based analysis was then employed to evaluate cell growth under various conditions. Subsequently, a flux ratio analysis was conducted to understand the mechanism of bifurcated hexanoic acid synthetic pathways, including the typical fatty acid synthetic pathway via acetyl-CoA and the TCA cycle in a counterclockwise direction through succinate. The resultant metabolic states showed that the highest hexanoic acid production could be achieved when the balanced fractional contribution via acetyl-CoA and succinate in reductive TCA cycle was formed in various cell growth rates. The highest hexanoic acid production was maintained in the most perturbed flux ratio, as phosphoenolpyruvate carboxykinase (pck) enables the bifurcated pathway to form consistent fluxes. Finally, organic acid consuming simulations suggested that succinate can increase both biomass formation and hexanoic acid production.

Engineering of a Microbial Cell Factory for the Extracellular Production of Catalytically Active Phospholipase A2 of Streptomyces violaceoruber.

Phospholipase A2 (PLA2) from Streptomyces violaceoruber is a lipolytic enzyme used in a wide range of industrial applications including production of lysolecithins and enzymatic degumming of edible oils. We have therefore investigated expression and secretion of PLA2 in two workhorse microbes, Pichia pastoris and Escherichia coli. The PLA2 was produced to an activity of 0.517 ± 0.012 U/ml in the culture broth of the recombinant P. pastoris. On the other hand, recombinant E. coli BL21 star (DE3), overexpressing the authentic PLA2 (P-PLA2), showed activity of 17.0 ± 1.3 U/ml in the intracellular fraction and 21.7 ± 0.7 U/ml in the culture broth. The extracellular PLA2 activity obtained with the recombinant E. coli system was 3.2-fold higher than the corresponding value reached in a previous study, which employed recombinant E. coli BL21 (DE3) overexpressing codon-optimized PLA2. Finally, we observed that the extracellular PLA2 from the recombinant E. coli P-PLA2 culture was able to hydrolyze 31.1 g/l of crude soybean lecithin, an industrial substrate, to a conversion yield of approximately 95%. The newly developed E. coli-based PLA2 expression system led to extracellular production of PLA2 to a productivity of 678 U/l·h, corresponding to 157-fold higher than that obtained with the P. pastoris-based system. This study will contribute to the extracellular production of a catalytically active PLA2.

Whole-Cell Photoenzymatic Cascades to Synthesize Long-Chain Aliphatic Amines and Esters from Renewable Fatty Acids.

Long-chain aliphatic amines such as (S,Z)-heptadec-9-en-7-amine and 9-aminoheptadecane were synthesized from ricinoleic acid and oleic acid, respectively, by whole-cell cascade reactions using the combination of an alcohol dehydrogenase (ADH) from Micrococcus luteus, an engineered amine transaminase from Vibrio fluvialis (Vf-ATA), and a photoactivated decarboxylase from Chlorella variabilis NC64A (Cv-FAP) in a one-pot process. In addition, long chain aliphatic esters such as 10-(heptanoyloxy)dec-8-ene and octylnonanoate were prepared from ricinoleic acid and oleic acid, respectively, by using the combination of the ADH, a Baeyer-Villiger monooxygenase variant from Pseudomonas putida KT2440, and the Cv-FAP. The target compounds were produced at rates of up to 37 U g-1 dry cells with conversions up to 90 %. Therefore, this study contributes to the preparation of industrially relevant long-chain aliphatic chiral amines and esters from renewable fatty acid resources.

Cofactor specificity engineering of a long-chain secondary alcohol dehydrogenase from Micrococcus luteus for redox-neutral biotransformation of fatty acids.

Structure-based engineering of a NAD+-dependent secondary alcohol dehydrogenase from Micrococcus luteus led to a 1800-fold increase in catalytic efficiency for NADP+. Furthermore, the engineered enzymes (e.g., D37S/A38R/V39S/T15I) were successfully coupled to a NADPH-dependent Baeyer-Villiger monooxygenase from Pseudomonas putida KT2440 for redox-neutral biotransformations of C18 fatty acids into C9 chemicals.

Multi-Step Enzymatic Synthesis of 1,9-Nonanedioic Acid from a Renewable Fatty Acid and Its Application for the Enzymatic Production of Biopolyesters.

1,9-Nonanedioic acid is one of the valuable building blocks for producing polyesters and polyamides. Thereby, whole-cell biosynthesis of 1,9-nonanedioic acid from oleic acid has been investigated. A recombinant Corynebacterium glutamicum, expressing the alcohol/aldehyde dehydrogenases (ChnDE) of Acinetobacter sp. NCIMB 9871, was constructed and used for the production of 1,9-nonanedioic acid from 9-hydroxynonanoic acid, which had been produced from oleic acid. When 9-hydroxynonanoic acid was added to a concentration of 20 mM in the reaction medium, 1,9-nonanedioic acid was produced to 16 mM within 8 h by the recombinant C. glutamicum. The dicarboxylic acid was isolated via crystallization and then used for the production of biopolyester by a lipase. For instance, the polyesterification of 1,9-nonanedioic acid and 1,8-octanediol in diphenyl ether by the immobilized lipase B from Candida antarctica led to formation of the polymer product with the number-average molecular weight (Mn) of approximately 21,000. Thereby, this study will contribute to biological synthesis of long chain dicarboxylic acids and their application for the enzymatic production of long chain biopolyesters.

Endocytosing Escherichia coli as a Whole-Cell Biocatalyst of Fatty Acids.

Whole cell biocatalysts can be used to convert fatty acids into various value-added products. However, fatty acid transport across cellular membranes into the cytosol of microbial cells limits substrate availability and impairs membrane integrity, which in turn decreases cell viability and bioconversion activity. Because these problems are associated with the mechanism of fatty acid transport through membranes, a whole-cell biocatalyst that can form caveolae-like structures was generated to promote substrate endocytosis. Caveolin-1 ( CAV1) expression in Escherichia coli increased both the fatty acid transport rate and intracellular fatty acid concentrations via endocytosis of the supplemented substrate. Furthermore, fatty-acid endocytosis alleviated substrate cytotoxicity in E. coli. These traits attributed to bacterial endocytosis resulted in dramatically elevated biotransformation efficiencies in fed-batch and cell-recycle reaction systems when caveolae-forming E. coli was used for the bioconversion of ricinoleic acid (12-hydroxyoctadec-9-enoic acid) to ( Z)-11-(heptanoyloxy) undec-9-enoic acid. We propose that CAV1-mediated endocytosing E. coli represents a versatile tool for the biotransformation of hydrophobic substrates.

Multi-level engineering of Baeyer-Villiger monooxygenase-based Escherichia coli biocatalysts for the production of C9 chemicals from oleic acid.

Whole-cell biotransformation is one of the promising alternative approaches to microbial fermentation for producing high-value chemicals. Baeyer-Villiger monooxygenase (BVMO)-based Escherichia coli biocatalysts have been engineered to produce industrially relevant C9 chemicals, such as n-nonanoic acid and 9-hydroxynonanoic acid, from a renewable long-chain fatty acid. The key enzyme in the biotransformation pathway (i.e., BVMO from Pseudomonans putida KT2440) was first engineered, using structure modeling-based design, to improve oxidative and thermal stabilities. Using a stable and tunable plasmid (STAPL) system, E. coli host cells were engineered to have increased plasmid stability and homogeneity of the recombinant E. coli population, as well as to optimize the level of BVMO expression. Multi-level engineering of the key enzyme in host cells, allowed recombinant E. coli expressing a fatty acid double-bond hydratase, a long-chain secondary alcohol dehydrogenase, and the engineered BVMO from P. putida KT2440 (i.e., E6BVMO_C302L/M340L), to ultimately produce C9 chemicals (i.e., n-nonanoic acid and 9-hydroxynonanoic acid) from oleic acid, with a yield of up to 6 mmoL/g dry cells. This yield was 2.4-fold greater than the yield in the control strain before engineering. Therefore, this study will contribute to the development of improved processes for the biosynthesis of industrially relevant medium chain fatty acids via whole-cell biocatalysis.

Structural basis for the selective addition of an oxygen atom to cyclic ketones by Baeyer-Villiger monooxygenase from Parvibaculum lavamentivorans.

Baeyer-Villiger monooxygenase (BVMO) catalyzes insertion of an oxygen atom into aliphatic or cyclic ketones with high regioselectivity. The BVMOs from Parvibaculum lavamentivorans (BVMOParvi) and Oceanicola batsensis (BVMOOcean) are interesting because of their homologies, with >40% sequence identity, and reaction with the same cyclic ketones with a methyl moiety to give different products. The revealed BVMOParvi structure shows that BVMOParvi forms a two-domain structure like other BVMOs. It has two inserted residues, compared with BVMOOcean, that form a bulge near the bound flavin adenine dinucleotide in the active site. Furthermore, this bulge is linked to a nearby α-helix via a disulfide bond, probably restricting access of the bulky methyl group of the substrate to this bulge. Another sequence motif at the entrance of the active site (Ala-Ser in BVMOParvi and Ser-Thr in BVMOOcean) allows a large volume in BVMOParvi. These minute differences may discriminate a substrate orientation in both BVMOs from the initial substrate binding pocket to the final oxygenation site, resulting in the inserted oxygen atom being in different positions of the same substrate.