Heterologous Biosynthesis Of Lovastatin And Monacolin J On Methanol And Ethanol By Engineered Pichia Pastoris

As a natural polyketide drug produced by Aspergillus terreus,lovastatin is used to treat hypercholesterolemia due to its inhibition in(3S)-hydroxy-3-methylglutaryl-coenzyme A(HMG-CoA)reductase,a key enzyme catalyzing cholesterol biosynthesis,and its intermediate,monacolin J,is a key precursor of the semi-synthetic drug,simvastatin(Zocor).Both lovastatin and simvastatin are widely prescribed antihypercholesterolemic drugs with commercial and medical value.Currently,nearly all commercial lovastatin drugs are produced through fungal fermentation,and monacolin J is obtained by chemical hydrolyzation of lovastatin for simvastatin production.However,fermentation by the native fungus such as A.terreus is still challenged by long fermentation period,producing multiple byproducts and environmental risks from waste discharges.Meanwhile,chemical hydrolysis for simvastatin production needs catalysts which are not environmentally friendly,and harsh conditions are also required by the chemical reaction.Recently,development of synthetic biology has paved a way for heterologous expression of biosynthetic pathways in chassis organisms to produce natural products,providing solutions to solve problems with natural production systems.Pichia pastoris has been widely used to express heterologous recombinant proteins.In comparison with prokaryote express systems,P.pastoris is capable of post-translational modifications but without excessive glycosylations as that observed in Saccharomyces cerevisiae.Meanwhile,P.pastoris can grow to a high cell density in simple synthetic media.It can also utilize broad carbon sources such as glycerol,methanol and ethanol for robust metabolism.Moreover,its genetic operations have been investigated for years,and host strains and expressing vectors have been commercialized.To present,a large number of recombinant proteins have been successfully expressed in P.pastoris,while only a few cases have been reported on the production of small molecule pharmaceuticals using this platform.In this work,the biosynthetic pathway of lovastatin was reconstructed and optimized in P.pastoris to improve the production of monacolin J and lovastatin with major progress below:(1)The native methanol-induced promoter PAOX1 from P.pastoris was used to direct the expression of key enzymes for lovastatin biosynthesis,and co-expression vectors were constructed for engineering the species to produce monacolin J and lovastatin.Condon optimization for lovA and coexpression of cpr enhanced the production of monacolin J from monacolin L,while condon optimization for lovD improved the transformation of monacolin J to lovastatin.All intermediates in the metabolic pathway including dihydromoacolin L,monacolin L and monacolin J were identified and characterized by HPLC,LC-MS and NMR.(2)Gene copies were optimized for the metabolic pathway using the antibiotics-stress based screening strategy,which improved lovastatin production to 19 mg/L in shake flasks with methanol as the feedstock.In 5-L bioreactor,the high gene-expression strain produced 288 mg/L lovastation,268%higher than that produced by the strain with single-copy genes.We further divided lovastatin synthetic pathway into two modules and engineered them separately into P.pastoris strains to construct a co-culture system,and when the inoculation ratio was 1:1,the consortium produced 25 mg/L lovastatin,71%higher than that produced by strain engineered with the whole pathway.The co-culture was further performed at a 5-L bioreactor.When inoculation ratio was controlled at 1:2(Strategy B),594 mg/L monacolin J was produced,and when inoculation ratio 1:1 was applied(Strategy D),255.5 mg/L lovastatin was produced,17%and 220%higher,respectively than that produced by strain engineered with the whole pathway.(3)An ethanol-induced transcription system was constructed for ethanol to be used as carbon source to improve the supply of precursor acetyl-CoA for the lovastatin synthetic pathway.We found that P.pastoris grew better with ethanol as the sole carbon source than methanol,and equivalent cell density was achieved as that with glucose or glycerol as the carbon source,indicating that P.pastoris could grow to high cell density with ethanol as the feedstock.Genes adh3,ald and acs1 encoding key enzymes for ethanol metabolism were deleted from P.pastoris,and mutant strains showed poor growth,demonstrating that ethanol-to-acetyl-CoA is the main metabolic pathway in P.pastoris.Green fluorescent protein was used as a reporter to examine the expression strength of genes,and we found that the eGFP expression of PICL1-LacI-Mitl/lacO-cPAOX1 in ethanol was 2.8-fold higher than that driven by PZOX1 in methanol.When cultured with glucose,the eGFP expressing was 6.7%of that observed in ethanol,indicating that the engineered ethanol-induced transcription system could be regulated by the switch of carbon source.(4)The engineered ethanol-induced transcription system was then further examined by the heterologous biosynthesis of 6-MSA,and its production reached 690 mg/L in flasks,which improved 305%than that produced with the PAOX1 native system in methanol.Meanwhile,63 mg/L dihydromonacolin L was produced using this system,which was 184%higher than that produced by the PAOX1 native system in methanol.Furthermore,adh2,ald6 and mutated acs1 from S.cerevisiae and the endogenous mutated accl from P.pastoris were over-expressed to enhance the transformation from ethanol to malonyl-CoA,which finally led to a high dihydromonacolin L production of 185 mg/L.Co-culturing this strain with P.pastoris strain engineered with sLovA and CPR by constitutive expression system produced 156 mg/L monacolin J in flasks.Finally,2.2 g/L monacolin J was produced in a 5-L bioreactor,which shows potentials for industrial applications.