-
Metabolic Engineering Jul 2023Modular polyketide synthases (PKS's) are promising platforms for the rational engineering of designer polyketides and commodity chemicals, yet their low productivities...
Modular polyketide synthases (PKS's) are promising platforms for the rational engineering of designer polyketides and commodity chemicals, yet their low productivities are a barrier to the practical biosynthesis of these compounds. Previously, we engineered triketide lactone synthases such as Pik167 using the recently updated module definition and showed they generate hundreds of milligrams of product per liter of Escherichia coli K207-3 shake flask culture. As the molar ratio between the 2 polypeptides of Pik167 is highly skewed, we sought to attenuate the strength of the T7 promoter controlling the production of the smaller, better-expressing polypeptide and thereby increase production of the first polypeptide under the control of an unoptimized T7 promoter. Through this strategy, a 1.8-fold boost in titer was obtained. After a further 1.5-fold boost obtained by increasing the propionate concentration in the media from 20 to 80 mM, a record titer of 791 mg L (627 mg L isolated) was achieved, a 2.6-fold increase overall. Spurred on by this result, the tetraketide synthase Pik1567 was engineered and the T7 promoter attenuation strategy was applied to its second and third genes. A 5-fold boost, from 20 mg L to 100 mg L, in the titer of its tetraketide product was achieved.
Topics: Polyketide Synthases; Polyketides; Lactones; Peptides
PubMed: 37257684
DOI: 10.1016/j.ymben.2023.05.008 -
Nature Communications Aug 2023Type I modular polyketide synthases (PKSs) are multi-domain enzymes functioning like assembly lines. Many engineering attempts have been made for the last three decades...
Type I modular polyketide synthases (PKSs) are multi-domain enzymes functioning like assembly lines. Many engineering attempts have been made for the last three decades to replace, delete and insert new functional domains into PKSs to produce novel molecules. However, inserting heterologous domains often destabilize PKSs, causing loss of activity and protein misfolding. To address this challenge, here we develop a fluorescence-based solubility biosensor that can quickly identify engineered PKSs variants with minimal structural disruptions. Using this biosensor, we screen a library of acyltransferase (AT)-exchanged PKS hybrids with randomly assigned domain boundaries, and we identify variants that maintain wild type production levels. We then probe each position in the AT linker region to determine how domain boundaries influence structural integrity and identify a set of optimized domain boundaries. Overall, we have successfully developed an experimentally validated, high-throughput method for making hybrid PKSs that produce novel molecules.
Topics: Polyketide Synthases; Amino Acid Sequence
PubMed: 37573440
DOI: 10.1038/s41467-023-40464-x -
BMC Biology May 2024Predation is a fundamental mechanism for organisms to acquire energy, and various species have evolved diverse tools to enhance their hunting abilities. Among protozoan...
BACKGROUND
Predation is a fundamental mechanism for organisms to acquire energy, and various species have evolved diverse tools to enhance their hunting abilities. Among protozoan predators, raptorial Haptorian ciliates are particularly fascinating as they possess offensive extrusomes known as toxicysts, which are rapidly discharged upon prey contact. However, our understanding of the genetic processes and specific toxins involved in toxicyst formation and discharge is still limited.
RESULTS
In this study, we investigated the predation strategies and subcellular structures of seven Haptoria ciliate species and obtained their genome sequences using single-cell sequencing technology. Comparative genomic analysis revealed distinct gene duplications related to membrane transport proteins and hydrolytic enzymes in Haptoria, which play a crucial role in the production and discharge of toxicysts. Transcriptomic analysis further confirmed the abundant expression of genes related to membrane transporters and cellular toxins in Haptoria compared to Trichostomatia. Notably, polyketide synthases (PKS) and L-amino acid oxidases (LAAO) were identified as potentially toxin genes that underwent extensive duplication events in Haptoria.
CONCLUSIONS
Our results shed light on the evolutionary and genomic adaptations of Haptorian ciliates for their predation strategies in evolution and provide insights into their toxic mechanisms.
Topics: Ciliophora; Genomics; Genome, Protozoan; Transcriptome
PubMed: 38715037
DOI: 10.1186/s12915-024-01904-2 -
The Science of the Total Environment Jan 2024Prymnesium parvum is a toxin-producing haptophyte that causes harmful algal blooms worldwide, which are often associated with massive fish-kills and subsequent economic...
Prymnesium parvum is a toxin-producing haptophyte that causes harmful algal blooms worldwide, which are often associated with massive fish-kills and subsequent economic losses. In here, we present nuclear and plastid genome assemblies using PacBio HiFi long reads and DNBseq short reads for the two P. parvum strains UTEX 2797 and CCMP 3037, representing producers of type A prymnesins. Our results show that the P. parvum strains have a moderate haptophyte genome size of 97.56 and 107.32 Mb. The genome assemblies present one of highest contiguous assembled contig sequences to date consisting of 463 and 362 contigs with a contig N50 of 596.99 kb and 968.39 kb for strain UTEX 2797 and CCMP 3037, respectively. The assembled contigs of UTEX 2797 and CCMP 3037 were anchored to 34 scaffolds, with a scaffold N50 of 5.35 Mb and 3.61 Mb, respectively, accounting for 93.2 % and 97.9 % of the total length. Each plastid genome comprises a circular contig. A total of 20,578 and 19,426 protein-coding genes were annotated for UTEX 2797 and CCMP 3037. The expanded gene family analysis showed that starch and sucrose metabolism, sulfur metabolism, energy metabolism and ABC transporters are involved in the evolution of P. parvum. Polyketide synthase (PKS) genes responsible for the production of secondary metabolites such as prymnesins displayed different expression patterns under nutrient limitation. Overlap with repeats and horizontal gene transfer may be two contributing factors to the high number of PKS genes found in this species. The two high quality P. parvum genomes will serve as valuable resources for ecological, genetic, and toxicological studies of haptophytes that can be used to monitor and potentially manage harmful blooms of ichthyotoxic P. parvum in the future.
Topics: Animals; Harmful Algal Bloom; Haptophyta; Fishes
PubMed: 37898203
DOI: 10.1016/j.scitotenv.2023.168042 -
Multiplex CRISPR-Cas9 knockout of EIL3, EIL4, and EIN2L advances soybean flowering time and pod set.BMC Plant Biology Oct 2023Ethylene inhibitor treatment of soybean promotes flower bud differentiation and early flowering, suggested that there is a close relationship between ethylene signaling...
BACKGROUND
Ethylene inhibitor treatment of soybean promotes flower bud differentiation and early flowering, suggested that there is a close relationship between ethylene signaling and soybean growth and development. The short-lived ETHYLENE INSENSITIVE2 (EIN2) and ETHYLENE INSENSITIVE3 (EIN3) proteins play central roles in plant development. The objective of this study was carried out gene editing of EIL family members in soybeans and to examine the effects on soybean yield and other markers of growth.
METHODS AND RESULTS
By editing key-node genes in the ethylene signaling pathway using a multi-sgRNA-in-one strategy, we obtained a series of gene edited lines with variable edit combinations among 15 target genes. EIL3, EIL4, and EIN2L were editable genes favored by the T0 soybean lines. Pot experiments also show that the early flowering stage R1 of the EIL3, EIL4, and EIN2L triple mutant was 7.05 d earlier than that of the wild-type control. The yield of the triple mutant was also increased, being 1.65-fold higher than that of the control. Comparative RNA-seq revealed that sucrose synthase, AUX28, MADS3, type-III polyketide synthase A/B, ABC transporter G family member 26, tetraketide alpha-pyrone reductase, and fatty acyl-CoA reductase 2 may be involved in regulating early flowering and high-yield phenotypes in triple mutant soybean plants.
CONCLUSION
Our results provide a scientific basis for genetic modification to promote the development of earlier-flowering and higher-yielding soybean cultivars.
Topics: Glycine max; CRISPR-Cas Systems; RNA, Guide, CRISPR-Cas Systems; Gene Editing; Ethylenes
PubMed: 37884905
DOI: 10.1186/s12870-023-04543-x -
Journal of Fungi (Basel, Switzerland) Dec 2023microsclerotia are fungal aggregates composed of compacted, pigmented hyphae. As they are highly tolerant to desiccation and produce infective conidia, they are...
microsclerotia are fungal aggregates composed of compacted, pigmented hyphae. As they are highly tolerant to desiccation and produce infective conidia, they are promising candidates to be formulated as bioinsecticides. Despite this potential, the nature of the pigments within these structures remains unclear. In this study, routine culture media used for the differentiation of microsclerotia were supplemented with four melanin inhibitors, and the resulting propagules were characterized. Inhibitors of the 1,8-dihydroxynaphthalene (DHN)-melanin biosynthetic pathway such as tricyclazole and guaiacol induced significant phenotypic and molecular modifications in the obtained propagules, which exhibited a more spherical shape, reduced size, and increased susceptibility to desiccation, heat, and oxidative stress than microsclerotia obtained without inhibitors. Additionally, genes encoding for a polyketide synthase (Mrpks2) and a putative 1,3,6,8-tetrahydroxynaphthalene reductase (Mrthnr), potentially involved in the DHN-melanin biosynthetic pathway, were upregulated in fungi grown in the inhibitor-added media. In conclusion, microsclerotia contain melanins of type DHN that might play a role in both microsclerotia differentiation and environmental stress tolerance.
PubMed: 38132763
DOI: 10.3390/jof9121162 -
Microbial Cell Factories Oct 2023Oxytetracycline which is derived from Streptomyces rimosus, inhibits a wide range of bacteria and is industrially important. The underlying biosynthetic processes are...
BACKGROUND
Oxytetracycline which is derived from Streptomyces rimosus, inhibits a wide range of bacteria and is industrially important. The underlying biosynthetic processes are complex and hinder rational engineering, so industrial manufacturing currently relies on classical mutants for production. While the biochemistry underlying oxytetracycline synthesis is known to involve polyketide synthase, hyperproducing strains of S. rimosus have not been extensively studied, limiting our knowledge on fundamental mechanisms that drive production.
RESULTS
In this study, a multiomics analysis of S. rimosus is performed and wild-type and hyperproducing strains are compared. Insights into the metabolic and regulatory networks driving oxytetracycline formation were obtained. The overproducer exhibited increased acetyl-CoA and malonyl CoA supply, upregulated oxytetracycline biosynthesis, reduced competing byproduct formation, and streamlined morphology. These features were used to synthesize bhimamycin, an antibiotic, and a novel microbial chassis strain was created. A cluster deletion derivative showed enhanced bhimamycin production.
CONCLUSIONS
This study suggests that the precursor supply should be globally increased to further increase the expression of the oxytetracycline cluster while maintaining the natural cluster sequence. The mutagenized hyperproducer S. rimosus HP126 exhibited numerous mutations, including large genomic rearrangements, due to natural genetic instability, and single nucleotide changes. More complex mutations were found than those typically observed in mutagenized bacteria, impacting gene expression, and complicating rational engineering. Overall, the approach revealed key traits influencing oxytetracycline production in S. rimosus, suggesting that similar studies for other antibiotics could uncover general mechanisms to improve production.
Topics: Streptomyces rimosus; Oxytetracycline; Systems Biology; Anti-Bacterial Agents; Mutation
PubMed: 37898787
DOI: 10.1186/s12934-023-02215-x -
Proceedings of the National Academy of... Oct 2023Cyanobacteria are infamous producers of toxins. While the toxic potential of planktonic cyanobacterial blooms is well documented, the ecosystem level effects of...
Cyanobacteria are infamous producers of toxins. While the toxic potential of planktonic cyanobacterial blooms is well documented, the ecosystem level effects of toxigenic benthic and epiphytic cyanobacteria are an understudied threat. The freshwater epiphytic cyanobacterium has recently been shown to produce the "eagle killer" neurotoxin aetokthonotoxin (AETX) causing the fatal neurological disease vacuolar myelinopathy. The disease affects a wide array of wildlife in the southeastern United States, most notably waterfowl and birds of prey, including the bald eagle. In an assay for cytotoxicity, we found the crude extract of the cyanobacterium to be much more potent than pure AETX, prompting further investigation. Here, we describe the isolation and structure elucidation of the aetokthonostatins (AESTs), linear peptides belonging to the dolastatin compound family, featuring a unique modification of the C-terminal phenylalanine-derived moiety. Using immunofluorescence microscopy and molecular modeling, we confirmed that AEST potently impacts microtubule dynamics and can bind to tubulin in a similar matter as dolastatin 10. We also show that AEST inhibits reproduction of the nematode . Bioinformatic analysis revealed the AEST biosynthetic gene cluster encoding a nonribosomal peptide synthetase/polyketide synthase accompanied by a unique tailoring machinery. The biosynthetic activity of a specific N-terminal methyltransferase was confirmed by in vitro biochemical studies, establishing a mechanistic link between the gene cluster and its product.
Topics: Animals; Eagles; Ecosystem; Cyanobacteria; Caenorhabditis elegans; Fresh Water
PubMed: 37751550
DOI: 10.1073/pnas.2219230120 -
Biotechnology For Biofuels and... Jun 2024Schizochytrium sp. is commercially used for production of docosahexaenoic acid (DHA). Schizochytrium sp. utilizes the polyketide synthase complex (PKS) and a single type...
BACKGROUND
Schizochytrium sp. is commercially used for production of docosahexaenoic acid (DHA). Schizochytrium sp. utilizes the polyketide synthase complex (PKS) and a single type I fatty acid synthase (FAS) to synthesize polyunsaturated fatty acids and saturated fatty acids, respectively. The acyl carrier protein (ACP) domains of FAS or PKS are used to load acyl groups during fatty acids biosynthesis. Phosphopantetheinyl transferase (PPTase) transfers the pantetheine moiety from Coenzyme A to the conserved serine residue of an inactive ACP domain to produce its active form.
RESULTS
In this study, in order to improve production and content of DHA, we decreased the expression of fas, strengthened the expression of the PKS pathway, and enhanced the supply of active ACP in Schizochytrium sp. ATCC20888. Weakening the expression of fas or disruption of orfA both led to growth defect and reduction of lipid yields in the resulting strains WFAS and DPKSA, indicating that both FAS and PKS were indispensable for growth and lipid accumulation. Although WFAS had a higher DHA content in total fatty acids than the wild-type strain (WT), its growth defect and low DHA yield hinders its use for DHA production. Overexpression of the orfAB, orfC, orfC-DH (truncated orfC), or ppt promoted DHA and lipid production, respectively. The yields and contents of DHA were further increased by combined overexpression of these genes. Highest values of DHA yield (7.2 g/L) and DHA content (40.6%) were achieved in a recombinant OPKSABC-PPT, ⁓56.5% and 15.3% higher than the WT values, respectively.
CONCLUSIONS
This study demonstrates that genetic engineering of the fatty acid biosynthetic pathways provides a new strategy to enhance DHA production in Schizochytrium.
PubMed: 38831337
DOI: 10.1186/s13068-024-02524-2 -
Frontiers in Fungal Biology 2024Fungal polyketides are a large group of secondary metabolites, valuable due to their diverse spectrum of pharmacological activities. Polyketide biosynthesis in...
Fungal polyketides are a large group of secondary metabolites, valuable due to their diverse spectrum of pharmacological activities. Polyketide biosynthesis in filamentous fungi presents some challenges: small yield and low-purity titers. To tackle these issues, we switched to the yeast , an easily cultivable heterologous host. As an oleaginous yeast, displays a high flux of acetyl- and malonyl-CoA precursors used in lipid synthesis. Likewise, acetyl- and malonyl-CoA are the building blocks of many natural polyketides, and we explored the possibility of redirecting this flux toward polyketide production. Despite its promising prospect, has so far only been used for heterologous expression of simple type III polyketide synthases (PKSs) from plants. Therefore, we decided to evaluate the potential of by targeting the more complex fungal polyketides synthesized by type I PKSs. We employed a CRISPR-Cas9-mediated genome editing method to achieve markerless gene integration of the genes responsible for bostrycoidin biosynthesis in Fusarium solani (, , and ) and 6-methylsalicylic acid (6-MSA) biosynthesis in Aspergillus hancockii (6MSAS). Moreover, we attempted titer optimization through metabolic engineering by overexpressing two enzymes, TGL4 and AOX2, involved in lipid β-oxidation, but we did not observe an effect on polyketide production. With maximum titers of 403 mg/L 6-MSA and 35 mg/L bostrycoidin, the latter being substantially higher than our previous results in (2.2 mg/L), this work demonstrates the potential of as a platform for heterologous production of complex fungal polyketides.
PubMed: 38586602
DOI: 10.3389/ffunb.2024.1327777