Construction of a Synthetic Metabolic Pathway for the Production of 2,4

Home , Homoserine dehydrogenase

Construction of a synthetic metabolic pathway for the production of 2,4-dihydroxybutyric acid from homoserine Thomas Walther, Florence Calvayrac, Yoann Malbert, Ceren Alkim, Clémentine Dressaire, Hélène Cordier, Jean Marie François

To cite this version:

Thomas Walther, Florence Calvayrac, Yoann Malbert, Ceren Alkim, Clémentine Dressaire, et al.. Construction of a synthetic metabolic pathway for the production of 2,4-dihydroxybutyric acid from homoserine. Metabolic Engineering, Elsevier, 2018, 10.1016/j.ymben.2017.12.005. hal-01886438

HAL Id: hal-01886438 https://hal.archives-ouvertes.fr/hal-01886438 Submitted on 27 May 2020

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Distributed under a Creative Commons Attribution| 4.0 International License Version postprint 2,4-dihydroxybutyric acidfrom homoserine. Metabolic Engineering. ,DOI :10.1016/j.ymben.2017.12.005 Walther, T.,Calvayrac, F.,Malbert,Y., Alkim,C.,Dressaire, C.,Cordier, H.,François, J.m. C A C A R R T R D P H M T h t C A c P r t p T h d h h e o o t u i I l (2018). Construction of asyntheticmetabolic pathway fortheproduction of o h h o i e e e o l c O é e e t v h e I a t u u k m b p o i v c f c n l n

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i f , i m n i c o 4 n o n g s d h f . - r , . , Version postprint 2,4-dihydroxybutyric acidfrom homoserine. Metabolic Engineering. ,DOI :10.1016/j.ymben.2017.12.005 Walther, T.,Calvayrac, F.,Malbert,Y., Alkim,C.,Dressaire, C.,Cordier, H.,François, J.m. fran_jm@insa thomas_walther@tu E * Present# 2 31077 Toulouse,France; 1 Affiliations TWB, 3TWB, rueAriane, 31520 Ramonville France Toulouse, INSA, INRA, CNRS, Toulouse, de Université LISBP, - Corresponding authors. mail adresses: Thomas Walther Thomas production of 2,4 of production (2018). Construction of asyntheticmetabolic pathway fortheproduction of Construction of Construction a synthetic address:

- toulouse.fr

Institute Natural of Materials Technology, TU Dresden, 01062 Dresden, Germany 1,2 - Dressaire dresden.de *# , Florence CalvayracFlorence,

Comment citer cedocument:

- 1,2 dihydroxybutyric fromhomoserine acid

, HélèneCordier -

St. Agne,France; 1,2 ,Yoann Malbert 1

metabolic 1,2 , Jean Marie FrançoisMarieJean

1,2 , pathway for the CerenAlkim 15 vne e agel F Rangueil, de Avenue 135 , 1,2 *

1,2 , Clémentine

- Version postprint 2,4-dihydroxybutyric acidfrom homoserine. Metabolic Engineering. ,DOI :10.1016/j.ymben.2017.12.005 Walther, T.,Calvayrac, F.,Malbert,Y., Alkim,C.,Dressaire, C.,Cordier, H.,François, J.m. OHB reductase enzyme homoserine A sterically on act which enzymes candidate several in activity reductase OHB and transaminase homoserine identified DHB to OHB of reduction the with continues transaminase homoserine the throughproceeds pathway The DHB. tohomoserine acid aminonatural the converts microbial analogue Abstract of (2018). Construction of asyntheticmetabolic pathway fortheproduction of atccu lactis Lactococcus 2,4 production from sugar, we have conceived a two a conceived have we sugar, from production 2 - - iyrxbtrt (H) s peusr o te hmcl ytei o te methionine the of synthesis chemical the for precursor a is (DHB) dihydroxybutyrate - hydroxy

overproducing - aaye daiain f ooeiet oti 2 obtain to homoserine of deamination catalyzed cognate - 4 - mtyti)uyae Sne o noae mtblc aha eit fr its for exists pathway metabolic annotated no Since (methylthio)butyrate. s

resulted in substrates, and impr and substrates, Comment citer cedocument:

by shrci coli Escherichia structure the

production of - ae ezm engineering enzyme based

tan hc expressed which strain , oved OHB reductase activity of lactate dehydrogenase lactate of activity reductase OHB oved

which is catalyzed by an OHB reductase enzyme. We enzyme. reductase OHB an by catalyzed is which 2

5.3

g/L atDHB a yield of - step synthetic metabolic pathway which pathway metabolic synthetic step - oxo

- ooeie transaminase homoserine . 4 - Fed hydroxyb - ac cliain f a of cultivation batch 0.1

utyrate g/g .

(OHB) and ,

and Version postprint 2,4-dihydroxybutyric acidfrom homoserine. Metabolic Engineering. ,DOI :10.1016/j.ymben.2017.12.005 Walther, T.,Calvayrac, F.,Malbert,Y., Alkim,C.,Dressaire, C.,Cordier, H.,François, J.m. substrate beor comme which succinate, or acetate that proposed was It methionine. yield to stage process enzymatic an in methanethiol sugars of fermentation O or acetylhomoserine producti addition, In source. sulfur the as sulfate replace to used is disulfide engineered using fermentations For alternative. promising a exampl therefore is is sulfur means, and (bio)chemical fermentation by by incorporated produced subsequently is precursor functionalized a first where processes, uncompetitive with chemical production. (L) pure (D/L) racemic produced chemically that fact 2006) al., the requires 2014) Willke, petroleum by dominated still is producedfermentation by 2014 in tons 2008) al., et Sauer diets poultry in supplements important are (HMTB) (methylthio)butyrate Introduction (2018). Construction of asyntheticmetabolic pathway fortheproduction of e, - rdcn methioni Producing sulfur The n rcs fr (L) for process on enantiomer . Zle e a. 2009) al., et (Zelder Comparatively low methionine yields methionine low Comparatively NADPH

. “edno es Service” News (“Feedinfo hs s any u t te ey ih eaoi cs o icroaig ufr which sulfur incorporating of cost metabolic high very the to due mainly is This is . The cumulated annual production volum productionannual cumulated The . -

otiig seta aio cd (L) acid amino essential containing eesd uig h ezmtc ovrin f h peusr cn e eyld as recycled be can precursor, the of conversion enzymatic the during released - (Dilger and Baker, 2007) Baker, and (Dilger dependent -

rcialized (Hong et al., 2014; Shim et al., 2016) al., et Shim 2014; al., et (Hong succinyl Comment citer cedocument: processesfrom renewable sugar,production the of methionineand HMTB e r MB y sn mr rdcd ufr ore, r i two via or sources, sulfur reduced more using by HMTB or ne - ehoie a rcnl dvlpd n hc te rcros O precursors the which in developed recently was methionine

- (Shim al., et 2016) ae shown have homoserine are produced by optimized microorganisms through the throughmicroorganisms optimized by produced are homoserine - reduction of t of reduction ae syntheses based oyeatru glutamicum Corynebacterium

) Wie ot f h 2 poengnc mn ais are acids amino proteinogenic 20 the of most While .

ht ehoie ils a b icesd n microbial in increased be can yields methionine that methionine has a very similar nutritional value as the as value nutritional similar very a has methionine renders methionine fermentation processesfermentation methionine renders he standard sulfur source sulfate to sulfide to sulfate source sulfur standard he of of

3 .

Fui e a. 20; ectnegr t l, 2005; al., et Leuchtenberger 2003; al., et (Faurie

microbial - ehoie n is nlge 2 analogue its and methionine . These precursors are then reacted withreacted then areprecursors These . e of both compounds reached ~1 million ~1 compoundsreached both of e

fermentations

hn h ls oiie dimethyl oxidized less the when

elegant two elegant

Dle ad ae, 2007; Baker, and (Dilger in combination with the with combination in - stage industrial stage - (Krö hydroxy at present at mer et mer - stage - 4 - - Version postprint 2,4-dihydroxybutyric acidfrom homoserine. Metabolic Engineering. ,DOI :10.1016/j.ymben.2017.12.005 Walther, T.,Calvayrac, F.,Malbert,Y., Alkim,C.,Dressaire, C.,Cordier, H.,François, J.m. transaminase homoserine the on homoserine precursor natural the converts which pathway metabolic amounts DHBof (<10 mg/L). characteristicintermedi alternative an constructed 2017) al., et (Walther enzyme coli sterically Escherichia on semi act by which enzymes obtained template were of engineering which activities enzymatic reductase semialdehyde malate and dehydrogenase, semialdehyde malate kinase, malate unknown previously employs pathway steps reduction successive two by followedphosphorylation by malate of pathwaysyntheticwhich metabolic production DHB for observed was urine patient’s the in deficiency, dehydrogenase semialdehyde succinic on study a to limited are organisms natural in DHB of occurrence simplify yield product % 100 near subsequent 2,4 of (2018). Construction of asyntheticmetabolic pathway fortheproduction of - dihydroxybutyrate (DHB) by microbial fermentation of sugar of fermentation microbial by (DHB) dihydroxybutyrate

In the present work we describe the design and construction of of construction and design the describe we work present the In DHB natural Annotated approach alternative An processdesign phosphoenolpyruvate carboxylase produced 1.8 g/L DHB from 20 g/L glucose in shake flaks shake in glucose g/L 20 from DHB g/L 1.8 produced carboxylase phosphoenolpyruvate chemical

strain which simultaneously expressed the optimized enzymes and the anaplerotic the and enzymes optimized the expressed simultaneously which strain

conversion of DHB and methanethiol to HMTB to methanethiol and DHB of conversion Sik e a. 2002) al., et (Shinka . and ate malyl n addition, In Comment citer cedocument:

Dc e a. 2009) al., et (Deck reduce three

- was - CoA rdcn mtblc ahas o o eit Rprs n the on Reports exist. not do pathways metabolic producing - step manufacturing converts the natural precursor malate into DHB via the activationthenatural convertsthe via precursorDHB into malate - without revealing without aaye daiain f ooeie o rdc 2 produce to homoserine of deamination catalyzed

recently . Thispathway, however, farreportedso was yield trace to only i t al. et Li

synthetic pathway which converts malate to DHB via via DHB to malate converts which pathway synthetic Teeoe w hv rcnl dvlpd three a developed recently have we Therefore, . proposed by our group our by proposed

( and 2014)

4 costs. where the accumulation of trace a trace of accumulation the where

os o poue n by any produce not does

the underlying metabolic pathway metabolicunderlying the n or group our and

cognate .

human (Walther et al., 2017) al., et (Walther T

and

he chemical process stage process chemical he into DHB. The pathway relies pathway The DHB. into

Wlhr t l, 2013) al., et (Walther sub

(Walther et al., 20 al., et (Walther consist tae. n engineered An strates. ains ufrn from suffering patients - a products

s two

in the production the in mounts of DHB of mounts - tp synthetic step ,

responsible which , and the and , - 17) rational - oxo

- . The . have step may

has the - 4 -

Version postprint 2,4-dihydroxybutyric acidfrom homoserine. Metabolic Engineering. ,DOI :10.1016/j.ymben.2017.12.005 Walther, T.,Calvayrac, F.,Malbert,Y., Alkim,C.,Dressaire, C.,Cordier, H.,François, J.m. cultivation of glucose on growing homoserine a in enzymes reductase OHB and transaminase homoserine the of Gln85Cy theintroducing (L) of activity reductase OHB respectively. activity, reductase OHB and transaminase homoserine significant had dehydrogenases organisms. natural 1986) Snell, 2005; al., organi reductase. DHB to OHB of reduction subsequent the and (OHB), hydroxybutyrate (2018). Construction of asyntheticmetabolic pathway fortheproduction of sms, as indicated by the serine the by indicated as sms, lhuh te aminotransferase other Although this strain

Our work showed that showed work Our ile 05 gL H atr 4 sae ls cliain Drn a During cultivation. flask shake h 24 after DHB g/L 0.58 yielded , we found no report on the operation of the homoserine the of operation the on report no found we , 5.3 s point mutation into the active site of the enzyme. Simultaneousexpressionenzyme.the of siteactive theinto pointmutation s Comment citer cedocument:

g/L g/L DHB wereproduced from glucoseat a molar yield of - att dehydrogenase lactate - to

- glycerate pathway which may operate in mice mice in operate may which pathway glycerate

several - re 5 natural uts ptwy apa t eit n natural in exist to appear pathways ductase

from A candidate transaminase and 2 and transaminasecandidate atccu lactis Lactococcus ,

which is catalyzed by an OHB an by catalyzed is which - overproducing strain overproducing - to

was - 0.1 DHB pathway in pathway DHB

g/g nrae by increased (Hagopian et (Hagopian - hydroxyacid . fed

- batch Version postprint 2,4-dihydroxybutyric acidfrom homoserine. Metabolic Engineering. ,DOI :10.1016/j.ymben.2017.12.005 Walther, T.,Calvayrac, F.,Malbert,Y., Alkim,C.,Dressaire, C.,Cordier, H.,François, J.m. All plasmids used in sequences.correctthe contain to sequencingDNA by shownand plasmidsisolatedwere constructed into transformed were products Ligation vectors. corresponding the into products PCR digested ligate to used was Biolabs) or (Fermentas vectors expression of Construction rpm. pre andGrowth mM by induced was enzymes pathway DHB and homoserine of expression and cultures OD initial an at inoculated were Cultures medium. mL 30 containing auxotrophies. 2017) al., et (Walther (Table S1 the of downstream and upstream hybridized that primers using PCR by collection Keio the of mutants method transduction phage the by constructed from derived were strains All conditions cultivation and Strains Methods and Materials

(2018). Construction of asyntheticmetabolic pathway fortheproduction of spoy β isopropyl ih ieiy oyeae hsoT (inye) a ue i al PCRs. all in used was (Finnzymes) PhusionTM polymerase fidelity High pre including cultivations flask shake All ).

Growth cultures for DHB production were carried out in 250 mL baffled shake flasks shake baffled mL 250 in out carried were production DHB for cultures Growth - cultures were incubated at 37 °C on a rotary shakerarotaryrunning (InforsMultitron)cultures200at incubated°C on were37 at - D - this studylisted are in Table 2 1 -

thiogalactopyranoside supplemented with 0.2 g/L methionine and threonine to complement strain complement to threonine and methionine g/L 0.2 with supplemented Comment citer cedocument: E. coli E.

(Baba et al., 2006) al., et (Baba

K -

12 substr 12

. coli E.

( PG we clue OD culture when IPTG) . MG1655. The mutant strains listed in Table in listed strains mutant The MG1655. . -

. 6 c

as the donor strains. Gene deletions were verified were deletions Gene strains. donor the as

lue wr crid u i M mnrl medium mineral M9 in out carried were ultures

Mle t l, 1992) al., et (Malke Hα el (E) sn standa using (NEB) cells DH5α 600

600

sn snl gn deletion gene single using f . rm vrih pre overnight from 0.2 of

a apoiaey 0.6. approximately was oiid eoi locus genomic modified

d protocols. rd the addition of 1 of addition the

4 N ligase DNA T4 1

were All -

Version postprint 2,4-dihydroxybutyric acidfrom homoserine. Metabolic Engineering. ,DOI :10.1016/j.ymben.2017.12.005 Walther, T.,Calvayrac, F.,Malbert,Y., Alkim,C.,Dressaire, C.,Cordier, H.,François, J.m. primers LLldhA KpnI and Xba with digested purified, was fragment PCR resulting The 823. and 820 primers contained which mix reaction the of µL 50 to fragment each of ng 150 adding by PCR extension overlap an in fused were pET28 and S1, Table in listed respectively, 822/823, and 820/821 pairs primer reverse and forward the using individually genes expression vector with digested were products of DNAGenomicrespectively. codon, stop the of downstream and RBS the upstreamof sites restriction respectively, and Construction of vectors for expression of the 28(a)+ vector (Novagen). of sites restriction corresponding the into ligated and S1 Table Supplementary in listed resul The S1. Table Supplementary in of or MG1655, EC purification: and expression protein for vectors of Construction

- E. coli E. aspC, EC aspC, LL (2018). Construction of asyntheticmetabolic pathway fortheproduction of Q85C - ldhA lsi pEXT20 Plasmid pEXT20Plasmid pEXT20 Plasmids 668

- by 12 substr. MG1655 or the pET28 the or MG1655 substr. 12 Q85C - ilvE, I, and ligated into th that introduced a ribosome binding site (RBS) in front of each gene,and each of front in (RBS) sitebinding ribosome a introducedthat and aspC L. lactis L.

genes by PCR using the forward and reverse primer couples primer reverse and forward the using PCR by genes and 7 (al S1) (Table 674 (Dykxhoorn et . Specifically, the Specifically, . LL

subsp. - - - ilvE ldhA, LL ldhA, aspC -

Comment citer cedocument: sC n pEXT20 and aspC - LLldhA - - ilvE or pET28 or ilvE lactis LLldhA BamHI

- al., 1996) e correspondinge sitespEXT20 of panE ht nrdcd RS n rn o te ee and gene, the of front in RBS a introduced that Q85C Il1403, respectively, using the forward and reverse primerslistedreverse and forward respectively,the Il1403,using aspC Q85C ting DNA fragments were digested with the restriction enzymesrestriction the with digested were fragmentsDNA ting

and

was constructed by PCR constructed was were

a otie b rpaig the replacing by obtained was gene was amplified by PCR using the forward and reverse and forward the using PCR by amplified was gene .

- SalI - LLldhA LLldh - PCR LL homoserine and the DHB pa lgtd no h crepnig ie o te pEXT20 the of sites corresponding the into ligated , - ldhA - AQ85C amplified from genomic DNA of of DNA genomic from amplified 7 Q85C

Q85C

as the template. the as ee osrce b apiyn the amplifying by constructed were

plasmid was us was plasmid - amplifying theamplifying . The genes The

The amplified PCR fragments PCR amplified The ed as the template. The PCR The template. the as ed thway: ilvE EC 609/610 and 679/680, and 609/610

EC - ee n pEXT20 in gene mdh, - ilvE

E. coli E. SacI

EC BamHI and -

ldh, EC ldh,

and K12 LL h pET the -

EC ldhA and - substr. BamHI - - - aspC tyrB, ilvE SalI Q85C - -

Version postprint 2,4-dihydroxybutyric acidfrom homoserine. Metabolic Engineering. ,DOI :10.1016/j.ymben.2017.12.005 Walther, T.,Calvayrac, F.,Malbert,Y., Alkim,C.,Dressaire, C.,Cordier, H.,François, J.m. Table Supplementary in additi in listedmutations) silent using (introduced site restriction new a contained PCR by mutated genes The 30 plasmid template each, TableSupplementary in listed pairoligonucleotide µM 0.04 primers reverse and direct LL of sequence Site SmaI of upstream reverse and the containing silent mutation inaddition to the functional for siterestriction new acontained PCR by createdPlasmids S1. Table Site (S345F). phe by positiondirectedin 345 replacedserine mutagenesiswhich site by constructed was threonine by inhibition for sensitivity decreased strongly with dehydrogenase kinase/homoserine SmaI from DNA SmaI primers reverse and forward sites theof SacI/BamHI template.theSacI/BamHIligatedusedfragmentas digestedi The PCR was pEXT20 respectively. codon, stop the of downstream and RBS the of upstream sites restriction - directed mutagenesis of OHB reductase LL reductase OHB of mutagenesis directed

, ligated into the correspothe intoligated , (2018). Construction of asyntheticmetabolic pathway fortheproduction of and restriction sites upstream of the RBS and downstream of the stop codon, respectively.codon,stop theGenomic downstreamof RBSand upstream the restriction sites of Plasmid pZA23 Plasmid pZA23 Plasmid KpnI E. coli E. - aspC ietd uaeei ws are ot s ecie aoe sn te rmr lse in listed primers the using above described as out carried was mutagenesis directed rmr (49 17, al S) hc itoue Sa ad pI etito sites restriction KpnI and SmaI introduced which S1) Table 1479, (1469, primers , ligated into the corresponding sites of thepZA23 - aspC dA ee nrdcd y C (hso 1, F ufr 0 (/) dTs . mM, 0.2 dNTPs (v/v), % 20 buffer HF 1U, (Phusion PCR by introduced were LdhA

MG1655 was used as the template. The PCR product was digested with digested was product PCR The template. the as used was MG1655

and downstream of of downstream and - LL - - - - thrA hA a cntutd y mlfig the amplifying by constructed was thrA digested and gel ldhA Comment citer cedocument: S345F Q85C 1250 1250 nding sites of the pZA23 expression vector (ExpressysexpressionvectorpZA23 the of sitesnding -

aspC operon using pEXT20 using operon and - LLldhA

LLldh - 1252 purified pEXT20 mutation facilitateto identification of mutated clones. Q85C on to the functional mutation to facilitate identification facilitate to mutation functional the to on AQ85C that introduced a RBS a introduced that

- was constructed by PCR by constructed was LdhA rsetvl.TePR rdc ws ietd with digested was product PCR The respectively. , 8 S1

and the pET28 the and - Q85 aspC - : ilvE

Point mutations to change the amino acid amino the change to mutations Point - LLldhA - LLldh - thrA . oi thrA coli E. AQ85C Q85C - S345 LLldhA in front of front in Xho

as the template and forward and template the as vector.

plasmid - - 0 n 50 amplifying a DNA fragment DNA a amplifying I which was introduced byintroduced was which I plasmid

ee y C uig the using PCR by gene nto the correspondingnto the , ae) sn the using water) g,

. thrA

TM as the templatethe as , and , ). An aspartateAn nylalanine EcoRI EcoRI - aspC

and and S1

Version postprint 2,4-dihydroxybutyric acidfrom homoserine. Metabolic Engineering. ,DOI :10.1016/j.ymben.2017.12.005 Walther, T.,Calvayrac, F.,Malbert,Y., Alkim,C.,Dressaire, C.,Cordier, H.,François, J.m. SDS was by verified enzymes eluted of Purity 7.5). pH Imidazole, mM 250 NaCl, mM 300 Hepes, mM (50 buffer elution of mL 0.5 with eluted were proteins before 7.5) pH Imidazole, mM 15 NaCl, mM 300 Hepes, bed 10 with washed was resin The and removed. centrifuge was top supernatant table a in g 700 at centrifuged was suspension The (Clontech). resin affinity Talon of volume) (bed mL 0.75 with °C 4 at h 1 for incubated was extract protein Clear supernatretainingandthe °C 4 streptomycinat min 10 for g 13000at (Sigma),centrifuging samples sulfate the mg/mL 15 adding by extracts the from removed were DNA and RNA supernatant. th centrifuging by removed VibraCell Scientific, Bioblock sonicator: %, 30 output: power s, 20 interval: (sonication sonication of rounds successive four by open broken and 5) 7. pH NaCl, mM 300 Hepes, enzymes: of Purification further analysis. Culture media contained 50 at stored were pellets Cell supernatant. the discarding and min 10 for °C 4 at g 4000 After medium. culture OD to grown and expresse were protocols genetic standard using enzymes: of Expression assays enzymatic and Protein expression mutations. individ competent into transformed and by digested was product PCR The clones. mutated of (2018). Construction of asyntheticmetabolic pathway fortheproduction of ual clones, and verified by restriction site analysis and DNA sequencing to carry the desired the carry to sequencing DNA and analysis site restriction by verified and clones, ual

d in 50 mL LB cultures that were inoculated from an overnight culture at OD at culture overnight an from inoculated were that cultures LB mL 50 in d - PAGE analysis. Protein c Protein analysis. PAGE 600

of 0.6 before protein expression was induced by addition of 1 mM IPTG to the to IPTG mM 1 of addition by induced was expression protein before 0.6 of 15

E. coli E.

Comment citer cedocument:

Frozen cell pellets were suspended in 0.5 0.5 in suspended were pellets cell Frozen h of protein expression at 25 °C, cells were harv were cells °C, 25 at expression protein of h cue xrcs o 1 mn t ° a 40 g n rtiig h clear the retaining and g 4000 at °C 4 at min 15 for extracts crude e

BL21 (DE3) star cells were transformed with the appropriate plasmidsappropriate the with transformed were cells star (DE3) BL21

(Sambrook et al., 1989) al., et (Sambrook . coli E.

oncentrations were estimated with the method of Bradford of method the with estimated were oncentrations

DH5 µg/mL α

el (E) Mttd lsis ee sltd from isolated were plasmids Mutated (NEB). cells 9

kanamycin. DpnI . Enzymes with an N an with Enzymes .

at 37 °C for 1 h to remove template DNA, template remove to h 1 for °C 37 at

oue o ws bfe (0 mM (50 buffer wash of volumes mL of breakage buffer (50 mM (50 buffer breakage of mL TM ested by centrifugation at centrifugation by ested

72437). Cell debris was debris Cell 72437). - terminal hexa terminal - 20 °C until °C 20 TM 600 -

His tag His

Cobalt of 0.1 of ant.

Version postprint 2,4-dihydroxybutyric acidfrom homoserine. Metabolic Engineering. ,DOI :10.1016/j.ymben.2017.12.005 Walther, T.,Calvayrac, F.,Malbert,Y., Alkim,C.,Dressaire, C.,Cordier, H.,François, J.m. NADH the monitoring by measured were rates Reaction acceptor. 2 with quantified was homoserine and substratepreferred their on activ transaminase of Estimation 1971) from adapted (method concentrations known using of calibrated solutions was ketonepyruvate the of concentration and absorbance between relation The OHB. absorban the and min 30 for temperature room at incubated was mixture The 6.5. pH at acid boric M 1 and arsenate sodium M 1 containing solution a of mL 1 with from Amicon Subsequently, °C. 37 at min 90 for 7.8 pH at buffer Tris venom snake homoserinesynthesized incubatingwasreader(BioRad125with mM microplate OHB 680XR).by a in µL. 250 volumeof final a in plates oxaloacetateor (OAA). Enzymaticassays werea carried out addingby started were Reactions extract. cell or enzymes purified of amounts appropriate and Sigma), from products (all enzymes) MgCl mM potassium chloride,5 mM 50 2 of Estimation di was proteinprocedure was repeated 4 times. The buffer. the remove to °C 4 at g 4000 Amicon an to transferred was sample protein The 7. pH to adjusted buffer phosphate mM 100 by exchanged systematically 1976) (Bradford, (2018). Construction of asyntheticmetabolic pathway fortheproduction of (Wellner and Lichtenberg, 1971) Lichtenberg, and (Wellner ). The typical OHB yield the of method was TM

Ultra centrifugal filter with a cut a with filter centrifugal Ultra (L) - - oxo amino acidamino . o tblz te att dhdoeae nye, h euin buffe elution the enzymes, dehydrogenase lactate the stabilize To - acid reductase activities: reductase acid Comment citer cedocument: TM

Ultra centrifugal filter (cut filter centrifugal Ultra oxidase (1.25 U/mL, Sigma) and catalase (4400 U/mL, Sigma) in 100 mM 100 in Sigma) U/mL, (4400 catalase Sigma)and U/mL, (1.25 oxidase

ities:

appropriate amounts of amounts appropriate The reactions were followed by the absorption of NADH at 340 nm 340 absorptionat NADH the of reactionsby werefollowed The ). OHB was quantified by mixing 100 µL of the tested solution tested the of µL 100 mixing by quantified was OHB ).

Transaminase activity of several candidate aminotransferasescandidate several of activity Transaminase 2 , 0.25 mM NADH, (5 mM fructose NADH, mM (5 mM 0.25 , - off of 10 kDa 10 of off

The reaction mixture contained 60 mM Hepes (pH 7), (pH Hepes mM 60 contained mixture reaction The 90 10

- off 10 kDa), and centrifuged during 8 min at min 8 during centrifuged and kDa), 10 off

to eliminate the enzymes the eliminate to

2 t 37t °Cin96 h rato mxue a prfe o an on purified was mixture reaction the - oxo ue it popae ufr n the and buffer phosphate into luted - 4 ce at 325 nm was used to quantify to used was nm 325 at ce - hydroxybutyrate - oxoglutarate as the amino groupamino the as oxoglutarate - eedn rdcin f h 2 the of reduction dependent - well flat bottomed microtiter Wlnr n Lichtenberg, and (Wellner - 1,6 - bisphosphate with bisphosphate (method adapted (method

(OHB), pyruvate, (OHB), was r Ldh - Version postprint 2,4-dihydroxybutyric acidfrom homoserine. Metabolic Engineering. ,DOI :10.1016/j.ymben.2017.12.005 Walther, T.,Calvayrac, F.,Malbert,Y., Alkim,C.,Dressaire, C.,Cordier, H.,François, J.m. 22 at held was column The ml/min. 1 was rate Flow methanol. % 15 and CuSO4 2mM containing phase mobile aqueous an with (Phenomenex) column 3126 Chirex a using by and detector RI the on determined 35 at heldwas temperature Column precolumn, Cartridge” Refill ( column exchange plus)depending the on measured compounds (SPD detector UV/VIS (WPS autosampler system 3000 Ultimate an on (HPLC) Chromatography Analytical methods 680XR). (BioRadreader microplate a in monitored consumptionwas NADH µL. 250 of volume final a in plates at out carried were assays The acid. amino the of amounts started were Reactions substrate. starting the as used was homoserine when (L) muscle rabbit and aspartate, of case in (Sigma) dehydrogenase lactis from PanE purified was enzyme dehydrogenase auxiliary The extract. cell or aminotransferase 2 auxiliary of Units/mL (PLP), MgCl mM 5 chloride, potassium mM 50 (pH 7),Hepes mM reactioncontained60reaction.the transaminase mixture The products of oxoacid

(2018). Construction of asyntheticmetabolic pathway fortheproduction of n ae f h aio cd phenylalanine acids amino the of case in .5 M AH (pinly m fructose mM 5 (optionally NADH, mM 0.25

Sugars and organic acids were measured with the UV and RI and UV the with measured were acids organic and Sugars Quantif °C. DHB °C. cto o guoe n mtblts a crid u b Hg Promne Liquid Performance High by out carried was metabolites and glucose of ication

concentrations were measured using an Aminex HPX Aminex an using measured were concentrations - mnx HPX Aminex 3000RS, Dionex 3000RS, - 0, hmdu, n/r a and/or Shimadzu), 20A,

Comment citer cedocument: - yrxai dhdoeae ad prpit aons f purified of amounts appropriate and dehydrogenase, hydroxyacid Biorad - 87H ). ), which was connected to a RI detector (RID 10A, Shimadzu), a Shimadzu), 10A, (RID detector RI a to connected was which ),

°C. Mobile phase was 1.25 mM H mM 1.25 was phase Mobile -

300 x 7.8 mm, 9 µm, 9 mm, 7.8 x 300

Homoserine, methionine and threonine concentrations werethreonineconcentrations and Homoserine, methionine 2 4 mM 4 , .

11 S ms setoee (ingn uvyr MSQ Surveyor (Finnigan spectrometer mass MSQ

n leucine and - 2 1,6 - xguaae 01 M pyridoxal mM 0.1 oxoglutarate, (Thermo scientific, France) equipped with equipped France) scientific, (Thermo - ipopae (al bisphosphate) 37 protected by a “Micro a by protected

°C in 96 in °C

Cablo e a. 2009) al., et (Chambellon 2 SO - detectors and by using a cation a using by and detectors att dhdoeae (Sigma) dehydrogenase lactate 4 -

well flat bottomed microtiter bottomed flat well - at a flow rate of 0.5 ml/min. 0.5 of rate flow a at 87H column (protected by a by (protected column 87H pout fo Sga, 4 Sigma), from products l

by adding appropriate adding by - ur cto H cation Guard - 5’ - phosphate malate , an L. - Version postprint 2,4-dihydroxybutyric acidfrom homoserine. Metabolic Engineering. ,DOI :10.1016/j.ymben.2017.12.005 Walther, T.,Calvayrac, F.,Malbert,Y., Alkim,C.,Dressaire, C.,Cordier, H.,François, J.m. Polypropyleneglycol (P 2,000) was usedas antifoaming agentduring the culture. ag appropriate the adjusting by concentration oxygen saturating the of % 30 above Dissolved maintained was tension vvm. oxygen 1.5 to 0.3 at air with aerated were reactors and KOH, M 5 of addition the by 7.0 of h 45 firstthe during glucoseconcentrations limiting fermentation andconcentrated a glucosesolutionstock g/L)was (500 manually assurenon added to the during monitored was concentration Glucose g/L. 45 was concentration glucose Initial MOPS. (NH g/l 6 contained additionally it that except experiments, flask shake the in used medium mineral the to similar was contained 1500 mL medium adjusting anOD initially that Sartorius) B (Biostat bioreactor L 2 a inoculate to used and harvested were cells The phase. exponential until above) indicated as (composition medium mineral mL 150 containing flasks cultures Bioreactor (28420 o Preparation sampler. auto spectrometer. with negative ionization (ionization temp kV) 0.25 at probe (ESI mode electrospray in spectrometer mass MSQ a by or Detector) Array Diode dual a by wereperformed DHB ofquantification concentratio the on Depending rate. flow isocratic ml/min 0.4 at acid formic “Micro (2018). Construction of asyntheticmetabolic pathway fortheproduction of - - ur cto H eil atig” rclm) ed t 8 C Mbl pae a 00 % (v/v) % 0.01 was phase Mobile °C. 48 at held precolumn) Cartridge” Refill H cation Guard 82, Nacalai Tesque Inc). a TOC of sample calculatedwas from threereplicate measurements. h pre The mass Pro Prima Scientific Thermo a by analyzed was culture bioreactor the of gas Exhaust Total Organic Carbon (TOC) was measured using a ShimadzuTOC measured usinga was Organic(TOC) TotalCarbon tto sed (300 speed itation - utrs o te nclto o te iratr wr cliae i cultivated were bioreactors the of inoculation the for cultures 4 ) 2 HPO

Comment citer cedocument: 4 , 0.4 g/l (NH g/l 0.4 , sadr sltos a md wt ptsim yrgn phthalate hydrogen potassium with made was solutions standard f - 50 p, uho rtr 2 m daee) n arto rate. aeration and diameter) mm 28 rotor, Rushton rpm, 1500

4 ) erature, 450 cone°C; voltage, 50 volts). 2 SO 6 4 00 , 1 g/L threonine and methionine, and threonine g/L 1 ,

of ~0.3.composition The of fermentation the of medium - waveUV 12

cultivation. The pH of the cultures was kept atkept was cultures the ofpH cultivation.The - VIS (200 nm and 210 nm) detector(Dionexnm) 210 and nm (200 VIS n range, detection and detection range, n -

L analyzer with ASI with analyzer L 6 g/L citrate g/L 6 10 m shake mL 1000 n and no and -

L - Version postprint 2,4-dihydroxybutyric acidfrom homoserine. Metabolic Engineering. ,DOI :10.1016/j.ymben.2017.12.005 Walther, T.,Calvayrac, F.,Malbert,Y., Alkim,C.,Dressaire, C.,Cordier, H.,François, J.m. synthetic homoserine µmol/(mg 0.082 with homoserine on EC Since homoserine. on activity poor comparativelytheir for indicator additional an was which mM, 50 to up of concentrations substrate at saturate not did homoserine lower tested transaminaseshadresidual only activity homoserine on which leastat was thanmore 450 enzymes. these of characterization the for appropriate were conditions assay our that reported results pheny and leucine, aspartate, substrates natural respective EC aminotransferases three the of values Km estimated The purityof purified the protein fraction was verified by SDS in expressed were enzymes pET the into cloned and DNA genomic from amplified correspond their and homoserine (EC phenylalanine from enzymes 1981) enzyme transaminase deleted a of function metabolic the and sterically on tr alternative that activity explains which substrates, different electrostatically significant have frequently transaminases that reported previously activity of Identification Results (2018). Construction of asyntheticmetabolic pathway fortheproduction of . We intended to mak to intended We . tha e is st u t ietf ezms with enzymes identify to out set first We

o ter aua substrate natural their on n

he dfeet lse o aiornfrss nml te satt (EC aspartate the namely aminotransferases, of classes different three (Lee - yB, n branched and TyrB), enzymes with with enzymes - Peng al., et 1979; Mavrides and Orr - DHB DHB pathway. Comment citer cedocument: e use of this pronounced promis pronounced this of use e E. coli E. ing preferred substrate. preferred ing

BL21(DE3) cells, and purified using affinity chromatography. The chromatography. affinity using purified and cells, BL21(DE3)

OHB reductase and and reductase OHB prot

- min) we chose this enzyme for the implementation of the of implementation the for enzyme this chose we min) chain Tbe 3 (Table

mn acid amino 13 ) Iiil ecin eoiis f h ezms on enzymes the of velocities reaction Initial .

ooeie rnaiae ciiy I hs been has It activity. transaminase homoserine , 1975; Yano et al., 1998) - PAGE (notshown) prior to enzymatic tests. - 8a+ xrsin etr Te His The vector. expression 28(a)+ The genes encoding these enzymes were enzymes these encoding genes The

cuity of transaminases and characterizedtransaminasesand of cuity

Br t l,18; esnad Calhoun, and Jensen 1988; al., et (Berg lalanine were very similar to previously to similar very were lalanine homoserine transaminase transaminase homoserine (EC - sC EC AspC, - IlvE) - transaminase ansaminases could take over take could ansaminases As - lE n EC and IlvE pC had the highest activity highest the had pC

(Table 3 s

- yB o their for TyrB of of ) indicating) . coli E. h three The - - tagged AspC), -

fold on

Version postprint 2,4-dihydroxybutyric acidfrom homoserine. Metabolic Engineering. ,DOI :10.1016/j.ymben.2017.12.005 Walther, T.,Calvayrac, F.,Malbert,Y., Alkim,C.,Dressaire, C.,Cordier, H.,François, J.m. continue our workwith this enzyme. smallest the also but OHB, specificities substrate saturating use we Therefore, OHB. mM 20 to up of concentrations n could OHB had enzyme LL dehydrogenase EC dehydrogenase lactate (L) was priorto enzymaticassays. His the and vectors, expression 28(a)+ pET into cloned were enzymes these encoding genes the aminotransferases, the of characterization from dehydrogenase by is from substrates. natural their anddehydrogenase enzymesOHB on dehydrogenase lactate OHB to cognatesterically are that substrates on reducta(OHB) encoded by encoded - att dehydro lactate

EC in (2018). Construction of asyntheticmetabolic pathway fortheproduction of Lactococcus lactis Lactococcus - qualitative mdh High OHB reductase activity reductase OHB High continued We

ocnrtos f h ntrl substrate natural the of concentrations

only only Steln ad McAlister and (Sutherland se activityse t e siae sne nta rato vlcte dd o strt a substrate at saturate not did velocities reaction initial since estimated be ot EC - hardly measurable activity on OHB activity on hardlymeasurable ldh agreement with an earlier work earlier an agreementwith - PanE genase A

. lactis L.

of the enzymes. the of (Bunch et al., 1997) al., et (Bunch , u work our . Ourscreen which is which

(2. Comment citer cedocument:

(Ldh) (Lane and Dekker, 1969) Dekker, and (Lane 0 - Ldh specificity 7 µmol/(mg 7 , hc is which mlt dhdoeae ad branched and dehydrogenase, malate , 05 µmol/(mg (0.56 encoded by encoded ih h ietfcto o ezms with enzymes of identification the with

of 11 µmol/(mg 11 of comprised

- fo tagged proteins were expressed and purifie and expressed were proteins tagged We found that found We prot noe by encoded is aua sbtae (Table substrate natural its r - , en 1985) Henn,

min)) (L) LL - malate dehydrogenase from dehydrogenase malate

- four . ldhA

14 Specifically, we estimated the kinetic parameters ofparameterskinetic the estimated weSpecifically, prot were reporting

Or eut show results Our . or 2 prot (

min)) (Llanos et al., 1992) al., et (Llanos Table LL -

hydroxy acid dehydrogenasesactiveacid hydroxywhich are

at LL - also active on OHB. In contrast, the contrast, In OHB. on active also min) ad h branched the and , panE d -

LdhA 0 M OHB mM 20 the ratio of of ratio the 4 n te rnhd hi 2 chain branched the and OHB reductase activity for rabbit musclerabbit for activity reductase OHB ) was found for LL for found was .

The selectedenzymesThe Cablo e a. 2009) al., et (Chambellon Substrate affinitiesSubstrate had not only the highest activity on activity highest the only not had the reaction the

ed 4 s rough a as . hrfr, e eie to decided we Therefore, ). ,

(D) furthermore 2 E. coli E. - - oxo Ldh from from Ldh - - - LdhA ( LdhA cha hi 2 chain - of the enzymes the of , 4 n 2 in

d to homogeneity to d which is which - rates attained rates estima

hydroxybutyrate included Table E. coli E. ht h (D) the that - - - . hydroxyacid hydroxyacid hydroxyacid

s o the for As e o the for te 4 encoded ), which ), EC

, (L) which - Mdh - Ldh for

at - - Version postprint 2,4-dihydroxybutyric acidfrom homoserine. Metabolic Engineering. ,DOI :10.1016/j.ymben.2017.12.005 Walther, T.,Calvayrac, F.,Malbert,Y., Alkim,C.,Dressaire, C.,Cordier, H.,François, J.m. enzymewas usedas the OHB reductasein exhibiting the enzyme, 4.5 thethat found show (not activity reductase OHB increased significantly asparagine and cysteine only 85, position in assayed residues acid amino alternative 19 the of Out Gln85 position in mutations of impact the Therefore, between studies The formation. productrespectively, duringdevelopingcharge, the stabilize to andcatalyst acid/base the pocketrestrictsactiveGln86thesitesizeandtheof undergoingpositioningpolar by additional charged positively charge oxamate fructose activator its to bound published previously a of inspection visual alignment sequence protein of (L) Engineering -

od erae atvt o te aua sbtae pyruvate substrate natural the on activity decreased fold pivotal role pivotal (2018). Construction of asyntheticmetabolic pathway fortheproduction of

( f h substrate the of Manual LL to specificity substrate provide that residues acid amino Key (Dunn et al.,1991)

(Wigley et al., 1992) al., et (Wigley

l8 (hc ain t Gn5 n LL in Gln85 to aligns (which Gln86 an almost equal affin equal almost an Gln85Cys mutant mutant Gln85Cys best

docking of

these residues these variant ie hi of chain side - lactate dehydrogenase to improve OHB reductase activity reductase OHB improve to dehydrogenase lactate

of of Comment citer cedocument: abxlc cd ru i nurlzd y lcrsai itrcin ih the with interaction electrostatic by neutralized is group acid carboxylic

and references therein) LL OHB into the into OHB of several (L) several of - . LdhA F was not was igure ity for both substrates (T substrates both for ity - 1,6

in substrate binding and catalysis has been reported in previous in reported been has catalysis and binding substrate in Gln r15 Thr Arg155. - ipopae te A cfco, n te usrt analogue substrate the and cofactor, NAD the bisphosphate, 2

85C

shows key residues in the active site of GS of site active the in residues key shows saturated by pyruvate concentrations of up to 20 mM thus mM 20 to up of concentrations pyruvate by saturated

active site c site active -

the syntheticDHB pathway. interactionsthe oxamate with lactate dehydrogenases of different biological ori biological different of dehydrogenases lactate ys

rsa srcue from structure crystal h - LdhA) ad a 4 ad a 3 and 233 15 .

T - fold increasedactivityfold and he catalytiche

onfiguration on O on n

able 5 able ) h lre snhtc substrate synthetic larger the . In a refined analysis of both mutants we mutants both of analysis refined a In . Asn HB reductase activity was characterized was activity reductase HB 126

Tbe 5 (Table ). residues Therefore, the LL the Therefore,

of Gs of otiue o correct to contribute ebclu stearothermophilus Geobacillus

. otay o h wild the to Contrary ). -

carboxyl and oxo functionscarboxyl oxo and Ldh His179 and Arg92His179and - dA ee dniid by identified were ldhA on OHB and a more thanmorea OHB and on revealed - Ldh. The ne The Ldh. - LdhA

a steric clash steric a

Q85C

(Figure

substrate

gin, serveas mutant mutant gative - type and 2 ) . . ,

Version postprint 2,4-dihydroxybutyric acidfrom homoserine. Metabolic Engineering. ,DOI :10.1016/j.ymben.2017.12.005 Walther, T.,Calvayrac, F.,Malbert,Y., Alkim,C.,Dressaire, C.,Cordier, H.,François, J.m. significantmetabolic burden. production DHB and homoserine reduced Eco4 into plasmids control empty amountssmall of under DHB conditions wherehomoserine activities reductase OHB and transaminasehomoserine endogenous 6 (Table and of deletion the that indicating homoserine mg/L 477 produced plasmids no harbored which with medium mineral were strains these LL reductase OHB the and with transformed deleted to increase productionDHB unde LL enzyme reductaseOHB optimized the of contribution positive the demonstrateproperly to strain production and respectively,deleted. were kinase homoserine the strain, production the in availability homoserine increase dehydrogenase kinase/homoserine increased was production homoserine and production homoserine Optimi which also bears some OHB reductase activity (Table activity reductase OHB some bears also which thrB 0.5 g/L 0.5 (2018). Construction of asyntheticmetabolic pathway fortheproduction of n hmsrn O homoserine and The resulting quadruple mutant mutant quadruple resulting The both pathway, synthetic described above the via production DHB high achieve To zation of DHB production by engineered engineered by production DHB zation of )

Te rdcin f 9 gL H b sri Eo show Eco4 strain by DHB mg/L 59 of production The . effectively blocked effectively - Ldh methionine and threonine and methionine A Q85C which contained which plasmids that expressed ThrA expressed that plasmids

determined to

DHB production. In addition, alcohol dehydrogenase, encoded by encoded dehydrogenase, alcohol addition, In production. DHB Comment citer cedocument: - - The solubledehydrogenaseThe lactate of LdhA ucnlrnfrs ezms wih r ecdd by encoded are which enzymes, succinyltransferase

homoserine y xrsig h threonine the expressing by Q85C fe 2 h f utvto udr eoi cniin o a defined a on conditions aerobic under cultivation of h 24 after

20 g/L g/L 20

yielded mutant ln o i combination. in or alone

to complement the auxotrophies of the strains. Strain Eco4 Strain strains. the of auxotrophies the complement to r oxygen glucose as the carbon source an source carbon the as glucose - to c onsumption by the methionine and threonine pathways threonine and methionine the by onsumption . oi ah ΔdA tr ΔmetA ΔthrB ΔldhA ΔadhE coli E.

- ThrA , H conve DHB (Tab tan Eco5 strains S345F 16 e 6 le - limited conditions (not shown in thispaper). S345F

, the homoserine transaminase homoserine the )

fo a medium a from , niaig ht hs pamd rpeet a represent plasmids these that indicating so need rsion E. coli E. - wih exhibit which 7 was 4 ), was deleted was ),

H ad ooeie rdcin by production homoserine and DHB overproduced -

nestv bfntoa aspartate bifunctional insensitive E. coli E. ed ed

which

that o e optimized. be to , which is which d - oy lsi. o further To plasmid. copy

ed . coli E. which was which lead to the production of production the to lead from the genome of the of genome the from - osmn homoserine consuming

. Thetransformation of mard rwh and growth impaired

encoded byencoded

osse residual possesses ( strain - bearing thrB supplemented

Homoserine and Eco4) adhE EC

- metA

AspC metA , was , - l dh was A , ,

Version postprint 2,4-dihydroxybutyric acidfrom homoserine. Metabolic Engineering. ,DOI :10.1016/j.ymben.2017.12.005 Walther, T.,Calvayrac, F.,Malbert,Y., Alkim,C.,Dressaire, C.,Cordier, H.,François, J.m. to reactor the to added manually was solution stock glucose concentrated a and fermentation, the culturemedium the maintain to used was HPLC medium. the ad Controlledcultivation. the duringconsumed not was citrate that inconfirmed analyses ions iron the stabilize to added were citrate g/L 6 and g/L, 1 to increased d g/L 6 of addition the by increased were concentrations phosphate) (and ammonium MOPS, by buffered the not was medium that except experiments flask shake the during as medium mineral same the on grown was Fed plasmid ThrA expressing 6 Table prod homoserine contributed positively results these together, Taken EC enzymes, Eco10. ThrA strain contrast, In Eco4. ThrA expressed was which homoserine mg/L 329 and DHB ThrA increased - S345F batch cultivation of DHB cultivation batch (2018). Construction of asyntheticmetabolic pathway fortheproduction of ). s expected, As . DHB production by the resulting DHB production by strain Eco19 was analyzedproductionstrainbyEco19 DHB was EC enzymes pathway DHB both expressed Eco10 Strain exhibited ,

To further To - sC n LL and AspC S345F S345F S345F uction

activity. Although growth of the strain was restored, it only produced 48 produced only it restored, was strain the of growth Although activity.

but no DHB pathway enzyme produced only 48 mg/L DHB and thus less than than less thus and DHB mg/L 48 only produced enzyme pathway DHB no but

prxmtl 2 approximately optimize DHB production we decreased the metabolic burden on the strain by strain the on burden metabolic the decreased we production DHB optimize H rdcin a frhr nrae o 9 m/ we bt H pathway DHB both when mg/L 190 to increased further was production DHB n te w DB aha ezms rm h sm medium same the from enzymes pathway DHB two the and iammonium phosphate, initial threonine and methionine concentrations were concentrationsmethionine and threonine initial phosphate,iammonium

o H pouto (opr Eo0 1, 2 1) ad ht increased that and 13), 12, 11, Eco10, (compare production DHB to s was

Comment citer cedocument: Eco11 and Eco12, which expressed EC expressed which Eco12, and Eco11 - LdhA

eurd o civ sgiiat H ttr (opr Eo3 n Eco9 and Eco13 (compare titers DHB significant achieve to required showed Q85C - producing strain producing were , - fold

ht ah individual each that

strain Eco18

at pH 7 pH at nrae DB rdcin hn oprd o c4 and Eco4 to compared when production DHB increased xrse smlaeul wt ThrA with simultaneously expressed es hn h c4sri. iial, tan c1 which Eco10 strain Similarly, strain. Eco4 the than less 17 .

Glucose concentration was monitored throughoutconcentrationmonitoredGlucosewas

in a fed in was increasedwas

nye f h synthetic the of enzyme - batch bioreactorcultivation.batch - - A sC n LL and AspC spC or LL or spC

to 410 mg/L - LdhA - ldhA S345F

(Table 6 Q85C

Q85C n tan Eco13. strain in

- in addition to to addition in H pathw DHB oy number copy dition of KOH of dition

u hd no had but ) .

The strainThe

mg/L ay , Version postprint 2,4-dihydroxybutyric acidfrom homoserine. Metabolic Engineering. ,DOI :10.1016/j.ymben.2017.12.005 Walther, T.,Calvayrac, F.,Malbert,Y., Alkim,C.,Dressaire, C.,Cordier, H.,François, J.m. accumulatedhave which products unidentifiedseveral or one in bound were carbon consumed total ca consumed alterations theof biomass composition. S2 Table spectrophotometric S2 (Table analyses HPLC our measurements with results these compared and cells without and with h 48 after medium fermentation the of content (TOC) Carbon Organic Total the CO and %), (11 HPLC by identifiedproducts fermentation soluble %), (9 biomass of form the inrecovered be could carbon (Figure 2). indicati phase production DHB the during decreased and phase growth exponential the homoserine of end the Interestingly, at g/L g/g. 0.3 only 0.1 at peaked of concentration yield product a to corresponding g/L 5.3 approximately up linearly increased concentration DHB and cultivation of h 39 following the during glucose consume to continued strain The arrest. growth the with coincided production cultivatio aerobic assure to concentration saturating conditions. the of % 40 above maintained was concentration non assure (2018). Construction of asyntheticmetabolic pathway fortheproduction of hn h cro blne a cmlmne b TC esrmns 86 measurements, TOC by complemented was balance carbon the When fed the of balance carbon A of h 9 approximately after growth exponential ceased Eco19 strain conditions, these Under , e ocue ta te paet ak f abn ol nt e xlie by explained be not could carbon of lack apparent the that concluded we ), , Please note that concentrations of OHB and lysine were found to be zero.). Since TOC and TOC Since zero.). be to found were lysine and OHB of concentrations that note Please , n due to the depletion of threonine in the culture medium (Figure 2). The onset of DHB of onset The 2). (Figure medium culture the in threonine of depletion the to due n

- limiting carbon source concentratio source carbon limiting

rbon could be recovered (Table 7 (Table recovered be could rbon . Wefound thatC 0.8

measurements of th of measurements 2 (39 %) (Table 7 (Table %) (39 Comment citer cedocument: - - mol in themol medium couldbeaccounte not ). In an attempt to identify unknown carbon sinks we measured we sinks carbon unknown identify to attempt an In ). batch fermentation revealed that only 59 % of the consumed the of % 59 only that revealed fermentation batch

e biomass e g ht h aio cd a drcl cnetd no DHB into converted directly was acid amino the that ng ns ). From t From ). 18 during the entire the during

- bound carbon gave identi gave carbon bound hese results we conclude that 27 that conclude we results hese

n spec and cultivation. The dissolved oxygen dissolved Thecultivation.

to a final concentration of concentration final a to rpooerc biomass trophotometric d for by HPLC by d analyses for cal results (0.3 C (0.3 results cal

o te total the of % unexpected

% of the of % - mol, Version postprint 2,4-dihydroxybutyric acidfrom homoserine. Metabolic Engineering. ,DOI :10.1016/j.ymben.2017.12.005 Walther, T.,Calvayrac, F.,Malbert,Y., Alkim,C.,Dressaire, C.,Cordier, H.,François, J.m. carbon metaboliunexpected of result the as medium the in (2018). Construction of asyntheticmetabolic pathway fortheproduction of most likelymost left the system intheform of Comment citer cedocument:

one one severalor 19

c activities and/or cell lysis, and lysis, cell and/or activities c

unidentified volatile product that 14 % of the of % 14 s .

Version postprint 2,4-dihydroxybutyric acidfrom homoserine. Metabolic Engineering. ,DOI :10.1016/j.ymben.2017.12.005 Walther, T.,Calvayrac, F.,Malbert,Y., Alkim,C.,Dressaire, C.,Cordier, H.,François, J.m. EC the of expression that found we Nevertheless, substrates. preferred the to compared smallrather were enzymes tested the of activities transaminasehomoserine contrast, In conversion. specifici equal almost with pyruvate substrate natural the ancysteine the smalleryielded to Gln85 pocket.of Mutationactive which LL of Activity testedinenzymes,behighest (L) reductaseto Among the foundOHB activitywas transa natural several been in identified have which of both activities, reductase OHB and transaminase homoserine of composed DHB of panel the extendwe work, present the al., 2014; Walther al.,et 2013, 2017) three conceived recently eventually yields thewater soluble methionine analogueHMTB thus pe DHB the producedenzymatically by described method the to incorporati chemical conserving sugar from DHB of production two fermentative a proposed recently have we problems, technological 5 approximately to pH neutral near and temperatures physiological at concentrations product limits which methionine of solubility low comparatively ha Discussion prd y oh h hg mtblc ot f ufr incorporation sulfur of cost metabolic high the both by mpered (2018). Construction of asyntheticmetabolic pathway fortheproduction of alleviated ic anttd DHB annotated Since cost Direct - ae poes a lat n hoy de nt gen not does theory, in least at process, based - dA n H ws ute ehne b structure by enhanced further was OHB on LdhA

a steric clash between the OHB substrate and Gln85 which restricts the size of the of size the restricts which Gln85 and substrate OHB the between clash steric a - fiin irba snhsso ehoie r t aaou HT rm ua is sugar from HMTB analogue its or methionine of synthesis microbial efficient from the activated precursorsO activatedthe from - Comment citer cedocument: step synthetic step - rdcn mtblc ahas o o eit w ad tes have others and we exist, not do pathways metabolic producing on of methanethiol to produce HMTB produce to methanethiol of on hm n coworkers and Shim iae r 2 or minase . By describingpathway a which

metabolic

- Wlhr t l, 2017) al., et (Walther producing metabolic routes. The synthetic pathwayis synthetic The routes. producingmetabolic - yrxai dhdoeae nye, respectively. enzymes, dehydrogenase hydroxyacid 20 pathways which produce DHB from malate from DHB produce which pathways

Si e a. 2016) al., et (Shim - 0 acetylhomoserine or O acetylhomoserine or

g/L y hs uprig fiin OBt DHB to OHB efficient supporting thus ty rt ay eaoi by metabolic any erate

Fcs t l, 2006) al., et (Fuchs - st - based engineering of of engineering based g poes which process age enzymewhich

derives DHB from homoserine in

ad h sbeun carbon subsequent the and ,

Koe e a. 2006) al., et (Kromer (Deck et al., 2009) al., et (Deck rmitting high producttiters. ,

where - succinylhomoserine, - from LdhA accepted OHB andOHB accepted T tcl these tackle To .

- methionine products includes h enzyme the . In contrast In .

n the and L. lactis L.

- (Li et (Li

AspC

and the

is - , . Version postprint 2,4-dihydroxybutyric acidfrom homoserine. Metabolic Engineering. ,DOI :10.1016/j.ymben.2017.12.005 Walther, T.,Calvayrac, F.,Malbert,Y., Alkim,C.,Dressaire, C.,Cordier, H.,François, J.m. the DHB pathway. the through flux Thus, respectively. pyruvate) concentrations unity to close commonly is acceptor group amino 2 use which reactions aminotransferase of ratio action mass the since sub the in be to expected is concentrationOHB intracellular The improved. strongly enzyme pathway. DHB synthetic the of performance homoserine activity. new the obtain to cells growing fast of analysis genetic subsequent and activity transaminasehomoserine for 2017) mutant double aminotransferase alanine the that shown recently very have coworkers and Bouzon that interesting flux. pathway limit to appear homoserine on transaminases tested all of activities homoserine the of performance the increase yields. and productivities pathw synthetic the via achieved fed h 48 after g/g 0.1 of EC enzymes reductase OHB and natural substrate aspartate. the with compete to high sufficiently were concentrations homoserine intracellular that suggesting homos bears which enzyme (2018). Construction of asyntheticmetabolic pathway fortheproduction of . These authors used long used authors These . , homoserine A

which currently which /glutamate in /glutamate

Buo e a. 2017) al., et (Bouzon lC A142P: AlaC

homoserine transaminase reaction transaminase homoserine

The f h crepnig usrt/rdc cul 2 couple substrate/product corresponding the of only an OHB reductase enzyme with very high substrate affinity can “pull” “pull” can affinity substrate high very withenzyme reductase OHB an only - lC A142P:Y275D AlaC overproducing E. coli E. -

Comment citer cedocument: batch cultivation batch cannot be saturated by OHB concentrations of up to 20 mM, mM, 20 to up of concentrations OHB by saturated be cannot The development of more efficient enzymes represents the major lever to lever major the represents enzymes efficient more of development The

Y275D

rn tasmns atvt sgiiaty nrae DB production DHB increased significantly activity transaminase erine are typically in the range of 0.4 mM and 90 mM 90 and mM 0.4 of range the in typically are - - term cultivations of of cultivations term AspC and LL and AspC ay.

a srnl increased strongly has However, much work is necessary to reach industrially relevant industrially reach to necessary is work much However, wih ae i a atatv cniae o mrv the improve to candidate attractive an it makes which ,

. coli E. . uat enzyme mutant

This result shows that significant DHB production can be can production DHB significant that shows result This

- - tan hc epesd the expressed which strain LdhA dependent DHB pathway. In particular, the very the particular, In pathway. DHB dependent n diin sbtae fiiy f h OB reductase OHB the of affinity substrate addition, In 21 Q85C

E. coli E. thereby enabling efficient enabling thereby , respectively, ,

Jnosi t l, 2008) al., et (Jankowski

cells under conditions that were selective were that conditions under cells a son o have to shown was ciiy n homoserine on activity produced - oxoglutarate o oxoglutarate

ooeie transaminase homoserine

5.3 g/L DHB at a yield a at DHB g/L 5.3 (Bennett et al., 2008) al., et (Bennett

K f . m for mM 1.7 of Km a in vivo in ad eas the because and , - oxoglutarate -

millimolar range millimolar Buo e al., et (Bouzon t s therefore is It

r pyruvate as pyruvate r operation of operation needs to be to needs carbon

small (or , Version postprint 2,4-dihydroxybutyric acidfrom homoserine. Metabolic Engineering. ,DOI :10.1016/j.ymben.2017.12.005 Walther, T.,Calvayrac, F.,Malbert,Y., Alkim,C.,Dressaire, C.,Cordier, H.,François, J.m. 41transformed Eco19 produ fermentation unknown of formation the of problem the tackle must optimization product prevent to to expected be can enzymes pathway strain. our by DHB of production limited rather co a as up build intracellularconcentrations,which homoserineelevated that suggest arguments we these with biosynthesis. own its inhibits strongly homoserineintracellular of accumulation that conclude concentrations, homoserine intracellular decreases RhtA of overexpression that respectively. the overexpressing additionally by optimized further homoserine was strain producer the when g/L 40 strain our by produced been fed a producedstrain This (MetL). Δ s engineered an by g/L 40 of demonstratedproduction have they that in interesting particularly 2007) al., et Lee 2014; al., et Hong 2003; (Debabov, homoserine other for shown as production homoserine improving decreasing by instance, for achieved, be could This strain. industrial an in removed be should ThrB and MetA of deletions the by caused were which threonine and methionine for auxotrophies strain Notably, ri. hi iiil tan abrd h sm efcie homoserine effective same the harbored strain initial Their train. thrB - (2018). Construction of asyntheticmetabolic pathway fortheproduction of ) batch cultivation batch as our strain our as The optimization of of optimization The nsequence of the very small homoserine transaminase activities, are a major cause for the for cause major a are transaminaseactivities,homoserine small very the of nsequence

the transcription of the corresponding genes rather than by deleting them. them. deleting by than rather genescorresponding the of transcription the - xotn permease exporting From the observations that homoserine inhibits aspartate kinase activity in vitro, and vitro, in activity kinase aspartate inhibits homoserine that observations the From - feedback inhibition on the homoserine the on inhibition feedback and overexpressed a bifunctional aspartate kinase/homoserine dehydrogenase kinase/homoserine aspartate bifunctional a overexpressed and

, which is very similar to the 5.3 g/L DHB and 0.1 g/g product yield which havewhich yield product g/g 0.1 and DHB g/L 5.3 the to similar very is which , % of the consumed carb consumed the of % Comment citer cedocument: the . only only

Li et al. et Li producer

6.8 g/L homoserine at a yield a homoserineat g/L 6.8 hA ad y eeig h homoserine the deleting by and RhtA, appears to be to appears - eie pout lk troie n O and threonine like products derived

( decrease

2016)

strain

reached m reached on into unidentified soluble (27 soluble unidentified into on

homoserine accumulation and accumulationhomoserine is another important factor to increase DHB yields. DHB increase to factor important another is

22 a promising approach to increase DHB production DHB increase to approach promising a

. In this context, the results of of results the context, this In . nraig h efcec o te ytei DHB synthetic the of efficiency the Increasing uch higher homoserine titers of 33.4 g/L and g/L 33.4 of titers homoserine higher uch - producing pathway. producing of 0.13 g/g from glucose after 48 h of h 48 after fromglucose g/g 0.13 of - nacn dltos ( deletions enhancing - motn permease importing

is, therefore, is, %) and volatile (14 %)(14 volatile and %)

- Furthermore, s Furthermore, succ Li et al. et Li i t al. et Li inylhomoserine Furthermore,

( t. Strain cts. 2016) required

Δ (

In line In metA, E. coli E. 2016) TdcC, train

are ,

Version postprint 2,4-dihydroxybutyric acidfrom homoserine. Metabolic Engineering. ,DOI :10.1016/j.ymben.2017.12.005 Walther, T.,Calvayrac, F.,Malbert,Y., Alkim,C.,Dressaire, C.,Cordier, H.,François, J.m. achieveto industrially relevant productivitiesDHB and yields. u of accumulation excessive avoid to engineering strain and pathway, to synthetic the engineeringof capacity catalytic enzymethe increase Further homoserine. from derives which pathway metabolic synthetic major these of formation the preventing and sinks carbon these Identifying products. (2018). Construction of asyntheticmetabolic pathway fortheproduction of requirement DH demonstrated have we summary, In nknown products and to redirect carbon flux towards flux carbonredirect to and products nknown

to improve production.DHB Comment citer cedocument:

23 pouto a a il o 01 / va two a via g/g 0.1 of yield a at production B

DHB

synthesis are necessary are synthesis by - rdcs is products - step a

Version postprint 2,4-dihydroxybutyric acidfrom homoserine. Metabolic Engineering. ,DOI :10.1016/j.ymben.2017.12.005 Walther, T.,Calvayrac, F.,Malbert,Y., Alkim,C.,Dressaire, C.,Cordier, H.,François, J.m. assistance during thiswork. Serrano Hélène Alkim, Ceren Spina Lucie Vax, Amélie Adisseo from grant industrial an by part in supported was work This Acknowledgements (2018). Construction of asyntheticmetabolic pathway fortheproduction of Comment citer cedocument:

- aale n Jle rdne fr technical for Fredonnet Julie and Bataille Te uhr ae rtfl to grateful are authors The . Version postprint 2,4-dihydroxybutyric acidfrom homoserine. Metabolic Engineering. ,DOI :10.1016/j.ymben.2017.12.005 Walther, T.,Calvayrac, F.,Malbert,Y., Alkim,C.,Dressaire, C.,Cordier, H.,François, J.m. K. Hong, 20 R., Weindruch, J.J., Ramsey, K., Hagopian, Fu Feedinfo NewsService [WWW Document], n.d. URL http://www.feedinfo.com/ (accessed 4.13.16). A., Marx, E., Kimura, M., Ikeda, S., Huebner, V.G., Debabov, B., Bathe, J., Thommel, R., Faurie, vectors. expression promoter tac compatible of set A 1996. T., Linn, R., Pierre, St D.M., Dykxhoorn, 1991. J.J., Holbrook, H., Muirhead, A.R., Clarke, T., Atkinson, D.J., Halsall, H.M., Wilks, C.R., Dunn, DL 2007. D.H., Baker, R.N., Dilger, 2009. B., Buschhaus, K.M., Exner, P., Deck, V.G., Debabov, B., Bathe, D.J., Thommel, D.R., Faurie, in: Story, Threonine The 2003. V.G., Debabov, indisruption Gene 1995. Wackernagel,W., P.P., Cherepanov, M., Yvon, J.A., Wouters, J.E.T., Vlieg, Hylckama van C., Gitton, F., Lorquet, L., Rijnen, E., Chambellon, Mat P.K., Bunch, of quantities microgram of quantitation the for method sensitive and rapid A 1976. M.M., Bradford, P., Marlière, F., Taran, J., Weissenbach, N., Perchat, V., Delmas, O., Loreau, A., Perret, M., Bouzon, capabilities: metabolic new of Acquisition 1988. L., Liu, N.B., Vartak, M.D., Wang, C.M., Berg, Kimball, J., Yuan, B.D., Bennett, Baba, Ara, T., T., Hasegawa, Takai M., References chs, D., Fischer, J., Tumakaka, F., Sadowski,2006.F.,G., Tumakaka,Fischer, J., D., chs, (2018). Construction of asyntheticmetabolic pathway fortheproduction of Butyric Acid. US2009318715 (A1). SpringerBerlin Heidelberg, pp. 113 L of Production Microbial E., Kimura, M., Ikeda, S., Huebner, 9 Flp of option the with system for branched 2009 dehydrogenase of protein utilizing theprinciple of protein 1533. ASynthetic2017. Alternative to Canonical One 202. mult metaboliteconcentrations by anisotoperatio mutants: theKeio collection. Syst. Mol. Biol. 2, 2006.0008. of Construction 2006. H., Mori, B.L., butanediol. J. Ind. Microbiol. Biotechnol. 41, 1517 lactone, homoserine acid, succinic 201 J.H., Lee, K.H., Cho, J.N., Kim, restriction and aging. FEBS Lett. 579, 2009 Res.45, 6578 Chem. Eng. Ind. Solubility. Aqueous on Cosolvents Alcoholic of Addition the and value in Biochemical Engineering/Biotechnology. Springer,Berlin, Heidelberg. Möcke Gene 177, 133 framework. dehydrogenase lactate the on based Philos. Trans. R.Soc. enzymes L new of synthesis and Design chicks.to Poult. Sci. 86, 2367 acid amino sulfur in anorexigenic are excesses - – K., Kim, J.H., Yoon, J.H., Park, H. Park, J.H., Yoon, J.H., Kim, K., 14.

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– - – 4 881. 1311. - Alkylthio - cystine ts fed ts

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1983. pham, C.M., Irague, R., Auriol, C., Baylac, A., Cordier, H., Dressaire, C., Lozano C., Dressaire,H., Cordier, A., Baylac, C., Auriol, R., Irague, C.M.,pham, ivatives: current status and prospects. Appl. Microbiol. Biotechnol. 69, 1 - Siméon, M., François, J.M., 2017. Construction of a synthetic met synthetic a Construction of 2017. J.M., François, M., Siméon, y tutr, amino structure, ry

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- Schrö threonineproduction. Mol. Syst. Biol. 3, 149. -

ketoglutarate activity. J. Bacteriol. 139, 339 - P., Lemme, A., Redshaw, M.S., Mosenthin, R., 2008. Meta2008. Redshaw,R., M.S., Mosenthin,Lemme,A., P., - keto der, - yrxbtrc cd n is eae cmons y gas by compounds related its and acid hydroxybutyric ldh -

emnl eune sbtae pcfct, n asne f a of absence and specificity, substrate sequence, terminal

- H., Heinzle, E., 2006. 2006. E., Heinzle, H.,

4 ein n cntuto o a non a of construction and Design te ee noig L encoding gene the , 31 fr L for W3110 - - yrxbtrt. ytei, hmcl rpris ad s a as and properties, chemical Synthesis, hydroxybutyrate. MethionineProduction. Adv. Biochem. Eng. B

and Related Bacteria.SpringHarborPress, LaboratoryRelated Cold and Isolation Crit. Rev. Microbiol. 8, 229 - – I2 regulatory elements. Nucleic Acids Res. 25, 1203 25, Res. Acids Nucleic elements. regulatory I2 – Escherichia regulation of transcriptional units in Escherichia coliEscherichia in unitstranscriptional of regulation 4133 26 - 6964. hydroxy

- ooeie production. homoserine - . n epeso o the of expression and . 21. Mto o Pouto o 2,4 of Production of Method A 2013. M.,

- analogue

Metabolic pathway analysis for rational for analysis pathway Metabolic coli – -

butanetriol from glucose. Sci. 4, Rep. 1079. and - (+) - notransferases: overlap enzymes free - att dhdoeae from dehydrogenase, lactate

oyeatru glutamicum Corynebacterium – - – 63. cd oprd ih DL with compared acid –

345. 266. - aua mlt t 1,2,4 to malate natural

Trends Biochem. Sci. 11, Sci. Biochem. Trends dehyde dehydrogenase dehyde –

shrci coli Escherichia Proc A Laboratory Manual Laboratory A

– 2031. abolic pathway for pathway abolic s Bohm 51, Biochem. ess shrci coli Escherichia – 1499. iotechnol. –

8. - analysis o

–

2966. - Huguet, gene

– - - f - : . Version postprint 2,4-dihydroxybutyric acidfrom homoserine. Metabolic Engineering. ,DOI :10.1016/j.ymben.2017.12.005 Walther, T.,Calvayrac, F.,Malbert,Y., Alkim,C.,Dressaire, C.,Cordier, H.,François, J.m. H S., Haefner, O., Zelder, with aminotransferase aspartate an of evolution Directed 1998. H., Kagamiyama, S., Oue, T., Yano, Meth 2014. T., Willke, S.J., Gamblin, D.B., Wigley, 1971. L.A., Lichtenberg, D., Wellner, (2018). Construction of asyntheticmetabolic pathway fortheproduction of Use dimethylof disulfidefor methionine production in microoragnisms. US2009281353 (A1). new substrate specificities. Proc. Natl. Acad. Sci. U.S. A. 95, 5511 9914. from dehydrogenase lactate stearothermophilus allosteric an of complex ternary a of Structure 1992. 596. non Commun. 8, 15828. the of biosynthesis

oie production ionine Comment citer cedocument: erold, A., Klopprogge, C., Schrö C., Klopprogge, A., erold,

at 2.5 A resolution. J. Mol. Biol. 223, 317

Turkenburg, J.P., Dodson, E.J., Piontek, K., Muirhead, H., Holbrook, J.J., Holbrook, H., Muirhead, K., Piontek, E.J., Dodson, J.P., Turkenburg, - aua mtinn peusr 2,4 precursor methionine natural Assay of amino acid oxidase. Methods Enzymol. 17, Part B, 593 B, Part 17, Enzymol. Methods oxidase. acid amino of Assay

-- ciia rve. pl Mcoil Boeho. 8 9893 98, Biotechnol. Microbiol. Appl. review. critical a 27

der, H., Yocum, R.R., Williams, M.K., 2009. M.K., Williams, R.R., Yocum, H., der, – 335. - iyrxbtrc acid. dihydroxybutyric –

5515.

Bacillus Nat. – –

Version postprint 2,4-dihydroxybutyric acidfrom homoserine. Metabolic Engineering. ,DOI :10.1016/j.ymben.2017.12.005 Walther, T.,Calvayrac, F.,Malbert,Y., Alkim,C.,Dressaire, C.,Cordier, H.,François, J.m. Subtext: Table6 not calculated. pyruvate Subtext: and 2 5 Table Mdh. nc mM. 20 to up of concentrations mM 20 (at substrate natural the Subtext: natural substrates Table4 concentration 50mM. of Ec mM): Subtext: Table3 Table2 Table1 Table Legends Figure and (2) (1) (3)

(2018). Construction of asyntheticmetabolic pathway fortheproduction of -

oxo : : Productformation by : Kinetic parameters several of transaminases on homoserineand their natural substrates. : Plasmids usedin this study. Strains: : Kinetic: parameters candi of : Impact of mutations in (L) in mutations of Impact : - and OHB at 20 mM substrate concentration. Ns concentration. substrate mM 20 at OHB and IlvE Data from a single a Data from experiment. alleviate to phenylalanine this replaced was variant,by enzyme cysteinereductase 85 glutamine increase to OHB activity. by replaced was 345 T ThrA. of threonineinhibition Serine respectively. enzymes, dehydrogenase homoserine coli E. wt

Data are presented as means ± STDV calculated from at least three replicates. (*) Ratio of activities measured on on measured activities of Ratio (*) replicates. three least at from calculated STDV ± means as presented are Data Data are presented as means ± STDV calculated from at least at from calculated STDV ± means presented areas Data Data are presented as means ± STDV calculated STDV ± means as presentedare Data - – 4 –

- wild

hydroxybutyrate ecn, Ec leucine,

genes - used in this study type strain,type .

aspC ns - TyrB –

Not saturated 50mMsubstrate concentration. to atup Not and , Comment citer cedocument: ∆4

pyruvate or 0.05 mM oxaloacetate) and OHB (at 20 mM). ns ns mM). 20 (at OHB and oxaloacetate) mM 0.05 or pyruvate – -

. ∆adhE – hnllnn, Ec phenylalanine,

E. coli

thrA not calculated. Natural substrates: Pyruvate for Ec for Pyruvate substrates: Natural calculated. not he LlldhA gene is from from is LlldhAgene he .

S345F

- ∆ldhA ∆thrB ∆metA∆ldhA ∆thrB lactate dehydrogenase A from A dehydrogenase lactate

date α

strains expressingsynthetic the DHB pathway

noe satt aiornfrs, n bfntoa aprae kinase aspartate bifunctional and aminotransferase, aspartate encode - oxo acid reductases on 2 acidreductasesoxo on - AspC –

from at least three least at from not saturated at substrate concentrations of up to 20 mM. nc mM. 20 to up of concentrations substrate at saturated not 28 –

Lactococcus lactis Lactococcus

satt. *) ciiy a etmtd t homoserine a at estimated was activity (**) aspartate. three

replicates. (*) Ratio of activities measured on on activitiesmeasured of Ratio (*) replicates. replicates. (*) Natural (*) replicates.

and encodes (L) encodes and L. lactis L. - oxo

- - 4 Ldh, Ll Ldh, -

on its activity on pyruvate on activity its on hydroxybutyrate and their – -

LdhA; Oxaloacetate for Ec for Oxaloacetate LdhA; - not saturated at substrate substrate at saturated not lactate dehydrogenase.In lactate .

substrates (each at 20 at (each substrates

– - -

Version postprint 2,4-dihydroxybutyric acidfrom homoserine. Metabolic Engineering. ,DOI :10.1016/j.ymben.2017.12.005 Walther, T.,Calvayrac, F.,Malbert,Y., Alkim,C.,Dressaire, C.,Cordier, H.,François, J.m. concentrations of > 10 g/L during to methionine and threonine g/L auxo strain 1 complement with supplemented was which medium mineral defined DHB the of profile Fermentation 3: Figure l red dashed as shown are residues acid amino enzyme and oxamate the Polar between interactions red. oxygen, blue; nitrogen, grey; carbon, type: element to according colored are anal substrate X experimental the into docked manually t in stearothermophilus region site Active 2: Figure OHB blue. in shown are pathway homoserine natural the in reactions catalyzing enzymes the of Names shown are reactions Synthetic 2,4 of production for pathway metabolic Synthetic 1: Figure Table7 ines.Thestructure overall shownis in pale brown. – (2018). Construction of asyntheticmetabolic pathway fortheproduction of

2 : Carbon: balance of the fed - oxo - 4 ogue oxamate (PDB code:1LDN).oxamate (PDB ogue - hydroxybutyrate, DHB .

h snhtc 2 synthetic The trophies. Glucose was manually added to assure non assure to added manually was Glucose trophies. Comment citer cedocument:

n e, aua hmsrn ptwy ecin ae hw i black. in shown are reactions pathway homoserine natural red, in the - batch fer

- fermentation. – e rsa srcue f (L) of structure crystal he oxo

2,4 - 4 - - a srcue f h ezm ope wt ND n the and NAD with complex enzyme the of structure ray dihydroxybutyrate. - mentation strain of Eco19. yrxbtrt (H) usrt son n re was green in shown substrate (OHB) hydroxybutyrate - rdcn Eo8 strain. Eco18 producing Atoms of key amino acid residues and substrates and residues acid amino key Atomsof 29

- iyrxbtrt fo homoserine. from dihydroxybutyrate - lactate dehydrogenase of of dehydrogenase lactate

The strain was cultivated on on cultivated was strain The - limiting carbon source carbon limiting G.

(2018). Construction of asyntheticmetabolic pathway fortheproduction of Comment citer cedocument:

Version postprint 2,4-dihydroxybutyric acidfrom homoserine. Metabolic Engineering. ,DOI :10.1016/j.ymben.2017.12.005 Walther, T.,Calvayrac, F.,Malbert,Y., Alkim,C.,Dressaire, C.,Cordier, H.,François, J.m. Table1 Strain name BL21(DE3) JW3973 JW0002 JW1375 JW1228 MG1655 NEB5 Eco18 Eco13 Eco12 Eco11 Eco10 (2018). Construction of asyntheticmetabolic pathway fortheproduction of Eco9 Eco7 Eco6 Eco5 Eco4 : Strains: usedin this study

- α

- - - -

1 3 1 1

Eco4 harboring empty pEXT20 empty harboring Eco4 pZA23 empty harboring Eco4 MG1655 hsdR514, F hsdR514, ( F hsdR514, gal F ompT hsdR51, F [lon] λ huA2 thi relA1 endA1 Δ(argF fhuA2 F Eco4 harboring Eco4 harboring Eco4 harboring Eco4 harboring Eco4 harboring Eco4 pEXT20 and pZA23 empty harboring Eco4 pEXT20 and pZA23 empty harboring Eco4 - - - - -

DE3 = DE3 , , , , λ -

Δ Δ Δ Δ ilvG (araD (araD (araD (araD - λ Δ

ΔadhE ΔldhA ΔthrB ΔmetA ΔmetA ΔthrB ΔldhA ΔadhE rfb sBamHIo ∆EcoRI sBamHIo Δ Δ Δ Comment citer cedocument: adhE748::kan - - - - metA780::kan thrB723::kan ldhA744::kan araB)567 araB)567 araB)567 araB)567 - 50 rph - lacZ)U169 phoA glnV44 Φ80Δ (lacZ)M15 gyrA96 recA1 recA1 gyrA96 (lacZ)M15 Φ80Δ glnV44 phoA lacZ)U169

- pZA23 pZA23 pZA23 pZA23 pZA23 1 hsdR17 .

- 1 , , , ,

- - - - - thrA thrA thrA thrA thrA Δ Δ Δ Δ

lacZ4787 lacZ4787 lacZ4787 lacZ4787

- B int::(lacI::PlacUV5::T7 S345F S345F S345F S345F S345F

Genotype - and pEXT20 and pEXT20 and pEXT20 and pEXT20 empty and aspC (::rrnB (::rrnB (::rrnB (::rrnB

31 - LLldhA

- - - - λ 3), 3), 3), 3),

aspC - - - Q85C aspC LLldhA aspC λ λ λ λ - - - - , , , , E) dm ∆hsdS [dcm] DE3) -

LLldhA rph rph rph rph -

LLldhA

Q85C ∆nin5 i21 gene1) - - - - 1 1 1 1 , , , , Q85C

Δ Δ Δ Δ Q85C (rhaD (rhaD (rhaD (rhaD

- - - - rhaB)568 rhaB)568 rhaB)568 rhaB)568

, , , ,

(Baba et al., 2006) etal., (Baba 2006) etal., (Baba 2006) etal., (Baba 2006) etal., (Baba

ATCC 47076 ATCC Reference This study This study This study This study This study This study This This study This study This study This NEB NEB study

TM TM

Version postprint 2,4-dihydroxybutyric acidfrom homoserine. Metabolic Engineering. ,DOI :10.1016/j.ymben.2017.12.005 Walther, T.,Calvayrac, F.,Malbert,Y., Alkim,C.,Dressaire, C.,Cordier, H.,François, J.m. mM. IlvE calculated STDV ± means as presented are Data Ec Ec Ec Enzyme Table3 pZA23 pEXT20 pEXT20 pEXT20 pEXT20 pZA23 pZA23 pET pZA23 pEXT20 pCP20 Name Table2 - - - AspC TyrB IlvE – -

28(a)+ ns leucine,Ec (2018). Construction of asyntheticmetabolic pathway fortheproduction of - - -

thrA thrA thrA – - - - -

: : Kinetic parameters of : Plasmids usedin this study.

ilvE aspC LLldhA aspC

Not saturated at up to 50mMsubstrate to atup Not saturated concentration.

S345F S345F

- LLldhA - LLldhA - Q85C TyrB TyrB - aspC

Q85C – Q85C

- phenylalanine,Ec LLldhA Homoserine**

0.082 0.057 0.077 Vmax [µmol/(min*mg prot)] [µmol/(min*mg Vmax Comment citer cedocument: Q85C ±0.01 ±0.01 ±0.02

several

- AspC

transaminases on homoserineand their natural substrates. pZA23 derivative carrying carrying LLldhA derivative pZA23 carrying derivative pEXT20 carrying derivative pEXT20 carrying derivative pEXT20 carrying derivative pEXT20 carrying derivative pZA23 carrying derivative pZA23 ori ori p15A ori cassette Kan remove to recombinase ori Relevant characteristics

–

from at least three least at from aspartate. (**) activity was estimated at a homoserine concentration of 50 homoserine of concentrationa estimated at was activity (**) aspartate. Natural Natural

colE1, Amp colE1, f1,Kan S11 Amp pSC101, Q85C 74 86.4 35.5 , Kan , .0 substrate*

R , T7 promoter T7 , 32 ±5.7 ± ±3.2 2.8

R , P R

, tac promoter tac ,

A1lacO

replicates. (*) Natural substrates (each at (each substrates Natural (*) replicates. R

, plasmid expressing Flp Flp expressing plasmid , - 1

promoter

thrA thrA

ilvE ilvE aspC LLldhA aspC Homoserine

S345F and and

and and

thrA ns ns ns Q85C

LLldhA

S345F

, Q85C Km [mM] Q85C aspC

, Natural substrate* Natural

Wackernagel, Wackernagel, Bujard, Bujard, (Cherepanov (Cherepanov et al., 1996) etal., (Dykxhoorn (Dykxhoorn Reference Novagen 1.59 1. 0.49 This study This study This study This study This study This study This study This (Lutz and and (Lutz 33 1995) and and 20

±0.69 ±0.84 ±0.2

1997) mM): Ec mM):

1 TM

- Version postprint 2,4-dihydroxybutyric acidfrom homoserine. Metabolic Engineering. ,DOI :10.1016/j.ymben.2017.12.005 Walther, T.,Calvayrac, F.,Malbert,Y., Alkim,C.,Dressaire, C.,Cordier, H.,François, J.m. Mdh. ns mM). 20 nc (at mM. 20 to OHB up of andconcentrations oxaloacetate) mM 0.05 or pyruvate mM 20 (at substrate natural presented are Data Ll Ec Ll Ec Enzyme natural substrates Table4 - - - - PanE LdhA Mdh Ldh

(2018). Construction of asyntheticmetabolic pathway fortheproduction of

: Kinetic: parameters candidate of α [µmol/(mg 405 105 466 Vmax min)]

as means ± STDV calculated from at least least at from calculated STDV ± means as ±22 ±72 129 .

Natural substrate Natural Comment citer cedocument: 0.0 2. 1.39 8 [mM] Km 3 6 – - ±0.02 ±0.47

±0.2 not ca not

lculated. Natural substrates: Pyruvate for Ec for Pyruvate substrates: Natural lculated.

Vmax/Km

3500 335 141 - - oxo acid reductases on 2 acidreductasesoxo on

0.0 [µmol/(mg 11 2. 0.56 three 0 Vmax min)] .33 2 7

2 ±0.00 ±0.69 ±0.01

±4 oxo replicates. (*) Ratio of activities measured on the the on measured activities of Ratio (*) replicates.

- 4 - hydroxybutyrate [mM] - Km ns ns ns ns oxo

- - 4 Ldh, Ll Ldh, - hydroxybutyrate and their Vmax/Km –

- o strtd t substrate at saturated not LdhA; Oxaloacetate for Ec for Oxaloacetate LdhA;

nc nc nc nc

Specificity 4748 718 41 -

- Version postprint 2,4-dihydroxybutyric acidfrom homoserine. Metabolic Engineering. ,DOI :10.1016/j.ymben.2017.12.005 Walther, T.,Calvayrac, F.,Malbert,Y., Alkim,C.,Dressaire, C.,Cordier, H.,François, J.m. calculated. Ns concentration. substrate mM 20 at OHB and presented are Data Q85 Q85 wt Mutation and 2 5 Table

N C

(2018). Construction of asyntheticmetabolic pathway fortheproduction of - oxo : Impact of mutations in mutations of Impact :

- 4 [µmol/(mg - hydroxybutyrate 216 101 466 Vmax min)]

as means ± STDV calculated from at least three replicates. (*) Ratio of activities measured on pyruvate on measured activities of Ratio (*) replicates. three least at from calculated STDV ± means as ±29 ±11 ±72

Pyruvate 1.39 Comment citer cedocument: [mM] Km ns ns ±0.2

(L) - Vmax/Km lactate dehydrogenase A from A dehydrogenase lactate 335

nc nc

–

o strtd t usrt cnetain o u t 2 m. nc mM. 20 to up of concentrations substrate at saturated not

[µmol/(mg 34 Vmax 16 47 11 min)]

2 ±3 ±7 ±4 - oxo

- 4 - hydroxybutyrate [mM] Km ns ns ns

L. lactis L. Vmax/Km nc nc nc

on its activity its on

Specificity 13 41 2

on pyruvate on *

–

not Version postprint 2,4-dihydroxybutyric acidfrom homoserine. Metabolic Engineering. ,DOI :10.1016/j.ymben.2017.12.005 Walther, T.,Calvayrac, F.,Malbert,Y., Alkim,C.,Dressaire, C.,Cordier, H.,François, J.m. Table MG1655 Eco10 Eco6 Eco5 Eco18 Eco13 Eco12 Eco11 Strain Strain Eco9 Eco7 Eco4 No (5) (4)

(2018). Construction of asyntheticmetabolic pathway fortheproduction of

(3) (3) 6

(3)

: Productformation by

ooeie eyrgns ezms rsetvl. eie 4 ws elcd y hnllnn t alleviate to phenylalanine by replaced was 345 Serine respectively. enzymes, dehydrogenase homoserine coli E. wt

Genotype –

wild Strain Strain ∆4

∆4 ∆4 ∆4 ∆4 ∆4 ∆4 ∆4 ∆4 ∆4 wt genes

- (1) type strain,type

(1)

aspC pZA23 pZA23 none none Plasmids pZA23 pZA23 pZA23 pZA23 pZA23 pZA23 and , Comment citer cedocument: ∆4

------thrA thrA thrA thrA thrA +pEXT20 empty +pEXT20 empty empty

∆adhE ∆metA∆ldhA ∆adhE ∆thrB (2) E. coli thrA

S345F S345F S345F S345F S345F S345F

aspC strains expressingsynthetic the DHB pathway

noe satt aiornfrs, n bfntoa aprae kinase aspartate bifunctional and aminotransferase, aspartate encode + pEXT20 + pEXT20 + pEXT20 + pEXT20 pEXT20 - LLldhA ------Q85C aspC empty LLldhA aspC aspC empty empty 35

- -

LLldhA LLldhA

Q85C

Q85C Q85C

5.6 ±0.45 7.2 ±0.42 5.4 ±0.1 5.5 ±0.1 6.1 ±0.1 5.3 ±0.1 5.2 ±1 OD 4.0 1.6 5.5

600

Homoserine 1040 ±350 1110 ±12 329 ±5.8 747 ±53 703 ±20 477 ±27 no growth no [mg/L] .

0 ±0 581 396

DHB 24h DHB 439 ±29 190 ±17 [mg/L] 81 ±1 87 ±8 48 ±2 59 ±4 0 ±0 48 11 45

Version postprint 2,4-dihydroxybutyric acidfrom homoserine. Metabolic Engineering. ,DOI :10.1016/j.ymben.2017.12.005 Walther, T.,Calvayrac, F.,Malbert,Y., Alkim,C.,Dressaire, C.,Cordier, H.,François, J.m. Sum Undetected Cin medium Sum CO Acetate Lactate Homoserine DHB Biomass Products Sum Met Thr Glucose Substrates Compound Table7 2

(6)

(2018). Construction of asyntheticmetabolic pathway fortheproduction of

: Carbon: balance of the fed

Data from a single a Data from experiment. this replaced was variant,by enzyme cysteinereductase 85 glutamine increase to OHB activity. from is LlldhAgene The ThrA. of threonineinhibition

Comment citer cedocument:

- batch fermentation strain of Eco19 C 2.549 0.800 1.749 1.149 0.019 0.030 0.003 0.280 0.268 2.977 0.034 0.037 2.906 - mol

36 Lactococcus lactis Lactococcus % Carbon 26.9 38.6 100 0.7 1.0 0.1 9.4 9.0 86 59

and encodes and .

(L) - lactate dehydrogenase.In lactate

Version postprint 2,4-dihydroxybutyric acidfrom homoserine. Metabolic Engineering. ,DOI :10.1016/j.ymben.2017.12.005 Walther, T.,Calvayrac, F.,Malbert,Y., Alkim,C.,Dressaire, C.,Cordier, H.,François, J.m. Highlights: (2018). Construction of asyntheticmetabolic pathway fortheproduction of     

An engineered OHB reductaseactivity was improved by Homoserinetransaminase activity identifiedwas by screening of natural pathwayThe employs homoserine transaminase and A

new synthetic meta Comment citer cedocument: E. coli

strain expressingpathway the produces atDHB a yield of 0.1 g/g bolic pathway

for production of 37

rational engineering of lactatedehydrogenase OHB

2,4 - dihydroxybutyrate

reductaseactivities transaminases

is presented