In vitro synthesis and biochemical characterization of acyl-homoserine lactone synthase and its deletion mutant.

Quorum sensing can induce density-dependent gene expressions that cause various problems. For quorum-sensing inhibition, fundamental solutions such as gene manipulation are required, and acyl-homoserine lactone synthase (AHL synthase), which synthesizes the universal quorum-sensing signal of gram-negative bacteria, can be used as a target. In this study, researchers synthesized His-tagged AHL synthase and its deletion mutant that lacks the active site and compared their biochemical characteristics. His-YpeI, the 6x His-tagged AHL synthase of Serratia fonticola, and His-ΔYpeI, its deletion mutant, were designed, and their property conservation were examined using in silico projection tools. For in vitro synthesis of enzymes, the His-YpeI CFPS template was synthesized by in vitro gene synthesis, and the His-ΔYpeI CFPS template was obtained by deletion PCR. CFPS was performed and the products were purified with the 6x His-tag. The enzymes' properties were compared using an enzymatic assay. The bioinformatic analysis confirmed the conservation of biochemical properties between 6x His-tagged and untagged enzymes, including helix-turn-helix interactions, hydropathy profiles, and tertiary structure between His-YpeI and YpeI and between His-ΔYpeI and ΔYpeI. His-YpeI and His-ΔYpeI synthesized by CFPS were found to have the expected molecular weights and demonstrated distinct differences in enzyme activity. The analyzed enzymatic constants supported a significant decrease in substrate affinity and reaction rate as a result of YpeI's enzyme active site deletion. This result showed that CFPS could be used for in vitro protein synthesis, and quorum sensing could be inhibited at the enzymatic level due to the enzyme active site's deletion mutation.

Structural insights into catalytic promiscuity of chalcone synthase from Glycine max (L.) Merr.: Coenzyme A-induced alteration of product specificity.

Catalytic promiscuity of enzymes plays a pivotal role in driving the evolution of plant specialized metabolism. Chalcone synthase (CHS) catalyzes the production of 2',4,4',6'-tetrahydroxychalcone (THC), a common precursor of plant flavonoids, from p-coumaroyl-coenzyme A (-CoA) and three malonyl-CoA molecules. CHS has promiscuous product specificity, producing a significant amount of p-coumaroyltriacetic lactone (CTAL) in vitro. However, mechanistic aspects of this CHS promiscuity remain to be clarified. Here, we show that the product specificity of soybean CHS (GmCHS1) is altered by CoA, a reaction product, which selectively inhibits THC production (IC50, 67 µM) and enhances CTAL production. We determined the structure of a ternary GmCHS1/CoA/naringenin complex, in which CoA is bound to the CoA-binding tunnel via interactions with Lys55, Arg58, and Lys268. Replacement of these residues by alanine resulted in an enhanced THC/CTAL production ratio, suggesting the role of these residues in the CoA-mediated alteration of product specificity. In the ternary complex, a mobile loop ("the K-loop"), which contains Lys268, was in a "closed conformation" placing over the CoA-binding tunnel, whereas in the apo and binary complex structures, the K-loop was in an "open conformation" and remote from the tunnel. We propose that the production of THC involves a transition of the K-loop conformation between the open and closed states, whereas synthesis of CTAL is independent of it. In the presence of CoA, an enzyme conformer with the closed K-loop conformation becomes increasingly dominant, hampering the transition of K-loop conformations to result in decreased THC production and increased CTAL production.

ZDHHC20-mediated S-palmitoylation of YTHDF3 stabilizes MYC mRNA to promote pancreatic cancer progression.

Post-translational modifications of proteins in malignant transformation and tumor maintenance of pancreatic ductal adenocarcinoma (PDAC) in the context of KRAS signaling remain poorly understood. Here, we use the KPC mouse model to examine the effect of palmitoylation on pancreatic cancer progression. ZDHHC20, upregulated by KRAS, is abnormally overexpressed and associated with poor prognosis in patients with pancreatic cancer. Dysregulation of ZDHHC20 promotes pancreatic cancer progression in a palmitoylation-dependent manner. ZDHHC20 inhibits the chaperone-mediated autophagic degradation of YTHDF3 through S-palmitoylation of Cys474, which can result in abnormal accumulation of the oncogenic product MYC and thereby promote the malignant phenotypes of cancer cells. Further, we design a biologically active YTHDF3-derived peptide to competitively inhibit YTHDF3 palmitoylation mediated by ZDHHC20, which in turn downregulates MYC expression and inhibits the progression of KRAS mutant pancreatic cancer. Thus, these findings highlight the therapeutic potential of targeting the ZDHHC20-YTHDF3-MYC signaling axis in pancreatic cancer.

Multiomics analysis identifies oxidative phosphorylation as a cancer vulnerability arising from myristoylation inhibition.

BACKGROUND: In humans, two ubiquitously expressed N-myristoyltransferases, NMT1 and NMT2, catalyze myristate transfer to proteins to facilitate membrane targeting and signaling. We investigated the expression of NMTs in numerous cancers and found that NMT2 levels are dysregulated by epigenetic suppression, particularly so in hematologic malignancies. This suggests that pharmacological inhibition of the remaining NMT1 could allow for the selective killing of these cells, sparing normal cells with both NMTs. METHODS AND RESULTS: Transcriptomic analysis of 1200 NMT inhibitor (NMTI)-treated cancer cell lines revealed that NMTI sensitivity relates not only to NMT2 loss or NMT1 dependency, but also correlates with a myristoylation inhibition sensitivity signature comprising 54 genes (MISS-54) enriched in hematologic cancers as well as testis, brain, lung, ovary, and colon cancers. Because non-myristoylated proteins are degraded by a glycine-specific N-degron, differential proteomics revealed the major impact of abrogating NMT1 genetically using CRISPR/Cas9 in cancer cells was surprisingly to reduce mitochondrial respiratory complex I proteins rather than cell signaling proteins, some of which were also reduced, albeit to a lesser extent. Cancer cell treatments with the first-in-class NMTI PCLX-001 (zelenirstat), which is undergoing human phase 1/2a trials in advanced lymphoma and solid tumors, recapitulated these effects. The most downregulated myristoylated mitochondrial protein was NDUFAF4, a complex I assembly factor. Knockout of NDUFAF4 or in vitro cell treatment with zelenirstat resulted in loss of complex I, oxidative phosphorylation and respiration, which impacted metabolomes. CONCLUSIONS: Targeting of both, oxidative phosphorylation and cell signaling partly explains the lethal effects of zelenirstat in select cancer types. While the prognostic value of the sensitivity score MISS-54 remains to be validated in patients, our findings continue to warrant the clinical development of zelenirstat as cancer treatment.

Lats1 and Lats2 regulate YAP and TAZ activity to control the development of mouse Sertoli cells.

Recent reports suggest that the Hippo signaling pathway regulates testis development, though its exact roles in Sertoli cell differentiation remain unknown. Here, we examined the functions of the main Hippo pathway kinases, large tumor suppressor homolog kinases 1 and 2 (Lats1 and Lats2) in developing mouse Sertoli cells. Conditional inactivation of Lats1/2 in Sertoli cells resulted in the disorganization and overgrowth of the testis cords, the induction of a testicular inflammatory response and germ cell apoptosis. Stimulated by retinoic acid 8 (STRA8) expression in germ cells additionally suggested that germ cells may have been preparing to enter meiosis prior to their loss. Gene expression analyses of the developing testes of conditional knockout animals further suggested impaired Sertoli cell differentiation, epithelial-to-mesenchymal transition, and the induction of a specific set of genes associated with Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ)-mediated integrin signaling. Finally, the involvement of YAP/TAZ in Sertoli cell differentiation was confirmed by concomitantly inactivating Yap/Taz in Lats1/2 conditional knockout model, which resulted in a partial rescue of the testicular phenotypic changes. Taken together, these results identify Hippo signaling as a crucial pathway for Sertoli cell development and provide novel insight into Sertoli cell fate maintenance.

Palmitoyl transferase ZDHHC20 promotes pancreatic cancer metastasis.

Metastasis is one of the defining features of pancreatic ductal adenocarcinoma (PDAC) that contributes to poor prognosis. In this study, the palmitoyl transferase ZDHHC20 was identified in an in vivo short hairpin RNA (shRNA) screen as critical for metastatic outgrowth, with no effect on proliferation and migration in vitro or primary PDAC growth in mice. This phenotype is abrogated in immunocompromised animals and animals with depleted natural killer (NK) cells, indicating that ZDHHC20 affects the interaction of tumor cells and the innate immune system. Using a chemical genetics platform for ZDHHC20-specific substrate profiling, a number of substrates of this enzyme were identified. These results describe a role for palmitoylation in enabling distant metastasis that could not have been detected using in vitro screening approaches and identify potential effectors through which ZDHHC20 promotes metastasis of PDAC.

Caveolin-2 palmitoylation turnover facilitates insulin receptor substrate-1-directed lipid metabolism by insulin receptor tyrosine kinase.

Here, we show that insulin induces palmitoylation turnover of Caveolin-2 (Cav-2) in adipocytes. Acyl protein thioesterases-1 (APT1) catalyzes Cav-2 depalmitoylation, and zinc finger DHHC domain-containing protein palmitoyltransferase 21 (ZDHHC21) repalmitoylation of the depalmitoylated Cav-2 for the turnover, thereby controlling insulin receptor (IR)-Cav-2-insulin receptor substrate-1 (IRS-1)-Akt-driven signaling. Insulin-induced palmitoylation turnover of Cav-2 facilitated glucose uptake and fat storage through induction of lipogenic genes. Cav-2-, APT1-, and ZDHHC21-deficient adipocytes, however, showed increased induction of lipolytic genes and glycerol release. In addition, white adipose tissues from insulin sensitive and resistant obese patients exhibited augmented expression of LYPLA1 (APT1) and ZDHHC20 (ZDHHC20). Our study identifies the specific enzymes regulating Cav-2 palmitoylation turnover, and reveals a new mechanism by which insulin-mediated lipid metabolism is controlled in adipocytes.

Conjugated linoleic acid (CLA) reduces intestinal fatty acid uptake and chylomicron formation in HFD-fed mice associated with the inhibition of DHHC7-mediated CD36 palmitoylation and the downstream ERK pathway.

The anti-obesity effect of conjugated linoleic acid (CLA) has been well elucidated, but whether CLA affects fat deposition by regulating intestinal dietary fat absorption remains largely unknown. Thus, this study aimed to investigate the effects of CLA on intestinal fatty acid uptake and chylomicron formation and explore the possible underlying mechanisms. We found that CLA supplementation reduced the intestinal fat absorption in HFD (high fat diet)-fed mice accompanied by the decreased serum TG level, increased fecal lipids and decreased intestinal expression of ApoB48 and MTTP. Correspondingly, c9, t11-CLA, but not t10, c12-CLA induced the reduction of fatty acid uptake and TG content in PA (palmitic acid)-treated MODE-K cells. In the mechanism of fatty acid uptake, c9, t11-CLA inhibited the binding of CD36 with palmitoyltransferase DHHC7, thus leading to the decreases of CD36 palmitoylation level and localization on the cell membrane of the PA-treated MODE-K cells. In the mechanism of chylomicron formation, c9, t11-CLA inhibited the formation of the CD36/FYN/LYN complex and the activation of the ERK pathway in the PA-treated MODE-K cells. In in vivo verification, CLA supplementation reduced the DHHC7-mediated total and cell membrane CD36 palmitoylation and suppressed the formation of the CD36/FYN/LYN complex and the activation of the ERK pathway in the jejunum of HFD-fed mice. Altogether, these data showed that CLA reduced intestinal fatty acid uptake and chylomicron formation in HFD-fed mice associated with the inhibition of DHHC7-mediated CD36 palmitoylation and the downstream ERK pathway.

Deacylases-structure, function, and relationship to diseases.

Reversible S-acylation plays a pivotal role in various biological processes, modulating protein functions such as subcellular localization, protein stability/activity, and protein-protein interactions. These modifications are mediated by acyltransferases and deacylases, among which the most abundant modification is S-palmitoylation. Growing evidence has shown that this rivalrous pair of modifications, occurring in a reversible cycle, is essential for various biological functions. Aberrations in this process have been associated with various diseases, including cancer, neurological disorders, and immune diseases. This underscores the importance of studying enzymes involved in acylation and deacylation to gain further insights into disease pathogenesis and provide novel strategies for disease treatment. In this Review, we summarize our current understanding of the structure and physiological function of deacylases, highlighting their pivotal roles in pathology. Our aim is to provide insights for further clinical applications.

Engineering Pseudomonas chlororaphis HT66 for the Biosynthesis of Copolymers Containing 3-Hydroxybutyrate and Medium-Chain-Length 3-Hydroxyalkanoates.

Polyhydroxyalkanoates (PHAs) are promising alternatives to petroleum-based plastics, owing to their biodegradability and superior material properties. Here, the controllable biosynthesis of scl-co-mcl PHA containing 3-hydroxybutyrate (3HB) and mcl 3-hydroxyalkanoates was achieved in Pseudomonas chlororaphis HT66. First, key genes involved in fatty acid ß-oxidation, the de novo fatty acid biosynthesis pathway, and the phaC1-phaZ-phaC2 operon were deleted to develop a chassis strain. Subsequently, an acetoacetyl-CoA reductase gene phaB and a PHA synthase gene phaC with broad substrate specificity were heterologously expressed for producing and polymerizing the 3HB monomer with mcl 3-hydroxyalkanoates under the assistance of native ß-ketothiolase gene phaA. Furthermore, the monomer composition of scl-co-mcl PHA was regulated by adjusting the amount of glucose and dodecanoic acid supplemented. Notably, the cell dry weight and scl-co-mcl PHA content reached 14.2 g/L and 60.1 wt %, respectively, when the engineered strain HT11Δ::phaCB was cultured in King's B medium containing 5 g/L glucose and 5 g/L dodecanoic acid. These results demonstrated that P. chlororaphis can be a platform for producing scl-co-mcl PHA and has the potential for industrial application.

Recurrent mutations in tumor suppressor FBXW7 bypass Wnt/ß-catenin addiction in cancer.

Pathologic Wnt/ß-catenin signaling drives various cancers, leading to multiple approaches to drug this pathway. Appropriate patient selection can maximize success of these interventions. Wnt ligand addiction is a druggable vulnerability in RNF43-mutant/RSPO-fusion cancers. However, pharmacologically targeting the biogenesis of Wnt ligands, e.g., with PORCN inhibitors, has shown mixed therapeutic responses, possibly due to tumor heterogeneity. Here, we show that the tumor suppressor FBXW7 is frequently mutated in RNF43-mutant/RSPO-fusion tumors, and FBXW7 mutations cause intrinsic resistance to anti-Wnt therapies. Mechanistically, FBXW7 inactivation stabilizes multiple oncoproteins including Cyclin E and MYC and antagonizes the cytostatic effect of Wnt inhibitors. Moreover, although FBXW7 mutations do not mitigate ß-catenin degradation upon Wnt inhibition, FBXW7-mutant RNF43-mutant/RSPO-fusion cancers instead lose dependence on ß-catenin signaling, accompanied by dedifferentiation and loss of lineage specificity. These FBXW7-mutant Wnt/ß-catenin-independent tumors are susceptible to multi-cyclin-dependent kinase inhibition. An in-depth understanding of primary resistance to anti-Wnt/ß-catenin therapies allows for more appropriate patient selection and use of alternative mechanism-based therapies.

Discovering a mitochondrion-localized BAHD acyltransferase involved in calystegine biosynthesis and engineering the production of 3ß-tigloyloxytropane.

Solanaceous plants produce tropane alkaloids (TAs) via esterification of 3α- and 3ß-tropanol. Although littorine synthase is revealed to be responsible for 3α-tropanol esterification that leads to hyoscyamine biosynthesis, the genes associated with 3ß-tropanol esterification are unknown. Here, we report that a BAHD acyltransferase from Atropa belladonna, 3ß-tigloyloxytropane synthase (TS), catalyzes 3ß-tropanol and tigloyl-CoA to form 3ß-tigloyloxytropane, the key intermediate in calystegine biosynthesis and a potential drug for treating neurodegenerative disease. Unlike other cytosolic-localized BAHD acyltransferases, TS is localized to mitochondria. The catalytic mechanism of TS is revealed through molecular docking and site-directed mutagenesis. Subsequently, 3ß-tigloyloxytropane is synthesized in tobacco. A bacterial CoA ligase (PcICS) is found to synthesize tigloyl-CoA, an acyl donor for 3ß-tigloyloxytropane biosynthesis. By expressing TS mutant and PcICS, engineered Escherichia coli synthesizes 3ß-tigloyloxytropane from tiglic acid and 3ß-tropanol. This study helps to characterize the enzymology and chemodiversity of TAs and provides an approach for producing 3ß-tigloyloxytropane.

Structural insights into the transporting and catalyzing mechanism of DltB in LTA D-alanylation.

DltB, a model member of the Membrane-Bound O-AcylTransferase (MBOAT) superfamily, plays a crucial role in D-alanylation of the lipoteichoic acid (LTA), a significant component of the cell wall of gram-positive bacteria. This process stabilizes the cell wall structure, influences bacterial virulence, and modulates the host immune response. Despite its significance, the role of DltB is not well understood. Through biochemical analysis and cryo-EM imaging, we discover that Streptococcus thermophilus DltB forms a homo-tetramer on the cell membrane. We further visualize DltB in an apo form, in complex with DltC, and in complex with its inhibitor amsacrine (m-AMSA). Each tetramer features a central hole. The C-tunnel of each protomer faces the intratetramer interface and provides access to the periphery membrane. Each protomer binds a DltC without changing the tetrameric organization. A phosphatidylglycerol (PG) molecule in the substrate-binding site may serve as an LTA carrier. The inhibitor m-AMSA bound to the L-tunnel of each protomer blocks the active site. The tetrameric organization of DltB provides a scaffold for catalyzing D-alanyl transfer and regulating the channel opening and closing. Our findings unveil DltB's dual function in the D-alanylation pathway, and provide insight for targeting DltB as a anti-virulence antibiotic.

Allosteric Modulation of the YAP/TAZ-TEAD Interaction by Palmitoylation and Small-Molecule Inhibitors.

The Hippo signaling pathway is a highly conserved signaling network that plays a central role in regulating cellular growth, proliferation, and organ size. This pathway consists of a kinase cascade that integrates various upstream signals to control the activation or inactivation of YAP/TAZ proteins. Phosphorylated YAP/TAZ is sequestered in the cytoplasm; however, when the Hippo pathway is deactivated, it translocates into the nucleus, where it associates with TEAD transcription factors. This partnership is instrumental in regulating the transcription of progrowth and antiapoptotic genes. Thus, in many cancers, aberrantly hyperactivated YAP/TAZ promotes oncogenesis by contributing to cancer cell proliferation, metastasis, and therapy resistance. Because YAP and TAZ exert their oncogenic effects by binding with TEAD, it is critical to understand this key interaction to develop cancer therapeutics. Previous research has indicated that TEAD undergoes autopalmitoylation at a conserved cysteine, and small molecules that inhibit TEAD palmitoylation disrupt effective YAP/TAZ binding. However, how exactly palmitoylation contributes to YAP/TAZ-TEAD interactions and how the TEAD palmitoylation inhibitors disrupt this interaction remains unknown. Utilizing molecular dynamics simulations, our investigation not only provides detailed atomistic insight into the YAP/TAZ-TEAD dynamics but also unveils that the inhibitor studied influences the binding of YAP and TAZ to TEAD in distinct manners. This discovery has significant implications for the design and deployment of future molecular interventions targeting this interaction.

Chalcone-Synthase-Encoding RdCHS1 Is Involved in Flavonoid Biosynthesis in Rhododendron delavayi.

Flower color is an important ornamental feature that is often modulated by the contents of flavonoids. Chalcone synthase is the first key enzyme in the biosynthesis of flavonoids, but little is known about the role of R. delavayi CHS in flavonoid biosynthesis. In this paper, three CHS genes (RdCHS1-3) were successfully cloned from R. delavayi flowers. According to multiple sequence alignment and a phylogenetic analysis, only RdCHS1 contained all the highly conserved and important residues, which was classified into the cluster of bona fide CHSs. RdCHS1 was then subjected to further functional analysis. Real-time PCR analysis revealed that the transcripts of RdCHS1 were the highest in the leaves and lowest in the roots; this did not match the anthocyanin accumulation patterns during flower development. Biochemical characterization displayed that RdCHS1 could catalyze p-coumaroyl-CoA and malonyl-CoA molecules to produce naringenin chalcone. The physiological function of RdCHS1 was checked in Arabidopsis mutants and tobacco, and the results showed that RdCHS1 transgenes could recover the color phenotypes of the tt4 mutant and caused the tobacco flower color to change from pink to dark pink through modulating the expressions of endogenous structural and regulatory genes in the tobacco. All these results demonstrate that RdCHS1 fulfills the function of a bona fide CHS and contributes to flavonoid biosynthesis in R. delavayi.

The fatty liver disease-causing protein PNPLA3-I148M alters lipid droplet-Golgi dynamics.

Nonalcoholic fatty liver disease, recently renamed metabolic dysfunction-associated steatotic liver disease (MASLD), is a progressive metabolic disorder that begins with aberrant triglyceride accumulation in the liver and can lead to cirrhosis and cancer. A common variant in the gene PNPLA3, encoding the protein PNPLA3-I148M, is the strongest known genetic risk factor for MASLD. Despite its discovery 20 y ago, the function of PNPLA3, and now the role of PNPLA3-I148M, remain unclear. In this study, we sought to dissect the biogenesis of PNPLA3 and PNPLA3-I148M and characterize changes induced by endogenous expression of the disease-causing variant. Contrary to bioinformatic predictions and prior studies with overexpressed proteins, we demonstrate here that PNPLA3 and PNPLA3-I148M are not endoplasmic reticulum-resident transmembrane proteins. To identify their intracellular associations, we generated a paired set of isogenic human hepatoma cells expressing PNPLA3 and PNPLA3-I148M at endogenous levels. Both proteins were enriched in lipid droplet, Golgi, and endosomal fractions. Purified PNPLA3 and PNPLA3-I148M proteins associated with phosphoinositides commonly found in these compartments. Despite a similar fractionation pattern as the wild-type variant, PNPLA3-I148M induced morphological changes in the Golgi apparatus, including increased lipid droplet-Golgi contact sites, which were also observed in I148M-expressing primary human patient hepatocytes. In addition to lipid droplet accumulation, PNPLA3-I148M expression caused significant proteomic and transcriptomic changes that resembled all stages of liver disease. Cumulatively, we validate an endogenous human cellular system for investigating PNPLA3-I148M biology and identify the Golgi apparatus as a central hub of PNPLA3-I148M-driven cellular change.

Targeting the GPI transamidase subunit GPAA1 abrogates the CD24 immune checkpoint in ovarian cancer.

CD24 is frequently overexpressed in ovarian cancer and promotes immune evasion by interacting with its receptor Siglec10, present on tumor-associated macrophages, providing a "don't eat me" signal that prevents targeting and phagocytosis by macrophages. Factors promoting CD24 expression could represent novel immunotherapeutic targets for ovarian cancer. Here, using a genome-wide CRISPR knockout screen, we identify GPAA1 (glycosylphosphatidylinositol anchor attachment 1), a factor that catalyzes the attachment of a glycosylphosphatidylinositol (GPI) lipid anchor to substrate proteins, as a positive regulator of CD24 cell surface expression. Genetic ablation of GPAA1 abolishes CD24 cell surface expression, enhances macrophage-mediated phagocytosis, and inhibits ovarian tumor growth in mice. GPAA1 shares structural similarities with aminopeptidases. Consequently, we show that bestatin, a clinically advanced aminopeptidase inhibitor, binds to GPAA1 and blocks GPI attachment, resulting in reduced CD24 cell surface expression, increased macrophage-mediated phagocytosis, and suppressed growth of ovarian tumors. Our study highlights the potential of targeting GPAA1 as an immunotherapeutic approach for CD24+ ovarian cancers.

Modulators for palmitoylation of proteins and small molecules.

As an essential form of lipid modification for maintaining vital cellular functions, palmitoylation plays an important role in in the regulation of various physiological processes, serving as a promising therapeutic target for diseases like cancer and neurological disorders. Ongoing research has revealed that palmitoylation can be categorized into three distinct types: N-palmitoylation, O-palmitoylation and S-palmitoylation. Herein this paper provides an overview of the regulatory enzymes involved in palmitoylation, including palmitoyltransferases and depalmitoylases, and discusses the currently available broad-spectrum and selective inhibitors for these enzymes.

Engineering of the Long-Main-Chain Monomer-Incorporating Polyhydroxyalkanoate Synthase PhaCAR for the Biosynthesis of Poly[(R)-3-hydroxybutyrate-co-6-hydroxyhexanoate].

Polyhydroxyalkanoate (PHA) synthases (PhaCs) are useful and versatile tools for the production of aliphatic polyesters. Here, the chimeric PHA synthase PhaCAR was engineered to increase its capacity to incorporate unusual 6-hydroxyhexanoate (6HHx) units. Mutations at positions 149 and 314 in PhaCAR were previously found to increase the incorporation of an analogous natural monomer, 3-hydroxyhexanoate (3HHx). We attempted to repurpose the mutations to produce 6HHx-containing polymers. Site-directed saturation mutants at these positions were applied for P(3HB-co-6HHx) synthesis in Escherichia coli. As a result, the N149D and F314Y mutants effectively increased the 6HHx fraction. Moreover, the pairwise NDFY mutation further increased the 6HHx fraction, which reached 22 mol %. This increase was presumably caused by altered enzyme activity rather than altered expression levels, as assessed based on immunoblot analysis. The glass transition temperature and crystallinity of P(3HB-co-6HHx) decreased as the 6HHx fraction increased.

Carbon dioxide valorization into resveratrol via lithoautotrophic fermentation using engineered Cupriavidus necator H16.

BACKGROUND: Industrial biomanufacturing of value-added products using CO2 as a carbon source is considered more sustainable, cost-effective and resource-efficient than using common carbohydrate feedstocks. Cupriavidus necator H16 is a representative H2-oxidizing lithoautotrophic bacterium that can be utilized to valorize CO2 into valuable chemicals and has recently gained much attention as a promising platform host for versatile C1-based biomanufacturing. Since this microbial platform is genetically tractable and has a high-flux carbon storage pathway, it has been engineered to produce a variety of valuable compounds from renewable carbon sources. In this study, the bacterium was engineered to produce resveratrol autotrophically using an artificial phenylpropanoid pathway. RESULTS: The heterologous genes involved in the resveratrol biosynthetic pathway-tyrosine ammonia lyase (TAL), 4-coumaroyl CoA ligase (4CL), and stilbene synthase (STS) -were implemented in C. necator H16. The overexpression of acetyl-CoA carboxylase (ACC), disruption of the PHB synthetic pathway, and an increase in the copy number of STS genes enhanced resveratrol production. In particular, the increased copies of VvSTS derived from Vitis vinifera resulted a 2-fold improvement in resveratrol synthesis from fructose. The final engineered CR-5 strain produced 1.9 mg/L of resveratrol from CO2 and tyrosine via lithoautotrophic fermentation. CONCLUSIONS: To the best of our knowledge, this study is the first to describe the valorization of CO2 into polyphenolic compounds by engineering a phenylpropanoid pathway using the lithoautotrophic bacterium C. necator H16, demonstrating the potential of this strain a platform for sustainable chemical production.