Heterologous Biosynthesis And Structural Innovation Of Polyketide-Nonribosomal Peptide Hybrid Macrolactone Thermolide From Fungi

Nematode infestations and parasitic plant diseases have led to significant damage in agricultural production and forests,resulting in an annual global economic loss of$150 billion.With the emergence of environmental pollution and drug resistance due to long-term use of chemical insecticides,there is a widespread consensus that the development of environmentally friendly and highly active natural drugs against nematodes is essential for biological control.Thermolide,a family of polyketide-nonribosomal peptide（PK-NRP）hybrid natural products containing a 13-membered macrolactone produced by thermophilic fungi Talaromyces thermophilus,exhibit strong nematocidal activity comparable to commercial avermectin to making them promising biopesticides.However,several factors have limited the development and application of thermolide-type biopesticides,including（1）the harsh culture conditions of T.thermophilus（45°C-55°C）,（2）the low yield of active components（0.05 mg/L）,and（3）the difficult chemical synthesis of the complex chiral center in the macrolactone skeleton.Fortunately,heterologous biosynthesis study based on pathway analysis can overcome these limitations,as well as clarify the molecular mechanism of thermolide synthesis.Structural innovation can be achieved through metabolic engineering or synthetic biology,which supports the further application of thermolide as valuable nematocidal agents for biological control in agriculture.In this study,we identified the thermolide biosynthetic gene cluster thm from T.thermophilus NRRL 2155 genome.The unique formation mechanism of the PK-NRP macrolactone skeleton and the complete biosynthetic pathway of thermolide were documented through bioinformatics analysis,heterologous biosynthesis,in vitro enzymatic characterization,and site-specific mutation.Additionally,we created a library of thermolide derivatives with diverse structural features by genome mining combined with combinatorial biosynthesis and metabolic engineering.The major results of this study are as follows:（1）Analysis and characterization of the tal gene cluster.Previous report suggested that a single module polyketide-nonribosomal peptide synthase（PKS-NRPS）hybrid gene cluster（tal cluster）was responsible for the biosynthesis of thermolide in T.thermophilus NRRL 2155.However,further bioinformatics analysis reveals that there are many contradictions between the tal gene cluster and the structural characteristics of thermolide,instead,the tal gene cluster shares high similarity/identity to the fusarin C biosynthetic gene cluster（fus cluster）in both gene organization and constitution.Heterologous expression of the tal gene cluster in Aspergillus nidulans did not detect thermolide but produced three dominant compounds with m/z 382[M+Na]⁺showing UV absorption closed to fusarin C,indicating that the tal gene cluster is not responsible for the biosynthesis of thermolide.（2）Identification of thm gene cluster and essential genes for thermolide biosynthesis.Upon reanalysis of the gene clusters from T.thermophilus NRRL 2155,another PKS-NRPS gene cluster（thm cluster）was found,in addition to the tal gene cluster.Seven conserved genes（thm A-thm G）encoding a PKS,a NRPS,a short-chain dehydrogenase/reductase（SDR）,a flavin-dependent oxidase,an acetyltransferase,a glycosyl hydrolase,and a glycosyl transferase were identified in the thm gene cluster.Heterologous expression of the thm gene cluster in A.nidulans resulted in efficient production of thermolides 158 and 160,and the production of thermolide derivatives192 and 193 with valine incorporation,confirming that the thm gene cluster is indeed responsible for the synthesis of thermolide.Further experiments showed that only four genes（thm ABCE）are essential for the production of thermolide.Subsequent hydrolysis and derivatization reactions also confirmed the L-configuration of the amino acid in the thermolide structure,which is in good agreement with the function and organization of the genes in the thm cluster.Therefore,compounds 158-164 was renamed 158’-164’.The identification of the thm gene cluster and essential genes（thm ABCE）laid a foundation for subsequent study on the thermolide biosynthesis pathway.The modification of the amino acid unit configuration in the thermolide structure provides a basis for further functional verification of Thm B.（3）Catalytic function of polyketide synthase Thm A and nonribosomal peptide synthase Thm B.Unlike traditional fungal PKS-NRPS,which is a single fusion protein,the single-module HRPKS and NRPS of thm gene cluster are separated into two independent enzymes.The HRPKS module（Thm A）consists of seven domains in the order KS-AT-DH-MT-ER-KR-ACP and the NRPS module（Thm B）contains four domains（C-A-T-C_T）.Co-expression of thm A and thm B in A.nidulans resulted in the formation of macrolactones 194 and 195,suggesting that Thm A and Thm B are sufficient to synthesize core macrolactone backbone.This conclusion was also confirmed by in vitro enzymatic characterization of recombinant Thm A and Thm B proteins expressed and purified from Saccharomyces cerevisiae.Site mutagenesis and complementation experiments demonstrated that the Thm B terminal condensation（Thm B-C_T）domain catalyzes the formation of ester bond to produce macrolactone backbone and release products,with H₁₂₃₂ playing a crucial role.Based on these results,the model for the formation of the thermolide macrolactone backbone was proposed as follows:Thm A catalyzes the formation ofα-methyl-β-ketoacyl-S-ACP polyketide chain 196 with 18 carbon atoms using acetyl-Co A as the starting unit and malonyl-Co A as the extension unit through eight extensions.The adenylation（A）domain of Thm B recognizes and activates L-alanine or L-valine loaded onto the thiolation（T）domain,while the condensation（C）domain catalyzes the condensation of polyketide chain and amino acids to form linear amide precursor 197.His residue(H₁₂₃₂)of the Thm B-C_T domain acting as a base,extracts hydrogen from the C-12 hydroxyl group to form the deprotonated hydroxyl group.The ester bond forms by nucleophilic attack from the deprotonated hydroxyl group to carbonyl carbon of thioester.Thm B-C_T domain-catalyzed macrocyclic esterification is a new function of fungal NRPS C domain,enriching the functional library of fungal NRPS C domain and providing a reference for further study of fungal C domain function.This also provides an important biological element for the formation of ester compounds.（4）Catalytic function study of Thm C.Bioinformatics analysis showed that Thm C belongs to the SDR family.Co-expression of thm ABC genes in A.nidulans resulted the production of compounds 161’and 162’with a hydroxyl group at the C-6position.Thm C protein was expressed and purified from E.coli,and its biochemical characterization showed that Thm C can indeed reduce the C-6 carbonyl group of 195to a hydroxyl group,thus confirming that Thm C is a C-6 carbonyl reductase.（5）Catalytic function study of acetyltransferase Thm E.Bioinformatics analysis predicted Thm E as an acetyltransferase.Heterologous co-expression of thm ABE genes in A.nidulans produced acetylation products 198-201,indicating that Thm E can also modify 194 and 195.Thm E was expressed and purified from E.coli,and its function was further confirmed by in vitro enzymatic characterization.Time course experiments of compound 200 in buffer C revealed that the acetylation at the C-18 position of 201 resulted from spontaneous transformation of the intramolecular acetyl group.Thm C and Thm E substrate competitive time course experiments in vitro showed that Thm E has no obvious preference for reducing and non-reducing substrates（162’and 195）;meanwhile,Thm C prefers the acetylated substrate 200.Based on the above results,the primary pathway of post-modification of thermolide was determined to be the acetylation of the C-16 position catalyzed by Thm E,followed by the reduction of the C-6 carbonyl group completed by Thm C.Furthermore,in vitro biochemical experiments revealed that Thm E has good substrate versatility.Thm E can catalyze the production of mono-acylated,di-acylated,or mixed acylated derivatives with different chain length using acetyl-Co A,butyryl-Co A,hexanoyl-Co A,and octanoyl-Co A as acyl donors.The engineered strain E.coli BL21-thm CE was obtained by co-expression of the thm E and thm C genes,and bioconversion of 195 led to various esterified thermolides.The production of different esterification products greatly enrichs the thermolide compounds library,which provides a material basis for further screening compounds with nematodetoxic activity,and also showed the potential application value of Thm E as a biocatalyst for acylation.（6）The proposed biosynthetic pathway of thermolide.Based on the above-mentioned functional characterization of four genes（thm ABCE）in the thm gene cluster,the biosynthetic pathway of thermolide was deduced as follows:Thm A and Thm B collaborate to catalyze the formation of the polyketide-amino acid linear long chain product 197,and the Thm B-C_T domain catalyzes the esterification and release of197 yielding macrolactones 194 and 195.Subsequently,Thm E catalyzes acetylation at C-16 position to form compounds 198 and 200,which can be spontaneously converted to compounds 199 and 201.All compounds 198-201 could be reduced by Thm C to produce final products 158’,160’,192 and 193.The elucidation of the biosynthetic pathway of thermolide supports the further study of fungal PK-NRP macrolactone natural products,as well as the metabolic regulation and synthetic biology modification of thermolide derivatives.（7）Genome mining and characterization of thm-like gene clusters.Eight related gene clusters from fungal genome in NCBI and in our laboratory were identified by genome mining using Thm A and Thm B as probes.Heterologous expression of acr and fca cluster produced 2-aminobutyric acid and phenylalanine type thermolide derivatives（205-213）,in addition to alanine and valine thermolide compounds.The discovery of unnatural amino acid and aromatic amino acid thermolide analogues greatly enriches the structural types of thermolide and provides novel backbone compounds for the development of natural drugs against nematodes.（8）High yield of thermolide products.We identified the AN-gpd A-acr A+gla A-acr B strain as the most efficient strain for producing thermolide products,after conducting gene combination screening and promoter modification.Further optimization of fermentation conditions showed that the maximum yield of the thermolide core backbone,reaching to 304.90±14.05 mg/L in solid medium fermentation at 30°C.This approach achieves green and efficient production of thermolide products.In this study,we achieved several important objectives.Firstly,we identified the biosynthetic gene cluster（thm cluster）of thermolide.Secondly,we clarified the mechanism of synthesis for fungal single module PK-NRP hybrid macrolactone natural products,in which the NRPS C_T domain catalyzes macrocyclic esterification and releases T domain-tethered PK-NRP intermediates.Thirdly,we synthesized a diverse range of esterified and amino acid-based thermolides.Finally,we succeeded in overproducing the core structure of thermolide using a heterologous system.Our study sheds light on the synthesis of natural products derived from microorganisms inhabiting extreme environments and supports the further application of thermolide as valuable nematocidal agents for biological control in agriculture.