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.

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.

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.

A nonsynonymous mutation in BhLS, encoding an acyl-CoA N-acyltransferase leads to fruit and seed size variation in wax gourd (Benincasa hispida).

Wax gourd (Benincasa hispida (Thunb.) Cogn., 2n = 2x = 24) is an economically important vegetable crop cultivated widely in many tropical and subtropical regions, including China, India, and Japan. Both fruit and seeds are prized agronomic attributes in wax gourd breeding and production. However, the genetic mechanisms underlying these traits remain largely unexplored. In this study, we observed a strong correlation between fruit size and seed size variation in our mapping population, indicating genetic control by a single gene, BhLS, with large size being dominant over small. Through bulk segregant analysis sequencing and fine mapping with a large F2 population, we precisely located the BhLS gene within a 47.098-kb physical interval on Chromosome 10. Within this interval, only one gene, Bhi10M000649, was identified, showing homology to Arabidopsis HOOKLESS1. A nonsynonymous mutation (G to C) in the second exon of Bhi10M000649 was found to be significantly associated with both fruit and seed size variation in wax gourd. These findings collectively highlight the pleiotropic effect of the BhLS gene in regulating fruit and seed size in wax gourd. Our results offer molecular insights into the variation of fruit and seed size in wax gourd and establish a fundamental framework for breeding wax gourd cultivars with desired traits.

Production and Evaluation of Ag85B:HspX:hFcγ1 Immunogenicity as an Fc Fusion Recombinant Multi-Stage Vaccine Candidate Against Mycobacterium tuberculosis.

An urgent need is to introduce an effective vaccine against Mycobacterium tuberculosis (M.tb) infection. In the present study, a multi-stage M.tb immunodominant Fcγ1 fusion protein (Ag85B:HspX:hFcγ1) was designed and produced, and the immunogenicity of purified protein was evaluated. This recombinant fusion protein was produced in the Pichia pastoris expression system. The HiTrap-rPA column affinity chromatography purified and confirmed the fusion protein using ELISA and Western blotting methods. The co-localisation assay was used to confirm its proper folding and function. IFN-γ, IL-12, IL-4, and TGF-ß expression in C57BL/6 mice then evaluated the immunogenicity of the construct in the presence and absence of BCG. After expression optimisation, medium-scale production and the Western blotting test confirmed suitable production of Ag85B:HspX:hFcγ1. The co-localisation results on antigen-presenting cells (APCs) showed that Ag85B:HspX:hFcγ1 properly folded and bound to hFcγRI. This strong co-localisation with its receptor can confirm inducing proper Th1 responses. The in vivo immunisation assay showed no difference in the expression of IL-4 but a substantial increase in the expression of IFN-γ and IL-12 (P ≤ 0.02) and a moderate increase in TGF-ß (P = 0.05). In vivo immunisation assay revealed that Th1-inducing pathways have been stimulated, as IFN-γ and IL-12 strongly, and TGF-ß expression moderately increased in Ag85B:HspX:hFcγ1 group and Ag85B:HspX:hFcγ1+BCG. Furthermore, the production of IFN-γ from splenocytes in the Ag85B:HspX:hFcγ1 group was enormously higher than in other treatments. Therefore, this Fc fusion protein can make a selective multi-stage delivery system for inducing appropriate Th1 responses and is used as a subunit vaccine alone or in combination with others.

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.

Characterization of a secondary hydroxy-acyltransferase for lipid A in Vibrio parahaemolyticus.

Lipid A plays a crucial role in Vibrio parahaemolyticus. Previously we have reported the diversity of secondary acylation of lipid A in V. parahaemolyticus and four V. parahaemolyticus genes VP_RS08405, VP_RS01045, VP_RS12170, and VP_RS00880 exhibiting homology to the secondary acyltransferases in Escherichia coli. In this study, the gene VP_RS12170 was identified as a specific lipid A secondary hydroxy-acyltransferase responsible for transferring a 3-hydroxymyristate to the 2'-position of lipid A. Four E. coli mutant strains WHL00, WHM00, WH300, and WH001 were constructed, and they would synthesize lipid A with different structures due to the absence of genes encoding lipid A secondary acyltransferases or Kdo transferase. Then V. parahaemolyticus VP_RS12170 was overexpressed in W3110, WHL00, WHM00, WH300, and WH001, and lipid A was isolated from these strains and analyzed by using thin-layer chromatography and high-performance liquid chromatography-tandem mass spectrometry. The detailed structural changes of lipid A in these mutant strains with and without VP_RS12170 overexpression were compared and conclude that VP_RS12170 can specifically transfer a 3-hydroxymyristate to the 2'-position of lipid A. This study also demonstrated that the function of VP_RS12170 is Kdo-dependent and its favorite substrate is Kdo-lipid IVA. These findings give us better understanding the biosynthetic pathway and the structural diversity of V. parahaemolyticus lipid A.

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.

A Cell Specific Effect of MBOAT7 MAFLD-risk Variant on Immune Cells.

BACKGROUND: Disease risk variants are likely to affect gene expression in a context- and cell-type specific manner. The membrane bound O-acyltransferase domain containing 7 (MBOAT7) rs8736 metabolic-dysfunction-associated fatty liver disease (MAFLD)-risk variant was recently reported to be a negative regulator of toll-like receptors (TLRs) signalling in macrophages. Whether this effect is generic or cell-type specific in immune cells is unknown. METHODS: We investigated the impact of modulating TLR signaling on MBOAT7 expression in peripheral blood mononuclear cells (PBMCs). We also examined whether the rs8736 polymorphism in MBOAT7 regulates this effect. Furthermore, we measured the allele-specific expression of MBOAT7 in various immune cell populations under both unstimulated and stimulated conditions. RESULTS: We show that MBOAT7 is down-regulated by TLRs in PBMCs. This effect is modulated by the MBOAT7 rs8736 polymorphism. Additionally, we provide evidence that MBOAT7 acts primarily as a modulator of TLR signalling in mononuclear phagocytes. CONCLUSION: Our results highlight the importance of studying Genome-Wide Association Studies (GWAS) signals in the specific cell types in which alterations of gene expression are found.

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.

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.

Membrane-bound O-acyltransferase 7 (MBOAT7) shapes lysosomal lipid homeostasis and function to control alcohol-associated liver injury.

Recent genome-wide association studies (GWAS) have identified a link between single-nucleotide polymorphisms (SNPs) near the MBOAT7 gene and advanced liver diseases. Specifically, the common MBOAT7 variant (rs641738) associated with reduced MBOAT7 expression is implicated in non-alcoholic fatty liver disease (NAFLD), alcohol-associated liver disease (ALD), and liver fibrosis. However, the precise mechanism underlying MBOAT7-driven liver disease progression remains elusive. Previously, we identified MBOAT7-driven acylation of lysophosphatidylinositol lipids as key mechanism suppressing the progression of NAFLD (Gwag et al., 2019). Here, we show that MBOAT7 loss of function promotes ALD via reorganization of lysosomal lipid homeostasis. Circulating levels of MBOAT7 metabolic products are significantly reduced in heavy drinkers compared to healthy controls. Hepatocyte- (Mboat7-HSKO), but not myeloid-specific (Mboat7-MSKO), deletion of Mboat7 exacerbates ethanol-induced liver injury. Lipidomic profiling reveals a reorganization of the hepatic lipidome in Mboat7-HSKO mice, characterized by increased endosomal/lysosomal lipids. Ethanol-exposed Mboat7-HSKO mice exhibit dysregulated autophagic flux and lysosomal biogenesis, associated with impaired transcription factor EB-mediated lysosomal biogenesis and autophagosome accumulation. This study provides mechanistic insights into how MBOAT7 influences ALD progression through dysregulation of lysosomal biogenesis and autophagic flux, highlighting hepatocyte-specific MBOAT7 loss as a key driver of ethanol-induced liver injury.

Two case reports of a novel missense mutation in the PNPLA6 gene in two siblings with chorioretinal dystrophy, hypogonadotropic hypogonadism, and cerebellar ataxia.

BACKGROUND: Boucher Neuhäuser Syndrome (BNS) is a rare disease with autosomal recessive inheritance defined by the classical triad; early-onset ataxia, hypogonadism and chorioretinal dystrophy. CASE PRESENTATION: We present two siblings diagnosed with BNS at midlife, identified with homozygous state of a novel PNPLA6 missense mutation. One healthy sibling and the mother were heterozygous carriers of the mutation. The proband presented with the classical triad and the other sibling presented with visual problems at first. The proband was referred to our department by a private Neurologist, in early adulthood, because of hypogonadism, cerebellar ataxia, axonal neuropathy, and chorioretinal dystrophy for further evaluation. The sibling was referred to our department for evaluation, at childhood, due to visual problems. Later, the patient displayed the triad of ataxia, hypogonadotropic hypogonadism, and chorioretinal dystrophy. The unusual medical history of the two siblings led to further examinations and eventually the diagnosis of the first BNS cases in Cyprus. WES-based ataxia in silico gene panel analysis revealed 15 genetic variants and further filtering analysis revealed the PNPLA6 c.3323G > A variant. Segregation analysis in the family with Sanger sequencing confirmed the PNPLA6 homozygous variant c.3323G > A, p.Arg1108Gln in exon 29. CONCLUSIONS: This highlights the importance of considering rare inherited causes of visual loss, spinocerebellar ataxia, or/and HH in a neurology clinic and the significant role of genetic sequencing in the diagnostic process.

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.

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.

Targeted degradation of zDHHC-PATs decreases substrate S-palmitoylation.

Reversible S-palmitoylation of protein cysteines, catalysed by a family of integral membrane zDHHC-motif containing palmitoyl acyl transferases (zDHHC-PATs), controls the localisation, activity, and interactions of numerous integral and peripheral membrane proteins. There are compelling reasons to want to inhibit the activity of individual zDHHC-PATs in both the laboratory and the clinic, but the specificity of existing tools is poor. Given the extensive conservation of the zDHHC-PAT active site, development of isoform-specific competitive inhibitors is highly challenging. We therefore hypothesised that proteolysis-targeting chimaeras (PROTACs) may offer greater specificity to target this class of enzymes. In proof-of-principle experiments we engineered cell lines expressing tetracycline-inducible Halo-tagged zDHHC5 or zDHHC20, and evaluated the impact of Halo-PROTACs on zDHHC-PAT expression and substrate palmitoylation. In HEK-derived FT-293 cells, Halo-zDHHC5 degradation significantly decreased palmitoylation of its substrate phospholemman, and Halo-zDHHC20 degradation significantly diminished palmitoylation of its substrate IFITM3, but not of the SARS-CoV-2 spike protein. In contrast, in a second kidney derived cell line, Vero E6, Halo-zDHHC20 degradation did not alter palmitoylation of either IFITM3 or SARS-CoV-2 spike. We conclude from these experiments that PROTAC-mediated targeting of zDHHC-PATs to decrease substrate palmitoylation is feasible. However, given the well-established degeneracy in the zDHHC-PAT family, in some settings the activity of non-targeted zDHHC-PATs may substitute and preserve substrate palmitoylation.

Evolution-guided engineering of trans-acyltransferase polyketide synthases.

Bacterial multimodular polyketide synthases (PKSs) are giant enzymes that generate a wide range of therapeutically important but synthetically challenging natural products. Diversification of polyketide structures can be achieved by engineering these enzymes. However, notwithstanding successes made with textbook cis-acyltransferase (cis-AT) PKSs, tailoring such large assembly lines remains challenging. Unlike textbook PKSs, trans-AT PKSs feature an extraordinary diversity of PKS modules and commonly evolve to form hybrid PKSs. In this study, we analyzed amino acid coevolution to identify a common module site that yields functional PKSs. We used this site to insert and delete diverse PKS parts and create 22 engineered trans-AT PKSs from various pathways and in two bacterial producers. The high success rates of our engineering approach highlight the broader applicability to generate complex designer polyketides.

Involvement of a putative acyltransferase gene in sporangium formation in Actinoplanes missouriensis.

The actinomycete Actinoplanes missouriensis forms branched substrate mycelia during vegetative growth and produces terminal sporangia, each of which contains a few hundred spherical flagellated spores, from the substrate mycelia through short sporangiophores. Based on the observation that remodeling of membrane lipid composition is involved in the morphological development of Streptomyces coelicolor A3(2), we hypothesized that remodeling of membrane lipid composition is also involved in sporangium formation in A. missouriensis. Because some acyltransferases are presumably involved in the remodeling of membrane lipid composition, we disrupted each of the 22 genes annotated as encoding putative acyltransferases in the A. missouriensis genome and evaluated their effects on sporangium formation. The atsA (AMIS_52390) null mutant (ΔatsA) strain formed irregular sporangia of various sizes. Transmission electron microscopy revealed that some ΔatsA sporangiospores did not mature properly. Phase-contrast microscopy revealed that sporangium dehiscence did not proceed properly in the abnormally small sporangia of the ΔatsA strain, whereas apparently normal sporangia opened to release the spores. Consistently, the number of spores released from ΔatsA sporangia was lower than that released from wild-type sporangia. These phenotypic changes were recovered by introducing atsA with its own promoter into the ΔatsA strain. These results demonstrate that AtsA is required for normal sporangium formation in A. missouriensis, although the involvement of AtsA in the remodeling of membrane lipid composition is unlikely because AtsA is an acyltransferase_3 (AT3) protein, which is an integral membrane protein that usually catalyzes the acetylation of cell surface structures.IMPORTANCEActinoplanes missouriensis goes through a life cycle involving complex morphological development, including mycelial growth, sporangium formation and dehiscence, swimming as zoospores, and germination to mycelial growth. In this study, we carried out a comprehensive gene disruption experiment of putative acyltransferase genes to search for acyltransferases involved in the morphological differentiation of A. missouriensis. We revealed that a stand-alone acyltransferase_3 domain-containing protein, named AtsA, is required for normal sporangium formation. Although the molecular mechanism of AtsA in sporangium formation, as well as the enzymatic activity of AtsA, remains to be elucidated, the identification of a putative acyltransferase involved in sporangium formation is significant in the study of morphological development of A. missouriensis. This finding will contribute to our understanding of a complex system for producing sporangia, a rare multicellular organism in bacteria.

Divergent evolution of the alcohol-forming pathway of wax biosynthesis among bryophytes.

The plant cuticle is a hydrophobic barrier, which seals the epidermal surface of most aboveground organs. While the cuticle biosynthesis of angiosperms has been intensively studied, knowledge about its existence and composition in nonvascular plants is scarce. Here, we identified and characterized homologs of Arabidopsis thaliana fatty acyl-CoA reductase (FAR) ECERIFERUM 4 (AtCER4) and bifunctional wax ester synthase/acyl-CoA:diacylglycerol acyltransferase 1 (AtWSD1) in the liverwort Marchantia polymorpha (MpFAR2 and MpWSD1) and the moss Physcomitrium patens (PpFAR2A, PpFAR2B, and PpWSD1). Although bryophyte harbor similar compound classes as described for angiosperm cuticles, their biosynthesis may not be fully conserved between the bryophytes M. polymorpha and P. patens or between these bryophytes and angiosperms. While PpFAR2A and PpFAR2B contribute to the production of primary alcohols in P. patens, loss of MpFAR2 function does not affect the wax profile of M. polymorpha. By contrast, MpWSD1 acts as the major wax ester-producing enzyme in M. polymorpha, whereas mutations of PpWSD1 do not affect the wax ester levels of P. patens. Our results suggest that the biosynthetic enzymes involved in primary alcohol and wax ester formation in land plants have either evolved multiple times independently or undergone pronounced radiation followed by the formation of lineage-specific toolkits.

Tailored polyhydroxyalkanoate production from renewable non-fatty acid carbon sources using engineered Cupriavidus necator H16.

As thermoplastic, nontoxic, and biocompatible polyesters, polyhydroxyalkanoates (PHAs) are considered promising biodegradable plastic candidates for diverse applications. Short-chain-length/medium-chain-length (SCL/MCL) PHA copolymers are flexible and versatile PHAs that are typically produced from fatty acids, which are expensive and toxic. Therefore, to achieve the sustainable biosynthesis of SCL/MCL-PHAs from renewable non-fatty acid carbon sources (e.g., sugar or CO2), we used the lithoautotrophic bacterium Cupriavidus necator H16 as a microbial platform. Specifically, we synthesized tailored PHA copolymers with varying MCL-3-hydroxyalkanoate (3HA) compositions (10-70 mol%) from fructose by rewiring the MCL-3HA biosynthetic pathways, including (i) the thioesterase-mediated free fatty acid biosynthetic pathway coupled with the beta-oxidation cycle and (ii) the hydroxyacyl transferase-mediated fatty acid de novo biosynthetic pathway. In addition to sugar-based feedstocks, engineered strains are also promising platforms for the lithoautotrophic production of SCL/MCL-PHAs from CO2. The set of engineered C. necator strains developed in this study provides greater opportunities to produce customized polymers with controllable monomer compositions from renewable resources.