Novel mutations in malonyl-CoA-acyl carrier protein transacylase provoke autosomal recessive optic neuropathy.

Inherited optic neuropathies are rare eye diseases of optic nerve dysfunction that present in various genetic forms. Previously, mutation in three genes encoding mitochondrial proteins has been implicated in autosomal recessive forms of optic atrophy that involve progressive degeneration of optic nerve and retinal ganglion cells (RGC). Using whole exome analysis, a novel double homozygous mutation p.L81R and pR212W in malonyl CoA-acyl carrier protein transacylase (MCAT), a mitochondrial protein involved in fatty acid biosynthesis, has now been identified as responsible for an autosomal recessive optic neuropathy from a Chinese consanguineous family. MCAT is expressed in RGC that are rich in mitochondria. The disease variants lead to structurally unstable MCAT protein with significantly reduced intracellular expression. RGC-specific knockdown of Mcat in mice, lead to an attenuated retinal neurofiber layer, that resembles the phenotype of optic neuropathy. These results indicated that MCAT plays an essential role in mitochondrial function and maintenance of RGC axons, while novel MCAT p.L81R and p.R212W mutations can lead to optic neuropathy.

Acquisition of exogenous fatty acids renders apicoplast-based biosynthesis dispensable in tachyzoites of Toxoplasma.

Toxoplasma gondii is a common protozoan parasite that infects a wide range of hosts, including livestock and humans. Previous studies have suggested that the type 2 fatty acid synthesis (FAS2) pathway, located in the apicoplast (a nonphotosynthetic plastid relict), is crucial for the parasite's survival. Here we examined the physiological relevance of fatty acid synthesis in T. gondii by focusing on the pyruvate dehydrogenase complex and malonyl-CoA-[acyl carrier protein] transacylase (FabD), which are located in the apicoplast to drive de novo fatty acid biosynthesis. Our results disclosed unexpected metabolic resilience of T. gondii tachyzoites, revealing that they can tolerate CRISPR/Cas9-assisted genetic deletions of three pyruvate dehydrogenase subunits or FabD. All mutants were fully viable in prolonged cultures, albeit with impaired growth and concurrent loss of the apicoplast. Even more surprisingly, these mutants displayed normal virulence in mice, suggesting an expendable role of the FAS2 pathway in vivo Metabolic labeling of the Δpdh-e1α mutant showed reduced incorporation of glucose-derived carbon into fatty acids with medium chain lengths (C14:0 and C16:0), revealing that FAS2 activity was indeed compromised. Moreover, supplementation of exogenous C14:0 or C16:0 significantly reversed the growth defect in the Δpdh-e1α mutant, indicating salvage of these fatty acids. Together, these results demonstrate that the FAS2 pathway is dispensable during the lytic cycle of Toxoplasma because of its remarkable flexibility in acquiring fatty acids. Our findings question the long-held assumption that targeting this pathway has significant therapeutic potential for managing Toxoplasma infections.

Elucidation of the crystal structure of FabD from the multidrug-resistant bacterium Acinetobacter baumannii.

Bacterial fatty acid synthesis (FAS) has been extensively studied as a potential target of antimicrobials. In FAS, FabD mediates transacylation of the malonyl group from malonyl-CoA to acyl-carrier protein (ACP). The mounting threat of nosocomial infection by multidrug-resistant Acinetobacter baumannii warrants a deeper understanding of its essential cellular mechanisms, which could lead to effective control of this highly competent pathogen. The molecular mechanisms involved in A. baumannii FAS are poorly understood, and recent research has suggested that Pseudomonas aeruginosa, a closely related nosocomial pathogen of A. baumannii, utilizes FAS to produce virulence factors. In this study, we solved the crystal structure of A. baumannii FabD (AbFabD) to provide a platform for the development of new antibacterial agents. Analysis of the structure of AbFabD confirmed the presence of highly conserved active site residues among bacterial homologs. Binding constants between AbFabD variants and A. baumannii ACP (AbACP) revealed critical conserved residues Lys195 and Lys200 involved in AbACP binding. Computational docking of a potential inhibitor, trifluoperazine, revealed a unique inhibitor-binding pocket near the substrate-binding site. The structural study presented herein will be useful for the structure-based design of potent AbFabD inhibitors.

Acyl-coenzyme A:(holo-acyl carrier protein) transacylase enzymes as templates for engineering.

This review will cover the structure, enzymology, and related aspects that are important for structure-based engineering of the transacylase enzymes from fatty acid biosynthesis and polyketide synthesis. Furthermore, this review will focus on in vitro characteristics and not cover engineering of the upstream or downstream reactions or strategies to manipulate metabolic flux in vivo. The malonyl-coenzyme A(CoA)-holo-acyl-carrier protein (holo-ACP) transacylase (FabD) from Escherichia coli serves as a model for this enzyme with thorough descriptions of structure, enzyme mechanism, and effects of mutation on substrate binding presented in the literature. Here, we discuss multiple practical and theoretical considerations regarding engineering transacylase enzymes to accept non-cognate substrates and form novel acyl-ACPs for downstream reactions.

The R117A variant of the Escherichia coli transacylase FabD synthesizes novel acyl-(acyl carrier proteins).

The commercial impact of fermentation systems producing novel and biorenewable chemicals will flourish with the expansion of enzymes engineered to synthesize new molecules. Though a small degree of natural variability exists in fatty acid biosynthesis, the molecular space accessible through enzyme engineering is fundamentally limitless. Prokaryotic fatty acid biosynthesis enzymes build carbon chains on a functionalized acyl carrier protein (ACP) that provides solubility, stability, and a scaffold for interactions with the synthetic enzymes. Here, we identify the malonyl-coenzyme A (CoA)/holo-ACP transacylase (FabD) from Escherichia coli as a platform enzyme for engineering to diversify microbial fatty acid biosynthesis. The FabD R117A variant produced novel ACP-based primer and extender units for fatty acid biosynthesis. Unlike the wild-type enzyme that is highly specific for malonyl-CoA to produce malonyl-ACP, the R117A variant synthesized acetyl-ACP, succinyl-ACP, isobutyryl-ACP, 2-butenoyl-ACP, and ß-hydroxybutyryl-ACP among others from holo-ACP and the corresponding acyl-CoAs with specific activities from 3.7 to 120 nmol min-1 mg-1. FabD R117A maintained K M values for holo-ACP (~ 40 µM) and displayed small changes in K M for acetoacetyl-CoA (110 ± 30 µM) and acetyl-CoA (200 ± 70 µM) when compared to malonyl-CoA (80 ± 30 µM). FabD R117A represents a novel catalyst that synthesizes a broad range of acyl-acyl-ACPs.

Identification of a malonyl CoA-acyl carrier protein transacylase and its regulatory role in fatty acid biosynthesis in oleaginous microalga Nannochloropsis oceanica.

Oleaginous microalgae hold great promises for biofuel production. However, commercialization of microalgal biofuels remains impracticable due to the lack of suitable industrial strains with high growth rate and lipid productivity. Engineering of metabolic pathways is a potential strategy for the improvement of microalgal strains for the production of lipids and also value-added products in microalgae. Malonyl CoA-acyl carrier protein transacylase (MCAT) has been reported to be involved in fatty acid biosynthesis. Here, we identified a putative MCAT in the oleaginous marine microalga Nannochloropsis oceanica. NoMCAT overexpressing N. oceanica showed a higher growth rate and photosynthetic efficiency. The neutral lipid content of engineered lines showed a significant increase by up to 31% compared to wild type. Gas chromatography-mass spectrometry analysis revealed that NoMCAT overexpression significantly altered the fatty acid composition. The composition of eicosapentaenoic acid (C20:5), which is a polyunsaturated fatty acid necessary for animal nutrition, increased by 8%. These results demonstrate the role of MCAT in enhancing fatty acid biosynthesis and growth in microalgae, and also provide an insight into metabolic engineering of microalgae with high industrial potential.

In Vitro Investigation of Crosstalk between Fatty Acid and Polyketide Synthases in the Andrimid Biosynthetic Assembly Line.

Andrimid (Adm) synthase, which belongs to the type II system of enzymes, produces Adm in Pantoea agglomerans. The adm biosynthetic gene cluster lacks canonical acyltransferases (ATs) to load the malonyl group to acyl carrier proteins (ACPs), thus suggesting that a malonyl-CoA ACP transacylase (MCAT) from the fatty acid synthase (FAS) complex provides the essential AT activity in Adm biosynthesis. Here we report that an MCAT is essential for catalysis of the transacylation of malonate from malonyl-CoA to AdmA polyketide synthase (PKS) ACP in vitro. Catalytic self-malonylation of AdmA (PKS ACP) was not observed in reactions without MCAT, although many type II PKS ACPs are capable of catalyzing self-acylation. This lack of self-malonylation was explained by amino acid sequence analysis of the AdmA PKS ACP and the type II PKS ACPs. The results show that MCAT from the organism's FAS complex can provide the missing AT activity in trans, thus suggesting a protein-protein interaction between the fatty acid and polyketide synthases in the Adm assembly line.

A novel process for obtaining pinosylvin using combinatorial bioengineering in Escherichia coli.

Pinosylvin as a bioactive stilbene is of great interest for food supplements and pharmaceuticals development. In comparison to conventional extraction of pinosylvin from plant sources, biosynthesis engineering of microbial cell factories is a sustainable and flexible alternative method. Current synthetic strategies often require expensive phenylpropanoic precursor and inducer, which are not available for large-scale fermentation process. In this study, three bioengineering strategies were described to the development of a simple and economical process for pinosylvin biosynthesis in Escherichia coli. Firstly, we evaluated different construct environments to give a highly efficient constitutive system for enzymes of pinosylvin pathway expression: 4-coumarate: coenzyme A ligase (4CL) and stilbene synthase (STS). Secondly, malonyl coenzyme A (malonyl-CoA) is a key precursor of pinosylvin bioproduction and at low level in E. coli cell. Thus clustered regularly interspaced short palindromic repeats interference (CRISPRi) was explored to inactivate malonyl-CoA consumption pathway to increase its availability. The resulting pinosylvin content in engineered E. coli was obtained a 1.9-fold increase depending on the repression of fabD (encoding malonyl-CoA-ACP transacylase) gene. Eventually, a phenylalanine over-producing E. coli consisting phenylalanine ammonia lyase was introduced to produce the precursor of pinosylvin, trans-cinnamic acid, the crude extraction of cultural medium was used as supplementation for pinosylvin bioproduction. Using these combinatorial processes, 47.49 mg/L pinosylvin was produced from glycerol.

Structural and biochemical characterization of MCAT from photosynthetic microorganism Synechocystis sp. PCC 6803 reveal its stepwise catalytic mechanism.

Malonyl-coenzyme A: acyl-carrier protein transacylase (MCAT) catalyzes the transfer of malonyl group from malonyl-CoA to the holo-acyl carrier protein (Holo-ACP), yielding malonyl-ACP. The overall reaction has been extensively studied in heterotrophic microorganisms, while its mechanism in photosynthetic autotrophs as well as the stepwise reaction information remains unclear. Here the 2.42 Å crystal structure of MCAT from photosynthetic microorganism Synechocystis sp. PCC 6803 is presented. It demonstrates that Arg113, Ser88 and His188 constitute catalytic triad. The second step involved ACP-MCAT-malonyl intermediate is speed-limited instead of the malonyl-CoA-MCAT intermediate in the first step. Therefore His87, Arg113 and Ser88 render different contributions for the two intermediates. Additionally, S88T mutant initializes the reaction by H87 deprotonating S88T which is different from the wild type.

Preparation of holo- and malonyl-[acyl-carrier-protein] in a manner suitable for analog development.

The fatty acid biosynthetic pathway generates highly reduced carbon based molecules. For this reason fatty acid synthesis is a target of pathway engineering to produce novel specialty or commodity chemicals using renewable techniques to supplant molecules currently derived from petroleum. Malonyl-[acyl carrier protein] (malonyl-ACP) is a key metabolite in the fatty acid pathway and donates two carbon units to the growing fatty acid chain during each step of biosynthesis. Attempts to test engineered fatty acid biosynthesis enzymes in vitro will require malonyl-ACP or malonyl-ACP analogs. Malonyl-ACP is challenging to prepare due to the instability of the carboxylate leaving group and the multiple steps of post-translational modification required to activate ACP. Here we report the expression and purification of holo- and malonyl-ACP from Escherichia coli with high yields (>15 mg per L of expression). The malonyl-ACP is efficiently recognized by the E. coli keto-acyl synthase enzyme, FabH. A FabH assay using malonyl-ACP and a coumarin-based fluorescent reagent is described that provides a high throughput alternative to reported radioactive assays.

Recessive pathogenic variants in MCAT cause combined oxidative phosphorylation deficiency.

Malonyl-CoA-acyl carrier protein transacylase (MCAT) is an enzyme involved in mitochondrial fatty acid synthesis (mtFAS) and catalyzes the transfer of the malonyl moiety of malonyl-CoA to the mitochondrial acyl carrier protein (ACP). Previously, we showed that loss-of-function of mtFAS genes, including Mcat, is associated with severe loss of electron transport chain (ETC) complexes in mouse immortalized skeletal myoblasts (Nowinski et al., 2020). Here, we report a proband presenting with hypotonia, failure to thrive, nystagmus, and abnormal brain MRI findings. Using whole exome sequencing, we identified biallelic variants in MCAT. Protein levels for NDUFB8 and COXII, subunits of complex I and IV respectively, were markedly reduced in lymphoblasts and fibroblasts, as well as SDHB for complex II in fibroblasts. ETC enzyme activities were decreased in parallel. Re-expression of wild-type MCAT rescued the phenotype in patient fibroblasts. This is the first report of a patient with MCAT pathogenic variants and combined oxidative phosphorylation deficiency.

Reconstitution of the FK228 biosynthetic pathway reveals cross talk between modular polyketide synthases and fatty acid synthase.

Functional cross talk between fatty acid biosynthesis and secondary metabolism has been discovered in several cases in microorganisms; none of them, however, involves a modular biosynthetic enzyme. Previously, we reported a hybrid modular nonribosomal peptide synthetase (NRPS)-polyketide synthase (PKS) pathway for the biosynthesis of FK228 anticancer depsipeptide in Chromobacterium violaceum strain 968. This pathway contains two PKS modules on the DepBC enzymes that lack a functional acyltransferase (AT) domain, and no apparent AT-encoding gene exists within the gene cluster or its vicinity. We report here that, through reconstitution of the FK228 biosynthetic pathway in Escherichia coli cells, two essential genes, fabD1 and fabD2, both encoding a putative malonyl coenzyme A (CoA) acyltransferase component of the fatty acid synthase complex, are positively identified to be involved in FK228 biosynthesis. Either gene product appears sufficient to complement the AT-less PKS modules on DepBC for polyketide chain elongation. Concurrently, a gene (sfp) encoding a putative Sfp-type phosphopantetheinyltransferase was identified to be necessary for FK228 biosynthesis as well. Most interestingly, engineered E. coli strains carrying variable genetic components produced significant levels of FK228 under both aerobic and anaerobic cultivation conditions. Discovery of the trans complementation of modular PKSs by housekeeping ATs reveals natural product biosynthesis diversity. Moreover, demonstration of anaerobic production of FK228 by an engineered facultative bacterial strain validates our effort toward the engineering of novel tumor-targeting bioagents.

MCAT Mutations Cause Nuclear LHON-like Optic Neuropathy.

Pathological variants in the nuclear malonyl-CoA-acyl carrier protein transacylase (MCAT) gene, which encodes a mitochondrial protein involved in fatty-acid biogenesis, have been reported in two siblings from China affected by insidious optic nerve degeneration in childhood, leading to blindness in the first decade of life. After analysing 51 families with negative molecular diagnostic tests, from a cohort of 200 families with hereditary optic neuropathy (HON), we identified two novel MCAT mutations in a female patient who presented with acute, sudden, bilateral, yet asymmetric, central visual loss at the age of 20. This presentation is consistent with a Leber hereditary optic neuropathy (LHON)-like phenotype, whose existence and association with NDUFS2 and DNAJC30 has only recently been described. Our findings reveal a wider phenotypic presentation of MCAT mutations, and a greater genetic heterogeneity of nuclear LHON-like phenotypes. Although MCAT pathological variants are very uncommon, this gene should be investigated in HON patients, irrespective of disease presentation.

Antisense RNA Elements for Downregulating Expression.

Antisense RNA (asRNA) technology is an important tool for downregulating gene expression. When applying this strategy, the asRNA interference efficiency is determined by several elements including scaffold design, loop size, and relative abundance. Here, we take the Escherichia coli gene fabD encoding malonyl-CoA-[acyl-carrier-protein] transacylase as an example to describe the asRNA design with reliable and controllable interference efficiency. Real-time PCR and fluorescence assay methods are introduced to detect the interference efficiency at RNA level and protein level, respectively.

The crystal structure of MCAT from Mycobacterium tuberculosis reveals three new catalytic models.

The malonyl coenzyme A (CoA)-acyl carrier protein (ACP) transacylase (MCAT) plays a key role in cell wall biosynthesis in Mycobacterium tuberculosis and other bacteria. The M. tuberculosis MCAT (MtMCAT) is encoded by the FabD gene and catalyzes the transacylation of malonate from malonyl-CoA to holo-ACP. Malonyl-ACP is the substrate in fatty acid biosynthesis and is a by-product of the transacylation reaction. This ability for fatty acid biosynthesis enables M. tuberculosis to survive in hostile environments, and thus understanding the mechanism of biosynthesis is important for the design of new anti-tuberculosis drugs. The 2.3 A crystal structure of MtMCAT reported here shows that its catalytic mechanism differs from those of ScMCAT and EcMCAT, whose structures have previously been determined. In MtMCAT, the C(beta)-O(gamma) bond of Ser91 turns upwards, resulting in a different orientation and thus an overall change of the active pocket compared to other known MCAT enzymes. We identify three new nucleophilic attack chains from the MtMCAT structure: His90-Ser91, Asn155-Wat6-Ser91 and Asn155-His90-Ser91. Enzyme activity assays show that His90A, Asn155A and His90A-Asn155A mutants all have substantially reduced MCAT activity, indicating that M. tuberculosis MCAT supports a unique means of proton transfer. Furthermore, His194 cannot form part of a His-Ser catalytic dyad and only stabilizes the substrate. This new discovery should provide a deeper insight into the catalytic mechanisms of MCATs.

Cloning, expression, crystallization and preliminary X-ray crystallographic analysis of malonyl-CoA-acyl carrier protein transacylase (FabD) from Xanthomonas oryzae pv. oryzae.

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight in rice, which is one of the most devastating diseases in rice-cultivating countries. The Xoo0880 (fabD) gene coding for a malonyl-CoA-acyl carrier protein transacylase (MCAT) from Xoo was cloned and expressed in Escherichia coli. MCAT is an essential enzyme that catalyzes a key reaction of fatty-acid synthesis in bacteria and plants: the conversion of malonyl-CoA to malonyl-acyl carrier protein. The FabD enzyme was purified and crystallized in order to elucidate its three-dimensional structure and to determine its enzymatic reaction mechanism and biological importance. The crystal obtained diffracted to 1.9 A resolution and belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 41.4, b = 74.6, c = 98.5 A. According to Matthews coefficient calculations, the crystallographic structure contains only one monomeric unit in the asymmetric unit with a V(M) of 2.21 A(3) Da(-1) and a solvent content of 44.3%.

Overexpression of Malonyl-CoA: ACP Transacylase in Schizochytrium sp. to Improve Polyunsaturated Fatty Acid Production.

Polyunsaturated fatty acids (PUFAs) have been widely applied in the food and medical industry. In this study, malonyl-CoA: ACP transacylase (MAT) was overexpressed through homologous recombination to improve PUFA production in Schizochytrium. The results showed that the lipid and PUFA concentration were increased by 10.1 and 24.5% with MAT overexpression, respectively. Metabolomics analysis revealed that the intracellular tricarboxylic acid cycle was weakened and glucose absorption was accelerated in the engineered strain. In the mevalonate pathway, intracellular carotene content was decreased, and the carbon flux was then redirected toward PUFA synthesis. Furthermore, a glucose fed-batch fermentation was finally performed with the engineered Schizochytrium. The total lipid yield was further increased to 110.5 g/L, 39.6% higher than the wild strain. Docosahexaenoic acid and eicosapentaenoic acid yield were enhanced to 47.39 g/L and 1.65 g/L with an increase of 81.5 and 172.5%, respectively. This study provided an effective metabolic engineering strategy for industrial PUFA production.

Combinatorial Optimization of Resveratrol Production in Engineered E. coli.

Resveratrol, a plant-derived polyphenolic compound with various health activities, is widely used in nutraceutical and food additives. Herein, combinatorial optimization of resveratrol biosynthetic pathway and intracellular environment of E. coli was carried out. By screening pathway genes from various species and exploring their expression pattern, we initially constructed resveratrol-producing strains. Further targeting at availability of malonyl-CoA through expressing ACC of Corynebacterium glutamicum and antisense inhibiting native fabD significantly increased resveratrol biosynthesis. Transport engineering for resveratrol secretion and molecular chaperones helping for folding heterologous enzymes were employed to improve the intracellular environments in remarkable degrees. By introducing PcTAL of Phanerochaete chrysosporium and tuning expression model of PcTAL, At4CL, and VvSTS, an engineered E. coli produced 57.77 mg/L of resveratrol from l-tyrosine. After integrating the above strategies, resveratrol titer reached to 238.71 mg/L from l-tyrosine. The combinatorial optimization in this study provides a promising strategy to produce valuable natural products in heterologous expression systems.

Malonyl-CoA: acyl carrier protein transacylase from Helicobacter pylori: Crystal structure and its interaction with acyl carrier protein.

Malonyl-CoA: acyl carrier protein transacylase (MCAT) is a critical enzyme responsible for the transfer of the malonyl moiety to holo-acyl carrier protein (ACP) forming the malonyl-ACP intermediates in the initiation step of type II fatty acid synthesis (FAS II) in bacteria. MCAT has been considered as an attractive drug target in the discovery of antibacterial agents. In this study, the crystal structure of MCAT from Helicobacter pylori (Hp) at 2.5 angstroms resolution is reported, and the interaction of HpMCAT with HpACP is extensively investigated by using computational docking, GST-pull-down, and surface plasmon resonance (SPR) technology-based assays. The crystal structure results reveal that HpMCAT has a compact folding composed of a large subdomain with a similar core as in alpha/beta hydrolases, and a similar ferredoxin-like small subdomain as in acylphosphatases. The docking result suggests two positively charged areas near the entrance of the active site of HpMCAT as the ACP-binding region. Binding assay research shows that HpMCAT demonstrates a moderately binding ability against HpACP. The solved 3D structure of HpMCAT is expected to supply useful information for the structure-based discovery of novel inhibitors against MCAT, and the quantitative study of HpMCAT interaction with HpACP is hoped to give helpful hints in the understanding of the detailed catalytic mechanisms for HpMCAT.

Characterization and inhibitor discovery of one novel malonyl-CoA: acyl carrier protein transacylase (MCAT) from Helicobacter pylori.

Type II fatty acid synthesis (FAS II) is an essential process for bacteria survival, and malonyl-CoA:acyl carrier protein transacylase (MCAT) is a key enzyme in FAS II pathway, which is responsible for transferring the malonyl group from malonyl-CoA to the holo-ACP by forming malonyl-ACP. In this work, we described the cloning, characterization and enzymatic inhibition of a new MCAT from Helicobacter pylori strain SS1 (HpMCAT), and the gene sequence of HpfabD was deposited in the GenBank database (Accession No. AY738332 ). Enzymatic characterization of HpMCAT showed that the K(m) value for malonyl-CoA was 21.01+/-2.3 microM, and the thermal- and guanidinium hydrochloride-induced unfolding processes for HpMCAT were quantitatively investigated by circular dichroism spectral analyses. Moreover, a natural product, corytuberine, was discovered to demonstrate inhibitory activity against HpMCAT with IC(50) value at 33.1+/-3.29 microM. Further enzymatic assay results indicated that corytuberine inhibits HpMCAT in an uncompetitive manner. To our knowledge, this is the firstly reported MCAT inhibitor to date. This current work is hoped to supply useful information for better understanding the MCAT features of H. pylori strain, and corytuberine might be used as a potential lead compound in the discovery of the antibacterial agents using HpMCAT as target.