Heterologous Reconstruction Of Monacolin J Biosynthetic Pathway In Aspergillus Niger And Fermentation Optimization

Monacolin J（MJ）,as a key precursor in the synthesis of hypolipidemic drug lovastatin,could be used to synthesize important statin drug simvastatin by hydrolyzing lovastatin and adding different side chains.At present,the synthesis of MJ is mainly done through chemical alkali hydrolysis,which has caused many problems such as cumbersome chemical process,generation of by-products,difficulties of separation,and serious environmental pollution from industrial sewage.In this study,four genes（lov B/lov C/lov G/lov A）related to the biosynthesis of lovastatin key precursor MJ in Aspergillus terreus ATCC20542 were successfully integrated into the genome of Aspergillus niger,and the MJ biosynthetic pathway was reconstructed.Through fermentation optimization,MJ was successfully synthesized in heterologous host.The main research contents and conclusions of this study were as follows:（1）Construction of recombinant expression vectors of MJ biosynthetic pathway genes based on Saccharomyces cerevisiae 2μhomologous recombination technology and In-fusion technology.To overcome the low efficiency of constructing long-fragment PKS gene lov B expression cassette,2μhomologous recombination technology was adopted and lov B expression cassette was successfully constructed.Through in-fusion technology,constitutive strong promoters Pgpda and Ptef were used in the construction of the expression vector of lov C/lov G/lov A which contained 500 bp homology arms.The target integration sites of the designed lov C/lov G/lov A were glucoamylase,pyr G_AN,acid amylase and neutral amylase sites respectively.The correct expression vectors were confirmed by enzyme digestion and sequencing verification.（2）Heterologous reconstruction of MJ biosynthetic pathway in A.niger and assay of MJ product.Based on the reuse of pyr G selection marker and CRISPR/Cas9 homology-directed recombination（CRISPR-HDR）technology,the constructed expression cassettes of lov B/lov C/lov G/lov A were successfully integrated into A.niger genome.The integration and expression of target genes were verified by PCR and RT-PCR.Finally,the A.niger engineered strain CBS-LovBCGA,in which the MJ biosynthetic pathway was reconstructed,was successfully obtained.After 7-day cultivation in the fermentation medium,the products were extracted from the medium and detected by HPLC.Results indicated that the engineered strain CBS-Lov BCGA could produce a compound with the same retention time as the MJ standard（retention time was 6.486 min）.LC-MS results showed that the ion peak of this compound was339.22[M-H]⁺,and the ion fragments information of LC-MS/MS tandem mass spectrometry were also consistent with the standard（the value of the main ion fragment:173.1327,199.1480,225.1620 and 285.1830）.Taken together,the compound with retention time of 6.486 min in the fermentation extract of A.niger strain CBS-Lov BCGA was determined as MJ.（3）Preliminary exploration of fermentation process optimization and metabolic engineering of MJ-producing strain A.niger CBS-Lov BCGA.By optimizing initial p H,carbon source,adding exogenous precursors etc.,the yield of MJ was improved.Under the conditions of p H 5.5,4%maltose as carbon source,and exogenous addition of 0.1%sodium citrate and0.1%sodium acetate precursor,the yield of MJ reached 132.12 mg/L,which was improved by42.2%than that in the initial fermentation condition（92.90 mg/L）.Based on this fermentation process,further optimization was performed by shake-flask fed-batch fermentation.On the 4^thday of fermentation,maltose was supplemented to the initial medium sugar concentration,and the fermentation was continued to the 9^th day.The yield of MJ reached 142.61 mg/L,which was improved by 53.5%than the yield that under the initial glucose culture condition.Moreover,in this study,strategies such as optimization of biosynthetic genes,double-copy expression,and free plasmid expression were applied for the heterologous synthesis of MJ.However,these strategies failed to effectively improve the synthesis level of MJ.The yield of MJ by codon optimization in the CBS-Lov BCHc GA strain was 112.82 mg/L,the yield of MJ in the double-copy lov A gene strain CBS-Lov BCGAA-5 was 116.55 mg/L,and the yield of MJ in the engineered strain by free plasmid expression of lov G/lov A was only 55.54 mg/L.In conclusion,in this study,an engineered MJ producing A.niger strain was successfully obtained by heterologous reconstruction of MJ biosynthetic pathway.By optimizing shake flask culture condition and fed-batch fermentation,the yield of MJ reached 142.61 mg/L.This study provided the potential of A.niger to heterologous synthesis of other secondary metabolites.It also provided novel insights and practical strategies for the synthesis of active derivatives based on MJ.