microbial fermentation ensures production of industrially important metabolites in large quantities . implication of rdna technology in microbial fermentations offers production of desired heterologous proteins at large scale .
apart from the proteins , nature offers diverse classes of complex metabolites ( isoprenoids ) that are utilized in the food , cosmetic , and pharmaceutical industries and so forth .
many of these complex metabolites are produced naturally in low quantities in plants that are difficult or expensive to cultivate .
metabolic engineering , systems , and synthetic biology principles and methods allowed easy transfer of heterologous pathways from natural plant producer to a suitable microbial host such as yeast and e. coli [ 24 ] .
e. coli was the most studied host for metabolic engineering of isoprenoid by modulating 1-deoxyxylulose-5-phosphate ( dxp ) and mevalonate ( mva ) pathways [ 58 ] .
other studies on mva pathway deregulation in yeasts have improved the biosynthesis of different isoprenoids [ 913 ] .
the vital role of mva pathway and eventual product ( ergosterol ) proportion from this pathway increased the interest for the engineering of isoprenoid pathway in yeast for the production of heterologous compounds .
ergosterol synthesis was carried out by squalene synthase ( erg9 ) , and conversion of farnesylpyrophosphate ( fpp ) to squalene was successfully regulated by replacing the native promoter with repressible promoters [ 2 , 15 ] .
atp sulfurylase ( met3 ) catalyzes the reduction of sulfate to sulfide , involved in methionine metabolism .
addition of methionine had the ability to repress the met3 promoter in s. cerevisiae and ashbya gossypii [ 16 , 17 ] , and met3 promoter was widely used as a molecular tool for yeast genetics .  
heterologous expression of pathways / enzymes in s. cerevisiae is complex due to the presence of numerous native host enzymes and tight regulation of the intermediate metabolites generated by the host machinery .
the non native product formation is not only affected by the host environment but also by the loss of intermediate metabolites through diffusion , degradation , or converted by competitive enzymes / pathways [ 19 , 20 ] . in order to avoid such intermediates loss and make heterologous expression more efficient ,
enzymes catalyzing successive reactions are often fused in close proximity to each other by using small linkers .
small linkers are the sequence codings for few amino acids , which separates the two proteins in space with a small distance allowing them to fold properly without constraints from each other . consequently , the substrate was channeled between active sites of two or more sequential enzymes of a pathway , without allowing free diffusion of the intermediates .
subsequently it reducing the transit time required for the intermediates to spread the enzyme that catalyzes the next step in the reaction .
several elucidations in vitro and in vivo recommended that this strategy can be used to improve the flux through a metabolic pathway [ 2125 ] . in the present study
, we used s. cerevisiae ( mtcc 3157 ) for heterologous expression of amorphadiene synthase ( ads ) for one - step conversion of fpp to amorphadiene . in order to increase the concentration and flux of fpp towards heterologous product , squalane synthase ( erg9 ) a key enzyme in ergosterol synthesis
further to avoid fpp loss through competitive pathways , enzyme fusion strategy was applied . here
, we composed a chimeric fusion protein between farnesyl diphosphate synthase ( fpps ) of yeast and amorphadiene synthase ( ads ) of artemisia annua l. and was expressed in erg9-repressed yeast strains .
all the reagents and media used in this study were of , analytical grade and procured either from himedia ( india ) or merck ( india ) or sigma ( india ) .
the strains used in this study were saccharomyces cerevisiae ( mtcc 3157 ) , and e. coli dh5 ( mtcc 1652 ) .
the met3 promoter was amplified from genomic dna of s. cerevisiae ( mtcc 3157 ) using the primer pairs 1 and 2 ( table 1 ) containing restriction sites bcui and cfr42i .
pcr was carried out in a total volume of 50  l with the following reagents : 1x ex taq dna polymerase buffer ( stratagene ) , 0.2  mm dntps ( pharmacia ) , 20  pmol of each primer , and 1  u ex taq dna polymerase ( takara , japan ) .
this pcr includes two cycles of 94c ( 0.5  min ) , 50c ( 1.0  min ) , and 72c ( 1.5  min ) ; 29 cycles of 94c ( 0.5  min ) , 56c ( 1  min ) , and 72c ( 1.5  min ) ; and 72c ( 5  min ) .
resultant pcr fragment and pug6 plasmid ( euroscarf , germany ) were digested with bcui - cfr42i restriction enzymes .
the vector and pcr fragments were purified with nucleospin gel and pcr clean - up kit ( macherey - nagel , germany ) and separated on 1% agarose gel ( merck biosciences , india ) , and gel was further purified using nucleospin gel and pcr clean - up kit ( macherey - nagel , germany ) .
ligation of the vector and pcr products was carried as per the standard protocol given for t4 dna ligase ( merck biosciences , india ) .
the ligated plasmid mix was transformed into competent e. coli ( dh5 ) mtcc 1652 , and transformants were selected on lb medium supplemented with ampicillin ( 50  mg / l ) , and the plasmid obtained was named as pug6 met3 . 
 erg9 promoter was replaced with methionine promoter by using fusion pcr , and four fragments were separately amplified before fusing them together in pairwise by using fusion pcr and a bipartite gene targeting method ( figure 1 ) [ 15 , 26 ] .
two fragments containing the met3 promoter and the kanmx selection cassette were amplified from plasmid pug6 met3 in two separate , but overlapping fragments using the two pairs of primers ( 3 ) a , b and ( 4 ) a , b ( table 1 ) .
also 500  bp upstream region of the erg9 promoter in the genome of s. cerevisiae was amplified using primers ( 5 ) and ( 6 ) .
subsequently , the first 500  bp of the erg9 open reading frame ( orf ) region was amplified using primer pairs ( 7 ) and ( 8) ( table 1 ) .
finally , pcr fragments were gel purified using the nucleospin gel and pcr clean - up kit ( macherey - nagel , germany ) and subsequently fused together in pairs using fusion pcr to obtain two fragments , first one with met3 promoter and 500  bp orf region of erg9 using primers ( 9 ) and ( 10 ) and a second fragment containing kanmx and first 500  bp upstream region of erg9 using primers ( 11 ) and ( 12 ) ( table 1 ) .
subsequently , fusion pcr fragments were gel purified with the nucleospin gel and pcr cleanup kit ( macherey - nagel , germany ) .  
an fpps ( erg 20 ) gene fragment was obtained by pcr amplification as mentioned above using genomic dna of s. cerevisiae mtcc 3157 with the primer pairs ( 13 ) and ( 14 ) .
the fragments were digested with ecori and clai and inserted into an ecori - clai vector fragment 2 based py01ura plasmid ( genecopeia usa ) and the resulting plasmid is designated as py01fpps . for construction of plasmid expressing fusion protein fpps - ads , first fpps and ads
were amplified separately using primer pairs ( 13 ) , ( 14 ) and ( 15 ) , ( 16 ) using py01fpps and prs425ads ( addgene#20119 ) as templates .
the two resulting pcr fragments were fused in the second round of pcr using primers ( 17 ) and ( 18 ) .
similarly , ads - fpps fragments were generated in the first round using the primer pairs ( 19 ) , ( 20 ) and ( 21 ) , ( 22 ) and the resulting fragments fused together in second round of pcr using primer pair ( 23 ) and ( 24 ) .
the resulting two pcr fragments were cut with ecori - clai and individually cloned into ecori - clai plasmid 2-based py01ura plasmid ( genecopeia , usa ) and the resulting plasmids were named as py01fpps - ads and py01ads - fpps , respectively .
transformation of all strains of s. cerevisiae was performed by lithium acetate and peg mediated transformation by using yeastmaker transformation system 2 kit ( clontech , usa ) .
s. cerevisiae strain ( mtcc 3157 ) , was used as the parent strain for all s. cerevisiae strains used in this study .
strain ycf - ad was constructed by the transformation of pesc - ura - ads plasmid ( constructed by using pesc - ura and addgene#20119 ) in to mtcc 3157 .
strain ycf-002 was generated by transforming the fusion pcr fragments in to strain ycf - ad and selected on kanamycin and synthetic defined ( sd)-ura drop out plates . finally strain ycf-001 obtained by exclusion of plasmid pesc - ura - ads from strain ycf-002 by selection on plates containing 5-fluoroorotic acid ( 5-foa ) .
strain ycf-004 , ycf-005 , and ycf-006 constructed by transforming the plasmids py01fpps , py01fpps - ads , py01ads - fpps , and the transformants were selected on sd - ura drop out plates ( table 2 ) .
optical density values of samples in triplicates were measured at 600  nm ( od600 ) by using uv - spectrophotometer ( thermo scientific , usa ) .
the dry weight was analyzed by using nitrocellulose filter papers ( pore size 0.45  m , whatman ) .
the filter papers were predried in a microwave oven at 60c for 10  min .
a known volume of the cell culture was filtered , and the residue was washed with distilled water and dried in an oven at 60c . an overnight culture grown in minimal medium supplemented with 1.5  mm methionine ( glucose 20 
g / l ) was centrifuged at 5,000  rpm for 5  min to get approximately 3  g of dry cells .
the cell pellet was washed with distilled water , and the cell suspension was centrifuged for another 5  min at 5,000  rpm .
further cell pellet was mixed with 300  ml of 25% alcoholic koh solution and vortexed for 1  min , and the suspension was saponified for 3  h at 90c in a reflux .
after cooling them to room temperature , nonsaponified sterols were extracted by adding 300  ml heptane followed by vortexing for 2  min .
a vortex mixture of 10  ml heptane and 10  ml of alcoholic koh solution is used as blank .
after clarification of heptane layer 0.5 ml of heptane from both sample and blank was diluted tenfolds with 4.5  ml absolute ethanol .
the absorbance of all samples were read against blank at 230 and 281.5  nm , respectively [ 14 , 28 ] .  
the ergosterol content was calculated as milligram ergosterol per gram dry weight using the following equation : 
 ( 1)ergosterol=%  ergosterols%24  ( 28)-dehydroergosterolergosterol  ( mggdw)=(od281.5290od230580)f , 
 where f is a correction factor for dilutions and sample sizes , and 290 and 580 are e of crystalline ergosterol and 24 ( 28)-dehydroergosterol , respectively . hundred milliliter medium was prepared with the following composition ( g / l ) : galactose , 20 ; kh2po4 , 14.4 ; ( nh4)2so4 , 7.5 ; mgso4 7h2o , 0.5 ; trace metal solution , 2 ; and vitamin solution , 1  ml and 50  l / l silicone antifoam .
the ph was adjusted to 6.20 using 1  m naoh and autoclaved separately from the carbon source solution .
variou concentrations of methionine ( 03  mm ) were used to know the minimal methionine concentration for the repression of erg9 expression .
flasks were further incubated in a shaking incubator ( remi , india ) at 30c with 150  rpm .
batch fermentation was carried out in a controlled bioreactor ( spectrochem , india ) containing 2  l mineral medium that consists of ( g / l ) galactose 20 ; ( nh4)2so4 , 5 ; kh2po4 , 3 ; mgso47h2o , 0.5 ; edta , 0.015 ; znso47h2o , 0.0045 ; coc126h2o , 0.0003 ; mnc12 4h2o , 0.001 ; cuso4 5h2o , 0.0003 ; cac122h2o , 0.0000045 ; feso47h2o , 0.0003 ; namoo4,2h2o , 0.0004 ; h3bo3 , 0.001 ; ki , 0.0001 ; and 0.025  ml silicone antifoam ( merck ) .
further filter sterilized vitamin solution containing ( mg / l ) : biotin , 0.05 ; calcium pantothenate , 1 ; nicotinic acid , 1 ; inositol , 25 ; thiamine hcl , 1 ; pyridoxine hcl , 1 ; and para - aminobenzoic acid , 0.2 was added to the autoclaved mineral medium .
finally , media were supplemented with 2  mm filter sterilized methionine . during the fermentation process
, the temperature was kept constant at 30  2c , and dissolved oxygen tension ( 50% ) was maintained with sterilized air ( 0.2   filter ) with airflow 1  l / min and with 250  rpm agitation and the off - gas passed through a outlet port .
ph was controlled between 6.20  0.5 by automatic addition of 1  m naoh and 1  m hcl .
after cells reaching 1 at od600 20% ( vol / vol ) isopropyl myristate ( merck millipore , germany ) was added aseptically to the media . this isopropyl myristate layer was sampled and diluted with ethylacetate for determination of amorphadiene concentration by gas chromatography coupled mass spectrometry ( gc - ms ) ( agilent technologies , usa ) .
amorpha-4,11-diene and farnesol were analysed by gas chromatography with flame - ionization detector ( gc / fid ) .
samples from fermentor were centrifuged at 5000  rpm for 5  min , diluted directly into ethyl acetate , and mixed for 30  min on a vortex mixer .
after phase separation 0.6  ml of the ethyl acetate , layer was transferred to a capped vial for analysis .
a 1  l sample was split 1  :  20 and separated using a db - wax column ( 50  m  200  m  0.2  m ) with hydrogen as the carrier gas with flow rate of 1.57  ml / min .
the column was initially held at 150c for 3.0  min , followed by a temperature gradient of 5c per min to a temperature of 250c .
amorpha-4,11-diene and farnesol peak areas were converted to concentration values from external standard calibrations using authentic compounds .
different yeast strains carrying the plasmids pads , pfpps , pfpps - ads , and pads - fpps were grown in appropriate sd drop out media at 37c .
after cells optical density values were reaching 2.5 at od600 , they were harvested and centrifuged at 5000  rpm for 5  min , and cell pellet was resuspended in 10  ml distilled water , and proteins were extracted according to cellytic y plus kit ( sigma , usa ) .
purification was carried out in a single step using immobilized metal affinity chromatography ( imac ) .
the supernatant was applied to a 5  ml nickel cl - agarose column ( merck biosciences , india ) , loaded with nickel .
elution of adsorbed recombinant proteins was achieved with elution buffer ( merck biosciences , india ) .
sds / page was carried out in 10% resolving and 5% stacking gels according to instructions given by the manufacturer ( merck biosciences , india ) and the samples along with protein marker were loaded on mini - protean tetra cell ( bio - rad , usa ) .
gel , visualized and analyzed in gel doc fire reader documentation system ( merck biosciences , india ) .
the minimum concentration of methionine for expression of erg9 was determined by growing the erg9 repressed yeast cells in shake flasks supplemented with varying quantities of methionine ( figure 2 ) . when yeast cells grown in the absence of methionine , cells propagated exponentially up to 10  h and followed by a slight drop in the growth and immediately after 12  h cells followed exponential phase up to 20  h. the methionine concentration at 1  mm elongated the lag phase and a lower final biomass concentration .
there was a drastic decrease in the growth of the yeast beyond 2.5  mm methionine concentration , and the consequences were observed by measuring the ergosterol content ( table 3 ) .
the final ergosterol content of control yeast strain ( mtcc 3157 ) was observed at 19.25  mg / g dw , and strain ycf-005 was given varied quantities of ergosterol at 0 , 1 , 2 , 2.5 , and 3  mm methionine as 19.22 , 14.05 , 13.98 , and 13.75 and 3.31  mg / g dw , respectively ( table 3 ) . transformed yeast strains harbouring amorphadiene synthase ( ads ) was able to produce amorpha,4 - 11-diene ( figure 3 ) .
strain ycf-005 analyzed for amorphadiene production by gc - ms as mentioned in materials and methods .
analysis of gc - ms for ethyl acetate extracts revealed the presence of amorphadiene major peak and other sesquiterpene as a minor peak ( figure 3(b ) ) .
retention time , mass spectrum of main component ( 19.75 and 14.21  min ) were almost matching with standard ( 19.70 and 14.18  min ) ( figures 3(a ) , 3(c ) , and 3(d ) ) .
l of amorphadiene , which was approximately 2-fold higher than ad ( 5.65  mg / l ) produced by ycf - ad respectively ( figure 4 ) .  
fpp conversion to farnesol in the strain ycf-004 was limited by expressing the fusion protein in yeast and to probe the effect of enzyme fusion on diversion of the flux more efficiently towards amorphadiene production .
different strains expressing the native proteins and chimeric proteins ads , fpps - ads , and ads - fpps were analyzed .
the strain transformed with the plasmid expressing ads - fpps produced amorphadiene ( 12.08  mg
/ l ) at the same level as that for the strain expressing free ads ( figure 4 ) .  
more interestingly , the strain transformed with the plasmid expressing fpps - ads produced amorphadiene at an almost 2-fold higher level ( 25.06  mg / l ) than that for the strain expressing free fpps and ads ( figure 4 ) .  
on sds - page the four proteins were compared across the protein molecular marker ( figure 5 ) . both fused proteins fpps - ads and ads - fpps molecular weights were approximately 105  kd which was almost equal to the sum of two individual proteins ( fpps 40  kd and 63  kd ) .
in the present study , we have improved the amorphadiene production by 4-fold in the yeast s. cerevisiae ( mtcc 3157 ) . in order to decrease
the fpp flux towards ergosterol and competitive pathways , erg9 promoter was replaced with repressible methionine ( met3 ) promoter , and strain ycf-005 growth rate was very low at 3.0  mm methionine concentration , due to low ergosterol production .
the ergosterol content of the erg9 repressed strain was quite low compared with control strain ( mtcc 3157 ) .
increasing concentration of methionine repressed the yeast strain and produced varied quantities of ergosterol ( table 3 ) upon increasing methionine , there was a tight regulation of the promoter .
farnesol accumulation observed in earlier studies with deletion and inhibition of erg9 gene [ 3032 ]
. moreover farnesol at low concentration inhibits the growth of the cells by cell cycle arrest in some yeast cells .
we observed that strain ycf-001 produced 12.03  mg / g dw of farnesol and final concentration 24.12  mg / l and disclosed the possibility of pooling the available fpp towards amorphadiene by enzyme fusion technology .
as mentioned in earlier studies , a discrepancy of methionine was not sufficient to repress erg9 during fermentation and 2.5  mm methionine was maintained during fermentation .  
we used isopropyl myristate as a solvent for trapping the volatile compounds and for phase separation , and that phase was analyzed for determination amorphadiene by gc - ms .
analysis of ethyl acetate extracts revealed the capability of yeast strains carrying ads gene for production of amorphadiene .
the major peak in the chromatogram confirmed as a amorphadiene and other minor peak as a farnesol with corresponding standards ( figures 3(a ) and 3(b ) ) [ 34 , 35 ] .
erg9 ( ycf-002 ) repression improved the amorphadiene production by increasing the fpp availability when compared with amorphadiene production in strain ycf - ad .
strains ycf - ad and ycf-002 were cultured in the fermentor and analyzed for amorphadiene and farnesol .
strain ycf - ad produced 5.65  mg / l of amorphadiene and 4.12  mg / l farnesol , as there was a 2-fold ( 11.02 
mg / l ) rise in amorphadiene production in strain ycf-002 ( figure 4 ) . to overcome
the natural loss of the metabolic intermediate fpp and its conversion towards farnesol , a chimeric protein with small gly - ser - gly linker was constructed as ads - fpps and fpps - ads .
l of amorphadiene which was almost close to amorphadiene produced by strain ycf-002 as ycf-005 produced 25.06  mg /
l of amorphadiene and which was 2-fold higher than than the ycf-002 and subsequently 5-fold higher than ycf-001 ( figure 4 ) .
strains ( ycf-005 ) expressing fpps - ads do not produce higher amorphadiene yields compared with strains ycf-005 and ycf-002 as the active sites were likely far apart to produce a beneficial proximity effect .
the fusion phenomenon of the enzyme further confirmed by isolation and purification of fpps , ads , ads - fpps , and fpps - ads enzymes with sds - page and fused proteins molecular weights ( 105  kd ) were almost equal to the sum of the individual enzymes ads ( 63  kd ) and fpps ( 40  kd ) ( figure 5 ) .
enzyme fusion technology will be an additive step to the metabolic engineering , and combination of metabolic engineering and enzyme fusion technology improves the flux of the intermediate metabolites in order to expand the heterologous components production .
further combinatorial expression of thmgr and upregulation of upc2 alleles with fusion technology accelerate the amorphadiene production . further channeling acetyl - coa into the mevalonate pathway
will make it possible to further increase the fpp production which is having tremendous significance in chemical industry as well as in medicine .
 the yeast strain ( saccharomyces cerevisiae ) mtcc 3157 
 was selected for combinatorial biosynthesis of plant sesquiterpene amorpha-4,11-diene . 
 our main objective was to overproduce amorpha 4 - 11-diene , which is a key precursor molecule of 
 artemisinin ( antimalarial drug ) produced naturally in plant artemisia annua through 
 mevalonate pathway . 
 farnesyl diphosphate ( fpp ) is a common intermediate metabolite of a variety 
 of compounds in the mevalonate pathway of yeast and leads to the production of ergosterols , 
 dolichol and ubiquinone , and so forth . in our studies 
 , fpp converted to amorphadiene ( ad ) by 
 expressing heterologous amorphadiene synthase ( ads ) in yeast . 
 first , 
 erg9 ( squalane synthase ) promoter of yeast was replaced with repressible 
 methionine ( met3 ) promoter by using bipartite gene fusion method . 
 further to overcome the loss of the 
 intermediate fpp through competitive pathways in yeast , fusion protein technology was adopted 
 and farnesyldiphosphate synthase ( fpps ) of yeast has been coupled with amorphadiene 
 synthase ( ads ) of plant origin ( artemisia annua l. ) where amorphadiene 
 production was improved by 2-fold ( 11.2  
 mg / l ) and 4-fold ( 25.02  mg 
 / l ) in yeast strains 
 ycf-002 and ycf-005 compared with control strain ycf - ad ( 5.5  mg / l ) , respectively . 
1. Introduction 
2. Materials and Methods
3. Results
4. Discussion
5. Conclusion