Extraction of the Anticancer and Antimicrobial Agent, Prodigiosin, from Vibrio gazogenes PB1 and Its Identification by 1D and 2D NMR.

Prodigiosin is a secondary metabolite produced in several species of bacteria. It exhibits antimicrobial and anticancer properties. Methods for the extraction and identification of prodigiosin and their related derivatives from bacterial cultures typically depend on solvent-based extractions followed by NMR spectroscopy. The estuarine bacterium, V. gazogenes PB1, was previously shown to produce prodigiosin. This conclusion, however, was based on analytical data obtained from ultraviolet-visible absorption spectrophotometry and infrared spectroscopy. Complete dependence on these techniques would be considered inadequate for the accurate identification of the various members of the prodiginine family of compounds, which possess very similar chemical structures and near-identical optical properties. In this study, we extracted prodigiosin from a culture of Vibrio gazogenes PB1 cultivated in minimal media, and for the first time, confirmed the synthesis of prodigiosin Vibrio gazogenes PB1 using NMR techniques. The chemical structure was validated by 1H and 13C NMR spectroscopy, and further corroborated by 2D NMR, which included 1H-1H-gDQFCOSY, 1H-13C-gHSQC, and 1H-13C-gHMBC, as well as 1H-1H-homonuclear decoupling experiments. Based on this data, previous NMR spectral assignments of prodigiosin are reaffirmed and in some cases, corrected. The findings will be particularly relevant for experimental work relating to the use of V. gazogenes PB1 as a host for the synthesis of prodigiosin.

Genetic and metabolic advances in the engineering of cyanobacteria.

Cyanobacteria are a group of photosynthetic microorganisms with high commercial potential. They can utilize sunlight directly to convert carbon dioxide or even nitrogen into a variety of industrially relevant chemicals. However, commercial platforms for the renewable and sustainable production of chemicals have yet to be demonstrated for cyanobacteria. Diverse strategies have therefore been employed in recent years to improve the production yields and efficiency of target chemicals. These include the use of CRISPR/Cas systems for mutant selection, synthetic RNA elements for controlling transcription, metabolic network modelling for understanding pathway fluxes, enzyme engineering, improving growth rates, alleviating product toxicity and microbial consortia. More elaborate strategies for engineering cyanobacteria, however, are still very much required if we are to meet the grand challenge of employing cyanobacteria as photosynthetic workhorses for large-scale industrial applications.

Hydrogen Peroxide-Based Fluorometric Assay for Real-Time Monitoring of SAM-Dependent Methyltransferases.

Methylated chemicals are widely used as key intermediates for the syntheses of pharmaceuticals, fragrances, flavors, biofuels and plastics. In nature, the process of methylation is commonly undertaken by a super-family of S-adenosyl methionine-dependent enzymes known as methyltransferases. Herein, we describe a novel high throughput enzyme-coupled assay for determining methyltransferase activites. Adenosylhomocysteine nucleosidase, xanthine oxidase, and horseradish peroxidase enzymes were shown to function in tandem to generate a fluorescence signal in the presence of S-adenosyl-L-homocysteine and Amplex Red (10-acetyl-3,7-dihydroxyphenoxazine). Since S-adenosyl-L-homocysteine is a key by-product of reactions catalyzed by S-adenosyl methionine-dependent methyltransferases, the coupling enzymes were used to assess the activities of EcoRI methyltransferase and a salicylic acid methyltransferase from Clarkia breweri in the presence of S-adenosyl methionine. For the EcoRI methyltransferase, the assay was sensitive enough to allow the monitoring of DNA methylation in the nanomolar range. In the case of the salicylic acid methyltransferase, detectable activity was observed for several substrates including salicylic acid, benzoic acid, 3-hydroxybenzoic acid, and vanillic acid. Additionally, the de novo synthesis of the relatively expensive and unstable cosubstrate, S-adenosyl methionine, catalyzed by methionine adenosyltransferase could be incorporated within the assay. Overall, the assay offers an excellent level of sensitivity that permits continuous and reliable monitoring of methyltransferase activities. We anticipate this assay will serve as a useful bioanalytical tool for the rapid screening of S-adenosyl methionine-dependent methyltransferase activities.

CRISPR-Based Technologies for Metabolic Engineering in Cyanobacteria.

In metabolic engineering, the production of industrially relevant chemicals, via rational engineering of microorganisms, is an intensive area of research. One particular group of microorganisms that is fast becoming recognized for their commercial potential is cyanobacteria. Through the process of photosynthesis, cyanobacteria can use CO2 as a building block to synthesize carbon-based chemicals. In recent years, clustered regularly interspaced short palindromic repeats (CRISPR)-dependent approaches have rapidly gained popularity for engineering cyanobacteria. Such approaches permit markerless genome editing, simultaneous manipulation of multiple genes, and transcriptional regulation of genes. The drastically shortened timescale for mutant selection and segregation is especially advantageous for cyanobacterial work. In this review, we highlight studies that have implemented CRISPR-based tools for the metabolic engineering of cyanobacteria.

Clinical diagnostic tools for vitamin D assessment.

Vitamin D deficiency has been implicated in a plethora of diseases including rheumatoid arthritis, Parkinson's disease, Alzheimer's disease, and osteoporosis. Deficiency of this vitamin is a global epidemic affecting both developing and developed nations. Within a clinical context, the qualitative and quantitative analysis of vitamin D is therefore vital. The main metabolic markers for assessing vitamin D status in humans are the hydroxylated forms of vitamin D, 25OHD3 and 25OHD2 on account of their long half-lives within the body and excellent stability. An adequate level for healthy individuals of these hydroxylated forms is estimated to be around 20-40ng/ml of blood. There are three main analytical techniques for determining the levels of 25OHD3 and 25OHD2. The first technique is immunoassay-based and can be performed in a rapid, high throughput, automated manner, allowing as many as 240 tests per hour with the duration of each assay as little as 18min. Furthermore, it offers excellent sensitivity with a detection range of 3.4-156ng/ml. A major downside of immunoassays is that they are unable to distinguish between the various forms of vitamin D. While HPLC is a highthroughput low cost instrument it is not a very sensitive technique and cannot quantify the down stream metabolites of vitamin D. The third technique, namely liquid chromatography-mass spectrometry (LC-MS/), provides excellent sensitivity with a wide dynamic range from 0.068pg/ml to 100ng/ml. Additionally, it offers a high level of separation and permits identification of vitamin D-related metabolites. However, a huge limitation with LC/MS/MS is their poor throughput for sample analyses. As yet, there is no analytical technique which combines the fine detection capabilities of LC/MS/MS and the rapid, automated format of immunoassay, for vitamin D analyses. Future attention therefore needs to be given to this area if the current clinical diagnostic tools for vitamin D analysis are to be further improved.

Modulating the import of medium-chain alkanes in E. coli through tuned expression of FadL.

BACKGROUND: In recent years, there have been intensive efforts to develop synthetic microbial platforms for the production, biosensing and bio-remediation of fossil fuel constituents such as alkanes. Building predictable engineered systems for these applications will require the ability to tightly control and modulate the rate of import of alkanes into the host cell. The native components responsible for the import of alkanes within these systems have yet to be elucidated. To shed further insights on this, we used the AlkBGT alkane monooxygenase complex from Pseudomonas putida GPo1 as a reporter system for assessing alkane import in Escherichia coli. Two native E. coli transporters, FadL and OmpW, were evaluated for octane import given their proven functionality in the uptake of fatty acids along with their structural similarity to the P. putida GPo1 alkane importer, AlkL. RESULTS: Octane import was removed with deletion of fadL, but was restored by complementation with a fadL-encoding plasmid. Furthermore, tuned overexpression of FadL increased the rate of alkane import by up to 4.5- fold. A FadL deletion strain displayed a small but significant degree of tolerance toward hexane and octane relative to the wild type, while the responsiveness of the well-known alkane biosensor, AlkS, toward octane and decane was strongly reduced by 2.7- and 2.9-fold, respectively. CONCLUSIONS: We unequivocally show for the first time that FadL serves as the major route for medium-chain alkane import in E. coli. The experimental approaches used within this study, which include an enzyme-based reporter system and a fluorescent alkane biosensor for quantification and real-time monitoring of alkane import, could be employed as part of an engineering toolkit for optimizing biological systems that depend on the uptake of alkanes. Thus, the findings will be particularly useful for biological applications such as bioremediation and biomanufacturing.

A Comparison of the Microbial Production and Combustion Characteristics of Three Alcohol Biofuels: Ethanol, 1-Butanol, and 1-Octanol.

Over the last decade, microbes have been engineered for the manufacture of a variety of biofuels. Saturated linear-chain alcohols have great potential as transport biofuels. Their hydrocarbon backbones, as well as oxygenated content, confer combustive properties that make it suitable for use in internal combustion engines. Herein, we compared the microbial production and combustion characteristics of ethanol, 1-butanol, and 1-octanol. In terms of productivity and efficiency, current microbial platforms favor the production of ethanol. From a combustion standpoint, the most suitable fuel for spark-ignition engines would be ethanol, while for compression-ignition engines it would be 1-octanol. However, any general conclusions drawn at this stage regarding the most superior biofuel would be premature, as there are still many areas that need to be addressed, such as large-scale purification and pipeline compatibility. So far, the difficulties in developing and optimizing microbial platforms for fuel production, particularly for newer fuel candidates, stem from our poor understanding of the myriad biological factors underpinning them. A great deal of attention therefore needs to be given to the fundamental mechanisms that govern biological processes. Additionally, research needs to be undertaken across a wide range of disciplines to overcome issues of sustainability and commercial viability.

A microbial platform for renewable propane synthesis based on a fermentative butanol pathway.

BACKGROUND: Propane (C3H8) is a volatile hydrocarbon with highly favourable physicochemical properties as a fuel, in addition to existing global markets and infrastructure for storage, distribution and utilization in a wide range of applications. Consequently, propane is an attractive target product in research aimed at developing new renewable alternatives to complement currently used petroleum-derived fuels. This study focuses on the construction and evaluation of alternative microbial biosynthetic pathways for the production of renewable propane. The new pathways utilize CoA intermediates that are derived from clostridial-like fermentative butanol pathways and are therefore distinct from the first microbial propane pathways recently engineered in Escherichia coli. RESULTS: We report the assembly and evaluation of four different synthetic pathways for the production of propane and butanol, designated a) atoB-adhE2 route, b) atoB-TPC7 route, c) nphT7-adhE2 route and d) nphT7-TPC7 route. The highest butanol titres were achieved with the atoB-adhE2 (473 ± 3 mg/L) and atoB-TPC7 (163 ± 2 mg/L) routes. When aldehyde deformylating oxygenase (ADO) was co-expressed with these pathways, the engineered hosts also produced propane. The atoB-TPC7-ADO pathway was the most effective in producing propane (220 ± 3 µg/L). By (i) deleting competing pathways, (ii) including a previously designed ADOA134F variant with an enhanced specificity towards short-chain substrates and (iii) including a ferredoxin-based electron supply system, the propane titre was increased (3.40 ± 0.19 mg/L). CONCLUSIONS: This study expands the metabolic toolbox for renewable propane production and provides new insight and understanding for the development of next-generation biofuel platforms. In developing an alternative CoA-dependent fermentative butanol pathway, which includes an engineered ADO variant (ADOA134F), the study addresses known limitations, including the low bio-availability of butyraldehyde precursors and poor activity of ADO with butyraldehyde. Graphical abstractPropane synthesis derived from a fermentative butanol pathway is enabled by metabolic engineering.

Microbial production of 1-octanol: A naturally excreted biofuel with diesel-like properties.

The development of sustainable, bio-based technologies to convert solar energy and carbon dioxide into fuels is a grand challenge. A core part of this challenge is to produce a fuel that is compatible with the existing transportation infrastructure. This task is further compounded by the commercial desire to separate the fuel from the biotechnological host. Based on its fuel characteristics, 1-octanol was identified as an attractive metabolic target with diesel-like properties. We therefore engineered a synthetic pathway specifically for the biosynthesis of 1-octanol in Escherichia coli BL21(DE3) by over-expression of three enzymes (thioesterase, carboxylic acid reductase and aldehyde reductase) and one maturation factor (phosphopantetheinyl transferase). Induction of this pathway in a shake flask resulted in 4.4 mg 1-octanol L-1 h-1 which exceeded the productivity of previously engineered strains. Furthermore, the majority (73%) of the fatty alcohol was localised within the media without the addition of detergent or solvent overlay. The deletion of acrA reduced the production and excretion of 1-octanol by 3-fold relative to the wild-type, suggesting that the AcrAB-TolC complex may be responsible for the majority of product efflux. This study presents 1-octanol as a potential fuel target that can be synthesised and naturally accumulated within the media using engineered microbes.

A synthetic O2 -tolerant butanol pathway exploiting native fatty acid biosynthesis in Escherichia coli.

Several synthetic metabolic pathways for butanol synthesis have been reported in Escherichia coli by modification of the native CoA-dependent pathway from selected Clostridium species. These pathways are all dependent on the O2 -sensitive AdhE2 enzyme from Clostridium acetobutylicum that catalyzes the sequential reduction of both butyryl-CoA and butyraldehyde. We constructed an O2 -tolerant butanol pathway based on the activities of an ACP-thioesterase, acting on butyryl-ACP in the native fatty acid biosynthesis pathway, and a promiscuous carboxylic acid reductase. The pathway was genetically optimized by screening a series of bacterial acyl-ACP thioesterases and also by modification of the physical growth parameters. In order to evaluate the potential of the pathway for butanol production, the ACP-dependent butanol pathway was compared with a previously established CoA-dependent pathway. The effect of (1) O2 -availability, (2) media, and (3) co-expression of aldehyde reductases was evaluated systematically demonstrating varying and contrasting functionality between the ACP- and CoA-dependent pathways. The yield of butanol from the ACP-dependent pathway was stimulated by enhanced O2 -availability, in contrast to the CoA-dependent pathway, which did not function well under aerobic conditions. Similarly, whilst the CoA-dependent pathway only performed well in complex media, the ACP-dependent pathway was not influenced by the choice of media except in the absence of O2 . A combination of a thioesterase from Bacteroides fragilis and the aldehyde reductase, ahr, from E. coli resulted in the greatest yield of butanol. A product titer of ~300 mg/L was obtained in 24 h under optimal batch growth conditions, in most cases exceeding the performance of the reference CoA-pathway when evaluated under equivalent conditions.

Cofactor engineering for enhancing the flux of metabolic pathways.

The manufacture of a diverse array of chemicals is now possible with biologically engineered strains, an approach that is greatly facilitated by the emergence of synthetic biology. This is principally achieved through pathway engineering in which enzyme activities are coordinated within a genetically amenable host to generate the product of interest. A great deal of attention is typically given to the quantitative levels of the enzymes with little regard to their overall qualitative states. This highly constrained approach fails to consider other factors that may be necessary for enzyme functionality. In particular, enzymes with physically bound cofactors, otherwise known as holoenzymes, require careful evaluation. Herein, we discuss the importance of cofactors for biocatalytic processes and show with empirical examples why the synthesis and integration of cofactors for the formation of holoenzymes warrant a great deal of attention within the context of pathway engineering.

An engineered pathway for the biosynthesis of renewable propane.

The deployment of next-generation renewable biofuels can be enhanced by improving their compatibility with the current infrastructure for transportation, storage and utilization. Propane, the bulk component of liquid petroleum gas, is an appealing target as it already has a global market. In addition, it is a gas under standard conditions, but can easily be liquefied. This allows the fuel to immediately separate from the biocatalytic process after synthesis, yet does not preclude energy-dense storage as a liquid. Here we report, for the first time, a synthetic metabolic pathway for producing renewable propane. The pathway is based on a thioesterase specific for butyryl-acyl carrier protein (ACP), which allows native fatty acid biosynthesis of the Escherichia coli host to be redirected towards a synthetic alkane pathway. Propane biosynthesis is markedly stimulated by the introduction of an electron-donating module, optimizing the balance of O2 supply and removal of native aldehyde reductases.

Renewable jet fuel.

Novel strategies for sustainable replacement of finite fossil fuels are intensely pursued in fundamental research, applied science and industry. In the case of jet fuels used in gas-turbine engine aircrafts, the production and use of synthetic bio-derived kerosenes are advancing rapidly. Microbial biotechnology could potentially also be used to complement the renewable production of jet fuel, as demonstrated by the production of bioethanol and biodiesel for piston engine vehicles. Engineered microbial biosynthesis of medium chain length alkanes, which constitute the major fraction of petroleum-based jet fuels, was recently demonstrated. Although efficiencies currently are far from that needed for commercial application, this discovery has spurred research towards future production platforms using both fermentative and direct photobiological routes.

Carboxylic acid reductase is a versatile enzyme for the conversion of fatty acids into fuels and chemical commodities.

Aliphatic hydrocarbons such as fatty alcohols and petroleum-derived alkanes have numerous applications in the chemical industry. In recent years, the renewable synthesis of aliphatic hydrocarbons has been made possible by engineering microbes to overaccumulate fatty acids. However, to generate end products with the desired physicochemical properties (e.g., fatty aldehydes, alkanes, and alcohols), further conversion of the fatty acid is necessary. A carboxylic acid reductase (CAR) from Mycobacterium marinum was found to convert a wide range of aliphatic fatty acids (C(6)-C(18)) into corresponding aldehydes. Together with the broad-substrate specificity of an aldehyde reductase or an aldehyde decarbonylase, the catalytic conversion of fatty acids to fatty alcohols (C(8)-C(16)) or fatty alkanes (C(7)-C(15)) was reconstituted in vitro. This concept was applied in vivo, in combination with a chain-length-specific thioesterase, to engineer Escherichia coli BL21(DE3) strains that were capable of synthesizing fatty alcohols and alkanes. A fatty alcohol titer exceeding 350 mg·L(-1) was obtained in minimal media supplemented with glucose. Moreover, by combining the CAR-dependent pathway with an exogenous fatty acid-generating lipase, natural oils (coconut oil, palm oil, and algal oil bodies) were enzymatically converted into fatty alcohols across a broad chain-length range (C(8)-C(18)). Together with complementing enzymes, the broad substrate specificity and kinetic characteristics of CAR opens the road for direct and tailored enzyme-catalyzed conversion of lipids into user-ready chemical commodities.

Physiological tolerance and stoichiometric potential of cyanobacteria for hydrocarbon fuel production.

Cyanobacteria are capable of directly converting sunlight, carbon dioxide and water into hydrocarbon fuel or precursors thereof. Many biological and non-biological factors will influence the ability of such a production system to become economically sustainable. We evaluated two factors in engineerable cyanobacteria which could potentially limit economic sustainability: (i) tolerance of the host to the intended end-product, and (ii) stoichiometric potential for production. Alcohols, when externally added, inhibited growth the most, followed by aldehydes and acids, whilst alkanes were the least inhibitory. The growth inhibition became progressively greater with increasing chain-length for alcohols, whilst the intermediate C6 alkane caused more inhibition than both C3 and C11 alkane. Synechocystis sp. PCC 6803 was more tolerant to some of the tested chemicals than Synechococcus elongatus PCC 7942, particularly ethanol and undecane. Stoichiometric evaluation of the potential yields suggested that there is no difference in the potential productivity of harvestable energy between any of the studied fuels, with the exception of ethylene, for which maximal stoichiometric yield is considerably lower. In summary, it was concluded that alkanes would constitute the best choice metabolic end-product for fuel production using cyanobacteria if high-yielding strains can be developed.

Construction of a synthetic YdbK-dependent pyruvate:H2 pathway in Escherichia coli BL21(DE3).

A synthetic pyruvate:H(2) pathway was constructed in Escherichia coli BL21(DE3) by co-expression of six proteins: E. coli YdbK, Clostridium pasteurianum [4Fe-4S]-ferredoxin, and Clostridium acetobutylicum HydF, HydE, HydG, and HydA. The effect of cofactor addition and host strain on H(2) yield and fermentation product accumulation was studied, together with in vitro reconstitution of the entire pathway. The deletion of iscR and/or the addition of thiamine pyrophosphate to the medium enhanced the total and specific activity of recombinant YdbK and increased the yield of H(2) per glucose. It was concluded that the introduced pathway outcompeted other pyruvate-consuming reactions, and that the ability to compete for pyruvate at least in part was determined by total YdbK activity. The results demonstrate the successful construction of a high-yielding H(2) pathway in a microorganism that effectively does not synthesize any H(2). The additional co-expression of Bacillus subtilis AmyE enabled starch-dependent H(2) synthesis in minimal media.

Deletion of iscR stimulates recombinant clostridial Fe-Fe hydrogenase activity and H2-accumulation in Escherichia coli BL21(DE3).

Proteins that catalyze H2-pathways often contain iron-sulfur (Fe-S) clusters and are sensitive to O2. We tested whether deletion of the gene encoding the transcriptional negative regulator, IscR, could enhance the ability of Escherichia coli BL21 to synthesize active recombinant H2-pathway components and stimulate ferredoxin-dependent H2-accumulation in the presence or absence of oxygen. Under anoxic conditions, deletion of iscR stimulated recombinant Fe-Fe hydrogenase activity threefold, whilst plasmid-based overexpression of the isc operon had no effect on hydrogenase activity. After cultivation with 21% (v/v) O2 in the headspace, no recombinant hydrogenase activity was observed in soluble extracts of wild-type BL21, although low levels of activity could be observed in the DeltaiscR strain (700-fold lower than anoxic conditions, 180-fold greater than the limit of detection). Under closed batch conditions starting with 5% (v/v) O2, DeltaiscR strains displayed fivefold greater levels of total hydrogenase activity and recombinant ferredoxin-dependent H2-accumulation relative to the control strain. In cultures starting with 10% (v/v) O2, H2-accumulation was stimulated 35-fold relative to the control. DeltaiscR strains displayed enhanced synthesis and activity of integral H2-pathway components under all tested conditions and enhanced H2-accumulation under partially oxic conditions. Deletion of iscR is, therefore, a useful strategy to stimulate H2-production, particularly if the hydrogenase catalyzes the rate-limiting reaction.

Constructing and testing the thermodynamic limits of synthetic NAD(P)H:H2 pathways.

NAD(P)H:H(2) pathways are theoretically predicted to reach equilibrium at very low partial headspace H(2) pressure. An evaluation of the directionality of such near-equilibrium pathways in vivo, using a defined experimental system, is therefore important in order to determine its potential for application. Many anaerobic microorganisms have evolved NAD(P)H:H(2) pathways; however, they are either not genetically tractable, and/or contain multiple H(2) synthesis/consumption pathways linked with other more thermodynamically favourable substrates, such as pyruvate. We therefore constructed a synthetic ferredoxin-dependent NAD(P)H:H(2) pathway model system in Escherichia coli BL21(DE3) and experimentally evaluated the thermodynamic limitations of nucleotide pyridine-dependent H(2) synthesis under closed batch conditions. NADPH-dependent H(2) accumulation was observed with a maximum partial H(2) pressure equivalent to a biochemically effective intracellular NADPH/NADP(+) ratio of 13:1. The molar yield of the NADPH:H(2) pathway was restricted by thermodynamic limitations as it was strongly dependent on the headspace:liquid ratio of the culture vessels. When the substrate specificity was extended to NADH, only the reverse pathway directionality, H(2) consumption, was observed above a partial H(2) pressure of 40 Pa. Substitution of NADH with NADPH or other intermediates, as the main electron acceptor/donor of glucose catabolism and precursor of H(2), is more likely to be applicable for H(2) production.

Export of a heterologous cytochrome P450 (CYP105D1) in Escherichia coli is associated with periplasmic accumulation of uroporphyrin.

This report suggests an important physiological role of a CYP in the accumulation of uroporphyrin I arising from catalytic oxidative conversion of uroporphyrinogen I to uroporphyrin I in the periplasm of Escherichia coli cultured in the presence of 5-aminolevulinic acid. A structurally competent Streptomyces griseus CYP105D1 was expressed as an engineered, exportable form in aerobically grown E. coli. Its progressive induction in the presence of 5-aminolevulinic acid-supplemented medium was accompanied by an accumulation of a greater than 100-fold higher amount of uroporphyrin I in the periplasm relative to cells lacking CYP105D1. Expression of a cytoplasm-resident engineered CYP105D1 at a comparative level to the secreted form was far less effective in promoting porphyrin accumulation in the periplasm. Expression at a 10-fold molar excess over the exported CYP105D1 of another periplasmically exported hemoprotein, the globular core of cytochrome b5, did not substitute the role of the periplasmically localized CYP105D1 in promoting porphyrin production. This, therefore, eliminated the possibility that uroporphyrin accumulation is merely a result of increased hemoprotein synthesis. Moreover, in the strain that secreted CYP105D1, uroporphyrin production was considerably reduced by azole-based P450 inhibitors. Production of both holo-CYP105D1 and uroporphyrin was dependent upon 5-aminolevulinic acid, except that at higher concentrations this resulted in a decrease in uroporphyrin. This study suggests that the exported CYP105D1 oxidatively catalyzes periplasmic conversion of uroporphyrinogen I to uroporphyrin I in E. coli. The findings have significant implications in the ontogenesis of human uroporphyria-related diseases.