Ancient Clostridium DNA and variants of tetanus neurotoxins associated with human archaeological remains.

The analysis of microbial genomes from human archaeological samples offers a historic snapshot of ancient pathogens and provides insights into the origins of modern infectious diseases. Here, we analyze metagenomic datasets from 38 human archaeological samples and identify bacterial genomic sequences related to modern-day Clostridium tetani, which produces the tetanus neurotoxin (TeNT) and causes the disease tetanus. These genomic assemblies had varying levels of completeness, and a subset of them displayed hallmarks of ancient DNA damage. Phylogenetic analyses revealed known C. tetani clades as well as potentially new Clostridium lineages closely related to C. tetani. The genomic assemblies encode 13 TeNT variants with unique substitution profiles, including a subgroup of TeNT variants found exclusively in ancient samples from South America. We experimentally tested a TeNT variant selected from an ancient Chilean mummy sample and found that it induced tetanus muscle paralysis in mice, with potency comparable to modern TeNT. Thus, our ancient DNA analysis identifies DNA from neurotoxigenic C. tetani in archaeological human samples, and a novel variant of TeNT that can cause disease in mammals.

C. difficile intoxicates neurons and pericytes to drive neurogenic inflammation.

Clostridioides difficile infection (CDI) is a major cause of healthcare-associated gastrointestinal infections1,2. The exaggerated colonic inflammation caused by C. difficile toxins such as toxin B (TcdB) damages tissues and promotes C. difficile colonization3-6, but how TcdB causes inflammation is unclear. Here we report that TcdB induces neurogenic inflammation by targeting gut-innervating afferent neurons and pericytes through receptors, including the Frizzled receptors (FZD1, FZD2 and FZD7) in neurons and chondroitin sulfate proteoglycan 4 (CSPG4) in pericytes. TcdB stimulates the secretion of the neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) from neurons and pro-inflammatory cytokines from pericytes. Targeted delivery of the TcdB enzymatic domain, through fusion with a detoxified diphtheria toxin, into peptidergic sensory neurons that express exogeneous diphtheria toxin receptor (an approach we term toxogenetics) is sufficient to induce neurogenic inflammation and recapitulates major colonic histopathology associated with CDI. Conversely, mice lacking SP, CGRP or the SP receptor (neurokinin 1 receptor) show reduced pathology in both models of caecal TcdB injection and CDI. Blocking SP or CGRP signalling reduces tissue damage and C. difficile burden in mice infected with a standard C. difficile strain or with hypervirulent strains expressing the TcdB2 variant. Thus, targeting neurogenic inflammation provides a host-oriented therapeutic approach for treating CDI.

Structural basis for botulinum neurotoxin E recognition of synaptic vesicle protein 2.

Botulinum neurotoxin E (BoNT/E) is one of the major causes of human botulism and paradoxically also a promising therapeutic agent. Here we determined the co-crystal structures of the receptor-binding domain of BoNT/E (HCE) in complex with its neuronal receptor synaptic vesicle glycoprotein 2A (SV2A) and a nanobody that serves as a ganglioside surrogate. These structures reveal that the protein-protein interactions between HCE and SV2 provide the crucial location and specificity information for HCE to recognize SV2A and SV2B, but not the closely related SV2C. At the same time, HCE exploits a separated sialic acid-binding pocket to mediate recognition of an N-glycan of SV2. Structure-based mutagenesis and functional studies demonstrate that both the protein-protein and protein-glycan associations are essential for SV2A-mediated cell entry of BoNT/E and for its potent neurotoxicity. Our studies establish the structural basis to understand the receptor-specificity of BoNT/E and to engineer BoNT/E variants for new clinical applications.

Functional Deimmunization of Botulinum Neurotoxin Protease Domain via Computationally Driven Library Design and Ultrahigh-Throughput Screening.

Botulinum neurotoxin serotype A (BoNT/A) is a widely used cosmetic agent that also has diverse therapeutic applications; however, adverse antidrug immune responses and associated loss of efficacy have been reported in clinical uses. Here, we describe computational design and ultrahigh-throughput screening of a massive BoNT/A light-chain (BoNT/A-LC) library optimized for reduced T cell epitope content and thereby dampened immunogenicity. We developed a functional assay based on bacterial co-expression of BoNT/A-LC library members with a Förster resonance energy transfer (FRET) sensor for BoNT/A-LC enzymatic activity, and we employed high-speed fluorescence-activated cell sorting (FACS) to identify numerous computationally designed variants having wild-type-like enzyme kinetics. Many of these variants exhibited decreased immunogenicity in humanized HLA transgenic mice and manifested in vivo paralytic activity when incorporated into full-length toxin. One variant achieved near-wild-type paralytic potency and a 300% reduction in antidrug antibody response in vivo. Thus, we have achieved a striking level of BoNT/A-LC functional deimmunization by combining computational library design and ultrahigh-throughput screening. This strategy holds promise for deimmunizing other biologics with complex superstructures and mechanisms of action.

Structure and activity of botulinum neurotoxin X.

Botulinum neurotoxins (BoNTs) are the most potent toxins known and are used to treat an increasing number of medical disorders. All BoNTs are naturally co-expressed with a protective partner protein (NTNH) with which they form a 300 kDa complex, to resist acidic and proteolytic attack from the digestive tract. We have previously identified a new botulinum neurotoxin serotype, BoNT/X, that has unique and therapeutically attractive properties. We present the cryo-EM structure of the BoNT/X-NTNH/X complex at 3.1 Å resolution. Unexpectedly, the BoNT/X complex is stable and protease resistant at both neutral and acidic pH and disassembles only in alkaline conditions. Using the stabilizing effect of NTNH, we isolated BoNT/X and showed that it has very low potency both in vitro and in vivo . Given the high catalytic activity and translocation efficacy of BoNT/X, low activity of the full toxin is likely due to the receptor-binding domain, which presents weak ganglioside binding and exposed hydrophobic surfaces.

Regioselective One-Pot Synthesis of Hydroxy-(S)-Equols Using Isoflavonoid Reductases and Monooxygenases and Evaluation of the Hydroxyequol Derivatives as Selective Estrogen Receptor Modulators and Antioxidants.

Several regiospecific enantiomers of hydroxy-(S)-equol (HE) were enzymatically synthesized from daidzein and genistein using consecutive reduction (four daidzein-to-equol-converting reductases) and oxidation (4-hydroxyphenylacetate 3-monooxygenase, HpaBC). Despite the natural occurrence of several HEs, most of them had not been studied owing to the lack of their preparation methods. Herein, the one-pot synthesis pathway of 6-hydroxyequol (6HE) was developed using HpaBC (EcHpaB) from Escherichia coli and (S)-equol-producing E. coli, previously developed by our group. Based on docking analysis of the substrate or products, a potential active site and several key residues for substrate binding were predicted to interpret the (S)-equol hydroxylation regioselectivity of EcHpaB. Through investigating mutations on the key residues, the T292A variant was verified to display specific mono-ortho-hydroxylation activity at C6 without further 3'-hydroxylation. In the consecutive oxidoreductive bioconversion using T292A, 0.95 mM 6HE could be synthesized from 1 mM daidzein, while 5HE and 3'HE were also prepared from genistein and 3'-hydroxydaidzein (3'HD or 3'-ODI), respectively. In the following efficacy tests, 3'HE and 6HE showed about 30∼200-fold higher EC50 than (S)-equol in both ERα and ERß, and they did not have significant SERM efficacy except 6HE showing 10% lower ß/α ratio response than that of 17ß-estradiol. In DPPH radical scavenging assay, 3'HE showed the highest antioxidative activity among the examined isoflavone derivatives: more than 40% higher than the well-known 3'HD. In conclusion, we demonstrated that HEs could be produced efficiently and regioselectively through the one-pot bioconversion platform and evaluated estrogenic and antioxidative activities of each HE regio-isomer for the first time.

Light-Triggered In Situ Biosynthesis of Artificial Melanin for Skin Protection.

Tyrosinase-mediated melanin synthesis is an essential biological process that can protect skin from UV radiation and radical species. This work reports on in situ biosynthesis of artificial melanin in native skin using photoactivatable tyrosinase (PaTy). The I41Y mutant of Streptomyces avermitilis tyrosinase (SaTy) shows enzymatic activity comparable to that of wild-type SaTy. This Y41 is replaced with photocleavable o-nitrobenzyl tyrosine (ONBY) using the introduction of amber codon and ONBY-tRNA synthetase/tRNA pairs. The ONBY efficiently blocks the active site and tyrosinase activity is rapidly recovered by the photo-cleavage of ONBY. The activated PaTy successfully oxidizes L-tyrosine and tyramine-conjugated hyaluronic acid (HA_T) to synthesize melanin particles and hydrogel, respectively. To produce artificial melanin in living tissues, PaTy is encapsulated into lipid nanoparticles as an artificial melanosome. Using liposomes containing PaTy (PaTy_Lip), PaTy is transdermally delivered into ex vivo porcine skin and in vivo mouse skin tissues, thus achieving the in situ biosynthesis of artificial melanin for skin tissue protection under UV irradiation. The results of this study demonstrate that this biomimetic system can recapitulate the biosynthetic analogs of naturally occurring melanin. It should therefore be considered to be a promising strategy for producing protective biological molecules within living systems for tissue protection.

Polyphenol-Hydroxylating Tyrosinase Activity under Acidic pH Enables Efficient Synthesis of Plant Catechols and Gallols.

Tyrosinase is generally known as a melanin-forming enzyme, facilitating monooxygenation of phenols, oxidation of catechols into quinones, and finally generating biological melanin. As a homologous form of tyrosinase in plants, plant polyphenol oxidases perform the same oxidation reactions specifically toward plant polyphenols. Recent studies reported synthetic strategies for large scale preparation of hydroxylated plant polyphenols, using bacterial tyrosinases rather than plant polyphenol oxidase or other monooxygenases, by leveraging its robust monophenolase activity and broad substrate specificity. Herein, we report a novel synthesis of functional plant polyphenols, especially quercetin and myricetin from kaempferol, using screened bacterial tyrosinases. The critical bottleneck of the biocatalysis was identified as instability of the catechol and gallol under neutral and basic conditions. To overcome such instability of the products, the tyrosinase reaction proceeded under acidic conditions. Under mild acidic conditions supplemented with reducing agents, a bacterial tyrosinase from Bacillus megaterium (BmTy) displayed efficient consecutive two-step monophenolase activities producing quercetin and myricetin from kaempferol. Furthermore, the broad substrate specificity of BmTy toward diverse polyphenols enabled us to achieve the first biosynthesis of tricetin and 3'-hydroxyeriodictyol from apigenin and naringenin, respectively. These results suggest that microbial tyrosinase is a useful biocatalyst to prepare plant polyphenolic catechols and gallols with high productivity, which were hardly achieved by using other monooxygenases such as cytochrome P450s.

Phage-assisted evolution of botulinum neurotoxin proteases with reprogrammed specificity.

Although bespoke, sequence-specific proteases have the potential to advance biotechnology and medicine, generation of proteases with tailor-made cleavage specificities remains a major challenge. We developed a phage-assisted protease evolution system with simultaneous positive and negative selection and applied it to three botulinum neurotoxin (BoNT) light-chain proteases. We evolved BoNT/X protease into separate variants that preferentially cleave vesicle-associated membrane protein 4 (VAMP4) and Ykt6, evolved BoNT/F protease to selectively cleave the non-native substrate VAMP7, and evolved BoNT/E protease to cleave phosphatase and tensin homolog (PTEN) but not any natural BoNT protease substrate in neurons. The evolved proteases display large changes in specificity (218- to >11,000,000-fold) and can retain their ability to form holotoxins that self-deliver into primary neurons. These findings establish a versatile platform for reprogramming proteases to selectively cleave new targets of therapeutic interest.

Circular permutation of a bacterial tyrosinase enables efficient polyphenol-specific oxidation and quantitative preparation of orobol.

Tyrosinase is a type 3 copper oxygenase that catalyzes a phenol moiety into ortho-diphenol, and subsequently to ortho-quinone. Diverse tyrosinases have been observed across the kingdom including Animalia, Bacteria, Plantae, and Fungi. Among the tyrosinases, bacterial, and mushroom tyrosinases have been extensively exploited to prepare melanin, ortho-hydroxy-polyphenols, or novel plant secondary metabolites during the past decade. And their use as a biocatalyst to prepare various functional biocompounds have drawn great attention worldwide. Herein, we tailored a bacterial tyrosinase from Bacillus megaterium (BmTy) using circular permutation (CP) engineering technique which is a novel enzyme engineering technique to covalently link original N and C termini and create new termini on the middle of its polypeptide. To construct a smart rationally-designed CP library, we introduced 18 new termini at the edge of each nine loops that link α-helical secondary structure in BmTy. Among the small library, seven functional CP variants were successfully identified and they represented dramatic change in their enzyme characteristics including kinetic properties and substrate specificity. Especially, cp48, 102, and 245 showed dramatically decreased tyrosine hydroxylase activity, behaving like a catechol oxidase. Exploiting the dramatic increased polyphenol oxidation activity of cp48, orobol (3'-hydroxy-genistein) was quantitatively synthesized with 1.48 g/L, which was a 6-fold higher yield of truncated wild-type. We examined their kinetic characters through structural speculation, and suggest a strategy to solubilize the insoluble artificial variants effectively.

Recent advances in the microbial hydroxylation and reduction of soy isoflavones.

Soy isoflavones are naturally occurring phytochemicals, which are biotransformed into functional derivatives through oxidative and reductive metabolic pathways of diverse microorganisms. Such representative derivatives, ortho-dihydroxyisoflavones (ODIs) and equols, have attracted great attention for their versatile health benefits since they were found from soybean fermented foods and human intestinal fluids. Recently, scientists in food technology, nutrition and microbiology began to understand their correct biosynthetic pathways and nutraceutical values, and have attempted to produce the valuable bioactive compounds using microbial fermentation and whole-cell/enzyme-based biotransformation. Furthermore, artificial design of microbial catalysts and/or protein engineering of oxidoreductases were also conducted to enhance production efficiency and regioselectivity of products. This minireview summarizes and introduces the past year's studies and recent advances in notable production of ODIs and equols, and provides information on available microbial species and their catalytic performance with perspectives on industrial application.

Polymeric solvent engineering for gram/liter scale production of a water-insoluble isoflavone derivative, (S)-equol.

A potent phytoestrogen, (S)-equol, is a promising isoflavone derivative drawing our great attention owing to its various biological and clinical benefits. Through selective activation of the estrogen receptor ERß or androgen receptor, (S)-equol reduces menopausal symptoms, osteoporosis, skin aging, hair loss, and incidence of prostate or ovarian cancers without adverse effects. Traditional biosynthesis of (S)-equol exploited non-productive natural equol-producing anaerobic bacteria that mainly belong to Coriobacteriaceae isolated from human intestine. Recently, we developed a recombinant Escherichia coli strain which could convert daidzein into (S)-equol effectively under an aerobic condition. However, the yield was limited up to about the 200 mg/L level due to unknown reasons. In this study, we identified that the bottleneck of the limited production was the low solubility of isoflavone (i.e., 2.4 mg/L) in the reaction medium. In order to solve the solubility problem without harmful effect to the whole-cell catalyst, we applied commercial hydrophilic polymers (HPs) and a polar aprotic co-solvent in the reaction medium. Among the examined water-soluble polymers, polyvinylpyrrolidone (PVP)-40k was verified as the most promising supplement which increased daidzein solubility by 40 times and (S)-equol yield up to 1.22 g/L, the highest ever reported and the first g/L level biotransformation. Furthermore, PVP-40k was verified to significantly increase the solubilities of other water-insoluble natural polyphenols in aqueous solution. We suggest that addition of both HP and polar aprotic solvent in the reaction mixture is a powerful alternative to enhance production of polyphenolic chemicals rather than screening appropriate organic solvents for whole-cell catalysis of polyphenols.

Rewiring FadR regulon for the selective production of ω-hydroxy palmitic acid from glucose in Escherichia coli.

ω-Hydroxy palmitic acid (ω-HPA) is a valuable compound for an ingredient of artificially synthesized ceramides and an additive for lubricants and adhesives. Production of such a fatty acid derivative is limited by chemical catalysis, but plausible by biocatalysis. However, its low productivity issue, including formations of unsaturated fatty acid (UFA) byproducts in host cells, remains as a hurdle toward industrial biological processes. In this study, to achieve selective and high-level production of ω-HPA from glucose in Escherichia coli, FadR, a native transcriptional regulator of fatty acid metabolism, and its regulon were engineered. First, FadR was co-expressed with a thioesterase with a specificity toward palmitic acid production to enhance palmitic acid production yield, but a considerable quantity of UFAs was also produced. In order to avoid the UFA production caused by fadR overexpression, FadR regulon was rewired by i) mutating FadR consensus binding sites of fabA or fabB, ii) integrating fabZ into fabI operon, and iii) enhancing the strength of fabI promoter. This approach led to dramatic increases in both proportion (48.3-83.0%) and titer (377.8 mg/L to 675.8 mg/L) of palmitic acid, mainly due to the decrease in UFA synthesis. Introducing a fatty acid ω-hydroxylase, CYP153A35, into the engineered strain resulted in a highly selective production of ω-HPA (83.5 mg/L) accounting for 87.5% of total ω-hydroxy fatty acids. Furthermore, strategies, such as i) enhancement in CYP153A35 activity, ii) expression of a fatty acid transporter, iii) supplementation of triton X-100, and iv) separation of the ω-HPA synthetic pathway into two strains for a co-culture system, were applied and resulted in 401.0 mg/L of ω-HPA production. For such selective productions of palmitic acid and ω-HPA, the rewiring of FadR regulation in E. coli is a promising strategy to develop an industrial process with economical downstream processing.

In vitro characterization of CYP102G4 from Streptomyces cattleya: A self-sufficient P450 naturally producing indigo.

Self-sufficient CYP102As possess outstanding hydroxylating activity to fatty acids such as myristic acid. Other CYP102 subfamily members share substrate specificity of CYP102As, but, occasionally, unusual characteristics of its own subfamily have been found. In this study, only one self-sufficient cytochrome P450 from Streptomyces cattleya was renamed from CYP102A_scat to CYP102G4, purified and characterized. UV-Vis spectrometry pattern, FAD/FMN analysis, and protein sequence comparison among CYP102s have shown that CYP102 from Streptomyces cattleya belongs to CYP102G subfamily. It showed hydroxylation activity toward fatty acids generating ω-1, ω-2, and ω-3-hydroxyfatty acids, which is similar to the general substrate specificity of CYP102 family. Unexpectedly, however, expression of CYP102G4 showed indigo production in LB medium batch flask culture, and high catalytic activity (kcat/Km) for indole was measured as 6.14±0.10min-1mM-1. Besides indole, CYP102G4 was able to hydroxylate aromatic compounds such as flavone, benzophenone, and chloroindoles. Homology model has shown such ability to accept aromatic compounds is due to its bigger active site cavity. Unlike other CYP102s, CYP102G4 did not have biased cofactor dependency, which was possibly determined by difference in NAD(P)H binding residues (Ala984, Val990, and Tyr1064) compared to CYP102A1 (Arg966, Lys972 and Trp1046). Overall, a self-sufficient CYP within CYP102G subfamily was characterized using purified enzymes, which appears to possess unique properties such as an only prokaryotic CYP naturally producing indigo.

Biosynthesis of (-)-5-Hydroxy-equol and 5-Hydroxy-dehydroequol from Soy Isoflavone, Genistein Using Microbial Whole Cell Bioconversion.

Equols are isoflavandiols formed by reduction of soy isoflavones such as daidzein and genistein by gut microorganisms. These phytoestrogens are of interest for their various biological effects. We report biosynthesis from genistein to (-)-5-hydroxy-equol in recombinant E. coli expressing three reductases (daidzein reductase DZNR, dihidrodaidzein reductase DHDR, tetrahydrodaidzein reductase THDR) and a racemase (dihydrodaidzein racemase, DDRC) originating from the gut bacterium, Slackia isoflavoniconvertens. The biosynthesized 5-hydroxy-equol proved as an optically negative enantiomer, nonetheless it displayed an inverse circular dichroism spectrum to (S)-equol. Compartmentalized expression of DZNR and DDRC in one E. coli strain and DHDR and THDR in another increased the yield to 230 mg/L and the productivity to 38 mg/L/h. If the last reductase was missing, the intermediate spontaneously dehydrated to 5-hydroxy-dehydroequol in up to 99 mg/L yield. This novel isoflavene, previously not known to be synthesized in nature, was also detected in this biotransformation system. Although (S)-equol favors binding to human estrogen receptor (hER) ß over hERα, (-)-5-hydroxy-equol showed the opposite preference. This study provides elucidation of the biosynthetic route of (-)-5-hydroxy-equol and measurement of its potent antagonistic character as a phytoestrogen for the first time.

P212A Mutant of Dihydrodaidzein Reductase Enhances (S)-Equol Production and Enantioselectivity in a Recombinant Escherichia coli Whole-Cell Reaction System.

(S)-Equol, a gut bacterial isoflavone derivative, has drawn great attention because of its potent use for relieving female postmenopausal symptoms and preventing prostate cancer. Previous studies have reported on the dietary isoflavone metabolism of several human gut bacteria and the involved enzymes for conversion of daidzein to (S)-equol. However, the anaerobic growth conditions required by the gut bacteria and the low productivity and yield of (S)-equol limit its efficient production using only natural gut bacteria. In this study, the low (S)-equol biosynthesis of gut microorganisms was overcome by cloning the four enzymes involved in the biosynthesis from Slackia isoflavoniconvertens into Escherichia coli BL21(DE3). The reaction conditions were optimized for (S)-equol production from the recombinant strain, and this recombinant system enabled the efficient conversion of 200 µM and 1 mM daidzein to (S)-equol under aerobic conditions, achieving yields of 95% and 85%, respectively. Since the biosynthesis of trans-tetrahydrodaidzein was found to be a rate-determining step for (S)-equol production, dihydrodaidzein reductase (DHDR) was subjected to rational site-directed mutagenesis. The introduction of the DHDR P212A mutation increased the (S)-equol productivity from 59.0 mg/liter/h to 69.8 mg/liter/h in the whole-cell reaction. The P212A mutation caused an increase in the (S)-dihydrodaidzein enantioselectivity by decreasing the overall activity of DHDR, resulting in undetectable activity for (R)-dihydrodaidzein, such that a combination of the DHDR P212A mutant with dihydrodaidzein racemase enabled the production of (3S,4R)-tetrahydrodaidzein with an enantioselectivity of >99%.

Fungal cytochrome P450 monooxygenases of Fusarium oxysporum for the synthesis of ω-hydroxy fatty acids in engineered Saccharomyces cerevisiae.

BACKGROUND: Omega hydroxy fatty acids (ω-OHFAs) are multifunctional compounds that act as the basis for the production of various industrial products with broad commercial and pharmaceutical implications. However, the terminal oxygenation of saturated or unsaturated fatty acids for the synthesis of ω-OHFAs is intricate to accomplish through chemocatalysis, due to the selectivity and controlled reactivity in C-H oxygenation reactions. Cytochrome P450, the ubiquitous enzyme is capable of catalyzing the selective terminal omega hydroxylation naturally in biological kingdom. RESULTS: To gain a deep insight on the biochemical role of fungal P450s towards the production of omega hydroxy fatty acids, two cytochrome P450 monooxygenases from Fusarium oxysporum (FoCYP), FoCYP539A7 and FoCYP655C2; were identified, cloned, and heterologously expressed in Saccharomyces cerevisiae. For the efficient production of ω-OHFAs, the S. cerevisiae was engineered to disrupt the acyl-CoA oxidase enzyme and the ß-oxidation pathway inactivated (ΔPox1) S. cerevisiae mutant was generated. To elucidate the significance of the interaction of redox mechanism, FoCYPs were reconstituted with the heterologous and homologous reductase systems--S. cerevisiae CPR (ScCPR) and F. oxysporum CPR (FoCPR). To further improve the yield, the effect of pH was analyzed and the homologous FoCYP-FoCPR system efficiently hydroxylated caprylic acid, capric acid and lauric acid into their respective ω-hydroxy fatty acids with 56%, 79% and 67% conversion. Furthermore, based on computational simulations, we identified the key residues (Asn106 of FoCYP539A7 and Arg235 of FoCYP655C2) responsible for the recognition of fatty acids and demonstrated the structural insights of the active site of FoCYPs. CONCLUSION: Fungal CYP monooxygenases, FoCYP539A7 and FoCYP655C2 with its homologous redox partner, FoCPR constitutes a promising catalyst due to its high regio- and stereo-selectivity in the hydroxylation of fatty acids and in the substantial production of industrially valuable ω-hydroxy fatty acids.

fadD deletion and fadL overexpression in Escherichia coli increase hydroxy long-chain fatty acid productivity.

A major problem of long-chain fatty acid (LCFA) hydroxylation using Escherichia coli is that FadD (long-chain fatty acyl-CoA synthetase), which is necessary for exogenous LCFA transport, also initiates cellular consumption of LCFA. In this study, an effective method to prevent the cellular consumption of LCFA without impairing its transport is proposed. The main idea is that a heterologous enzyme which consumes LCFA can replace FadD in LCFA transport. For the model heterologous enzyme, CYP153A from Marinobacter aquaeolei, which converts palmitic acid into ω-hydroxy palmitic acid, was expressed in E. coli. When fadD was deleted from an E. coli strain, CYP153A indeed maintained the ability to transport LCFA. A disadvantage of fadD deletion mutant is the fact that FadD deficiency downregulates the transcription of fadL (outer membrane LCFA transporter) via FadR (fatty acid metabolism regulator protein), was solved by fadL overexpression from a plasmid. In addition, the overexpression of fadL was able to offset catabolite repression on fadL, allowing glucose to be used as the primary carbon source. In conclusion, the strain with fadD deletion and fadL overexpression showed 5.5-fold increase in productivity compared to the wild-type strain, converting 2.6 g/L (10.0 mM) of palmitic acid into 2.4 g/L (8.8 mM) of ω-hydroxy palmitic acid in a shake flask. This simple genetic manipulation can be applied to any LCFA hydroxylation using E. coli.