Characterization of the off-flavor from Pichia pastoris GS115 during the overexpression of an α-l-rhamnosidase.

The off-flavor of Pichia pastoris strains is a negative characteristic of proteins overexpressed with this yeast. In the present study, P. pastoris GS115 overexpressing an α-l-rhamnosidase was taken as the example to characterize the off-flavor via sensory evaluation, gas chromatography-mass spectrometer, gas chromatography-olfaction, and omission test. The result showed that the off-flavor was due to the strong sweaty note, and moderate metallic and plastic notes. Four volatile compounds, that is, tetramethylpyrazine, 2,4-di-tert-butylphenol, isovaleric acid, and 2-methylbutyric acid, were identified to be major contributors to the sweaty note. Dodecanol and 2-acetylbutyrolactone were identified to be contributors to the metallic and plastic notes, respectively. It is the first study on the off-flavor of P. pastoris strains, helping understand metabolites with off-flavor of this yeast. Interestingly, it is the first study illustrating 2-acetylbutyrolactone and dodecanol with plastic and metallic notes, providing new information about the aromatic contributors of biological products. IMPORTANCE: The methylotrophic yeast Pichia pastoris is an important host for the industrial expression of functional proteins. In our previous studies, P. pastoris strains have been sniffed with a strong off-flavor during the overexpression of various functional proteins, limiting the application of these proteins. Although many yeast strains have been reported with off-flavor, no attention has been paid to characterize the off-flavor in P. pastoris so far. Considering that P. pastoris has advantages over other established expression systems of functional proteins, it is of interest to identify the compounds with off-flavor synthesized in the overexpression of functional proteins with P. pastoris strains. In this study, the off-flavor synthesized from P. pastoris GS115 was characterized during the overexpression of an α-l-rhamnosidase, which helps understand the aromatic metabolites with off-flavor of P. pastoris strains. In addition, 2-acetylbutyrolactone and dodecanol were newly revealed with plastic and metallic notes, enriching the aromatic contributors of biological products. Thus, this study is important for understanding the metabolites with off-flavor of P. pastoris strains and other organisms, providing important knowledge to improve the flavor of products yielding with P. pastoris strains and other organisms. ONE-SENTENCE SUMMARY: Characterize the sensory and chemical profile of the off-flavor produced by one strain of P. pastoris in vitro.

Delineation of the CYP505E subfamily of fungal self-sufficient in-chain hydroxylating cytochrome P450 monooxygenases.

Cytochrome P450 monooxygenases (CYP450s) are abundant in eukaryotes, specifically in plants and fungi where they play important roles in the synthesis and degradation of secondary metabolites. In eukaryotes, the best studied "self-sufficient" CYP450s, with a fused redox partner, belong to the CYP505 family. Members of the CYP505 family are generally considered sub-terminal fatty acid hydroxylases. CYP505E3 from Aspergillus terreus, however, gives remarkable in-chain hydroxylation at the ω-7 position of C10 to C16 alkanes and C12 and C14 fatty alcohols. Because CYP505E3 is a promising catalyst for the synthesis of δ-dodecalactone, we set out to delineate the unique ω-7 hydroxylase activity of CYP505E3. CYP505E3 and six additional CYP505Es as well as four closely related CYP505s from four different subfamilies were expressed in Pichia pastoris. Only the CYP505Es, sharing more than 70% amino acid identity, displayed significant ω-7 hydroxylase activity toward 1-dodecanol, dodecanoic acid, and tetradecanoic acid giving products that can readily be converted to δ-dodecalactone. Concentrations of δ-dodecalactone, directly extracted from dodecanoic acid biotransformations, were higher than previously obtained with E. coli. Searches of the UniProt and NCBI databases yielded a total of only 23 unique CYP505Es, all from the Aspergillaceae. Given that CYP505Es with this remarkable activity occur in only a few Aspergillus and Penicillium spp., we further explored the genetic environments in which they occur. These were found to be very distinct environments which include a specific ABC transporter but could not be linked to apparent secondary metabolite gene clusters. KEY POINTS: • Identified CYP505Es share > 70% amino acid identity. • CYP505Es hydroxylate 1-dodecanol, dodecanoic, and tetradecanoic acid at ω-7 position. • CYP505E genes occur in Aspergillus and Penicillium spp. near an ABC transporter.

Green Chemistry Production of Codlemone, the Sex Pheromone of the Codling Moth (Cydia pomonella), by Metabolic Engineering of the Oilseed Crop Camelina (Camelina sativa).

Synthetic pheromones have been used for pest control over several decades. The conventional synthesis of di-unsaturated pheromone compounds is usually complex and costly. Camelina (Camelina sativa) has emerged as an ideal, non-food biotech oilseed platform for production of oils with modified fatty acid compositions. We used Camelina as a plant factory to produce mono- and di-unsaturated C12 chain length moth sex pheromone precursors, (E)-9-dodecenoic acid and (E,E)-8,10-dodecadienoic acid, by introducing a fatty acyl-ACP thioesterase FatB gene UcTE from California bay laurel (Umbellularia californica) and a bifunctional ∆9 desaturase gene Cpo_CPRQ from the codling moth, Cydia pomonella. Different transgene combinations were investigated for increasing pheromone precursor yield. The most productive Camelina line was engineered with a vector that contained one copy of UcTE and the viral suppressor protein encoding P19 transgenes and three copies of Cpo_CPRQ transgene. The T2 generation of this line produced 9.4% of (E)-9-dodecenoic acid and 5.5% of (E,E)-8,10-dodecadienoic acid of the total fatty acids, and seeds were selected to advance top-performing lines to homozygosity. In the T4 generation, production levels of (E)-9-dodecenoic acid and (E,E)-8,10-dodecadienoic acid remained stable. The diene acid together with other seed fatty acids were converted into corresponding alcohols, and the bioactivity of the plant-derived codlemone was confirmed by GC-EAD and a flight tunnel assay. Trapping in orchards and home gardens confirmed significant and specific attraction of C. pomonella males to the plant-derived codlemone.

Dodecanol, metabolite of entomopathogenic fungus Conidiobolus coronatus, affects fatty acid composition and cellular immunity of Galleria mellonella and Calliphora vicina.

One group of promising pest control agents are the entomopathogenic fungi; one such example is Conidiobolus coronatus, which produces a range of metabolites. Our present findings reveal for the first time that C. coronatus also produces dodecanol, a compound widely used to make surfactants and pharmaceuticals, and enhance flavors in food. The main aim of the study was to determine the influence of dodecanol on insect defense systems, i.e. cuticular lipid composition and the condition of insect immunocompetent cells; hence, its effect was examined in detail on two species differing in susceptibility to fungal infection: Galleria mellonella and Calliphora vicina. Dodecanol treatment elicited significant quantitative and qualitative differences in cuticular free fatty acid (FFA) profiles between the species, based on gas chromatography analysis with mass spectrometry (GC/MS), and had a negative effect on G. mellonella and C. vicina hemocytes and a Sf9 cell line in vitro: after 48 h, almost all the cells were completely disintegrated. The metabolite had a negative effect on the insect defense system, suggesting that it could play an important role during C. coronatus infection. Its high insecticidal activity and lack of toxicity towards vertebrates suggest it could be an effective insecticide.

A machine learning Automated Recommendation Tool for synthetic biology.

Synthetic biology allows us to bioengineer cells to synthesize novel valuable molecules such as renewable biofuels or anticancer drugs. However, traditional synthetic biology approaches involve ad-hoc engineering practices, which lead to long development times. Here, we present the Automated Recommendation Tool (ART), a tool that leverages machine learning and probabilistic modeling techniques to guide synthetic biology in a systematic fashion, without the need for a full mechanistic understanding of the biological system. Using sampling-based optimization, ART provides a set of recommended strains to be built in the next engineering cycle, alongside probabilistic predictions of their production levels. We demonstrate the capabilities of ART on simulated data sets, as well as experimental data from real metabolic engineering projects producing renewable biofuels, hoppy flavored beer without hops, fatty acids, and tryptophan. Finally, we discuss the limitations of this approach, and the practical consequences of the underlying assumptions failing.

Lessons from Two Design-Build-Test-Learn Cycles of Dodecanol Production in Escherichia coli Aided by Machine Learning.

The Design-Build-Test-Learn (DBTL) cycle, facilitated by exponentially improving capabilities in synthetic biology, is an increasingly adopted metabolic engineering framework that represents a more systematic and efficient approach to strain development than historical efforts in biofuels and biobased products. Here, we report on implementation of two DBTL cycles to optimize 1-dodecanol production from glucose using 60 engineered Escherichia coli MG1655 strains. The first DBTL cycle employed a simple strategy to learn efficiently from a relatively small number of strains (36), wherein only the choice of ribosome-binding sites and an acyl-ACP/acyl-CoA reductase were modulated in a single pathway operon including genes encoding a thioesterase (UcFatB1), an acyl-ACP/acyl-CoA reductase (Maqu_2507, Maqu_2220, or Acr1), and an acyl-CoA synthetase (FadD). Measured variables included concentrations of dodecanol and all proteins in the engineered pathway. We used the data produced in the first DBTL cycle to train several machine-learning algorithms and to suggest protein profiles for the second DBTL cycle that would increase production. These strategies resulted in a 21% increase in dodecanol titer in Cycle 2 (up to 0.83 g/L, which is more than 6-fold greater than previously reported batch values for minimal medium). Beyond specific lessons learned about optimizing dodecanol titer in E. coli, this study had findings of broader relevance across synthetic biology applications, such as the importance of sequencing checks on plasmids in production strains as well as in cloning strains, and the critical need for more accurate protein expression predictive tools.

Molecular and Functional Characterization of pheromone binding protein 1 from the Oriental Fruit Moth, Grapholita molesta (Busck).

Pheromone binding protein (PBP) is thought primarily to bind and transport the sex pheromone in moths. The accumulated studies suggest that three PBPs were identified in moth species. In Grapholita molesta, the functions of GmolPBP2 and GmolPBP3 have been previously studied. However, the function of GmolPBP1 is still unclear. Furthermore, the Cydia pomonella sex pheromone Codlemone can act as a sex pheromone synergist of G. molesta. In C. pomonella, CpomPBP1 specifically bind the Codlemone. CpomPBP1 displays high identity with GmolPBP1 (70%), indicating that the two PBPs may share a similar 3D structure thus can bind the similar or same ligands. In this study, we explored the molecular and functional characterization of GmolPBP1. GmolPBP1, bearing the typical characteristics of Lepidopteran odorant binding proteins, was closest phylogenetically to CpomPBP1. Binding studies demonstrated that GmolPBP1 exhibited strong binding affinities with (Z)-8-dodecenyl alcohol, 1-dodecanol and Codlemone. Molecular docking showed that GmolPBP1 has different ligand recognition mechanism for the three ligands. Our results suggest that GmolPBP1 functions as recognizer of (Z)-8-dodecenyl alcohol and 1-dodecanol of the female sex pheromone blend, and may be the potential transporter of Codlemone, which contributes to the synergism of the pheromone response of G. molesta by Codlemone.

Production of 1-Dodecanol, 1-Tetradecanol, and 1,12-Dodecanediol through Whole-Cell Biotransformation in Escherichia coli.

Medium- and long-chain 1-alkanol and α,ω-alkanediols are used in personal care products, in industrial lubricants, and as precursors for polymers synthesized for medical applications. The industrial production of α,ω-alkanediols by alkane hydroxylation primarily occurs at high temperature and pressure using heavy metal catalysts. However, bioproduction has recently emerged as a more economical and environmentally friendly alternative. Among alkane monooxygenases, CYP153A from Marinobacter aquaeolei VT8 (CYP153A M.aq ; the strain is also known as Marinobacter hydrocarbonoclasticus VT8) possesses low overoxidation activity and high regioselectivity and thus has great potential for use in terminal hydroxylation. However, the application of CYP153A M.aq is limited because it is encoded by a dysfunctional operon. In this study, we demonstrated that the operon regulator AlkR M.aq is functional, can be induced by alkanes of various lengths, and does not suffer from product inhibition. Additionally, we identified a transposon insertion in the CYP153A M.aq operon. When the transposon was removed, the expression of the operon genes could be induced by alkanes, and the alkanes could then be oxyfunctionalized by the resulting proteins. To increase the accessibility of medium- and long-chain alkanes, we coexpressed a tunable alkane facilitator (AlkL) from Pseudomonas putida GPo1. Using a recombinant Escherichia coli strain, we produced 1.5 g/liter 1-dodecanol in 20 h and 2 g/liter 1-tetradecanol in 50 h by adding dodecane and tetradecane, respectively. Furthermore, in 68 h, we generated 3.76 g/liter of 1,12-dodecanediol by adding a dodecane-1-dodecanol substrate mixture. This study reports a very efficient method of producing C12/C14 alkanols and C12 1,12-alkanediol by whole-cell biotransformation.IMPORTANCE To produce terminally hydroxylated medium- to long-chain alkane compounds by whole-cell biotransformation, substrate permeability, enzymatic activity, and the control of overoxidability should be considered. Due to difficulties in production, small amounts of 1-dodecanol, 1-tetradecanol, and 1,12-dodecanediol are typically produced. In this study, we identified an alkane-inducible monooxygenase operon that can efficiently catalyze the conversion of alkane to 1-alkanol with no detection of the overoxidation product. By coexpressing an alkane membrane facilitator, high levels of 1-dodecanol, 1-tetradecanol, and 1,12-dodecanediol could be generated. This study is significant for the bioproduction of medium- and long-chain 1-alkanol and α,ω-alkanediols.

Customized microscale approach for optimizing two-phase bio-oxidations of alkanes with high reproducibility.

BACKGROUND: Numerous challenges remain to achieve industrially competitive space-time yields for bio-oxidations. The ability to rapidly screen bioconversion reactions for characterization and optimization is of major importance in bioprocess development and biocatalyst selection; studies at conventional lab scale are time consuming and labor intensive with low experimental throughput. The direct ω-oxyfunctionalization of aliphatic alkanes in a regio- and chemoselective manner is efficiently catalyzed by monooxygenases such as the AlkBGT enzyme complex from Pseudomonas putida under mild conditions. However, the adoption of microscale tools for these highly volatile substrates has been hindered by excessive evaporation and material incompatibility. RESULTS: This study developed and validated a robust high-throughput microwell platform for whole-cell two-liquid phase bio-oxidations of highly volatile n-alkanes. Using microwell plates machined from polytetrafluoroethylene and a sealing clamp, highly reproducible results were achieved with no significant variability such as edge effects determined. A design of experiment approach using a response surface methodology was adopted to systematically characterize the system and identify non-limiting conditions for a whole cell bioconversion of dodecane. Using resting E. coli cells to control cell concentration and reducing the fill volume it is possible to operate in non-limiting conditions with respect to oxygen and glucose whilst achieving relevant total product yields (combining 1-dodecanol, dodecanal and dodecanoic acid) of up to 1.5 mmol g DCW-1 . CONCLUSIONS: Overall, the developed microwell plate greatly improves experimental throughput, accelerating the screening procedures specifically for biocatalytic processes in non-conventional media. Its simplicity, robustness and standardization ensure high reliability of results.

Transcriptomic analysis of the highly efficient oil-degrading bacterium Acinetobacter venetianus RAG-1 reveals genes important in dodecane uptake and utilization.

The hydrocarbonoclastic bacterium Acinetobacter venetianus RAG-1 has attracted substantial attention due to its powerful oil-degrading capabilities and its potential to play an important ecological role in the cleanup of alkanes. In this study, we compare the transcriptome of the strain RAG-1 grown in dodecane, the corresponding alkanol (dodecanol), and sodium acetate for the characterization of genes involved in dodecane uptake and utilization. Comparison of the transcriptional responses of RAG-1 grown on dodecane led to the identification of 1074 genes that were differentially expressed relative to sodium acetate. Of these, 622 genes were upregulated when grown in dodecane. The highly upregulated genes were involved in alkane catabolism, along with stress response. Our data suggest AlkMb to be primarily involved in dodecane oxidation. Transcriptional response of RAG-1 grown on dodecane relative to dodecanol also led to the identification of permease, outer membrane protein and thin fimbriae coding genes potentially involved in dodecane uptake. This study provides the first model for key genes involved in alkane uptake and metabolism in A. venetianus RAG-1.

Pathway Compartmentalization in Peroxisome of Saccharomyces cerevisiae to Produce Versatile Medium Chain Fatty Alcohols.

Fatty alcohols are value-added chemicals and important components of a variety of industries, which have a >3 billion-dollar global market annually. Long chain fatty alcohols (>C12) are mainly used in surfactants, lubricants, detergents, pharmaceuticals and cosmetics while medium chain fatty alcohols (C6-C12) could be used as diesel-like biofuels. Microbial production of fatty alcohols from renewable feedstock stands as a promising strategy to enable sustainable supply of fatty alcohols. In this study, we report, for the first time, that medium chain fatty alcohols could be produced in yeast via targeted expression of a fatty acyl-CoA reductase (TaFAR) in the peroxisome of Saccharomyces cerevisiae. By tagging TaFAR enzyme with peroxisomal targeting signal peptides, the TaFAR could be compartmentalized into the matrix of the peroxisome to hijack the medium chain fatty acyl-CoA generated from the beta-oxidation pathway and convert them to versatile medium chain fatty alcohols (C10 &C12). The overexpression of genes encoding PEX7 and acetyl-CoA carboxylase further improved fatty alcohol production by 1.4-fold. After medium optimization in fed-batch fermentation using glucose as the sole carbon source, fatty alcohols were produced at 1.3 g/L, including 6.9% 1-decanol, 27.5% 1-dodecanol, 2.9% 1-tetradecanol and 62.7% 1-hexadecanol. This work revealed that peroxisome could be engineered as a compartmentalized organelle for producing fatty acid-derived chemicals in S. cerevisiae.

Parallel pathways of ethoxylated alcohol biodegradation under aerobic conditions.

Non-ionic surfactants (NS) are a major component of the surfactant flux discharged into surface water, and alcohol ethoxylates (AE) are the major component of this flux. Therefore, biodegradation pathways of AE deserve more thorough investigation. The aim of this work was to investigate the stages of biodegradation of homogeneous oxyethylated dodecanol C12E9 having 9 oxyethylene subunits, under aerobic conditions. Enterobacter strain Z3 bacteria were chosen as biodegrading organisms under conditions with C12E9 as the sole source of organic carbon. Bacterial consortia of river water were used in a parallel test as an inoculum for comparison. The LC-MS technique was used to identify the products of biodegradation. Liquid-liquid extraction with ethyl acetate was selected for the isolation of C12E9 and metabolites from the biodegradation broth. The LC-MS/MS technique operating in the multiple reaction monitoring (MRM) mode was used for quantitative determination of C12E9, C12E8, C12E7 and C12E6. Apart from the substrate, the homologues C12E8, C12E7 and C12E6, being metabolites of C12E9 biodegradation by shortening of the oxyethylene chain, as well as intermediate metabolites having a carboxyl end group in the oxyethylene chain (C12E8COOH, C12E7COOH, C12E6COOH and C12E5COOH), were identified. Poly(ethylene glycols) (E) having 9, 8 and 7 oxyethylene subunits were also identified, indicating parallel central fission of C12E9 and its metabolites. Similar results were obtained with river water as inoculum. It is concluded that AE, under aerobic conditions, are biodegraded via two parallel pathways: by central fission with the formation of PEG, and by Ω-oxidation of the oxyethylene chain with the formation of carboxylated AE and subsequent shortening of the oxyethylene chain by a single unit.

The heat sensitive factor (HSF) of Yersinia ruckeri is produced by an alkyl sulphatase involved in sodium dodecyl sulphate (SDS) degradation but not in virulence.

BACKGROUND: The heat sensitive factor (HSF) of the fish pathogen Yersinia ruckeri was previously identified as an unusual band on SDS-PAGE. According to this, Y. ruckeri strains were classified in HSF+ and HSF - in terms of the presence/absence of the factor. Experiments carried out by injection challenge with HSF + strains caused high mortalities in rainbow trout. In contrast, HSF - strains did not cause mortality. In conclusion, HSF appeared to be a relevant virulence factor in Y. ruckeri. RESULTS: We report here the identification and study of the gene coding for the enzyme involved in the production of HSF. Culture medium containing SDS and Coomassie brilliant blue dye was used to screen a mini-Tn5 Km2 mutant library of Y. ruckeri 150. Blue colonies lacking a surrounding creamy deposit, a phenotype described in former studies as HSF - , were identified. DNA sequence analysis of a selected mutant revealed that this had a transposon interruption in a chromosome-located gene which codes for a heat sensitive alkyl sulphatase of 78.7 kDa (YraS; Yersinia ruckeri alkyl sulphatase) which is able to degrade SDS to 1-dodecanol. As it was expected, the introduction of the yraS gene into an HSF - strain turned this into HSF + . Surprisingly, although the protein allows Y. ruckeri to degrade SDS, the bacterium could not use this compound as the sole carbon source. Moreover, the yraS mutant showed a similar level of SDS resistance to the parental strain. It was the interruption of the acrA gene which made Y. ruckeri sensitive to this compound. LD50 experiments showed a similar virulence of the yraS mutant and parental strain. CONCLUSIONS: The HSF of Y. ruckeri is the product of the alkyl sulphatase YraS, able to degrade SDS to 1-dodecanol. This degradation is not linked to the utilization of SDS as a carbon source and surprisingly, the enzyme is not involved in bacterial virulence or in the high SDS resistance displayed by the bacterium. This role is played by the AcrAB-TolC system.

Production of medium chain length fatty alcohols from glucose in Escherichia coli.

Metabolic engineering offers the opportunity to produce a wide range of commodity chemicals that are currently derived from petroleum or other non-renewable resources. Microbial synthesis of fatty alcohols is an attractive process because it can control the distribution of chain lengths and utilize low cost fermentation substrates. Specifically, primary alcohols with chain lengths of 12 to 14 carbons have many uses in the production of detergents, surfactants, and personal care products. The current challenge is to produce these compounds at titers and yields that would make them economically competitive. Here, we demonstrate a metabolic engineering strategy for producing fatty alcohols from glucose. To produce a high level of 1-dodecanol and 1-tetradecanol, an acyl-ACP thioesterase (BTE), an acyl-CoA ligase (FadD), and an acyl-CoA/aldehyde reductase (MAACR) were overexpressed in an engineered strain of Escherichia coli. Yields were improved by balancing expression levels of each gene, using a fed-batch cultivation strategy, and adding a solvent to the culture for extracting the product from cells. Using these strategies, a titer of over 1.6 g/L fatty alcohol with a yield of over 0.13 g fatty alcohol/g carbon source was achieved. These are the highest reported yield of fatty alcohols produced from glucose in E. coli.

The effect of lauryl capping group on protein release and degradation of poly(D,L-lactic-co-glycolic acid) particles.

The aim of this study was to investigate the effect of a specific and frequently used end group (lauryl alcohol) on the protein release and degradation kinetics of poly(DL-lactic-co-glycolic acid) particles of different sizes. Lauryl-capped PLGA and uncapped PLGA (referred to as PLGA-capped and PLGA-COOH, respectively) particles (0.3, 1 and 20 µm) were prepared by a double emulsion solvent evaporation technique. Bovine serum albumin (BSA) was used as a model protein for release studies. During degradation (PBS buffer, pH7.4 at 37°C), a slower dry mass loss was observed for 0.3 µm particles than for particles of 1 and 20 µm. It was further shown that PLGA-capped particles showed slower mass loss likely due to its more hydrophobic nature. It was found that the ester bond hydrolysis rate was substantially slower for PLGA-capped particles and that the rate increased with particle size. Particles showed enrichment in lactic acid content (and thus a decrease in glycolic acid content) in time, and interestingly PLGA-capped particles showed also an enrichment of the lauryl alcohol content. No difference was observed in degradation kinetics between BSA loaded and blank particles. Independent of size, PLGA-COOH based particles showed, after a small burst, a sustained and nearly complete release of BSA during 60-80 days. On the other hand, particles based on PLGA-capped showed a much slower release and exhibited incomplete release, accompanied by the presence of an insoluble residue remaining even after 180 days. FTIR analysis of this residue showed that it contained both polymer and protein. Considering the polymer enrichment in lauryl alcohol, the incomplete release observed for PLGA-capped is likely attributed to interactions between the protein and the lauryl end group. In conclusion, since PLGA-COOH, in contrast to the capped derivative, shows complete degradation as well as quantitative release of an entrapped protein, this polymer is preferred for the design of protein formulations.

Tools for characterizing the whole-cell bio-oxidation of alkanes at microscale.

This article describes the first reported microwell whole-cell bioconversion using a water immiscible substrate that matches the specific activity and yield achieved in a 1.2 L stirred tank bioreactor. Maximum yields of 0.6 g/L(total) 1-dodecanol achieved in 24 h compare favorably to 0.28 g/L(total) 1-dodecanol after 48 h obtained in a stirred tank reactor. Using the microwell platform we present a rapid and systematic approach to identify the key bottlenecks in the bio-oxidation of long-chain alkanes using Escherichia coli expressing the alkane hydroxylase (alkB) complex. The results indicate that mass transfer rates limit productivity in the n-dodecane bio-oxidation system, rather than inherent enzyme activity. Furthermore, substrate solubility, oxygen availability and glucose concentration act cooperatively to affect the amount of by-product, dodecanoic acid. Optimizing these factors using response surface methodology enabled specific yields of 1-dodecanol to increase eightfold and overoxidation to dodecanoic acid to be reduced from 95% to 55%. This resulted in specific activities of 10.4 µmol/min/g(dcw) on n-dodecane; approximately 50% of the 21 µmol/min/g(dcw) obtained with n-octane. For the first time, this in vivo rate difference is within the range reported for the purified enzyme. Finally, the results obtained also provide strong evidence that the mechanism of E. coli interaction with alkanes is mainly via uptake of alkanes dissolved in the aqueous phase rather than by direct cell-droplet contact.

Whole-cell bio-oxidation of n-dodecane using the alkane hydroxylase system of P. putida GPo1 expressed in E. coli.

The alkane-1-monoxygenase (alkB) complex of Pseudomonas putida GPo1 has been extensively studied in the past and shown to be capable of oxidising aliphatic C(5)-C(12) alkanes to primary alcohols both in the wild-type organism by growth on C(5)-C(12) alkanes as sole carbon source and in vitro. Despite this, successful n-dodecane oxidation for the production of 1-dodecanol or dodecanoic acid has proven elusive in the past when using alkB-expressing recombinants. This article demonstrates, for the first time in vivo, by using the Escherichia coli GEC137 pGEc47ΔJ strain, that n-dodecane oxidation using this enzyme for the production of primary alcohols and carboxylic acids is feasible and in fact potentially more promising than n-octane oxidation due to lower product and substrate toxicity. Yields are reported of 1-dodecanol of up to 2 g/L(organic) and dodecanoic acid up to 19.7 g/L(organic) in a 2 L stirred tank reactor with 1L aqueous phase and 200 mL of n-dodecane as a second phase. The maximum volumetric rate of combined alcohol and acid production achieved was 1.9 g/L(organic)/h (0.35 g/L(total)/h). The maximum specific activity of combined alcohol and acid production was 7-fold lower on n-dodecane (3.5 µmol/min/g(dcw)) than on n-octane (21 µmol/min/g(dcw)); similar to the 5-fold difference observed between wild-type growth rates using the two respective alkanes as sole carbon source. Despite this, both total volumetric rate and final yield exceeded n-octane oxidation by 3.5-fold under the same conditions, due to the lower toxicity of n-dodecane and its oxidation products to E. coli compared to the 8-carbon equivalents. Substrate access limitations and the overoxidation of 1-dodecanol to dodecanoic acid were identified as the most important limitations to be addressed.

Production of new-to-nature sophorolipids by cultivating the yeast Candida bombicola on unconventional hydrophobic substrates.

The naturally occurring sophorolipids synthesized by Candida bombicola possess--despite their overall heterogeneity--little variation in the length of the lipid tail. The range is limited to C16-C18 fatty acids and is governed by the specificity of a cytochrome P450 monooxygenase. However, incorporation of fatty acids differing from the conventional C16-C18 range could broaden up the application potential of sophorolipids. The incorporation of medium-chain fatty acids should render the molecules more hydrophilic and consequently improve their water solubility. Two strategies to circumvent this C16-C18 preference are described in this paper. The first one skips the controlling action of the cytochrome P450 enzyme by supplying the yeast with already hydroxylated substrates, while the other method is based on the deception of the enzyme by presenting it substrates structurally resembling stearic acid. This later strategy can be applied to create very specific tailor-made sophorolipids when combined with post-fermentive modification.

Microbial biodegradation of a novel fluorotelomer alcohol, 1H,1H,2H,2H,8H,8H-perfluorododecanol, yields short fluorinated acids.

The accumulation of perfluorooctanoic acid (PFOA) has been detected in wildlife, soil, and water. Further, 8:2 fluorotelomer alcohol (8:2 FTOH) is used for the industrial synthesis of other fluorotelomer compounds, surfactants, and polymeric materials; however, it was recently found to be a potential source of PFOA contamination in the environment. 1H,1H,2H,2H,8H,8H-perfluorododecanol (degradable telomer fluoroalcohol (DTFA)), which is a newly developed fluorotelomer, contains the -CH2- group in the fluorinated carbon backbone, making it potentially degradable through biological reactions. In this study, we investigated the biodegradation of DTFA in a mixed bacterial culture obtained from activated sludge. Optimized quantitative liquid chromatography-mass spectrometry analysis of the predicted metabolites generated in the culture revealed accumulations of the transformation products from DTFA to 2H,2H,8H,8H-PFDoA and 2H,8H,8H-2-PFUDoA via multiple processes. Furthermore, the production of short fluorinated compounds, perfluorobutanoic acid, perfluoropentanoic acid, and perfluoropentanedioic acid, which are believed to have lower accumulation potential and toxicity toward organisms than PFOA, was determined.

Knocking out the MFE-2 gene of Candida bombicola leads to improved medium-chain sophorolipid production.

The nonpathogenic yeast Candida bombicola synthesizes sophorolipids. These biosurfactants are composed of the disaccharide sophorose linked to a long-chain hydroxy fatty acid and have potential applications in the food, pharmaceutical, cosmetic and cleaning industries. In order to expand the range of application, a shift of the fatty acid moiety towards medium-chain lengths would be recommendable. However, the synthesis of medium-chain sophorolipids by C. bombicola is a challenging objective. First of all, these sophorolipids can only be obtained by fermentations on unconventional carbon sources, which often have a toxic effect on the cells. Furthermore, medium-chain substrates are partially metabolized in the beta-oxidation pathway. In order to redirect unconventional substrates towards sophorolipid synthesis, the beta-oxidation pathway was blocked on the genome level by knocking out the multifunctional enzyme type 2 (MFE-2) gene. The total gene sequence of the C. bombicola MFE-2 (6033 bp) was cloned (GenBank accession number EU371724), and the obtained nucleotide sequence was used to construct a knock-out cassette. Several knock-out mutants with the correct geno- and phenotype were evaluated in a fermentation on 1-dodecanol. All mutants showed a 1.7-2.9 times higher production of sophorolipids, indicating that in those strains the substrate is redirected towards the sophorolipid synthesis.