Yarrowia lipolytica growth, lipids, and protease production in medium with higher alkanes and alkenes.

Two strains of Yarrowia lipolytica (CBS 2075 and DSM 8218) were first studied in bioreactor batch cultures, under different controlled dissolved oxygen concentrations (DOC), to assess their ability to assimilate aliphatic hydrocarbons (HC) as a carbon source in a mixture containing 2 g·L-1 of each alkane (dodecane and hexadecane), and 2 g·L-1 hexadecene. Both strains grew in the HC mixture without a lag phase, and for both strains, 30 % DOC was sufficient to reach the maximum values of biomass and lipids. To enhance lipid-rich biomass and enzyme production, a pulse fed-batch strategy was tested, for the first time, with the addition of one or three pulses of concentrated HC medium. The addition of three pulses of the HC mixture (total of 24 g·L-1 HC) did not hinder cell proliferation, and high protease (> 3000 U·L-1) and lipids concentrations of 3.4 g·L-1 and 4.3 g·L-1 were achieved in Y. lipolytica CBS 2075 and DSM 8218 cultures, respectively. Lipids from the CBS 2075 strain are rich in C16:0 and C18:1, resembling the composition of palm oil, considered suitable for the biodiesel industry. Lipids from the DSM 8218 strain were predominantly composed of C16:0 and C16:1, the latter being a valuable monounsaturated fatty acid used in the pharmaceutical industry. Y. lipolytica cells exhibited high intrinsic surface hydrophobicity (> 69 %), which increased in the presence of HC. A reduction in surface tension was observed in both Y. lipolytica cultures, suggesting the production of extracellular biosurfactants, even at low amounts. This study marks a significant advancement in the valorization of HC for producing high-value products by exploring the hydrophobic compounds metabolism of Y. lipolytica.

Metabolic engineering of Saccharomyces cerevisiae for enhanced taxadiene production.

BACKGROUND: Metabolic engineering enables the sustainable and cost-efficient production of complex chemicals. Efficient production of terpenes in Saccharomyces cerevisiae can be achieved by recruiting an intermediate of the mevalonate pathway. The present study aimed to evaluate the engineering strategies of S. cerevisiae for the production of taxadiene, a precursor of taxol, an antineoplastic drug. RESULT: SCIGS22a, a previously engineered strain with modifications in the mevalonate pathway (MVA), was used as a background strain. This strain was engineered to enable a high flux towards farnesyl diphosphate (FPP) and the availability of NADPH. The strain MVA was generated from SCIGS22a by overexpressing all mevalonate pathway genes. Combining the background strains with 16 different episomal plasmids, which included the combination of 4 genes: tHMGR (3-hydroxy-3-methylglutaryl-CoA reductase), ERG20 (farnesyl pyrophosphate synthase), GGPPS (geranyl diphosphate synthase) and TS (taxadiene synthase) resulted in the highest taxadiene production in S. cerevisiae of 528 mg/L. CONCLUSION: Our study highlights the critical role of pathway balance in metabolic engineering, mainly when dealing with toxic molecules like taxadiene. We achieved significant improvements in taxadiene production by employing a combinatorial approach and focusing on balancing the downstream and upstream pathways. These findings emphasize the importance of minor gene expression modification levels to achieve a well-balanced pathway, ultimately leading to enhanced taxadiene accumulation.

Systematic Engineering to Enhance ß-Myrcene Production in Yeast.

ß-Myrcene is an important monoterpene compound widely used in the fragrance, agricultural, and food industries. The microbial production of ß-myrcene conforms to the trend of green biological manufacturing, which has great potential for development. The poor catalytic activity of ß-myrcene synthase (MS) and the insufficient supply of precursors are considered to be the bottlenecks of ß-myrcene production. Here, source screening, subcellular localization, enzyme fusion, and precursor-enhancing strategies were integrated for ß-myrcene biosynthesis with Saccharomyces cerevisiae. The ß-myrcene titer gradually increased by 218-fold (up to 63.59 mg/L) compared to that of the initial titer of the shake flask. Moreover, the titer reached 66.82 mg/L after the addition of antioxidants (1 mM glutathione, GSH, and 1% butylated hydroxytoluene, BHT). Ultimately, 142.64 mg/L ß-myrcene in S. cerevisiae was achieved in 5.0 L of fed-batch fermentation under a carbon restriction strategy, which was the highest reported titer in yeast thus far. This study not only established a platform for ß-myrcene production but also provided a reference for the efficient biosynthesis of other monoterpene compounds.

Genome and metabolome analysis of Bacillus sp. Hex-HIT36: A newly screened functional microorganism for the degradation of 1-hexadecene in industrial wastewater.

1-Hexadecene has been detected at a level of mg/L in both influent and effluent of wastewater treatment plants situated in chemical/pharmaceutical industrial parks, which poses a potential threat to the environment. However, few reports are available on aerobic metabolic pathways and microorganisms involved in 1-Hexadecene degradation. In this study, a new strain of 1-Hexadecene-degrading bacteria, Bacillus sp. Hex-HIT36 (HIT36), was isolated from the activated sludge of a wastewater treatment plants located in an industrial park. The physicochemical properties and degradation efficacy of HIT36 were investigated. HIT36 was cultured on a medium containing 1-Hexadecene as a sole carbon source; it was found to remove ∼67% of total organic carbon as confirmed by mass spectrometric analysis of intermediate metabolites. Metabolomic and genomic analysis showed that HIT36 possesses various enzymes, namely, pyruvate dehydrogenase, dihydropolyhydroxyl dehydrogenase, and 2-oxoglutarate-2-oxoiron oxidoreductase (subunit alpha), which assist in the metabolization of readily available carbon source or long chain hydrocarbons present in the growth medium/vicinity. This suggests that HIT36 has efficient long-chain alkane degradation efficacy, and understanding the alkane degradation mechanism of this strain can help in developing technologies for the degradation of long-chain alkanes present in wastewater, thereby assisting in the bioremediation of environment.

Unlocking the function promiscuity of old yellow enzyme to catalyze asymmetric Morita-Baylis-Hillman reaction.

Exploring the promiscuity of native enzymes presents a promising strategy for expanding their synthetic applications, particularly for catalyzing challenging reactions in non-native contexts. In this study, we explore the promiscuous potential of old yellow enzymes (OYEs) to facilitate the Morita-Baylis-Hillman reaction (MBH reaction), leveraging substrate similarities between MBH reaction and reduction reaction. Using mass spectrometry and spectroscopic techniques, we confirm promiscuity of GkOYE in both MBH and reduction reactions. By blocking H- and H+ transfer pathways, we engineer GkOYE.8, which loses its reduction ability but enhances its MBH activity. The structural basis of MBH reaction catalyzed by GkOYE.8 is obtained through mutation studies and kinetic simulations. Furthermore, enantiocomplementary mutants GkOYE.11 and GkOYE.13 are obtained by directed evolution, exhibiting the ability to accept various aromatic aldehydes and alkenes as substrates. This study demonstrates the potential of leveraging substrate similarities to unlock enzyme functionalities, enabling the catalysis of new-to-nature reactions.

Improved taxadiene production by optimizing DXS expression and fusing short-chain prenyltransferases.

This study highlights the significance of overexpressing 1-deoxy-d-xylulose-5-phosphate synthase (DXS) from the MEP (methylerythritol 4-phosphate) pathway, in addition to short-chain prenyltransferase fusions for the improved production of the diterpene, taxa-4,11-diene, the first committed intermediate in the production of anti-cancer drug paclitaxel. The results showed that the strain which has (i) the taxadiene synthase (txs) gene integrated into the genome, (ii) the MEP pathway genes overexpressed, (iii) the fpps-crtE prenyltransferases fusion protein and (iv) additional expression of 1-deoxy-d-xylulose-5-phosphate synthase (DXS), yielded the highest production of taxa-4,11-diene at 390 mg/L (26 mg/L/OD600). This represents a thirteen-fold increase compared to the highest reported concentration in B. subtilis. The focus on additional overexpression of DXS and utilizing short-chain prenyltransferase fusions underscores their pivotal role in achieving significant titer improvements in terpene biosynthesis.

BioLindlar Catalyst: Ene-Reductase-Promoted Selective Bioreduction of Cyanoalkynes to Give (Z)-Cyanoalkenes.

The direct synthesis of alkenes from alkynes usually requires the use of transition-metal catalysts. Unfortunately, efficient biocatalytic alternatives for this transformation have yet to be discovered. Herein, the selective bioreduction of electron-deficient alkynes to alkenes catalysed by ene-reductases (EREDs) is described. Alkynes bearing ketone, aldehyde, ester, and nitrile moieties have been effectively reduced with excellent conversions and stereoselectivities, observing clear trends for the E/Z ratios depending on the nature of the electron-withdrawing group. In the case of cyanoalkynes, (Z)-alkenes were obtained as the major product, and the reaction scope was expanded to a wide variety of aromatic substrates (up to >99 % conversion, and Z/E stereoselectivities of up to >99/1). Other alkynes containing aldehyde, ketone, or ester functionalities also proved to be excellent substrates, and interestingly gave the corresponding (E)-alkenes. Preparative biotransformations were performed on a 0.4 mmol scale, producing the desired (Z)-cyanoalkenes with good to excellent isolated yields (63-97 %). This novel reactivity has been rationalised through molecular docking by predicting the binding poses of key molecules in the ERED-pu-0006 active site.

A chimeric membrane enzyme and an engineered whole-cell biocatalyst for efficient 1-alkene production.

Bioproduction of 1-alkenes from naturally abundant free fatty acids offers a promising avenue toward the next generation of hydrocarbon-based biofuels and green commodity chemicals. UndB is the only known membrane-bound 1-alkene-producing enzyme, with great potential for 1-alkene bioproduction, but the enzyme exhibits limited turnovers, thus restricting its widespread usage. Here, we explore the molecular basis of the limitation of UndB activity and substantially improve its catalytic power. We establish that the enzyme undergoes peroxide-mediated rapid inactivation during catalysis. To counteract this inactivation, we engineered a chimeric membrane enzyme by conjugating UndB with catalase that protected UndB against peroxide and enhanced its number of turnovers tremendously. Notably, our chimeric enzyme is the only example of a membrane enzyme successfully engineered with catalase. We subsequently constructed a whole-cell biocatalytic system and achieved remarkable efficiencies (up to 95%) in the biotransformation of a wide range of fatty acids (both aliphatic and aromatic) into corresponding 1-alkenes with numerous biotechnological applications.

Convergence and molecular evolution of floral fragrance after independent transitions to self-fertilization.

Studying the independent evolution of similar traits provides valuable insights into the ecological and genetic factors driving phenotypic evolution.1 The transition from outcrossing to self-fertilization is common in plant evolution2 and is often associated with a reduction in floral attractive features such as display size, chemical signals, and pollinator rewards.3 These changes are believed to result from the reallocation of the resources used for building attractive flowers, as the need to attract pollinators decreases.2,3 We investigated the similarities in the evolution of flower fragrance following independent transitions to self-fertilization in Capsella.4,5,6,7,8,9 We identified several compounds that exhibited similar changes in different selfer lineages, such that the flower scent composition reflects mating systems rather than evolutionary history within this genus. We further demonstrate that the repeated loss of ß-ocimene emission, one of the compounds most strongly affected by these transitions, was caused by mutations in different genes. In one of the Capsella selfing lineages, the loss of its emission was associated with a mutation altering subcellular localization of the ortholog of TERPENE SYNTHASE 2. This mutation appears to have been fixed early after the transition to selfing through the capture of variants segregating in the ancestral outcrossing population. The large extent of convergence in the independent evolution of flower scent, together with the evolutionary history and molecular consequences of a causal mutation, suggests that the emission of specific volatiles evolved as a response to changes in ecological pressures rather than resource limitation.

Applications of biocatalytic CC bond reductions in the synthesis of flavours and fragrances.

Industrial biotechnology and biocatalysis can provide very effective synthetic tools to increase the sustainability of the production of fine chemicals, especially flavour and fragrance (F&F) ingredients, the market demand of which has been constantly increasing in the last years. One of the most important transformations in F&F chemistry is the reduction of CC bonds, typically carried out with metal-catalysed hydrogenations or hydride-based reagents. Its biocatalytic counterpart is a competitive alternative, showcasing a range of advantages such as excellent chemo-, regio- and stereoselectivity, ease of implementation, mild reaction conditions and modest environmental impact. In the present review, the application of biocatalysed alkene reductions (from microbial fermentations with wild-type strains to engineered isolated ene-reductase enzymes) to synthetic processes useful for the F&F industry will be described, highlighting not only the exquisite stereoselectivity achieved, but also the overall improvement when chirality is not involved. Multi-enzymatic cascades involving CC bioreductions are also examined, which allow much greater chemical complexity to be built in one-pot biocatalytic systems.

Design and Evolution of an Enzyme for the Asymmetric Michael Addition of Cyclic Ketones to Nitroolefins by Enamine Catalysis.

Consistent introduction of novel enzymes is required for developing efficient biocatalysts for challenging biotransformations. Absorbing catalytic modes from organocatalysis may be fruitful for designing new-to-nature enzymes with novel functions. Herein we report a newly designed artificial enzyme harboring a catalytic pyrrolidine residue that catalyzes the asymmetric Michael addition of cyclic ketones to nitroolefins through enamine activation with high efficiency. Diverse chiral γ-nitro cyclic ketones with two stereocenters were efficiently prepared with excellent stereoselectivity (up to 97 % e.e., >20 : 1 d.r.) and good yield (up to 86 %). This work provides an efficient biocatalytic strategy for cyclic ketone functionalization, and highlights the usefulness of artificial enzymes for extending biocatalysis to further non-natural reactions.

Molecular Basis for Chemoselectivity Control in Oxidations of Internal Aryl-Alkenes Catalyzed by Laboratory Evolved P450s.

P450 enzymes naturally perform selective hydroxylations and epoxidations of unfunctionalized hydrocarbon substrates, among other reactions. The adaptation of P450 enzymes to a particular oxidative reaction involving alkenes is of great interest for the design of new synthetically useful biocatalysts. However, the mechanism that these enzymes utilize to precisely modulate the chemoselectivity and distinguishing between competing alkene double bond epoxidations and allylic C-H hydroxylations is sometimes not clear, which hampers the rational design of specific biocatalysts. In a previous work, a P450 from Labrenzia aggregata (P450LA1) was engineered in the laboratory using directed evolution to catalyze the direct oxidation of trans-ß-methylstyrene to phenylacetone. The final variant, KS, was able to overcome the intrinsic preference for alkene epoxidation to directly generate a ketone product via the formation of a highly reactive carbocation intermediate. Here, additional library screening along this evolutionary lineage permitted to serendipitously detect a mutation that overcomes epoxidation and carbonyl formation by exhibiting a large selectivity of 94 % towards allylic C-H hydroxylation. A multiscalar computational methodology was applied to reveal the molecular basis towards this hydroxylation preference. Enzyme modelling suggests that introduction of a bulky substitution dramatically changes the accessible conformations of the substrate in the active site, thus modifying the enzymatic selectivity towards terminal hydroxylation and avoiding the competing epoxidation pathway, which is sterically hindered.

The Chemical Reactivity of Membrane Lipids.

It is well-known that aqueous dispersions of phospholipids spontaneously assemble into bilayer structures. These structures have numerous applications across chemistry and materials science and form the fundamental structural unit of the biological membrane. The particular environment of the lipid bilayer, with a water-poor low dielectric core surrounded by a more polar and better hydrated interfacial region, gives the membrane particular biophysical and physicochemical properties and presents a unique environment for chemical reactions to occur. Many different types of molecule spanning a range of sizes, from dissolved gases through small organics to proteins, are able to interact with membranes and promote chemical changes to lipids that subsequently affect the physicochemical properties of the bilayer. This Review describes the chemical reactivity exhibited by lipids in their membrane form, with an emphasis on conditions where the lipids are well hydrated in the form of bilayers. Key topics include the following: lytic reactions of glyceryl esters, including hydrolysis, aminolysis, and transesterification; oxidation reactions of alkenes in unsaturated fatty acids and sterols, including autoxidation and oxidation by singlet oxygen; reactivity of headgroups, particularly with reactive carbonyl species; and E/Z isomerization of alkenes. The consequences of reactivity for biological activity and biophysical properties are also discussed.

A novel soluble di-iron monooxygenase from the soil bacterium Solimonas soli.

Soluble di-iron monooxygenase (SDIMO) enzymes enable insertion of oxygen into diverse substrates and play significant roles in biogeochemistry, bioremediation and biocatalysis. An unusual SDIMO was detected in an earlier study in the genome of the soil organism Solimonas soli, but was not characterized. Here, we show that the S. soli SDIMO is part of a new clade, which we define as 'Group 7'; these share a conserved gene organization with alkene monooxygenases but have only low amino acid identity. The S. soli genes (named zmoABCD) could be functionally expressed in Pseudomonas putida KT2440 but not in Escherichia coli TOP10. The recombinants made epoxides from C2 C8 alkenes, preferring small linear alkenes (e.g. propene), but also epoxidating branched, carboxylated and chlorinated substrates. Enzymatic epoxidation of acrylic acid was observed for the first time. ZmoABCD oxidised the organochlorine pollutants vinyl chloride (VC) and cis-1,2-dichloroethene (cDCE), with the release of inorganic chloride from VC but not cDCE. The original host bacterium S. soli could not grow on any alkenes tested but grew well on phenol and n-octane. Further work is needed to link ZmoABCD and the other Group 7 SDIMOs to specific physiological and ecological roles.

Functional production and biochemical investigation of an integral membrane enzyme for olefin biosynthesis.

Integral membrane enzymes play essential roles in a plethora of biochemical processes. The fatty acid desaturases (FADS)-like superfamily is an important group of integral membrane enzymes that catalyze a wide array of reactions, including hydroxylation, desaturation, and cyclization; however, due to the membrane-bound nature, the majority of these enzymes have remained poorly understood. UndB is a member of the FADS-like superfamily, which catalyzes fatty acid decarboxylation, a chemically challenging reaction at the membrane interface. UndB reaction produces terminal olefins that are prominent biofuel candidates and building blocks of polymers with widespread industrial applications. Despite the great importance of UndB for several biotechnological applications, the enzyme has eluded comprehensive investigation. Here, we report details of the expression, solubilization, and purification of several constructs of UndB to achieve the optimally functional enzyme. We gained important insights into the biochemical, biophysical, and catalytic properties of UndB, including the thermal stability and factors influencing the enzyme activity. Additionally, we established the ability and kinetics of UndB to produce dienes by performing di-decarboxylation of diacids. We found that the reaction proceeds by forming a mono-carboxylic acid intermediate. Our findings shed light on the unexplored biochemical properties of the UndB and extend opportunities for its rigorous mechanistic and structural characterization.

Engineering cytochrome P450s for selective alkene to carbonyl oxidation.

The Wacker-Tsuji oxidation is an important aerobic oxidation process to synthesize ethanal from ethene and methyl ketones from 1-alkenes. Current challenges in aerobic alkene oxidation include selective carbonyl product formation beyond methyl ketones. This includes the regioselective oxidation of the terminal carbon atom of 1-alkenes, the regioselective ketone formation with internal alkenes as well as the enantioselective alkene to carbonyl oxidation. Recently, the potential of high-valent metal-oxo species for direct alkene to carbonyl oxidation was explored as carbonyl product formation is frequently reported as a side reaction of alkene epoxidation by cytochrome P450s. It was shown that such promiscuous P450s can be engineered via directed evolution to perform alkene to carbonyl oxidation reactions with high activity and selectivity. Here, we report a protocol to convert promiscuous P450s into efficient and selective enzymes for Wacker-type alkene oxidation. One round of directed evolution is described in detail, which includes the generation and handling of site-saturation libraries, recombinant protein expression, library screening in a 96-well plate format and the rescreening of variants with beneficial mutations. These protocols might be useful to engineer various P450s for selective alkene to carbonyl oxidation, and to engineer enzymes in general.

P450 fatty acid decarboxylase.

P450 fatty acid decarboxylases are able to utilize hydrogen peroxide as the sole cofactor to decarboxylate free fatty acids to produce α-olefins with abundant applications as drop-in biofuels and important chemical precursors. In this chapter, we review diverse approaches for discovery, characterization, engineering, and applications of P450 fatty acid decarboxylases. Information gained from structural data has been advancing our understandings of the unique mechanisms underlying alkene production, and providing important insights for exploring new activities. To build an efficient olefin-producing system, various engineering strategies have been proposed and applied to this unusual P450 catalytic system. Furthermore, we highlight a select number of applied examples of P450 fatty acid decarboxylases in enzyme cascades and metabolic engineering.

The 2-methylpropene degradation pathway in Mycobacteriaceae family strains.

Mycolicibacterium gadium IBE100 and Mycobacterium paragordonae IBE200 are aerobic, chemoorganoheterotrophic bacteria isolated from activated sludge from a wastewater treatment plant. They use 2-methylpropene (isobutene, 2-MP) as the sole source of carbon and energy. Here, we postulate a degradation pathway of 2-methylpropene derived from whole genome sequencing, differential expression analysis and peptide-mass fingerprinting. Key genes identified are coding for a 4-component soluble diiron monooxygenase with epoxidase activity, an epoxide hydrolase, and a 2-hydroxyisobutyryl-CoA mutase. In both strains, involved genes are arranged in clusters of 61.0 and 58.5 kbp, respectively, which also contain the genes coding for parts of the aerobic pathway of adenosylcobalamin synthesis. This vitamin is essential for the carbon rearrangement reaction catalysed by the mutase. These findings provide data for the identification of potential 2-methylpropene degraders.

Functional Characterization of a (E)-ß-Ocimene Synthase Gene Contributing to the Defense against Spodoptera litura.

Soybean is a worldwide crop that offers valuable proteins, fatty acids, and phytonutrients to humans but is always damaged by insect pests or pathogens. Plants have captured sophisticated defense mechanisms in resisting the attack of insects and pathogens. How to protect soybean in an environment- or human-friendly way or how to develop plant-based pest control is a hotpot. Herbivore-induced plant volatiles that are released by multiple plant species have been assessed in multi-systems against various insects, of which (E)-ß-ocimene has been reported to show anti-insect function in a variety of plants, including soybean. However, the responsible gene in soybean is unknown, and its mechanism of synthesis and anti-insect properties lacks comprehensive assessment. In this study, (E)-ß-ocimene was confirmed to be induced by Spodoptera litura treatment. A plastidic localized monoterpene synthase gene, designated as GmOCS, was identified to be responsible for the biosynthesis of (E)-ß-ocimene through genome-wide gene family screening and in vitro and in vivo assays. Results from transgenic soybean and tobacco confirmed that (E)-ß-ocimene catalyzed by GmOCS had pivotal roles in repelling a S. litura attack. This study advances the understanding of (E)-ß-ocimene synthesis and its function in crops, as well as provides a good candidate for further anti-insect soybean improvement.

Systematic In Vitro Metabolic Profiling of the OXIZID Synthetic Cannabinoids BZO-4en-POXIZID, BZO-POXIZID, 5F-BZO-POXIZID, BZO-HEXOXIZID and BZO-CHMOXIZID.

A new class of synthetic cannabinoids termed OXIZIDs has recently emerged on the recreational drug market. In order to continue the detection of new drugs in biological specimens, the identification of metabolites is essential. The aim of this study was to elucidate the metabolites of BZO-4en-POXIZID produced in human liver microsomes (HLMs) and human hepatocyte incubations and to compare the results with closely related analogs using the same experimental setup. Each drug was incubated for 1 h in HLM and BZO-4en-POXIZID was also incubated in human hepatocytes for up to 3 h. Subsequently, the incubates were analyzed by liquid chromatography-high-resolution mass spectrometry. BZO-4en-POXIZID metabolites were obtained in the incubation with HLMs and human hepatocytes, via the metabolic pathways of dihydrodiol formation, hydroxylation, reduction of the alkene bond and glucuronidation. The major metabolic pathway was found to be dihydrodiol formation at the pentenyl tail moiety. BZO-POXIZID, 5 F-BZO-POXIZID, BZO-HEXOXIZID and BZO-CHMOXIZID underwent similar metabolism to those reported in the literature, via the metabolic pathways of N-dealkylation, hydroxylation, ketone formation and oxidative defluorination (to alcohol or carboxylic acid). The results suggest that OXIZIDs are mainly metabolized at the N-alkyl moiety and the major metabolic pathways are hydroxylation when the N-alkyl moiety is a simple hydrocarbon, whereas functional-group-specific pathways (dihydrodiol formation and oxidative defluorination) are preferred when the moiety contains specific functional groups (alkene or fluoro), as has been observed for other synthetic cannabinoids. The major metabolites generated via these major metabolic pathways should serve as useful analytical targets for urine analysis. Furthermore, the higher abundance of glucuronidated metabolite suggests that enzymatic hydrolysis of glucuronides may be necessary for urine analysis to increase phase I metabolite concentration and improve detection.