Functional Characterization Of Proline Hydroxylase In Echinocandin Biosynthesis And Developing CRISPR/Cas9 Gene Editing System In Aspergillus Pachycristatus

Filamentous fungi are capable of producing large amounts of biologically active secondary metabolites,which are important medicine precusors.However,yield of secondary metabolites with specific biological activities produced by filamentous fungi are generally very low,making it difficult for commercial application.Thus,many efforts have been made to increase the yield and activity of fungi secondary metabolites.Echinocandins synthesized by certain filamentous fungi are the most effective drugs for the treatment of invasive diseases caused by fungi such as Candida.However,the echinocandins are relatively expensive,which limits its clinical application.Therefore,it is urgent to study the biosynthesis mechanism of echinocandin aiming to increase their yield and activity and reduce the cost.Echinocandin B,the precursor of anidulafungin,is a secondary metabolite produced by the filamentous fungus Aspergillus pachycristatus.It is a cyclic hexapeptides synthesized by a non-ribosomal polypeptide synthetase(NRPS).Notably,its synthesis process involves multiple hydroxylation reactions catalyzed by different oxidases.Thereinto,the hydroxylation of proline and 4-methyl-proline in echinocandin B has not been investigated in detail.The echinocandin B biosynthesis mechanism also has not been completely elucidated.In the present study,the biosynthesis of echinocandin B in A.pachycristatus was physiologically and genetically characterized.Specifically,HtyE was identified as the enzyme responsible for the hydroxylation of proline and 4-methyl-proline.Its enzymatic properties,functions and key sites for catalyzation were investigated in vivo and in vitro.In view of the difficulty in genetic manipulation of Aspergillus pachycristatus,CRISPR/Cas9 system for genome editing was established the in this strain.The main findings of the present study are summarized as below:1.Physiological and genetic characterization of the biosynthesis of echinocandin B in A.pachycristatusThe production of echinocandin B by A.pachycristatus was found to be related to the growth.In the late growth stage(day 5),the yield of echinocandin B maximally reached 0.7 mg/g.Genome sequencing revealed that both the echinocandin B and aculeacin A of A.aculeatus were synthesised by two subcluster,ecd and hty cluster.Ecd cluster composed of 7 genes,containing the non ribosomal polypepetide synthase gene ecdA while hty cluster consisted of 6 genes(htyA-F),being responsible for the synthesis of L-homotyrosine in the fourth position of hexacyclopeptide.Transcription analysis indicated that the expression of ecdA and ecdk were probably the limiting factors in the synthesis of echinocandin B.2.Ap-htyE plays critical roles in the biosynthesis and activity of echinocandin B in A.pachycristatus.HtyE can catalyze the synthesis of trans-3-hydroxylproline and trans-4-hydroxylprolime in vitro with the proline as substrates.However,the ratio of traps-3/traps-4 produced by two different HtyE is significantly different.Ap-htyE gene was knocked out in A.pachycristatus strain and the fermentative products of Ap-htyE deletion strain was examined by HPLC,LC-MS and anti-Candida albicans assays.The results showed that deletion of Ap-htyE abolished the production of echinocandin B and anti-Candida activity,suggesting that Ap-htyE is essential for the synthesis and acitivity of echinocandin B.The putative proline hydroxylase in A.pachycristatus and A.aculeatus echinocandins biosynthetic gene clusters were identified as Ap-HtyE and Aa-HtyE,respectively.The identity of Ap-HtyE and Aa-HtyE were 69.9%.Ap-HtyE and Aa-HtyE are heterologously expressed and purified.The enzymatic properties of Ap-HtyE and Aa-HtyE were investigated in vitro.Our results showed that both Ap-HtyE and Aa-HtyE converted L-proline to trans-4-hydroxylproline and trans-3-hydroxyproline.Interestingly,the ratio of two different forms of hydroxyproline produced by Ap-HtyE and Aa-HtyE is 2.5:1 and 7.2:1,respectively,suggesting their different selectivity on the position of hydroxylating proline.3.Leu182 was identified as a key residue in determining the regioselectivity of Ap-HtyE through site-directed mutagenesis studies.Notably,the efficiency in C-3 hydroxylation of 4R-methyl-proline has no direct correlation with the ratio of trans-4-hydroxylproline totrans-3-hydroxylproline catalyzed by HtyE.The active site residues of Ap_HtyE were identified by homology modeling.Our further site-directed mutagenesis revealed that mutant L182N converted L-proline to trans-4-hydroxylproline andtrans-3-hydroxylproline with a ratio of 7.4:1,which is very different from that of wildtype.Therefore,Leu182 is a key residue in determining the regioselectivity of Ap-HtyE.Our biochemical characterization showed that 4-methyl-proline was specifically hydroxylated at position 3 by Ap-HtyE producing 3-hydroxyl-methylproline.However,our mutagenesis study showed that the efficiency in C-3 hydroxylation of 4R-methyl-proline has no direct correlation with the ratio of trans-4-hydroxylproline totrans-3-hydroxylproline catalyzed by HtyE.4.The CRISPR/Cas9 system was established in A.pachycristatus to facilliate its genetic engineering.Due to the low efficiency of homologous recombination in A.pachycristatus,the genetic manipulation of A.pachycristatus is proven to be difficult.The CRISPR/Cas9 system is an effective tool for genome editing.In order to establish the CRISPR/Cas9 in A.pachycristatus,we constructed the plasmid,containg both Cas9 and guide RNA expresson cassete.The expression of Cas9 was dirven by the gpdA promoter and gpdAterminor of A.pachycristatus while the expression of guide RNA was driven by promoter of 5s rRNA of Aspergillus niger.As a proof of concept,the pyrG gene was successfully knocked out using this system.Three different guide RNAs at different positions of pyrG gene were slected to test their positional effects.Our results showed that the CRISPR/Cas9 system can be used in A.pachycristatus genome editing.Furthermore,the homologous recombinantion with the constructed CRISPR/Cas9 was tested by cotransforming both the donor DNA and the guide RNA.As proof of concept,deletion efficiency of conidia pigment polyketone synthase gene pksP of A.pachycristatus was 93.75%when only 40 bp of homologous region was used in the donor DNA.These results fully supported that the CRISPR/Cas9 is valid and feasible in genome editing of A.pachycristatus.