Methods for producing l-pipecolic acid

Abstract

The present invention relates to a method for the production of L-pipecolic acid by a bacterial host cell in culture at a temperature of more than 32 °C. The present invention further relates to a bacterial host cell being genetically engineered to express lysine 6-aminotransferase to convert L-lysine to the intermediate delta-1-piperideine-6-carboxylic acid and express pyrroline-5-carboxylate reductase and being further characterized by overproduction of lysine.

Description

[0001] ^^^^^^^^application Univ^rsita^t^d^s^Saarland^s Our^R^f.:^SAR17989^^^ ^ ^^^^^^^^F^r^Pr^^ucing^L-Pip^c^lic^Aci^ ^h^^pr^s^nt^inv^ntion^r^lat^s^to a^m^thod for^th^^production^of^L^pip^colic^acid^by^a^bact^rial^ host^c^ll in^cultur^ at^a^t^mp^ratur^^of^mor^^than^32^°^.^^h^^pr^s^nt^inv^ntion^furth^r^r^lat^s^ to^ a^ bact^rial^ host^ c^ll^ b^ing^ g^n^tically^ ^ngin^^r^d^ to^ ^xpr^ss^ lysin^^ 6^aminotransf^ras^^^ and^pyrrolin^^5^carboxylat^^ r^ductas^ and^b^ing^ furth^r^charact^riz^d^by^ov^rproduction^of^ lysin^. L^pip^colic^acid^is^a^non^prot^inog^nic^amino^acid^^hich^is^found^in^many^organisms^among^ plants,^ animals^ incl.^ human^ b^ings,^ and^ bact^ria.^ In^ plants,^ it^ is^ kno^n^ to^ induc^^ plant^^^ immunity^ and^ initiat^^ r^sistanc^^ m^chanisms^ upon^ contact,^ and^ it^ accumulat^s^ in^ r^surr^cting^plants^^hich^us^^L^pip^colic^acid^ as^cons^rvation^compound^ (^avarova^^t^al.,^ ^lant^ ^^ll^ (2012)^ 24:^ 5123^5141;^ B^rnsdorff^ ^t^ al.,^ ^lant^ ^^ll^ (2016)^ 28:^ 102^129).^ In^ humans,^ L^pip^colic^ acid^ is^ produc^d^ in^ diff^r^nt^ organs^ and^ tissu^s^ and^ also^ kno^n^ as^ diagnostic^ mark^r^ for^ diff^r^nt^ dis^as^s^ such^ as,^ ^.g.,^ ^pil^psy,^ acut^^ isch^mic^ strok^,^ or^^^ liv^r^and^gallbladd^r^dis^as^s^(Mihalik^^t^al.,^^^diatric^R^s^(1989)^25:^548^552,^^l^cko^^t^al.,^ ^^urop^diatrics^(2005) 36:^200^205,^Qi^^t^al.,^Appli^d^Bioch^m^Biot^chnol^(2022)^194:^5443^ 5455).^ L^pip^colic^ acid^ is^ furth^r^ us^d^ as^a^pr^cursor^ for^ producing^pharmac^uticals,^ int^r^ alia^ as^ anticanc^r^ drug,^ immunosuppr^ssants,^ and^ antibiotics^ (s^^,^ ^.g.,^ G^rmann^ ^t^ al.,^ Anticanc^r^Drugs^(1997)^8:^125^140,^H^^^t^al.,^J^Ind^Microbiol^Biot^chnol^(2006)^33:^401^407,^^^ Ir^land^^t^al.,^J^Org^^h^m^(1996)^61:^6856^6872,^Gatto^^t^al., J^Am^^h^m^Soc (2006)^128:^ 3838^3847,^ and^ Bog^r^ ^t^ al.,^ J^ Am^ ^h^m^ Soc^ (1996) 188:^ 1629^1644).^ In^ bact^ria,^ L^ pip^colic^acid^has^b^^n^d^scrib^d^as^osmoprot^ctant^and^prot^ctant^against^^xt^rnal^str^ss^ factors^(s^^,^^.g.,^Gouffi^^t^al.,^Appl^Environ^Microbiol^(2000)^66:^2358^2364,^^^r^z^Garcia ^t^ al.,^Front^Microbiol^(2019)^10:^340,^and^B^ck^r^^t^al.,^Microb^^^ll^Fact^(2020)^12:^110). ^^ ^omm^rcial^ us^^ of^ L^pip^colic^ acid,^ ^.g.^ in^ th^^ pharmac^utical^ ar^a,^ food^ industry^ or^ cosm^tics,^r^quir^s^a^high^optical^purity^(Madd^ss^^t^al.,^^rog^Drug^R^s^(2008)^66:^15^186).^ Whil^^ ch^mical^ synth^sis^ m^thods^ can^ b^^ us^d^ to^ yi^ld^ larg^^ amounts^ of^ pip^colic^ acid,^ s^l^ction^ of^ prop^r^ rac^mat^s^ among^ D^ and^ L^pip^colic^ acid^ isom^rs^ is^ cost^ and^ tim^^^^ int^ns^,^ making^ L^pip^colic^ acid^ v^ry^ ^xp^nsiv^^ (^h^ng^ ^t^ al.,^ J^ Ind^Microbiol^ Biot^chnol^ (2018)^45:^719^734).^^h^^production^of^L^pip^colic^acid^dir^ctly^from^natural^microorganisms^ is^ too^ lo^^ to^ b^^ comm^rcially^ r^asonabl^.^ On^ th^^ oth^r^ hand,^ ^nzymatic / ^ biological^ L^ ^ pip^colic^synth^sis^m^thods^allo^^st^r^os^l^ctiv^^production^of^L^pip^colic^acid,^but^r^sult^in^ limit^d^and^non^satisfying^production^yi^lds^(s^^,^^.g.,^L^mir^^^t^al.,^J^Org^^h^m^(2010),^75:^ 2077^2080;^Fuji^^t al.,^Biosci^Biot^chnol^Bioch^m^(2002),^66(3):^622^627,^^^r^z^Garcia^^t^al.,^ Biot^chnol^J^(2017)^12,^doi:^10.1002 / biot.201600646).^ ^ ^h^s^^ and^ furth^r^ disadvantag^s^ n^^d^ to^ b^^ ov^rcom^.^ ^h^^ pr^s^nt^ inv^ntion^ th^r^for^^ addr^ss^s^th^s^^n^^ds^and^t^chnical^obj^ctiv^s^and^provid^s^a^solution^as^d^scrib^d^h^r^in^ and^as^d^fin^d^in^th^^claims. ^^ ^h^^ pr^s^nt^ inv^ntion^ thus^ r^lat^s^ to^ a m^thod for^ th^^production^of^ L^pip^colic^ acid^ by^a^ bact^rial^ host^ c^ll in^ cultur^,^ comprising^ conv^rting^ L^lysin^^ (may^ also^ b^^ ^quival^ntly^ r^f^rr^d^ to^ h^r^in^ as^ ^ “lysin^”)^ by^ th^^ us^^ of^ lysin^^6^aminotransf^ras^ (also^ r^f^rr^d^ to^ h^r^in^as^LA^)^to^d^lta^1^pip^rid^in^^6^carboxylic^acid (also^r^f^rr^d^to^h^r^in^as^^6^);^and^ r^ducing^ d^lta^1^pip^rid^in^^6^carboxylic^ acid^ by^ th^^ us^^ of^ pyrrolin^^5^carboxylat^^^^ r^ductas^^(also^r^f^rr^d^to^h^r^in^as^^5^R)^to^L^pip^colic^acid, ^h^r^in^ said^c^ll^cultur^^ is^ carri^d^out^ by^a^ t^mp^ratur^^of^mor^^ than about 32°^,^^.g.,^at^ about^33^°^,^about^34^°^,^about^35^°^,^about^36^°^,^about^37^°^,^or^about^38^°^,^or^about^ 39^°^,^or^about^40^°^,^or^about^45^°^,^or^about^50^°^. For^^xampl^,^th^^c^ll^cultur^^is^carri^d^ out^at^a^t^mp^ratur^^of^mor^^than^about^32^°^^to^about^60 °^,^pr^f^rably^from^about^32^°^^to^^^ about^ 40^ °^, pr^f^rably^ from^about^ 32^ °^^ to^ about^ 39^ °^,^ pr^f^rably^ from^about^ 32 °^^ to^ about^38^°^,^mor^^pr^f^rably^from about^33^°^^to^about^37^°^,^mor^^pr^f^rably^from^about^ 34^°^^to^about^37^°^,^or^from^about^34^°^^ to^about^35^°^. In^a^sp^cific^^mbodim^nt^of^ th^^ pr^s^nt^inv^ntion,^th^^c^ll^cultur^^is^carri^d^out^at^a^t^mp^ratur^^of^about^34^°^. ^^ ^h^^ t^rm^ "about"^ or^ "approximat^ly"^ as^ us^d^ h^r^in^ m^ans^ ^ithin^ 20%,^ pr^f^rably^ ^ithin^ 10%,^and^mor^^pr^f^rably^^ithin^5% or^2% of^a^giv^n^ valu^^or^ rang^,^and^^ach^of^ th^s^s^ t^rms^also^compris^s th^^r^sp^ctiv^^^xact^num^ric^valu^. ^h^^t^rm “mor^^than”^^h^n^us^d^in^th^^cont^xt^of^t^mp^ratur^^valu^s^or^rang^s^m^ans^that^^^ th^^^xact^ t^mp^ratur^^valu^^ is^also^ includ^d,^^.g.,^a^t^mp^ratur^^of^“mor^^than^about^32°^”^ m^ans^that^th^^t^mp^ratur^^also^includ^s^32°^. L^pip^colic^acid^(or,^as^may^b^^som^tim^s^us^d^^quival^ntly^h^r^in^“pip^colic^acid”)^has^th^^ follo^ing^structural^formula: ^

[0002] Formula^(1). Its^ch^mical^formula^is^^6H11^O2.^Its^^AS^numb^r^is^3105^95^1. ^ Synonyms^of^L^pip^cilic^acid^^hich^may^also^b^^us^d^in^th^^cont^xt^of^th^^pr^s^nt^inv^ntion^ ar^^L^Homoprolin^,^^ip^ridin^^2^carboxylic^acid or^(S)^(^)^2^^ip^ridin^carboxylic^acid. As^has^ turn^d^out^ in^ cont^xt^^ith^ th^^ pr^s^nt^ inv^ntion,^ applying^an^adapt^d^approach^as^ kno^n^in^th^^art^(cf.^^^r^z^Garcia^^t^al.,^(2017),^^^^^^^^^.),^L^pip^colic^acid^production^using^L^^^ lysin^^6^d^hydrog^nas^^(^.g.,^from^^^^p^mer^y^;^also^r^f^rr^d^to^h^r^in^as^LysDH)^ in^a^first^ st^p^and^d^lta^ pyrrolin^^5^carboxylat^^ r^ductas^ (^.g.,^ from^C^^g^u^am^^um) ^as^us^d^ in^a^ s^cond^st^p did^not^r^sult^in^satisfying yi^lds,^^v^n^though^application^of^high^r^t^mp^ratur^s (^.g.,^ 34^ °^)^ l^d^ to^ improv^d^ production^ yi^lds (cf.^ Exampl^s^ 1.1^ to^ 2.3^ h^r^in).^ Oth^r^ approach^s^kno^n^in^th^^art^^h^r^^lysin^^6^aminotransf^ras^ (LA^)^(^.g.,^from^F^^^u^es^ens)^^^ ^as^ us^d^ in^ a^ first^ st^p^and^ d^lta^pyrrolin^^5^carboxylat^^ r^ductas^ (^5^R)^ (^.g.,^ from^E^^ ^^^^) in^ a^ s^cond^ st^p r^sult^d^ in^ ^v^n^ lo^^r^ yi^lds of^ L^pip^colic acid compar^d^ to^ th^^ approach^ using^ L^lysin^^6^d^hydrog^nas^ (LysDH) and^ d^lta^pyrrolin^^5^carboxylat^^ r^ductas^ (^5^R) abov^ (s^^^Fuji^ ^t^ al.,^ (2002),^ ^^^^^ ^^^.). Also^ anoth^r^approach^using^L^ lysin^^ cyclod^aminas^^ (rapL;^ cf.^ also^ Gatto^ ^t^ al.,^ (2006),^ ^^^^^ ^^^.) ^hich^ catalys^s^ both^^^ r^actions,^ oxidation^ of^ L^lysin^^ and^ r^duction^ of^ th^^ int^rm^diat^^ d^lta^1^pip^rid^in^^6^ carboxylic^acid^to^L^pip^colic^acid, r^sult^d^only^in^poor production^yi^lds (data^not^sho^n). Ho^^v^r,^as^has^b^^n^surprisingly^found in^cont^xt^^ith^th^^pr^s^nt^inv^ntion,^^mploying^a^ bact^rial^host^c^ll^ to^conv^rt L^lysin^^by^th^^us^^of^ lysin^^6^aminotransf^ras^ (also^r^f^rr^d^^^ to^h^r^in^as^LA^)^to^d^lta^1^pip^rid^in^^6^carboxylic^acid (also^r^f^rr^d^to^h^r^in^as^^6^)^in^ a^ first^ st^p^ and^ r^ducing^ d^lta^1^pip^rid^in^^6^carboxylic^ acid^ by^ th^^ us^^ of^ pyrrolin^^5^ carboxylat^^r^ductas^^(also^r^f^rr^d^to^h^r^in^as^^5^R)^to^L^pip^colic^acid in^a^s^cond^st^p,^ ^h^r^^ th^^ bact^rial^ host^ c^ll^ is^ cultur^d^ at^ a^ t^mp^ratur^^ of^ mor^^ than^ 32^ °^,^ l^ads^ to^ significantly^ incr^as^d^ yi^lds^of^ L^pip^colic^acid^production (s^^,^^.g.,^Exampl^^2.4^h^r^in).^^^ ^his^finding^is^ind^^d^surprising^sinc^^th^^us^^of^LA^^and^^5^R at^t^mp^ratur^s^of^about^30^ ^ °^^l^d^to^much^lo^^r^yi^lds^of^L^pip^colic^acid^compar^d^to^th^^us^ of^LysDH and^^5^R^(cf.^ Fuji^^t^al.,^(2002)^ ^^^^^^^^.^vs^^^r^z^Garcia^^t^al.,^(2017),^ ^^^^^^^^.),^and^– as^found^ in^cont^xt^ ^ith^th^^pr^s^nt^ inv^ntion^ ^ ^v^n^^l^vation^of^ th^^t^mp^ratur^^(^.g.,^ to^34^°^)^for^ th^^prima^ faci^^mor^^promising^approach^using^LysDH^and^^5^R^did^not^l^ad^to^satisfying^yi^lds^of^L^ ^ pip^colic^ acid^ production. ^hus,^ as^ is^ sho^n^ in^ th^^ pr^s^nt^ inv^ntion,^ it^ is^ ind^^d^ an^ incr^as^d^ t^mp^ratur^^^hich^provid^s^ for^an^ incr^as^d^production^of^L^pip^colic^acid.^^his^ could^not^hav^^b^^n^^xp^ct^d,^ l^t^alon^^sugg^st^d^ in^th^^prior^art,^sinc^ prior^art^att^mpts^ ^h^n^actually^producing^L^pip^colic^acid^ constantly^apply a^ t^mp^ratur^^of^at^most^around^ 30°^. ^^ Also,^ as^ ^as^ found^ in^ cont^xt^ ^ith^ th^^ pr^s^nt^ inv^ntion,^ ^h^n^^mploying^ th^^m^thod^ as^ d^scrib^d^and^provid^d^h^r^in,^production^s^l^ctivity^of^pip^colic^acid^isom^rs^sho^s^a^v^ry^ high^s^l^ction^of^th^^L^pip^colic^acid^form^^hich^is^favourabl^^for^comm^rcial^applications.^ ^^ In^on^^^mbodim^nt^of^th^^pr^s^nt^inv^ntion,^pyridoxal^phosphat^^is^add^d^to^cultur^. As^has^ furth^rmor^^b^^n^ found^ in^ cont^xt^^ith^ th^^pr^s^nt^ inv^ntion and^as^ provid^d^ and^ sho^n^ h^r^in,^ th^^ addition^ of^ pyridoxal^ phosphat^^ to^ th^^ c^ll^ cultur^^ l^d^ to^ incr^as^d^ production^of^L^pip^colic^acid (s^^,^^.g.,^Exampl^^2.5^h^r^in). Also,^as^has^surprisingly^b^^n^^^ sho^n^ in^ cont^xt^ ^ith^ th^^ pr^s^nt^ inv^ntion,^ th^^ stimulating^ ^ff^ct^ of^ pyridoxal^ phosphat^^ addition^to^th^^c^ll^cultur^^^as^^v^n^strong^r^at^t^mp^ratur^s^of^mor^^than^about^32^°^,^^.g.,^ at^ t^mp^ratur^s^ of^ about^ 34^ °^^ to^37^ °^,^ ^.g.,^ at^ a^ t^mp^ratur^^of^ about^ 34^ °^.^^yridoxal^ phosphat^^can^b^^add^d^to^ th^^c^ll^cultur^^ in^any^suitabl^^amount.^For^^xampl^,^ it^may^b^^ add^d^ in^ a^ conc^ntration^of^ about^ 1 to^about^ 50^mg / L,^ 2^ to^ about^ 50^mg / L,^3^ to^about^ 50^^^ mg / L,^4^to^about^50^mg / L,^5^to^about^50^mg / L,^6^to^about^50^mg / L,^7^to^about^50^mg / L,^8^to^ about^50^mg / L,^9^to^about^50^mg / L,^10^to^about^50^mg / L,^11^to^about^50^mg / L,^12^to^about^50^ mg / L,^13^ to^about^ 50^mg / L,^14^ to^about^ 50^mg / L^or^15^ to^about^ 50^mg / L,^pr^f^rably^ 10^ to^ about^15^mg / L,^mor^^pr^f^rably^13^to^15^mg / L.^In^on^^^mbodim^nt^of^th^^pr^s^nt^inv^ntion,^ pyridoxal^ phosphat^^ is^ add^d^ to^ th^^ c^ll^ cultur^^ in^ a^ conc^ntration^ of^about^ 5^ to^ about^ 40^^^ mg / L,^pr^f^rably^about^10^to^about^25^mg / L,^pr^f^rably^about^10^to^about^15^mg / L. In^on^^^mbodim^nt^of^th^^pr^s^nt^inv^ntion,^said c^ll cultur^^is^a^f^d^batch^cultur^. As^has^ furth^rmor^^b^^n^ found^ in^ cont^xt^^ith^ th^^pr^s^nt^ inv^ntion^and^as^ provid^d^ and^^^ sho^n^ h^r^in,^ applying^ a^ f^d^batch^ production^ proc^ss^ r^sult^d^ in^ v^ry^ high^ yi^lds^ of^ L^ pip^colic^ acid^at^ t^mp^ratur^s^of^mor^^ than^ about^ 32^ °^^ (^.g.,^ about^ 34^ °^).^ As^ could^b^^ obs^rv^d^in^cont^xt^^ith^th^^pr^s^nt^inv^ntion,^th^^yi^ld^of^L^pip^colic^acid^vs.^th^^yi^ld^of^L^ ^ lysin^^incr^as^d^significantly^during^th^^f^^ding^phas^^(s^^,^^.g.,^Exampl^^2.6^h^r^in).^Also,^ s^l^ctivity^of^production^of^th^^L^pip^colic^acid^isom^r^incr^as^d^^v^n^furth^r.^^h^s^ findings ^^r^ particularly^s^^n^in^combination^^ith^addition^of^pyridoxal^phosphat^^to^th^^c^ll^cultur^. ^ In^on^^^mbodim^nt^of^th^^pr^s^nt^inv^ntion,^said^bact^rial^host^c^ll^^hich^is^^mploy^d^in^th^^ c^ll^cultur^^of^th^^m^thod^d^scrib^d^and^provid^d^h^r^in^is^charact^riz^d^by^having^(natural)^ pyrrolin^^5^carboxylat^^r^ductas^^activity^and^b^ing^g^n^tically^^ngin^^r^d^to^^xpr^ss^lysin^^ 6^aminotransf^ras^. ^^ In^accordanc^^^ith^th^^pr^s^nt^inv^ntion,^any^suitabl^^bact^rial^c^ll^can^b^^us^d^as^host^c^ll^ in^ th^^m^thod^ d^scrib^d^ and^provid^d^ h^r^in.^ In^ on^^ ^mbodim^nt,^ such^ bact^rial^ c^ll^ has^ (natural)^ pyrrolin^^5^carboxylat^^ r^ductas^^ (^5^R)^activity^ and^ is^ g^n^tically^ ^ngin^^r^d^ to^ ^xpr^ss^ lysin^^ 6^aminotransf^ras^ (LA^).^ “^5^R”^ activity^ as^ us^d^ h^r^in^ g^n^rally^ m^ans^ that^ such^ bact^rial^ host^ c^ll^ (stably)^ ^xpr^ss^s^^5^R,^ pr^f^rably^ naturally,^ i.^.^ ^ithout^ th^^^^ n^^d^ to^ g^n^tically^ ^ngin^^r^ such^ bact^rial^ host^ c^ll^ to^ ^xpr^ss^ ^5^R.^ Ho^^v^r,^ in^ accordanc^^ ^ith^ this^ inv^ntion,^ it^ is^ also^ possibl^^ that^ a^ bact^rial^ host^ c^ll^ having^ ^5^R^ activity^ has^ that^ activity^ as^ a^ r^sult^ of^ having^ acquir^d^ a^ g^n^ (polynucl^otid^) ^ncoding^ ^5^R^ (^.g.,^ is^ g^n^tically^ ^ngin^^r^d^ to^ hav^^ ^5^R^ activity).^ ^5^R^ is^ nativ^ly^ part^ of^ L^ prolin^^biosynth^sis.^^hat^is,^for^^xampl^^and^in^accordanc^^^ith^this^inv^ntion,^any^bact^rial^^^ c^ll^that^is^capabl^^of^producing^L^prolin^^including^th^^^mploym^nt^of^^5^R^can^b^^us^d^in^ th^^m^thod^d^scrib^d^and^provid^d^ h^r^in,^ ^hich^ is^ furth^rmor^^g^n^tically^ ^ngin^^r^d^ to^ ^xpr^ss^LA^.^Exampl^s^for^bact^rial^c^lls^having^^5^R^activity^includ^^C^^g^u^am^^um.^ In^a^ sp^cific^ ^mbodim^nt^ of^ th^^ pr^s^nt^ inv^ntion,^ a^ bact^rial^ c^ll^ having^ ^5^R^ activity^ is^C^^ g^u^am^^um.^ ^hat^ is,^ in^ on^^ ^mbodim^nt^ of^ th^^ pr^s^nt^ inv^ntion,^ said^ bact^rial^ host^ c^ll^^^ ^hich^ is^ ^mploy^d^ in^ th^^ c^ll^ cultur^^ of^ th^^ m^thod^ d^scrib^d^ and^ provid^d^ h^r^in^ is^C^^ g^u^am^^um ^hich^ is^ g^n^tically^ ^ngin^^r^d^ to^ ^xpr^ss^ lysin^^ 6^aminotransf^ras^.^ In^ a^ particular^ ^mbodim^nt^ of^ th^^ pr^s^nt^ inv^ntion,^ th^^ bact^rial^ c^ll^ b^ing^ charact^riz^d^ by^ having^(natural)^pyrrolin^^5^carboxylat^^r^ductas^^(^5^R)^and^b^ing^g^n^tically^^ngin^^r^d^ to^^xpr^ss^lysin^^6^aminotransf^ras^^activity^(LA^)^^is^g^n^tically^^ngin^^r^d^by^introduction^^^ of^a^polynucl^otid^^^ncoding^LA^^d^riv^d^from^F^^^u^es^ens,^or^variants^or^d^viations of^such^ polynucl^otid^^or^prot^in, as^d^scrib^d^h^r^in. In^accordanc^^^ith^th^^pr^s^nt^inv^ntion,^an^ ^xampl^^for^a^polynucl^otid^^^ncoding^LA^^is^sho^n^in^SEQ^ID^^O:^4. ^h^^ t^rm^ “pyrrolin^^5^carboxylat^^ r^ductas^^ (^5^R)”,^ unl^ss^ oth^r^is^^ sp^cifi^d^ h^r^in,^ is^^^ us^d^h^r^in^in^accordanc^^^ith^its^m^aning^as^g^n^rally^kno^n^in^th^^art.^Int^r^alia,^^5^R^is^ kno^n^to^b^^an^^nzym^^that^participat^s^in^m^tabolic^path^ays^for^synth^sizing^prolin^^from^ arginin^^or^glutamic^acid.^For^^xampl^,^^5^R^has^an^activity^for^r^ducing^d^lta^1^pyrrolin^^5^ ^ carboxylic^acid^ to^prolin^ ^ith^th^^aid^of^th^^r^duc^d^form^of^^ADH^or^th^^r^duc^d^form^of^ ^AD^H.^ Also^ and^ pr^f^rably,^ ^5^R^ has^ an^ activity^ for^ r^ducing^ d^lta^1^pip^rid^in^^6^ carboxylic^acid^(^6^)^to^L^pip^colic^acid.^In^on^^^mbodim^nt^of^th^^pr^s^nt^inv^ntion,^^5^R^ corr^sponds^to^a^prot^in^according^to^E^^1.5.1.2^or^variants^or^d^viations th^r^of. In^anoth^r^ ^ ^mbodim^nt^ of^ th^^ pr^s^nt^ inv^ntion,^ th^^ ^5^R^ is^ d^riv^d^ from^ E^^ ^^^^ or^ coryn^form^ bact^rium,^for^^xampl^^C^^g^u^am^^um. In^accordanc^^^ith^th^^pr^s^nt^inv^ntion,^an^^xampl^^ for^a^polynucl^otid^^^ncoding^^5^R^(also^r^f^rr^d^to^as^^ro^) is^sho^n^in^SEQ^ID^^O:^5. “D^viations”^ from^ s^qu^nc^s^ (^.g.,^ amino^ acid^ or^ nucl^ic^ acid^ s^qu^nc^s)^ as^us^d^h^r^in^^^ may^ compris^,^ ^.g.,^d^l^tions,^ substitutions,^ additions,^ ins^rtion^ and / or^ r^combination.^ ^h^^ t^rm^"addition" r^f^rs^to^adding^a^nucl^ic^acid^r^sidu^ / amino^acid^to^th^^^nd^or^b^ginning^of^ th^^giv^n^s^qu^nc^,^^h^r^as^"ins^rtion"^r^f^rs to^ins^rting^a^nucl^ic^acid^r^sidu^ / amino^acid^ ^ithin^a^giv^n^s^qu^nc^.^^h^^t^rm^"d^l^tion"^r^f^rs^ to^d^l^ting^or^r^moval^of^a^nucl^ic^acid^ r^sidu^^ or^ amino^ acid^ r^sidu^^ in^ a^ giv^n^ s^qu^nc^.^ ^h^^ t^rm^ "substitution"^ r^f^rs^ to^ th^^^^ r^plac^m^nt^of^a^nucl^ic^acid^r^sidu^ / amino^acid^r^sidu^^in^a^giv^n^s^qu^nc^.^Again,^th^s^^ d^finitions^as^us^d^h^r^^apply,^mu^a^^s^mu^and^s,^ for^all^s^qu^nc^s^provid^d^and^d^scrib^d^ h^r^in^ unl^ss^ sp^cifi^d^ oth^r^is^. As^ us^d^ h^r^in,^ a^ “d^viation”^ of^ a^ particular^ g^n^^ or^ prot^in^still^^xhibits^th^^r^sp^ctiv^^activiti^s of^th^^r^sp^ctiv^^prot^in^(^ncod^d^by^th^^g^n^).^ For^^xampl^,^ in^accordanc^^^ith^th^^pr^s^nt^ inv^ntion,^a^d^viation^of^^5^R^still^has^^5^R^^^ activity^kno^n^in^th^^art,^^.g.,^th^^d^viation^of^^5^R^still^has^an^activity^for^r^ducing^d^lta^1^ pip^rid^in^^6^carboxylic^acid^ (^6^)^ to^L^pip^colic^acid. As^anoth^r^ ^xampl^,^ in^accordanc^^ ^ith^th^^pr^s^nt^inv^ntion,^a^d^viation^of^LA^^still^has^LA^^activity^kno^n^in^th^^art,^^.g.,^th^^ d^viation^of^LA^^still^has^an^activity^for^L^lysin^ to^^6^. ^^ As^ us^d^ h^r^in,^ th^^ t^rm “g^n^tically^ ^ngin^^r^d”^ g^n^rally^ m^ans^ (unl^ss^ sp^cifi^d^ oth^r^is^^ h^r^in)^ that^ th^^ r^sp^ctiv^^ c^ll^ ^ncod^s^ and^ is^ capabl^^ to (stably) ^xpr^ss^ a^ polynucl^otid^^ that^ it^ do^s^ naturally^ not^ contain^ and / or^ ^xpr^ss.^ For^ ^xampl^,^ such^ polynucl^otid^^has^b^^n^ introduc^d^ into^th^^r^sp^ctiv^^c^ll.^^r^f^rably,^such^polynucl^otid^^ r^pr^s^nts^an^^ntir^^ g^n^^or^ORF,^^ncoding^a^ compl^t^^or^ r^l^vant^ fragm^nt^of^a^prot^in.^^^ Such^polynucl^otid^^may^b^^brought^ into^th^^c^ll^via^t^chnical or^biological m^ans,^^.g.,^via transduction,^transformation,^ transf^ction,^or^any^oth^r^m^ans^that^ is^suitabl^^to^ introduc^^a^ polynucl^otid^^ into^ a^ c^ll,^ allo^ing^ (pr^f^rably^ stabl^)^ ^xpr^ssion^ of^ that^ polynucl^otid^^ in^ said^ c^ll.^ Such^m^thods^ ar^^ ^^ll^ kno^n^ in^ th^^ art^ and^ d^scrib^d,^ int^r^ alia,^ in Mol^cular^ ^loning ^ A^ Laboratory^Manual,^ 2nd^ Ed.^ by^ Sambrook,^ Fritsch^ and^Maniatis^ (^old^ Spring^^^ Harbor^ Laboratory^ ^r^ss,^ 1989);^ D^A^ ^loning,^ Volum^s^ I^ and^ II (D.^.^ Glov^r^ ^d.,^ 1985);^ Oligonucl^otid^^Synth^sis^(M.J.^Gait^^d.,^1984);^Mullis^^t^al.,^U.S.^^at^nt^^o.^4,683,195;^and ^ucl^ic^Acid^Hybridization^ (B.D.^Ham^s^&^S.J.^Higgins^^ds.,^1984). In accordanc^^^ith^ th^^ ^ pr^s^nt^ inv^ntion,^such^polynucl^otid^^may^b^^ introduc^d^ into^ th^^c^ll^alon^,^ i.^.^sol^ly^ th^^ d^sir^d^ polynucl^otid^,^ or^ tog^th^r^ ^ith^ oth^r^ polynucl^otid^s^ (^.g.,^ oth^r^ g^n^s,^ ORFs,^ promot^r^,^ r^gulator^ or^ ^nhanc^r^s^qu^nc^s,^ or^ oth^r^ functional^ or^ non^functional^ nucl^otid^s),^^ith^r^^ithin^a^singl^^mol^cul^^(^.g.,^a^plasmid,^cosmid,^v^ctor,^or^oth^r^forms)^ ^ or^ on^ s^parat^^ mol^cul^s. ^h^^ r^sp^ctiv^^ c^ll^ ^hich^ is^ “g^n^tically^ ^ngin^^r^d”^ in^ accordanc^^ ^ith^ this^ inv^ntion^ may^ contain^ th^^ introduc^d^ polynucl^otid^^ ^ith^r^ on^ an^ ^xtrachromosomal^ mol^cul^^ (^.g.,^ on^ a^ plasmid)^ or^ th^^ polynucl^otid^^ may^ b^^ (stably)^ int^grat^d^into^th^^or^a^c^ll^chromosom^.^^h^^introduc^d^polynucl^otid^^may^stand^und^r^th^^ control^ of^ its^ o^n^ promot^r or^ r^gulator^ or^ th^^ lik^^ (introduc^d^ tog^th^r^ ^ith^ th^^^^ polynucl^otid^),^ or^ und^r^ th^^ control^ of^ a^ promot^r^ or^ r^gulator^ or^ th^^ lik^^ of^ th^^host^ c^ll^ (^ith^r^alon^^or^und^common^control^tog^th^r^^ith^an^^ndog^nous^g^n^^of^th^^host^c^ll). G^n^rally,^ as^ us^d^ h^r^in,^ th^^ t^rms^ „polynucl^otid^“,^ „nucl^ic^ acid“^ and „nucl^ic^ acid^ mol^cul^“^ ar^^ to^ b^^ constru^d^ synonymously.^ G^n^rally,^ nucl^ic^ acid^ mol^cul^s^ may^^^ compris^^ ^n^er^ a^^a D^A^ mol^cul^s,^ R^A^ mol^cul^s,^ oligonucl^otid^^ thiophosphat^s,^ substitut^d^ ribo^oligonucl^otid^s^ or^ ^^A^ mol^cul^s.^ Furth^rmor^,^ th^^ t^rm^ "nucl^ic^ acid^ mol^cul^"^may^ r^f^r^ to^ D^A^ or^R^A^ or^ hybrids^ th^r^of^ or^ any^modification^ th^r^of^ that^ is^ kno^n^ in th^^ art^ (s^^,^ ^.g.,^ US^ 5525711,^ US^ 471^ 1955,^ US^ 5792608^ or^ E^^ 302175^ for^ ^xampl^s^ of^ modifications).^ ^h^^ polynucl^otid^^ s^qu^nc^^ may^ b^^ singl^^ or^ doubl^^^^ strand^d,^lin^ar^or^circular,^natural^or^synth^tic,^and^^ithout^any^siz^^limitation.^For^instanc^,^ th^^ polynucl^otid^^ s^qu^nc^^ may^ b^^ g^nomic^ D^A,^ cD^A,^ mitochondrial^ D^A,^ mR^A,^ antis^ns^^R^A,^ribozymal^R^A^or^a^D^A^^ncoding^such^R^As^or^chim^roplasts^(Gamp^r,^ ^ucl^ic^Acids^R^s^arch,^ 2000,^ 28,^4332^ ^ 4339).^Said^polynucl^otid^^s^qu^nc^^may^b^^ in^ th^^ form of^ a^ v^ctor,^ plasmid,^ cosmid or^ of^ viral^ D^A^ or^R^A.^ Also^ d^scrib^d^h^r^in^ ar^^^^ nucl^ic^ acid^ mol^cul^s^ ^hich^ ar^^ compl^m^ntary^ to^ th^^ nucl^ic^ acid^ mol^cul^s^ d^scrib^d^ abov^^ and^ nucl^ic^ acid^ mol^cul^s^ ^hich^ ar^^ abl^^ to^ hybridiz^^ to^ nucl^ic^ acid^ mol^cul^s^ d^scrib^d^h^r^in.^A^nucl^ic^ acid^mol^cul^^d^scrib^d^h^r^in^may^also^b^^a^ fragm^nt^of^ th^^ nucl^ic^acid^mol^cul^s^in^cont^xt^of^ th^^pr^s^nt^ inv^ntion.^^articularly,^such^a^fragm^nt^ is^a^ functional^ fragm^nt.^ Exampl^s^ for^ such^ functional^ fragm^nts^ ar^^ nucl^ic^ acid^ mol^cul^s^^^ ^hich^can^s^rv^^as^prim^rs. In^on^^^mbodim^nt^of^th^^pr^s^nt^inv^ntion, said^bact^rial^host^c^ll^^hich^is^^mploy^d^in^th^^ c^ll^cultur^^of^th^^m^thod^d^scrib^d^and^provid^d^h^r^in^is^charact^riz^d^by^having (natural) lysin^^ 6^aminotransf^ras^^ activity^ (LA^)^ and^ b^ing^ g^n^tically^ ^ngin^^r^d^ to^ ^xpr^ss^^^ pyrrolin^^5^carboxylat^^r^ductas^ (^5^R). In^accordanc^^^ith^th^^pr^s^nt^inv^ntion,^any^suitabl^^bact^rial^c^ll^can^b^^us^d^as^host^c^ll^ in^ th^^m^thod^ d^scrib^d^ and^provid^d^ h^r^in.^ In^ on^^ ^mbodim^nt,^ such^ bact^rial^ c^ll^ has^ (natural)^ lysin^^ 6^aminotransf^ras^^ activity^ (LA^)^ activity^ and^ is^ g^n^tically^ ^ngin^^r^d^ to^ ^xpr^ss^ pyrrolin^^5^carboxylat^^ r^ductas^ (^5^R).^ “LA^”^ activity^ as^ us^d^h^r^in^ g^n^rally^ ^ m^ans^ that^such^bact^rial^host^c^ll^ (stably)^^xpr^ss^s^LA^,^pr^f^rably^naturally,^i.^.^^ithout^ th^^ n^^d^ to^ g^n^tically^ ^ngin^^r^ such^ bact^rial^ host^ c^ll^ to^ ^xpr^ss^ LA^.^ Ho^^v^r,^ in^ accordanc^^^ith^this^inv^ntion,^it^is^also^possibl^^that^a^bact^rial^host^c^ll^having^LA^ activity^ has^ that^ activity^ as^ a^ r^sult^ of^ having^ acquir^d^ a^ g^n^^ ^ncoding^ LA^ (^.g.,^ is^ g^n^tically^ ^ngin^^r^d^ to^ hav^^ LA^ activity).^ As^ kno^n^ in^ th^^ art,^ LA^^ can^ conv^rt^ L^lysin^^ to^ ^6^.^^^ Exampl^s^ for^ bact^rial^ c^lls^ having^ LA^ activity^ includ^^ F^av^ba^^er^um^ ^u^es^ens, ^^en^^r^ph^m^nas^ g^nseng^s^^^,^ ^^en^^r^ph^m^nas^ k^reens^s, ^^en^^r^ph^m^nas^ sp.^ W1S232, ^^en^^r^ph^m^nas^ hum^, ^^en^^r^ph^m^nas^ ^errae, ^^en^^r^ph^m^nas^ pana^^hum^, ^^en^^r^ph^m^nas^ a^^dam^n^ph^^a, ^^en^^r^ph^m^nas^ sp.^ L^af70, ^^en^^r^ph^m^nas^sp.^Mars^ill^^Q4652, ^^en^^r^ph^m^nas^ma^^^ph^^^a,^F^av^ba^^er^um sp.^^^ MXW15,^ Xan^h^m^nas^ ^ryzae pv.^ Oryzicola,^ Xan^h^m^nas^ perf^rans,^ Xan^h^m^nas^ ^ampes^r^s pv.^ ^amp^stris^ (strain^ 8004),^ Xan^h^m^nas^ ^ryzae,^ Xan^h^m^nas^ f^^r^dens^s,^ Xan^h^m^nas^sp.^X^M01,^Dye^^a^jap^n^^a A8,^Dye^^a^^h^^^xydans, Pseud^xan^h^m^nas sp.,^ Pseud^xan^h^m^nas k^reens^s,^ Pseud^xan^h^m^nas suw^nens^s,^ Pseud^xan^h^m^nas ka^hs^ungens^s,^ Pseud^xan^h^m^nas daeje^nens^s,^ Lu^e^m^nas sp.^ JM171,^ Lu^e^ba^^er^^^ rh^z^v^^^nus DSM^ 16549,^ Rh^dan^ba^^er^ ^h^^^xydans,^ Pseud^m^nas sp.^ Hp2,^ F^av^ba^^er^um fus^um (Soda^ ^t^ al.,^ Bioch^mistry^ (1968),^ 7:^ 4102^4109),^ ^^rep^^my^es ^^avu^^gerus (Wu^ ^t^ al.,^ J^ Agric^ Food^ ^h^m^ (2007),^ 56:^ 1767^1772),^ and^My^^ba^^er^um ^uber^u^^s^s (Bioorg^ M^d^ ^h^m^ (2017),^ 25:^ 2761^2771). In^ a^ sp^cific^ ^mbodim^nt^ of^ th^^ pr^s^nt^ inv^ntion,^ a^ bact^rial^ c^ll^ having^ LA^ activity^ is^ F^^ ^u^es^ens.^ ^hat^ is,^ in^ on^^^^ ^mbodim^nt^ of^ th^^pr^s^nt^ inv^ntion,^ said^bact^rial^ host^ c^ll^^hich^ is^ ^mploy^d^ in^ th^^c^ll^ cultur^^ of^ th^^ m^thod^ d^scrib^d^ and^ provid^d^ h^r^in^ is^ F^^ ^u^es^ens ^hich^ is^ g^n^tically^ ^ngin^^r^d^ to^ ^xpr^ss^ ^5^R. In^ a^ particular^ ^mbodim^nt^ of^ th^^ pr^s^nt^ inv^ntion,^ th^^ bact^rial^c^ll^b^ing^charact^riz^d^by^having^(natural)^lysin^^6^aminotransf^ras^^activity^(LA^)^ and^ b^ing^ g^n^tically^ ^ngin^^r^d^ to^ ^xpr^ss^ pyrrolin^^5^carboxylat^^ r^ductas^^ (^5^R)^ is^^^ g^n^tically^ ^ngin^^r^d^ by^ introduction^ of^ a^polynucl^otid^^^ncoding^a^prot^in^ according^ to^ E^^1.5.1.2^or^variants^or^d^viations th^r^of as^d^scrib^d^h^r^in. ^h^^ t^rm^ “lysin^^ 6^aminotransf^ras^^ activity^ (LA^)”,^ unl^ss^ oth^r^is^^ sp^cifi^d^ h^r^in,^ is^ us^d^h^r^in^in^accordanc^^^ith^its^m^aning^as^g^n^rally^kno^n^in^th^^art.^ Int^r^alia,^LA^ is^^^ kno^n^to^b^^an^^nzym^^having an^activity^for^conv^rting^L^lysin^^to^^6^.^In^on^^^mbodim^nt^ of^th^^pr^s^nt^ inv^ntion,^LA^ or^ ^a^ may^b^ a^prot^in^or^polynucl^otid^ (g^n^),^r^sp^ctiv^ly,^ d^riv^d^ from^F^^ ^u^es^ens (^.g.,^ strain^ IFO3084;^ cf.^Soda^ ^t^ al.,^ Bioch^m^ (1968),^ 7:^ 4102^ 4109;^4110^4119) or^variants^or^d^viations th^r^of. In^ on^^ ^mbodim^nt^ of^ th^^ pr^s^nt^ inv^ntion,^ said^ host^ c^ll^ ^hich^ is^ ^mploy^d^ in^ th^^ c^ll^ ^ cultur^^ of^ th^^m^thod^ d^scrib^d^ and^ provid^d^ h^r^in^ is^ g^n^tically^ ^ngin^^r^d^ to^ ^xpr^ss^ lysin^^6^aminotransf^ras^ (^.g.,^a^polynucl^otid^^^ncoding^LA^^d^riv^d^from^F^ ^u^es^ens,^or^ variants^or^d^viations of^such^polynucl^otid^^or^prot^in,^as^d^scrib^d^h^r^in) and^pyrrolin^^5^ carboxylat^^r^ductas^ (^.g.,^by^introduction^of^a^polynucl^otid^^^ncoding^a^prot^in^according^ to^ E^^ 1.5.1.2^ or^ variants^ or^ d^viations th^r^of as^ d^scrib^d^ h^r^in).^ ^r^f^rr^d^ host^ c^lls^^^ includ^^Es^her^^h^a ^^^^,^Ba^^^^us^sub^^^^s,^or^Pseud^m^nas^pu^^da.^Mor^^pr^f^rr^d^host^c^lls^ includ^^^oryn^bact^rium^ sp^ci^s,^ mor^^pr^f^rably^C^^ g^u^am^^um,^ such^ as^C^^ g^u^am^^um LYS^12^(B^ck^r,^M^tab^Eng^(2011),^13: 159^168). In^ accordanc^^ ^ith^ th^^ pr^s^nt^ inv^ntion,^ th^^ g^n^s^ for^ pyrrolin^^5^carboxylat^^ r^ductas^^^ and / or^ lysin^^ 6^aminotransf^ras^ may^ b^^ ^xpr^ss^d^ on^ s^parat^^ or^ th^^ sam^^ op^ron,^ r^gardl^ss^ ^h^th^r^ said^ g^n^(s)^ is^ (ar^)^ pr^s^nt^ in^ th^^ host^ c^ll^ as^ naturally^ contain^d^ g^n^(s)^or^ introduc^d^ (i.^.^c^lls^ar^^g^n^tically^^ngin^^r^d^to^contain^such^g^n^(s)).^ In^on^^ ^mbodim^nt^of^ th^^pr^s^nt^ inv^ntion,^lysin^^6^aminotransf^ras^ and^pyrrolin^^5^carboxylat^^ r^ductas^ ar^^^xpr^ss^d^in^on^^op^ron. ^^ In^ accordanc^^ ^ith^ th^^ pr^s^nt^ inv^ntion,^ th^^ polynucl^otid^s^ ^ncoding^ pyrrolin^^5^ carboxylat^^ r^ductas^ and / or^ lysin^^ 6^aminotransf^ras^ may^ stand^ und^r^ th^^ control^ of^ a^ constitutiv^^ or^ r^gulat^d^ promot^r.^ ^r^f^rably,^ th^^ polynucl^otid^s^ ^ncoding^ pyrrolin^^5^ carboxylat^^ r^ductas^^ and / or^ lysin^^ 6^aminotransf^ras^^ ar^ und^r^ th^^ control^ of^ a^^^ constitutiv^^promot^r.^Mor^^pr^f^rably,^ th^^polynucl^otid^s^^ncoding^pyrrolin^^5^carboxylat^^ r^ductas^^ and^ lysin^^ 6^aminotransf^ras^^ ar^ und^r^ th^^ control^ of^ a^ constitutiv^^ promot^r,^ ^ith^r^ und^r^s^parat^^promot^rs^or^ (pr^f^rably)^ th^^ sam^^promot^r,^mor^^pr^f^rably^ und^r^ th^^common^control^of^ th^^sam^^promot^r.^In^on^^^mbodim^nt^of^ th^^pr^s^nt^ inv^ntion,^ th^^ polynucl^otid^s^^ncoding^pyrrolin^^5^carboxylat^^r^ductas^^and / or^lysin^^6^aminotransf^ras^^^^ ar^ und^r^ th^^ control^ of^ a^ tuf^promot^r^ or^ sod^promot^r. For^ ^xampl^,^ th^^polynucl^otid^s^ ^ncoding^ pyrrolin^^5^carboxylat^^ r^ductas^^and^ lysin^^ 6^aminotransf^ras^^ stand^und^r^ th^^ (pr^f^rably^common)^control^of a^tuf^promot^r^or^sod^promot^r. In^a^sp^cific^^mbodim^nt^of th^^pr^s^nt^inv^ntion,^th^^tuf^promot^r^ is sho^n^in^SEQ^ID^^O:^1 or^und^r^th^^control^of^th^^ sod^promot^r^sho^n^in^SEQ^ID^^os:^2^or^3. ^^ A^“tuf^promot^r”^(“tuf”^stands^for^translational^^longation^factor)^as^us^d^h^r^in^includ^s^th^^ nativ^^ tuf^promot^r,^ ^.g.^ th^^ s^qu^nc^^ as^ sho^n^ in^ SEQ^ ID^ ^O:^ 1^ or^ modifications,^ ^.g.^ mutations^or^fragm^nts th^r^of^as^long^as^th^y^function^as^promot^r.^Modifi^d^tuf^promot^rs^ ar^,^^.g.^d^scrib^d^in^WO 2017 / 037013. A^ “sod^promot^r”^ (“sod”^ stands^ for^ sup^roxid^^ dismutas^)^ as^ us^d^ h^r^in^ includ^s^ th^^ ^ s^qu^nc^^as^sho^n^in^SEQ^ID^^O:^2^or^modifications,^^.g.^mutations^or^fragm^nts^th^r^of^as^ long^as^th^y^function^as^promot^r.^A^modifi^d^sod^promot^r^is^sho^n^in^SEQ^ID^^O:^3. In^ accordanc^^^ith^ th^^pr^s^nt^ inv^ntion,^ th^^m^thod^ d^scrib^d^ and^ provid^d^ h^r^in^ may^ furth^r^ compris^^ a^ st^p^of^ conv^rting^ th^^ produc^d^L^pip^colic^ acid^ to^ L^hydroxy^ pip^colic^^^ acid.^ Accordingly,^ in^ ^mbodim^nt^ of^ th^^ pr^s^nt^ inv^ntion,^ th^^ m^thod^ d^scrib^d^ and^ provid^d^h^r^in^may^furth^r^compris^s^a^st^p^of^conv^rting^th^^produc^d^L^pip^colic^acid^to^ L^hydroxy^pip^colic^acid.^M^thods^to^conv^rt^L^pip^colic^acid^to^L^hydroxy^pip^colic^acid^ar^^ g^n^rally^kno^n^in^th^^art,^cf.,^^.g.,^E^2873730.^In^on^^^mbodim^nt^of^th^^pr^s^nt^inv^ntion,^ th^^ conv^rsion^ of^ L^pip^colic^ acid^ to^ L^hydroxy^ pip^colic^ acid^ is^ also^ carri^d^ out^ at^ a^^^ t^mp^ratur^^of^mor^^than^about^32^°^, ^.g.,^at^about^33^°^,^about^34^°^,^about^35^°^,^about^ 36^°^,^about^37^°^,^or^about^38^°^,^or^about^39^°^,^or^about^40^°^,^or^about^45^°^,^or^about^ 50^°^. For^^xampl^,^th^^c^ll^cultur^^is^carri^d^out^at^a^t^mp^ratur^^of^mor^^than^about^32^°^^ to^about^60^°^,^pr^f^rably^from^about^32^°^^to^about^40^°^, pr^f^rably^from^about^32^°^^to^ about^39^°^,^pr^f^rably^from^about^32^°^^to^about^38^°^,^mor^^pr^f^rably^from^about^33^°^^^^ to^about^ 37^°^,^mor^^pr^f^rably^ from^about^ 34^ °^^ to^about^ 37^ °^,^or^ from^about^34^ °^^ to^ about^35^°^.^In^a^sp^cific^^mbodim^nt^of^th^^pr^s^nt^inv^ntion,^th^^conv^rsion^of^L^pip^colic^ acid^to^L^hydroxy^pip^colic^acid^is^carri^d^out^at^a^t^mp^ratur^^of^about^34^°^.^^r^f^rably,^in^ accordanc^^^ith^th^^pr^s^nt^ inv^ntion,^conv^rsion^of^L^pip^colic^acid^to^L^hydroxy^pip^colic^ acid^is^carri^d^out^using^an^^nzym^ b^ing^capabl^^of^conv^rting^L^pip^colic^acid^to^L^hydroxy^^^ pip^colic^acid,^^.g.,^a^hydroxylas^^(^.g., a^cis^5^hydroxylas^,^^.g.,^as^sho^n^in E^2873730)^ or^flavin^monooxyg^nas^.^^hat^is,^ th^^bact^rial^host^c^ll^^mploy^d^ in^th^^m^thod^d^scrib^d^ and^provid^d^h^r^in^may^furth^r^b^^g^n^tically^^ngin^^r^d^to^^xpr^ss^a^hydroxylas^^or^flavin^ monooxyg^nas^.^ In^ this^ cont^xt,^ as^ d^scrib^d^ h^r^in,^ th^^ bact^rial^ host^ c^ll^ g^n^tically^ ^ngin^^r^d^ to^ ^xpr^ss^ a^ hydroxylas^^ or^ flavin^ monooxyg^nas^^ may^ b^^ charact^riz^d^ by^^^ (naturally)^ having^ ^5^R^ activity^ and^ b^ing^ g^n^tically^ ^ngin^^r^d^ to^ hav^^ LA^^ activity.^ Alt^rnativ^ly,^ in^ this^ cont^xt,^ as^ d^scrib^d^ h^r^in,^ th^^ bact^rial^ host^ c^ll^ g^n^tically^ ^ngin^^r^d^ to^ ^xpr^ss^ a^ hydroxylas^^ or^ flavin^ monooxyg^nas^^ may^ b^^ charact^riz^d^ by^ (naturally)^ having^ LA^^ activity^ and^ b^ing^ g^n^tically^ ^ngin^^r^d^ to^ hav^^ ^5^R^ activity. Alt^rnativ^ly,^ in^ this^ cont^xt,^ as^ d^scrib^d^ h^r^in,^ th^^ bact^rial^ host^ c^ll^ g^n^tically^^^ ^ngin^^r^d^ to^ ^xpr^ss^ a^ hydroxylas^^ or^ flavin^ monooxyg^nas^^ may^ b^^ charact^riz^d^ by^ b^ing^g^n^tically^^ngin^^r^d^ to^hav^^^5^R^and^LA^^activity.^ In^this^cont^xt,^ th^^^xpr^ssion^ of^ th^^hydroxylas^^or^ flavin^monooxyg^nas^^may^b^^on^ th^^sam^^op^ron^as^^5^R^and / or^ ^^ LA^,^ or^on^ s^parat^^op^rons,^ r^gardl^ss^^h^th^r^^5^R^and / or^ is^ (ar^)^pr^s^nt^ in^ th^^host^ c^ll^ as^ naturally^ contain^d^ g^n^(s)^ or^ introduc^d^ (i.^.^ c^lls^ ar^^ g^n^tically^ ^ngin^^r^d^ to^ contain^^5^R^and / or^LA^).^Lik^^is^,^th^^polynucl^otid^s^^ncoding^th^^hydroxylas^^or^flavin^ monooxyg^nas^^and^th^^pyrrolin^^5^carboxylat^^r^ductas^^and / or^lysin^^6^aminotransf^ras^^ ^ may^b^ und^r^th^^control^of^a^constitutiv^^or^r^gulat^d^promot^r^as^d^scrib^d^h^r^in,^^.g.,^tuf^ or^sod^promot^r. ^h^^bact^rial^host^c^ll^^mploy^d^in^th^^m^thod^d^scrib^d^and^provid^d^h^r^in^may^furth^r^b^^ charact^riz^d^by^lysin^^(pr^f^rably^L^lysin^)^ov^rproduction.^As^us^d^h^r^in,^unl^ss^sp^cifi^d^^^ oth^r^is^,^ th^^ t^rm^ “ov^rproduction”^ m^ans^ that^ th^^ r^sp^ctiv^^ c^ll^ is^ abl^^ to^ synth^siz^^ lysin^^ (pr^f^rably^ L^lysin^).^ ^r^f^rably,^ said^ host^ c^ll^ produc^s^ mor^^ lysin^^ (pr^f^rably L^ lysin^)^ than^ it^n^^ds^for^ its^o^n^prot^in^synth^sis^and / or^said^host^c^ll^ is^not^d^p^nd^nt^on^ ^xt^rnal^ addition^ lysin^^ addition^ to^ produc^^^6^^ from^L^lysin^^ (pr^f^rably^ by^ conv^rting^L^ lysin^^ to^ ^6^^ using^ LA^^ as^ d^scrib^d^ h^r^in).^ Exampl^s^ for^ bact^rial host^ c^lls^ b^ing^^^ charact^riz^d^by^ lysin^ (pr^f^rably^L^lysin^) ov^rproduction ar^^ kno^n^ in^th^^art^ (s^^,^^.g.,^ E^2582815^or^E^2872730)^and includ^^coryn^form^bact^rium,^^.g.,^C^^g^u^am^^um,^such^as^ C^^g^u^am^^um LYS^12^(B^ck^r,^M^tab^Eng (2011), 13: 159^168). Alt^rnativ^ly^ or^ in^ addition,^ in^ accordanc^^^ith^ th^^ pr^s^nt^ inv^ntion,^ it is^ possibl^^ to^ add^^^ lysin^^(pr^f^rably^L^lysin^)^to^th^^c^ll^cultur^^to^incr^as^^production^yi^lds^of^L^pip^colic^acid.^ Addition^of^L^lysin^^to^th^^c^ll^cultur^^is^particularly^us^ful^if^th^^bact^rial^c^ll^^mploy^d^in^th^^ m^thod^d^scrib^d^and^provid^d h^r^in^is^not^its^lf^abl^^to^produc^^(significant^amounts^of)^L^ lysin^,^but^may^also^b^^appli^d^if^a^bact^rial^host^c^ll^is^us^d^^hich^is^charact^riz^d^by^lysin^^ (pr^f^rably^L^lysin^)^ov^rproduction^as^d^scrib^d^h^r^in. ^^ In^ a^ sp^cific^ ^mbodim^nt^ of^ th^^ pr^s^nt^ inv^ntion,^ th^^bact^rial^ host^ c^ll^ ^mploy^d^ in^ th^^ m^thod^ d^scrib^d^ and^ provid^d^ h^r^in^ is a^ ^oryn^bact^rium^ sp^ci^s,^ pr^f^rably C^^ g^u^am^^um. ^^ In^ a^ furth^r^ ^mbodim^nt^ of^ th^^ pr^s^nt^ inv^ntion,^ th^^ bact^rial^ host^ c^ll^ ^mploy^d^ in^ th^^ m^thod^d^scrib^d^and^provid^d^h^r^in^– particularly^if^th^^bact^rial^host^c^ll^is^charact^riz^d^ by^ lysin^^ (pr^f^rably^L^lysin^)^ov^rproduction^as^d^scrib^d^h^r^in^– is^furth^r^charact^riz^d^ by^a^lack^of^a^or^inhibit^d^lysin^^(pr^f^rably^L^lysin^)^^xport^out^of^th^^c^ll.^In^accordanc^^^ith^ th^^pr^s^nt^ inv^ntion,^such^ lack^or^ inhibition^of^a^ lysin^^ (pr^f^rably^L^lysin^)^^xport^may^b^^^^ achi^v^d^ by,^ ^.g.,^ a^ natural^ lack^ of^ a^ lysin^ (pr^f^rably^ L^lysin^)^ ^xport^r^ or^ by^ g^n^tic^ d^l^tion^of^or^oth^r^is^^ inhibiting^an^^xisting^ lysin^^(pr^f^rably^L^lysin^)^^xport^r,^^.g.,^^ith^ inhibiting,^ int^rf^ring^ or^ sil^ncing^ R^A^ mol^cul^s,^ g^n^tic^ modifications^ of^ th^^ ^^ polynucl^otid^s^ ^ncoding^ th^^ lysin^^ ^xport^r,^ or^ dir^ctly^ inhibiting^ or^ blocking^ th^^ lysin^^ (pr^f^rably^L^lysin^)^^xport^r.^ ^h^^pr^s^nt^ inv^ntion^furth^r^r^lat^s^to^a bact^rial^host^c^ll^b^ing^g^n^tically^^ngin^^r^d^to^ ^ ^xpr^ss^ in^ on^^ op^ron^ lysin^^ 6^aminotransf^ras^ and^ pyrrolin^^5^carboxylat^^ r^ductas^^ is^ und^r^th^^control^of^th^^tuf^promot^r,^pr^f^rably^th^^tuf^promot^r^sho^n^in^SEQ^ID^^O:^1 and^ b^ing furth^r^charact^riz^d^by^ov^rproduction^of^lysin^. ^h^^pr^s^nt^ inv^ntion^furth^r^r^lat^s^to^a bact^rial^host^c^ll^b^ing^g^n^tically^^ngin^^r^d^to^^^ ^xpr^ss^ in^ on^^ op^ron^ lysin^^ 6^aminotransf^ras^ and^ pyrrolin^^5^carboxylat^^ r^ductas^^ is und^r^th^^control^of^th^^sod^promot^r,^pr^f^rably^th^^sod^promot^r sho^n^SEQ^ID^^O:^2^or^ 3, and^b^ing furth^r^charact^riz^d^by^ov^rproduction^of^lysin^. As^is^sho^n^in^Figur^^8,^th^^choic^^of^th^^promot^r^^^ h^r^^th^^tuf^promot^r^or^sod^promot^r^ – provid^s^ for^ an^ advantag^^ insofar^ as^ th^^ tuf^promot^r^ or^ sod^promot^r^ sho^^ a^ b^tt^r^^^ p^rformanc^^than^an^alt^rnativ^^promot^r,^^.g.^th^^dapB^promot^r^(controlling^^xpr^ssion^of^ th^^g^n^^^ncoding^4^hydroxy^t^trahydrodipicolinat^^r^ductas^^in^C^^g^u^am^^um). ^h^^^mbodim^nts^^hich^ charact^riz^^ th^^pr^s^nt^ inv^ntion^ar^^d^scrib^d^h^r^in,^ sho^n^ in^^^ th^^Figur^s,^illustrat^d^in^th^^Exampl^s,^and^r^fl^ct^d^in^th^^claims. It^must^b^^not^d^ that^as^us^d^h^r^in,^ th^^singular^ forms^ “a”,^ “an”,^ and^ “th^”,^ includ^^plural^ r^f^r^nc^s^unl^ss^th^^cont^xt^cl^arly^indicat^s^oth^r^is^.^^hus,^for^^xampl^,^r^f^r^nc^^to^“a^ r^ag^nt”^ includ^s^ on^^ or^ mor^^ of^ such^ diff^r^nt^ r^ag^nts^ and^ r^f^r^nc^^ to^ “th^^ m^thod”^^^ includ^s^r^f^r^nc^^to^^quival^nt^st^ps^and^m^thods^kno^n^to^thos^^of^ordinary^skill^in^th^^art^ that^could^b^^modifi^d^or^substitut^d^for^th^^m^thods^d^scrib^d^h^r^in.^ Unl^ss^ oth^r^is^^ indicat^d,^ th^^ t^rm^ "at^ l^ast"^ pr^c^ding^ a^ s^ri^s^ of^ ^l^m^nts^ is^ to^ b^^ und^rstood^to^r^f^r^ to^^v^ry^^l^m^nt^ in^th^^s^ri^s.^^hos^^skill^d^ in^th^^art^^ill^ r^cogniz^,^or^^^ b^^ abl^^ to^asc^rtain^ using^no^mor^^ than^ routin^^^xp^rim^ntation,^many^ ^quival^nts^ to^ th^^ sp^cific^^mbodim^nts^of^th^^inv^ntion^d^scrib^d^h^r^in.^Such^^quival^nts^ar^^int^nd^d^to^b^^ ^ncompass^d^by^th^^pr^s^nt^inv^ntion.^ ^h^^t^rm^"and / or"^^h^r^v^r^us^d^h^r^in^includ^s^th^^m^aning^of^"and",^"or"^and^"all^or^any^^^ oth^r^combination^of^th^^^l^m^nts^conn^ct^d^by^said^t^rm". ^^ ^hroughout^ this^ sp^cification^ and^ th^^ claims^ ^hich^ follo^,^ unl^ss^ th^^ cont^xt^ r^quir^s^ oth^r^is^,^th^^^ord^“compris^”,^and^variations^such^as^“compris^s”^and^“comprising”,^^ill^b^^ und^rstood^to^imply^th^^inclusion^of^a^stat^d^int^g^r^or^st^p^or^group^of^int^g^rs^or^st^ps^but^ not^th^^^xclusion^of^any^oth^r^int^g^r^or^st^p^or^group^of^ int^g^r^or^st^p.^Wh^n^us^d^h^r^in^ ^ th^^ t^rm^ “comprising”^ can^ b^^ substitut^d^ ^ith^ th^^ t^rm^ “containing”^ or^ “including”^ or^ som^tim^s^^h^n^us^d^h^r^in^^ith^th^^t^rm^“having”. Wh^n^us^d^h^r^in^ “consisting^of"^^xclud^s^any^^l^m^nt,^st^p,^or^ ingr^di^nt^not^sp^cifi^d^ in^ th^^claim^^l^m^nt.^Wh^n^us^d^h^r^in,^"consisting^^ss^ntially^of"^do^s^not^^xclud^^mat^rials^^^ or^st^ps^that^do^not^mat^rially^aff^ct^th^^basic^and^nov^l^charact^ristics^of^th^^claim. In^ ^ach^ instanc^^ h^r^in^ any^ of^ th^^ t^rms^ "comprising",^ "consisting^ ^ss^ntially^ of"^ and^ "consisting^of"^may^b^^r^plac^d^^ith^^ith^r^of^th^^oth^r^t^o^t^rms. ^^ It^ should^ b^^ und^rstood^ that^ this^ inv^ntion^ is^ not^ limit^d^ to^ th^^ particular^ m^thodology,^ protocols,^and^r^ag^nts,^^tc.,^d^scrib^d^h^r^in^and^as^such^can^vary.^^h^^t^rminology^us^d^ h^r^in^ is^ for^ th^^purpos^^of^ d^scribing^particular^^mbodim^nts^only,^and^ is^ not^ int^nd^d^to^ limit^th^^scop^^of^th^^pr^s^nt^inv^ntion,^^hich^is^d^fin^d^sol^ly^by^th^^claims. ^^ All^ publications^ and^ pat^nts^ cit^d^ throughout^ th^^ t^xt^ of^ this^ sp^cification^ (including^ all^ pat^nts,^ pat^nt^ applications,^ sci^ntific^ publications,^ manufactur^r’s^ sp^cifications,^ instructions,^ ^tc.),^ ^h^th^r^ supra^ or^ infra,^ ar^^ h^r^by^ incorporat^d^ by^ r^f^r^nc^^ in^ th^ir^ ^ntir^ty.^^othing^h^r^in^is^to^b^^constru^d^as^an^admission^that^th^^inv^ntion^is^not^^ntitl^d^to^ ant^dat^^such^disclosur^^by^virtu^^of^prior^inv^ntion.^^o^th^^^xt^nt^th^^mat^rial^incorporat^d^^^ by^ r^f^r^nc^^ contradicts^ or^ is^ inconsist^nt^ ^ith^ this^ sp^cification,^ th^^ sp^cification^ ^ill^ sup^rs^d^^any^such^mat^rial.^ SEQ^ID^^O:^1 tuf^promot^r ^^ D^A C^^g^u^am^^um ^GG^^G^^A^^^^G^GAA^G^^^A^AGGG^AG^^GG^AG^^^GAAAA^^AA^G^^G^^G ^^^^^AGGA^^^AG^AA^^GG^A^A^^^^G^AA^G^G^^AGA^^^G^G^G^^^AG^^^^^ ^AGG^^G^^^A^^A^AG^GAAAG^AAAA^^AA^^^G^GG^^G^GAAAG^^G^AG^^A^^^^ A^GAAG^^^AGGAGGA^A^A^A SEQ^ID^^O:^2 ^ativ^^sod^promot^r ^^ D^A C^^g^u^am^^um ^AG^^G^^AA^^A^^^^GGG^^^G^GA^^^G^^A^^^GA^AAA^AGG^^GG^^GAAAAA ^^^^G^^G^AA^A^^AA^AAAAAGG^^^A^^A^^GGGAGG^G^^G^A^^AAG^A^^^^^G ^ ^GAAG^G^^A^^^GA^GGA^^^^^AAAAGA^G^A^A^G^^^GG^G^GGAAA^^^A^GAA AGGA^^^^^^A^^^ SEQ^ID^^O:^3 Modifi^d^sod^promot^r ^^ D^A Artificial ^AG^^G^^AA^^A^^^^GGG^^^G^GA^^^A^^A^^^GA^AAG^AGG^^GG^^GAGAAA ^^^^G^^G^AG^A^^AA^AGGGAGG^^^A^^A^^GGGGGG^G^^G^G^^AAG^A^^^^ ^G^GGAA^G^^A^^^GA^GGA^^^^^AGAAGA^G^A^A^G^^^GG^G^GGAGA^^^A^^^ GAAAGGA^^^^^^A^^^ SEQ^ID^^O:^4 ^a^ D^A ^^ F^ ^u^es^ens A^G^^^^^^^^^G^^^^G^^^G^^^^G^^^^G^G^^^A^G^^GG^A^^^G^^^^A^^^ AGGG^^^G^^^GA^^^G^AGG^^GAG^AG^^GG^^G^^AA^^A^^^^GA^^^G^G^G ^^G^^A^^GA^G^^G^^G^^GA^GAA^A^G^G^G^A^^AAA^^G^AGG^^G^GG^A^ ^G^^GGA^^^GGA^GAAAG^G^G^AGA^^G^^G^^G^G^AGGA^GG^^^^G^^AA^^^^^ ^^A^G^^GA^GA^G^GG^GG^G^^^^A^A^^G^^^^GG^^G^^^G^GGG^^G^GGG^ GG^^AG^^^GAAGGG^G^GG^G^^G^A^GA^G^^GG^GG^^A^GG^A^G^^^GG^^^ ^GG^^A^A^^^^GG^^GA^A^^^^GGAGG^GG^^GG^AAG^^G^AGG^GA^GG^^AA ^A^^A^GA^^^^^^^G^^GG^^^AGGG^^G^^^^A^^G^^G^AA^G^G^^G^GAAA^^ GG^^A^A^^^G^GG^GG^^G^^^G^^^^^G^A^^^^A^G^G^^^GAA^^^^GG^^^^G^^ AAG^GG^^GGG^^GG^^G^G^G^A^^G^^GA^A^^AA^G^^AAG^^GA^GA^^GA^^ ^GGG^G^^^GG^A^G^^GG^G^^A^GA^^AAG^G^G^GG^GA^^AAGGG^AG^^^^^ A^GG^^G^A^^GA^^G^^^GG^G^^G^A^^^^GA^^^^A^^^G^AAGG^^^A^GA^G^ G^A^^^GG^^AG^^A^^G^GA^GAG^A^AG^G^^A^^G^^A^^G^^^^G^A^GA^^AG ^AGG^^^^G^G^^AGG^G^^^G^^GA^G^^^AGG^^AA^^A^^GG^^^A^^GAGG^G^^ G^G^^^^^GGAG^^GG^GA^GGG^GAAGG^GA^^^GGG^^G^G^GG^G^^GG^GGA ^^^^^A^^G^^^GG^^^G^GAG^^GA^^^G^GAA^A^GG^AG^^^G^^G^^GA^^GA^ ^^GA^^^AGG^^G^G^^G^G^G^G^A^GG^A^^^^G^^^^^^G^^GA^^A^^^^GG^ ^A^^AGGAG^^GGAGG^A^^GGA^A^GGAGA^^^A^^^^AAGG^^^^GAA^GG^G^^ ^AG^^^^^G^^G^^GG^AG^GG^^G^GA^^GAG^A^G^^G^^G^G^^G^A^^G^AAG^^ GG^G^G^A^GG^AA^A^^A^GA^^A^^AA^^^G^GGG^G^^GGA^G^GG^^^G^G^^ A^^^^GG^A^G^^^GGA^GAG^^GG^^^G^AA^AA^A^^^G^^^G^G^GG^^AG^AG G^GA^G^AGAAG^^GGAAG^A^^GAAGGAA^GG^^GGGGGG^G^GA^^A^^AAGG^G ^AGGG^A^^GG^^^G^^G^^^^^^^G^GAG^^GG^^^^G^AG^A^AAG^G^^A^GGG G^^GG^^^^A^^GAGGAG^GG^^G^G^A^G^A^GGGG^^AA^G^GA^^^A^GG^GG^ ^^ GAGAA^^^G^^G^G^^^^A^^^^G^A^^^^GG^A^GGA^GAGG^^GAA^^GGA^^^G^ ^GG^GGAGA^GG^^GGG^G^G^G^^GG^^GAAGG^^^A^G^^GGG^^^GA SEQ^ID^^O:^5 ^ pro^^(^5^R) D^A C^^g^u^am^^um A^GA^AA^AA^^G^^G^AA^^GG^GG^GGA^AAA^^GG^GAGG^^^^AG^^^^AGG^^^ GA^^G^GG^^AA^A^GAA^^^A^AAAA^A^^^G^G^^A^^AA^^G^^^GGAAGAG^G^^^ GG^^AAGAG^^G^G^GA^^G^^A^GG^A^^^^^AA^A^GA^GGA^AA^^^^^AAG^^G ^AGA^GAAG^^GA^G^GG^G^^^^^G^G^G^GAAG^^GAAA^^^A^^G^^GAAG^G^^ ^^^^GAAA^^A^^GG^A^^^^GGA^AA^AA^^^^G^A^AAAG^G^^G^GG^^AG^A^GG ^^G^AGG^A^^AG^A^^G^^G^^A^GGAAGAAAG^G^^^^^G^GGGG^^^^^^G^^G ^G^G^G^^A^G^^GAA^A^^^^AA^G^^^G^GGG^AAGGG^A^G^^GA^^G^^A^^AA^^ AGG^^G^^A^G^^GA^G^GGAA^AG^^GGAA^AAG^^AAGGA^^^G^^GAG^A^^G^^ GGAGA^G^^^^^GAAG^^G^GGAA^^AGA^A^^GA^G^AG^^A^^G^GA^G^^^GGA^ ^^^^^^^^G^A^A^^^G^^^^^^G^GA^^GAAG^G^^^A^^GAGG^AGGAG^^AA^^^A GG^^^G^^^^G^G^GA^^G^^AAAAAG^^^G^^G^GG^^^^A^^^GAAGG^G^^G^AA ^^A^GA^GAAGGAAA^^GG^AAAGAA^^^^^AGAA^^G^G^G^AGG^G^^^^^^^A^^^^ ^G^AGG^A^^A^^G^^G^AG^^A^^^GAGAA^^^GAAGAAAG^GGAA^^^GAGG^G^ ^^^^^A^^G^G^AG^^^AAG^^^G^G^^GA^^GA^^^GAAGAA^^^GGAAAG^G^^AG SEQ^ID^^O:^44 dapB^promot^r D^A ^^ C^^g^u^am^^um AAGGG^AA^^^AAG^^^^A^A^^^^AAA^A^AG^^^^A^^^G^G^GA^^AA^^^^^AGAA ^GGAA^AAA^^GA^GAA^AA^^G^^AA^AA^A^AGA^^AAAA^GG^^AG^^AGG^A^GG A^A^^AG^A^^^^^^GAA^GGG^A^G^^^AGA^^GG^GGG^G^^^GAAAAA^^^^^^G^ ^^^A^GAAAA^GAAGGAG^A^A ^^ Figur^^ ^h^^Figur^s^sho^: Figur^ 1:^ ^^^ab^lic^ pa^^way^ ^^^ign^ f^r^ ^^^^^^^ pr^^uc^i^n^ ^f^ L-pip^c^lic^ aci^^ in^^^ ^^ry^^bact^rium^glutamicum.^^h^^ov^rvi^^^illustrat^s^th^^g^n^tic^layout^of^th^^^IA^strain^ family,^ cr^at^d^ in^ this^ ^ork.^ For^ ^ach^ produc^r,^ ^^^ sho^^ th^^ implant^d^ g^n^tic^ chang^s^ r^lat^d^to^th^^cor^^carbon^m^tabolism,^th^^synth^sis^of^L^lysin^,^its^furth^r^conv^rsion^into^L^ pip^colic^acid^ (^IA),^and^n^^ly^ introduc^d^supporting^path^ays.^^h^^g^n^s^ ^ysDH from^R^^ p^mer^y^,^ ^ncoding^L^lysin^^ d^hydrog^nas^^ (LysDH),^ and^ pr^C from^ C^^ g^u^am^^um,^^^ ^ncoding^pyrrolin^^5^carboxylat^^ r^ductas^ (^ro^),^^^r^^us^d^ to^^stablish^ th^^biosynth^tic^ ^IA^modul^.^^h^^ t^o^g^n^s^^^r^^^pisomally^ ^xpr^ss^d^ as^a^monocistronic^ op^ron^ und^r^ ^^ control^of^th^^constitutiv^^^uf^promotor^ in^th^^L^lysin^^hyp^r^produc^r^C^^g^u^am^^um LYS^12^ (A).^^h^^LYS^12^strain^had^b^^n^d^riv^d^from^th^^^ild^typ^^b^for^^by^th^^impl^m^ntation of^ t^^lv^^ g^nomic^ modifications,^ including^ ov^r^xpr^ssion^ of^ th^^ ^k^ op^ron^ (Ps^d ^k^),^ ov^r^xpr^ssion^ of^ fructos^^ 1,6^bisphosphatas^^ (P^uf fbp),^ modification^ and^ amplification^ of^ ^ pyruvat^^ carboxylas^^ (^s^dpy^AP458^),^ d^l^tion^ of^ phospho^nolpyruvat^^ carboxykinas^^ (Dp^k),^ att^nuation^ of^ isocitrat^^ d^hydrog^nas^^ (^^dGTG),^ modification^ and^ amplification^ of^ aspartokinas^^ (P ^ys^T311I),^ att^nuation^ of^ homos^rin^^ d^hydr V59A s^dog^nas^^ (h^m ),^ amplification^of^4^hydroxy^t^trahydrodipicolinat^^r^ductas^ (Ps^ddapB),^duplication^of^m^so^ diaminopim^lat^^ d^hydrog^nas^^ (2x^ ddh),^ and^ duplication^ of^ m^so^diaminopim^lat^^^^ d^carboxylas^^ (2x^ ^ysA)^ (B^ck^r^ ^t^al.,^2011,^ ^^^^^^^^.).^Diff^r^nt^ g^n^tic^modifications^^^r^^ impl^m^nt^d^ to^ards^ improv^d^ L^pip^colic^ acid^ production^ cr^ating^ a^ nov^l^ family^ of^ produc^rs^ (B).^ ^h^^ optimization^ includ^d^ r^balancing^ of^ ^xpr^ssion^ of^ th^^P^uf^ysDH^ pr^C op^ron^by^adaptation^of^th^^codon^usag^^and^^xpr^ssion^of^a^s^cond^pr^C copy.^In^addition,^ G^^s^ear^^herm^ph^^us and^A^ ^umefa^^ens ^^r^^consid^r^d^as^alt^rnativ^^^ysDH donors^and^^^ pro^^ ^as^ r^plac^d^ by^ th^^ str^ss^r^lat^d^ iso^nzym^^ pr^H^ from^ B^^ sub^^^^s,^ r^sp^ctiv^ly.^ Furth^rmor^,^alt^rnativ^^path^ays^for^L^pip^colic^acid^synth^sis^^^r^^impl^m^nt^d,^including^ th^^ cyclod^aminas^^ rapL from^ ^^^ hygr^s^^p^^us and^ L^lysin^^ 6^aminotransf^ras^^ from^ F^ ^u^es^ens.^Furth^r^strain^^ngin^^ring^aim^d^at^m^tabolic^^ngin^^ring^of^th^^r^dox^supply,^ by^ g^nomic^ ^xpr^ssion^ of^ th^^m^mbran^^bound^ transhydrog^nas^^ pn^AB^ from^E^ ^^^^ and^^^ gapN from^ ^^^ mu^ans,^ ^ncoding^ glyc^rald^hyd^^3^phosphat^^ d^hydrog^nas^,^ both^ und^r^ control^of^P^uf.^ Figur^^2:^P^rf^rmanc^^^f^ba^ic^L-pip^c^lic^aci^^pr^^ucing^^.^glutamicum ^^rain^.^All^ strains^ ^^r^^bas^d^on^ ^pisomal^ ^xpr^ssion^ of^ ^ysDH^ from R^^ p^mer^y^^and^ pr^C from C^^^^ g^u^am^^um.^ ^IA^1A^ (A)^ ^xpr^ss^d^ th^^ t^o^ g^n^s^ on^ a^ monocistronic^ op^ron^ ^ith^ nativ^^ codon^usag^,^^h^r^as^^IA^1B^ (B)^^xpr^ss^d^a^codon^optimiz^d^ v^rsion.^Strains^^IA^4^ (^)^ and^^IA^5^(D)^carri^d^additional^supporting^modifications^for^^nhanc^d^r^dox^supply^(^abl^ 1).^ ^ultivations^ ^^r^^ p^rform^d^ in^ shak^^ flasks^ at^ 30^ °^^ using^ minimal^ using^ glucos^^ m^dium.^n^=^3. ^^ Figur^^ 3:^ Sy^^^m^^ m^^ab^lic^ ^ngin^^ring^ ^f^ ^.^ glutamicum f^r^ L-pip^c^lic^ aci^^ pr^^uc^i^n.^During^ strain^ d^v^lopm^nt,^ th^^production^p^rformanc^^ of^ th^^nov^l^ family^ of^ produc^rs^ ^as^ ^valuat^d^ in^ batch^ proc^ss^s^ at^ miniaturiz^d^ scal^^ on^ glucos^^ minimal^ m^dium^(n=3)^(A). In^addition,^th^^b^st^produc^r^C^^g^u^am^^um ^IA^7^^as^b^nchmark^d^in^a^^^ f^d^batch^proc^ss,^^h^r^by^th^^data^sho^n^r^f^r^to^th^^diff^r^nt^proc^ss^stag^s^(n=2)^(B).^ ^^ Figur^^4:^ Impr^v^^^L-pip^c^lic^aci^^pr^^uc^i^n^in^^.^glutamicum PIA-1A^a^^ incr^a^^^^ ^^mp^ra^ur^.^^^mp^ratur^^d^p^nd^nc^^of^th^^activity^of^th^^L^lysin^^6^d^hydrog^nas^^from R^^p^mer^y^^(A).^^h^^assay^^as^conduct^d^at^pH^10.0^^ith^30 mM^ + L^lysin^^and^2^mM^^AD ,^ using^crud^^c^ll^^xtract^from^C^^g^u^am^^um ^IA^1A,^harv^st^d^during^th^^^xpon^ntial^gro^th^ ^ phas^^ (n=3).^Gro^th^and L^pip^colic^acid production^of^C^^g^u^am^^um ^IA^1A^at^34^°^^ (B).^ ^h^^cultivation^^as^conduct^d^in^shak^^flasks^on^minimal^glucos^^m^dium^(n=3). Figur^^5: P^rf^rmanc^^^f^^^^^a^vanc^^^L-pip^c^lic^aci^^pr^^uc^r^^.^glutamicum PIA-7^ in^^^ak^^ fla^k^cul^ur^^.^^h^^cultivations ^^r^^on^minimal^using^glucos^^m^dium^at^30^°^^^^ (A)^ and^ 34^ °^^ (^).^ Additionally,^ th^^ m^dium^ ^as^ ^nrich^d^ ^ith^ 12.5^ mg^ L^1 pyridoxal^ phosphat^,^and^th^^c^lls^^^r^^incubat^d^again^at^30^°^^(B)^and^34^°^^(D).^^n^=^3. Figur^^ 6:^Ac^ivi^y^ ^f^ L-ly^in^^ 6-amin^^ran^f^ra^^^ fr^m^F.^ lut^sc^^s^^xpr^^^^^^ in^ ^^^^ a^vanc^^^L-pip^c^lic^aci^^pr^^uc^r^^.^glutamicum PIA-7.^^h^^assay^^as^conduct^d^at^^^ 30°^^using^diff^r^nt^pH^valu^s^(7.8^and^8.5)^and^l^lysin^^conc^ntrations^(10^mM^and^30^mM)^ using^crud^^c^ll^^xtract^from^^xpon^ntially^gro^ing^C^^g^u^am^^um ^IA^7^ n=3. Figur^^ 7:^ F^^-ba^c^^ pr^^uc^i^n^ ^f^ L-pip^c^lic^ aci^^ by^ m^^ab^lically^ ^ngin^^r^^^ ^.^ glutamicum PIA-7. Aft^r^d^pl^tion^of^th^^initial^sugar^at^ th^^^nd^of^ th^^batch^phas^,^puls^s^ of^ f^^d^^^r^^add^d^automatically,^using^an^ incr^as^^of^ th^^ l^v^l^ of^ dissolv^d^oxyg^n^ (DO)^^^ abov^^50%^as^a^trigg^r^(A).^R^garding^th^^cultur^^profil^,^batch^and^f^^d^phas^^ar^^d^not^d^ by^a^dott^d^lin^.^^h^^batch^m^dium^contain^d^glucos^^(90^g^L^1)^and^y^ast^^xtract^(15^g^L^1)^ as^carbon^sourc^.^In^th^^f^^d,^th^^l^v^l^of^glucos^^(600^g^L^1 ,^pr^f^rably 500^g^L^1)^^as^7.5^ fold^ incr^as^d,^ ^hil^^ th^^ y^ast^ ^xtract^ l^v^l^ (15^ g^ L^1)^ r^main^d^ th^^ sam^.^ ^h^^ yi^ld^ and^ s^l^ctivity^of^L^pip^colic^acid^production^improv^d^during^th^^f^rm^ntation,^^hich^^as^ link^d^^^ to^ th^^ availability^ and^ f^^ding^ of^ glucos^^ (B).^ ^ ^h^^ data^ r^pr^s^nt^ m^an^ valu^s^ and^ d^viations^from^t^o^r^plicat^s. Figur^^ 8:^ P^rf^rmanc^^ ^f^ PIA-7,^ PIA-9,^ PIA-10,^ PIA-11^ an^^ PIA-12^ a^^ vari^u^^ ^^mp^ra^ur^^ ^^ In^ ^IA^7^ th^^ tuf^promot^r^ controls^ ^xpr^ssion^ of^ th^^ op^ron^ (pyrrolin^^5^carboxylat^^ r^ductas^^and^lysin^^6^aminotransf^ras^),^in^^IA^9,^th^^sod^promot^r^controls^^xpr^ssion^of^ th^^op^ron,^in^^IA^10^a^modifi^d^sod^promot^r^controls^^xpr^ssion^of^th^^op^ron,^^h^r^as^in^ ^IA_11^ th^^ dapB^promot^r^ controls^ ^xpr^ssion^ of^ th^^ op^ron^ and^ in^ ^IA^12^ th^^ dapB^ promot^r^ controls^ ^xpr^ssion^ of^ lysin^^ 6^aminotransf^ras^^ and^ th^^ tuf^promot^r^ controls^^ ^xpr^ssion^of^pyrrolin^^5^carboxylat^^r^ductas^,^both^g^n^s^ar^^in^on^^op^ron,^but^driv^n^by^ diff^r^nt^promot^rs.^(A)^28^°^;^(B)^30^°^;^(^)^32^°^;^(D)^34^°^ ^h^^pr^s^nt^inv^ntion^may^also^b^^charact^riz^d^by^th^^follo^ing^it^ms: ^7 (1) A^ m^thod for^ th^^ production^ of^ L^pip^colic^ acid^ by^ a^ bact^rial^ host^ c^ll in^ cultur^,^ comprising^conv^rting^L^lysin^^by^th^^us^^of^lysin^^6^aminotransf^ras^ (LA^)^to^d^lta^ 1^pip^rid^in^^6^carboxylic^acid;^and^ ^ r^ducing^ d^lta^1^pip^rid^in^^6^carboxylic^ acid^ by^ th^^ us^^ of^ pyrrolin^^5^carboxylat^^ r^ductas^^(^5^R)^to^L^pip^colic^acid, ^h^r^in^said^c^ll^cultur^^is^carri^d^out^by^a^t^mp^ratur^^of^mor^^than^32°^. (2) ^h^^m^thod^of^it^m^1,^^h^r^in^pyridoxal^phosphat^^is^add^d^to^cultur^. ^^ (3) ^h^^ m^thod^ of^ any^ on^^ of^ th^^ pr^c^ding^ it^ms,^ ^h^r^in^ said^ cultur^^ is^ f^d^batch^ cultur^. (4) ^h^^ m^thod^ of^ any^ on^^ of^ th^^ pr^c^ding^ it^ms,^ ^h^r^in^ said^ bact^rial^ host^ c^ll^ is^^^ charact^riz^d^ by^ having^ pyrrolin^^5^carboxylat^^ r^ductas^^ activity^ and^ b^ing^ g^n^tically^^ngin^^r^d^to^^xpr^ss^lysin^^6^aminotransf^ras^. (5) ^h^^ m^thod^ of^ any^ on^^ of^ th^^ pr^c^ding^ it^ms,^ ^h^r^in^ said^ bact^rial^ host^ c^ll^ is^ charact^riz^d^ by^ having^ lysin^^ 6^aminotransf^ras^^ activity^ and^ b^ing^ g^n^tically^^^ ^ngin^^r^d^to^^xpr^ss pyrrolin^^5^carboxylat^^r^ductas^. (6) ^h^^m^thod^of^any^on^^of^ th^^pr^c^ding^ it^ms,^^h^r^in^said^host^c^ll^ is^g^n^tically^ ^ngin^^r^d^ to^ ^xpr^ss^ lysin^^ 6^aminotransf^ras^ and^ pyrrolin^^5^carboxylat^^ r^ductas^. ^^ (7) ^h^^ m^thod^ of^ it^m^ 6,^ ^h^r^in^ said^ lysin^^ 6^aminotransf^ras^ and^ pyrrolin^^5^ carboxylat^^r^ductas^ ar^^^xpr^ss^d^in^on^^op^ron. (8) ^h^^ m^thod^ of^ any^ on^^ of^ it^ms^ 4^ to^ 7,^ ^h^r^in^ ^xpr^ssion^ of^ said^ lysin^^ 6^^^ aminotransf^ras^^ and / or^ said^ pyrrolin^^5^carboxylat^^ r^ductas^^ is^ und^r^ th^^ control^ of^ th^^tuf^promot^r,^pr^f^rably^ th^^tuf^promot^r^sho^n^in^SEQ^ID^^O:^1^or^und^r^th^^ control^of^th^^sod^promot^r,^pr^f^rably^th^^sod^promot^r sho^n^SEQ^ID^^os:^2^or^3.^ (9) ^h^^ m^thod^ of^ any^ on^^ of^ th^^ pr^c^ding^ it^ms,^ ^h^r^in^ said lysin^^ 6^^^ aminotransf^ras^^is^d^riv^d^from^F^av^ba^^er^um^^u^es^ens^ ^8 (10) ^h^^m^thod^of^any^on^^of^ th^^pr^c^ding^ it^ms,^^h^r^in^said pyrrolin^^5^carboxylat^^ r^ductas^^is^d^riv^d^from Es^her^^h^a^^^^^ or^a^coryn^form^bact^rium. (11) ^h^^ m^thod^ of^ any^ on^^ of^ th^^ pr^c^ding^ it^ms,^ ^h^r^in^ said^ m^thod^ furth^r^ ^ compris^s^conv^rting^L^pip^colic^acid to^L^hydroxy^pip^colic^acid. (12) ^h^^m^thod^of^any^on^^of^ th^^pr^c^ding^ it^ms,^^h^r^in^said^host^c^ll^ is^g^n^tically^ ^ngin^^r^d^to^^xpr^ss^a^Flavin^monooxyg^nas^. ^^ (13) ^h^^ m^thod^ of^ any^ on^^ of^ th^^ pr^c^ding^ it^ms,^ ^h^r^in^ said^ bact^rial^ host^ c^ll^ is^ charact^riz^d^by^lysin^^ov^rproduction. (14) ^h^^m^thod^of^any^on^^of^ th^^pr^c^ding^ it^ms,^^h^r^in^said^bact^rial^host^c^ll^ is^a^ ^oryn^bact^rium^sp^ci^s,^pr^f^rably^C^ryneba^^er^um^g^u^am^^um. ^^ (15) A^bact^rial^host^c^ll^b^ing^g^n^tically^^ngin^^r^d^to^^xpr^ss^ in^on^^op^ron^ lysin^^6^ aminotransf^ras^ and^ pyrrolin^^5^carboxylat^^ r^ductas^^ is^ und^r^ th^^ control^ of^ th^^ tuf^promot^r,^pr^f^rably^th^^tuf^promot^r^sho^n^in^SEQ^ID^^O:^1^or^und^r^th^^control^ of^ th^^ sod^promot^r,^ pr^f^rably^ th^^ sod^promot^r sho^n^ SEQ^ ID^^os:^ 2^or^ 3, and^^^ b^ing furth^r^charact^riz^d^by^ov^rproduction^of^lysin^. ^h^^pr^s^nt^inv^ntion^is^furth^r^illustrat^d^by^th^^follo^ing^^xampl^s.^Y^t,^th^^^xampl^s^and^ sp^cific^ ^mbodim^nts^ d^scrib^d th^r^in^must^ not^ b^^ constru^d^as^ limiting^ th^^ inv^ntion^ to^ such^sp^cific^^mbodim^nts. ^^ Exampl^^ 1.^^a^^rial^^an^^m^^^^^^ 1.1.^^icr^^rgani^m^,^pla^mi^^,^an^^^yn^^^^ic^g^n^^ C^ryneba^^er^um^g^u^am^^um LYS^12^(B^ck^r^^t^al.,^M^tab^Eng^(2011),^13:^159^168) and^C^^^^ g^u^am^^um^ ^ysCfbr Δ^ysE (Gi^ss^lmann^ ^t^ al.,^ Biot^chnol^ J^ (2008),^ 14:^ ^1800417)^ ^^r^^ obtain^d^ from^ pr^vious^ ^ork.^E^ ^^^^ DH10B^ and^ ^M522^ (Invitrog^n,^ ^arlsbad,^ ^A,^ USA)^ ^^r^^us^d^as^ hosts^ to^ amplify^ and^m^thylat^^ plasmids^ (B^ck^r^ ^t^ al.,^ 2011,^ ^ ^^^ ^^^.).^ For^ g^n^^ cloning,^ D^A^ of^ Rueger^a^ p^mer^y^ DSS^3^ (DSM^ 15171)^ and^ ^^rep^^my^es^ hygr^s^^p^^us^ ^RRL^ 5491 (DSM^ 41530)^ ^as^ obtain^d^ from^ th^^ G^rman^ ^oll^ction^ of^^^ Microorganisms^ and^ ^^ll^ ^ultur^s^ (DSMZ,^ Braunsch^^ig,^ G^rmany),^ ^hil^^ D^A^ from^ Ba^^^^us^ sub^^^^s K168^ ^as^ tak^n^ from pr^vious^ ^ork^ (Kohlst^dt^ ^t^ al.,^ Eniron^ Microbiol^ (2014),^ 16:^ 1898^1917).^ In^ addition,^ s^l^ct^d^ g^n^s^ from^Ge^ba^^^^us^ s^ear^^herm^ph^^us^ ^9 U^B1103,^Agr^ba^^er^um^^umefa^^ens,^F^av^ba^^er^um^^u^es^ens IFO^3084,^Es^her^^h^a^^^^^ K12^MG1655,^ and^ ^^rep^^my^es^ mu^ans^ UA159^ ^^r^^ synth^tiz^d,^ bas^d^ on^ digital^ s^qu^nc^^ information^ (G^nScript,^ ^iscata^ay^ ^o^nship,^ ^J,^ USA)^ or^ tak^n^ from^ pr^vious^ ^ork (Hoffmann^^t^al.,^M^tab^Eng^(2018),^47:^475^487;^Hoffmann^^t^al.,^M^tab^Eng^(2021),^ ^ 67:^ 293^307). ^h^^ plasmid^ pC^^k5a^ MC^ ^as^ us^d^ for^ ^pisomal^ g^n^^ ^xpr^ssion^ in^ C^^ g^u^am^^um (Buschk^^ ^t^ al.,^ Biot^chnol^ J^ (2011),^ 6:^ 306^317).^ For^ g^nom^^bas^d^ modification^of^ th^^microb^,^ th^^ int^grativ^^plasmid^pC^^k^ ^n^^sa^B ^as^us^d^ (B^ck^r ^t^al.,^ Appl^ Environ^Microbiol^ (2005),^ 71:^ 8587^8596).^ During^ cloning,^ pTC ^as^ co^^xpr^ss^d^ in^ E^ ^^^^ ^M522^for^appropriat^^plasmid^m^thylation.^All^strains^and^plasmids^us^d^in^this^study^^^ ar^^list^d^in^^abl^^1. Tabl^^1:^Pla^mi^^^an^^^^rain^^u^^^^in^^^i^^^^u^y. S^rain^ D^^crip^i^n R^f^r^nc^ E^ ^^^^ DH10B H^at^ shock^comp^t^nt^ c^lls^ for^ v^ctor^ Invitrog^n amplification^ ^M522 H^at^ shock^comp^t^nt^ c^lls^ for^ v^ctor^ Invitrog^n amplification^&^m^thylation^ C^^g^u^am^^um A^^^^13032 Wild^typ^ B^ck^r^ ^t^ al.,^ 2011,^ ^^^^^^^^^ LYS^12 L^Lysin^^ produc^r^ ^ith^ 12^ g^nom^^bas^d^ B^ck^r^ ^t^ al.,^ 2011,^ modifications ^^^^^^^^^ ^ysCfbr D^ysE ^on^L^lysin^^s^cr^ting^chassis^strain^ Gi^ss^lmann^ ^t^ al.,^ 2019,^^^^^^^^^^ Pla^mi^^ pTC ^loning^v^ctor,^ORI^for^E^ ^^^^,^^e^R B^ck^r^ ^t^ al.,^ 2011,^ ^^^^^^^^^ pC^^k5a^ Episomal^ v^ctor,^ ORI^ for^ E^ ^^^^ and^ Buschk^^ ^t^ al.,^ C^ g^u^am^^um, kanR Biot^chnol^J^(2011),^6:^ 306^317 ^^ pC^^k5a^P^ufEpisomal^^xpr^ssion^of^^ysDH^from^R^^p^mer^y^ ^his^^ork ^ysDHRp^m pr^CCg^u DSS^3^ (Acc.^^o.^ S^O0234)^ and^pr^C from^C^^ g^u^am^^um (Acc.^^o. ^0^1E4)^und^r^control^of^ P^ufpC^^k5a^ P^ufEpisomal^ ^xpr^ssion^ of ^ysDH from^ A^^ ^his^^ork ^ysDHA^um pr^CCg^u ^umefa^^ens (Acc.^^o.^^5^M90)^and^pr^C from^ C^^g^u^am^^um und^r^control^of^P^uf^pC^^k5a^ P^ufEpisomal^ ^xpr^ssion^ of ^ysDH from^ G^^ ^his^^ork ^ysDHGs^e pr^CCg^u s^ear^^herm^ph^^us (Acc.^ ^o.^ Q9AJ^6)^ and^ pr^C from^C^^g^u^am^^um und^r^control^of P^uf,Episomal^^xpr^ssion^of^codon^optimiz^d ^ysDH^ ^his^^ork from^R^^p^mer^y^ DSS^3^ and^ codon^optimiz^d pr^C from^C^^g^u^am^^um und^r^control^of P^ufEpisomal^^xpr^ssion^of^^ysDH^from^R^^p^mer^y^ ^his^^ork DSS^3^ and^ 2^ copi^s^ of^ pr^C from^ C^^ g^u^am^^um und^r^control^of^P^ufEpisomal^^xpr^ssion^of^^ysDH^from^R^^p^mer^y^ ^his^^ork DSS^3^and^pr^H from^B^^sub^^^^s K168^(Acc.^^o.^ ^0^I77) und^r^control^of^P^ufpC^^k5a^ P^uf^a^F^u^ Episomal^ ^xpr^ssion^ of^ ^a^^ from^ F^^ ^u^es^ens ^his^^ork pr^CCg^u IFO^3084^(Acc.^^o.^Q9EVJ7)^and^pr^C from^C^^ g^u^am^^um und^r^control^of^P^ufpC^^k5a^ Ps^d^a^F^u^ Episomal^ ^xpr^ssion^ of^ ^a^^ from^ F^^ ^u^es^ens ^his^^ork pr^CCg^u IFO^3084^(Acc.^^o.^Q9EVJ7)^and^pr^C from^C^^ g^u^am^^um und^r^control^of^Ps^d pC^^k5a^ Ps^d,^ m^dEpisomal^ ^xpr^ssion^ of^ ^a^^ from^ F^^ ^u^es^ens ^his^^ork ^a^F^u^ pr^CCg^u IFO^3084^(Acc.^^o.^Q9EVJ7)^and^pr^C from^C^^ g^u^am^^um und^r^control^of^Ps^d,^m^dpC^^k^ ^n^^ sa^B^ P^ufG^nomic^int^gration^of^ ^ysDH^from^R^^p^mer^y^ ^his^^ork ^ysDHRp^m pr^CCg^u DSS^3^ and^ pr^C from^ C^^ g^u^am^^um und^r^ control^of^P^ufinto^b^^A (^^g2604l) pC^^k^ ^n^^ sa^B^ P^ufG^nomic^ int^gration^of^pn^AB^ from^E^ ^^^^ K12^ Hoffmann^ ^t^ al.,^ 2021,^ pn^ABE^^^ MG1655^ (Acc.^ ^o.^ ^07001, ^0AB67) und^r^ ^^^^^^^^^ control^of^P^ufinto^^r^B (^^gl0598) pC^^k^ ^n^ sa^B^ P^ufG^nomic^ int^gration^ of^ codon^optimiz^d^ gapN Hoffmann^ ^t^ al.,^ 2021,^ gapN^mu^ from^ ^^ mu^ans UA159^ (Acc.^ ^o.^ Q59931)^ ^^^^^^^^^ und^r^control^of P^uf into^^r^I2 (^^gl0597) pC^^k5a^ P F^u dapB^a^ ^ Episomal^ ^xpr^ssion^ of^ ^a^^ from^ F^^ ^u^es^ens ^his^^ork pr^CCg^u IFO^3084^(Acc.^^o.^Q9EVJ7)^and^pr^C from^C^^ g^u^am^^um und^r^control^of^P^ufpC^^k5a^ PdapB^a^F^u^ Episomal^ ^xpr^ssion^ of^ ^a^^ from^ F^^ ^u^es^ens ^his^^ork P^ufpr^CCg^u IFO^3084^(Acc.^^o.^Q9EVJ7)^and^pr^^ from^C^^ g^u^am^^um und^r^control^of^P^uf1.2.^^^l^cular^^^^ign^an^^g^n^^ic^^ngin^^ring ^loning^strat^gi^s^^^r^^d^sign^d^using^th^^soft^ar^^SnapG^n^^(V^rsion^5.3.2^GSL^Biot^ch,^ ^hicago,^ IL,^ USA).^ ^lasmid^ D^A^ ^as^ synth^siz^d,^ purifi^d,^ and^ analyz^d^ as^ d^scrib^d^^ b^for^^ (Rohl^s^^t^al.,^Microb^^^ll^Fact^ (2016),^15:^154).^ In^short,^^^R^^as^us^d^to amplify^ d^sir^d^D^A^fragm^nts^from^g^nomic^D^A^(^husion^High^Fid^lity^^^R^Mast^r^Mix^^ith^HF^ Buff^r,^^^^^England^Biolabs,^Frankfurt^am^Main,^G^rmany)^using^sp^cific^prim^rs^(^abl^^2).^ V^ctors^^^r^^ lin^ariz^d^using^^maI,^XbaI,^and^KpnI^ (FastDig^st,^ ^h^rmo^Fish^r^Sci^ntific,^ Waltham,^ MA,^ USA).^ ^h^^ obtain^d^ fragm^nts^ and^ th^^ lin^ariz^d^ v^ctor^ backbon^^ ^^r^^ ^^ ass^mbl^d^ ^n^ v^^r^ (Gibson^ ^t^ al.,^ ^at^ M^thods^ (2009),^ 6:^ 343^345).^ ^lasmids^ ^^r^^ transform^d^ into^ E^ ^^^^ by^ h^at^shock^ (Okayama,^ G^n^^ (1990),^ 96:^ 23^28) and^ into^ C^ g^u^am^^um by^^l^ctroporation (B^ck^r^ ^t^ al.,^Eng^Lif^^Sci^ (2010),^10:^430^438).^^orr^ct^ clon^s^ ^^r^^ v^rifi^d^ by^ ^^R^ (^hir^^ Gr^^n^ Hot^ Start^ II^ ^^R^ Mast^r^ Mix,^ ^h^rmo^ Fish^r^ ^ Sci^ntific)^and^Sang^r^s^qu^ncing^(Az^nta,^^h^lmsford,^MA,^USA). ^o^^nabl^^L^pip^colic^acid^production,^diff^r^nt^op^rons,^varying^in^combination^and^layout^of^ th^^biosynth^tic^g^n^s,^^^r^^cr^at^d^and^^xpr^ss^d^using^^pisomal^v^ctors^und^r^control^of^ constitutiv^^promot^rs such^as^P^uf,^Ps^d,^and^Ps^d,^m^dand PdapB. G^nom^^bas^d^^xpr^ssion^of^^^ g^n^s^ of^ int^r^st^ for^ improv^d^ r^dox^ supply,^ including^ th^^ nativ^^ transhydrog^nas^^ g^n^^ pn^AB from^E^^ ^^^^ K12^MG1655^ (Hoffmann^ ^t^ al.,^ 2021,^ ^ ^^^ ^^^.) and^ a^ codon^ optimiz^d^ v^rsion^ of^ gapN from^ ^^rep^^^^^^us^ mu^ans UA159,^ ^ncoding^ ^AD^^d^p^nd^nt^ glyc^rald^hyd^^3^phosphat^^d^hydrog^nas^^(Hoffmann^^t^al.,^2018,^ ^ ^^^^^^.),^^as^bas^d^on^ th^^int^grativ^^v^ctor^pC^^k^^n^^sa^B. ^^ Mutat^d^ variants^ of^ th^^ constitutiv^^ promot^r^ Ps^d^of^ th^^ s^d g^n^^ (^^Gl286,^ ^GL_RS14560),^ ^ncoding^ sup^roxid^^dismutas^^ (B^ck^r^ ^t^ al.,^ J^ Biot^chnol^ (2007),^ 132:^ 99^109),^ ^^r^^ g^n^rat^d^ by^ random^ mutag^n^sis^ (JBS^ d^^^^Mutag^n^sis^ Kit,^ J^na^ Biosci^nc^,^J^na,^G^rmany). ^^ Tabl^^2:^Prim^r^ S^qu^ncing^of pC^^k5a p^lik5a_s^qF^G^GG^^GA^AA^^AA^GAAG^^A^G^(SEQ^ID^^O:^6) p^lik5a_s^qR^^^GGAGAA^^^G^G^G^AA^^^A^^(SEQ^ID^^O:^7) ^^ S^qu^ncing^of^pC^^k^^n^^^a^B RB06_S^q_antis^ns^^A^^G^^^G^^G^G^^^AG^^A^AG^(SEQ^ID^^O:^8) SK_p^lik_int_sacB^s^ns^^AA^AA^AG^GAA^GG^AGG (SEQ^ID^^O:^9) Fragm^nt^amplification^for^^pisomal^^ysDH^pr^C^op^ron ^r_p^liK_^ftu_Sma_f^d^A^^GGGA^^^^^^AGA^^^^GG^^G^^A^^^^G^GAA^G^(SEQ^^^ ID^^O:^10) ^r_^ftu_lysdh_r^v^^GA^A^AAA^G^^^^AG^G^A^^G^A^G^^^^^^^GGA^^^^ (SEQ^ ID^^O:^11) ^r_lysdh_^ftu_f^d^GAAG^^^AGGAGGA^A^A^AA^G^G^^GGAA^A^^^G^G^^G (SEQ^ ID^^O:^12) ^^ ^r_lysdh_RBS_r^v^^G^A^G^^^^^^^GGA^^^^^^AAG^GG^^^^G^^^^GGG (SEQ^ID^ ^O:^13) ^^ ^r_RBS_pro^_f^d^GAAG^^^AGGAGGA^A^A^AA^GA^AA^AA^^G^^G^AA^^ (SEQ^ID^ ^O:^14) ^r_pro^_p^liK_Sma_r^v^^^G^^AG^GA^^^AAA^^^^^^AG^G^^^^^^GAG^^^ (SEQ^ ID^^O:^15) ^ Fragm^nt^amplification^for^s^cond^pr^C^copy ^R_^^ftu_p^lik5aM^S^^^^GA^G^^GGG^^^GG^A^^GG^^G^^A^^^^G^GAA^G^ (SEQ^ID^^O:^16) ^R_^^ftu_pro^_r^v^A^^A^AG^AA^^G^^G^^A^^G^A^G^^^^^^^GGA^^^^ (SEQ^ID^ ^O:^17) ^^ ^R_^^ftu_pro^_f^d^GAAG^^^AGGAGGA^A^A^AA^GA^AA^AA^^G^^G^AA^ (SEQ^ID^ ^O:^18) ^R_pro^_p^lik5aM^S_r^v^AA^^AG^^A^A^GA^G^G^G^^AG^G^^^^^^GAG^^^^^ (SEQ^ID^^O:^19) Fragm^nt^amplification^for^pr^H ^^ ^R_RBS_proH_f^d^GAAG^^^AGGAGGA^A^A^AA^G^GAA^AAAAAAG^GAA^^(SEQ^ID^ ^O:^20) ^R_proH_r^v^^AG^GGA^^A^^^AAA^^^^^^A^^^^A^^A^^^^^GA^AG (SEQ^ID^^O:^ 21) Fragm^nt^amplification^for^int^grativ^^^ysDH^pr^C^op^ron ^^ ^R1_^S1^BioA_nat_f^d^AA^^GGGA^^^^^^AGA^^^G^^^G^G^GAG^^^GA^^A^^ (SEQ^ID^^O:^22) ^R2_^S1^BioA_nat_r^v^^A^^^G^AGGG^AA^GG^^AG^G^AGG^A^^^GGAAA^^G (SEQ^ID^^O:^23) ^R3_BioA_Op^ron^nat_f^d^^GA^^^^^GAG^A^^^G^G^^GG^^G^^A^^^^G^GAA^G^^ (SEQ^ID^^O:^24) ^R4_BioA_Op^ron^nat_r^v^^AG^GG^AA^^^^A^^GA^G^^AG^G^^^^^^GAG^^^^^^ (SEQ^ID^^O:^25) ^R5_^S2 BioA_nat_f^d^GAAGAA^^^GGAAAG^G^^AG^G^^GA^GAAA^^G^^A^^G (SEQ^ID^^O:^26) ^^ ^R6_^S2^BioA_nat_r^v^^^G^^AG^GA^^^AAA^^^^^^A^^^^^^^^^AA^^G^AG^ (SEQ^ID^^O:^27) Fragm^nt^amplification^for^int^grativ^^pn^AB^construct SH53_^R_Fusion_^ftu_pntab_f^^ GAAG^^^AGGAGGA^A^A^AA^G^GAA^^GG^A^A^^AAG^(SEQ^ID^^O:^28) ^^ SH54_^R_Fusion_^ftu_pntab_r^v^ ^^^GG^A^G^^AA^^^G^A^^G^A^G^^^^^^^GGA^^^^ (SEQ^ID^^O:^29) ^^ SH55_^R_Fusion_p^lik_^S1(crtB)^ AA^^GGGA^^^^^^AGA^^^GG^AG^AGG^GG^G^^A^^G (SEQ^ID^^O:^30) SH56_^R_Fusion_pntab_^S2(crtB)^f^^ ^AA^^^^GAAAG^^^^G^AA^GAAGG^^^^GA^^AAAA^^ (SEQ^ID^^O:^31) ^ SH57_^R_Fusion_pntab_^S2(crtB)^r^v^ AG^^^^AG^^GAGA^^^^^A^^A^AGAG^^^^^AGGA^^G (SEQ^ID^^O:^32) SH58_^R_Fusion_^S1(crtB)_^ftu^f^^ ^A^^AGAAG^AG^^^^AG^^^GG^^G^^A^^^^G^GAA^G (SEQ^ID^^O:^33) SH59_^R_Fusion_^S1(crtB)_^ftu^r^v^ ^^ ^A^^^G^AGGG^AA^GG^^AAG^^GAGG^^G^^^^^GG^A (SEQ^ID^^O:^34) SH60_^R_Fusion_^S2(crtB)_p^lik^r^v^ ^^G^^AG^GA^^^AAA^^^^^^AGGA^^^GG^G^AA^A^^ (SEQ^ID^^O:^35) Fragm^nt^amplification^for^rapL ^r1_p^liK5A_^ftu_f^d^GA^^AG^^^GGA^^^AGGGA^^GG^^G^^A^^^^G^GAA^G^^^ (SEQ^ID^^O:^36) ^r2_^ftu_rapL_r^v^^A^AGAA^^^^GG^^^G^A^^G^A^G^^^^^^^GGA^^^^ (SEQ^ID^ ^O:^37) ^r3_^ftu_rapL_f^d^GAAG^^^AGGAGGA^A^A^AA^G^AGA^^AAGG^^^^G^G (SEQ^ID^ ^O:^38) ^^ ^r4_rapL_p^liK5A_r^v^GAAGAG^A^^GA^G^^GA^GA^^^A^AG^GAG^A^GGA^^GAG G (SEQ^ID^^O:^39) Fragm^nt^amplification^for^promot^r^^xchang^ (Ps^d,^Ps^d,^m^d) ^R1_^sod_lat_f^d:^AA^^GGGA^^^^^^AGA^^^^AG^^G^^AA^^A^^^^GGG^(SEQ^ID^ ^O:^40) ^^ ^R2_^sod_lat_r^v:^G^GGGG^AAGAAGGGA^A^GGG^AAAAAA^^^^^^^G^A^(SEQ^ID^ ^O:^41) ^R3_^sod_lat_f^d:^^^A^GAAAGGA^^^^^^A^^^A^G^^^^^^^^^G^^^^G^^^(SEQ^ID^ ^O:^42) ^R4_^sod_lat_r^v:^^AG^GGA^^A^^^AAA^^^^^^AG^G^^^^^^GAG^^^^(SEQ^ID^^O:^^^ 43) Fragm^nt^amplification^for^promot^r^^xchang^ (PdapB) ^R1_^sch^ach_f^d:^AA^^GGGA^^^^^^AGA^^^AAGGG^AA^^^AAG^^^^A^ (SEQ^ ID^^O:^45) ^R2_^sch^ach_r^v:^AG^GGGG^AAGAAGGGA^A^^A^G^^^^^^^A^^^^^G^G^(SEQ^^^ ID^^O:^46) ^R3_^sch^ach_f^d:^^A^GAAAA^GAAGGAG^A^AA^G^^^^^^^^^G^^^^G^^^(SEQ^ ID^^O:^47) ^^ ^R4_^sch^ach_r^v:^^A^^^G^AGGG^AA^GG^^A^^AGG^^^GG^G^GGG^^^^^(SEQ^ ID^^O:^48) ^R5_^sch^ach_f^d:^AAGG^^^A^G^^GGG^^^GA^GG^^G^^A^^^^G^GAA^G^(SEQ^ ID^^O:^49) ^ ^R6_^sch^ach_r^v:^A^^A^AG^AA^^G^^G^^A^^G^A^G^^^^^^^GGA^^^^^(SEQ^ID^ ^O:^50) ^R7_^sch^ach_f^d:^GAAG^^^AGGAGGA^A^A^AA^GA^AA^AA^^G^^G^AA^^(SEQ^ ID^^O:^51) ^R8_^sch^ach_r^v:^^AG^GGA^^A^^^AAA^^^^^^AG^G^^^^^^GAG^^^^(SEQ^ID^^^ ^O:^52) 1.3.^^^^ia In^a^first^st^p,^C^^g^u^am^^um ^as^pr^^cultur^d^in^BHI^m^dium^(37^g^L^1 ,^Brain^h^art^Infusion,^ B^cton^Dickinson,^H^id^lb^rg,^G^rmany).^For^subs^qu^nt^pr^^cultur^s^and^main^cultur^s,^a^^^ minimal^m^dium^^as us^d.^ It^contain^d^p^r^ lit^r:^10^g^of^glucos^,^15 g^of^(^H4)2SO4,^1^g^of^ ^a^l,^200^mg^of^MgSO4·7H2O,^55^mg^of^^a^l2,^20^mg^of^F^SO4·7H2O,^1^mg^of^thiamin^H^l,^ 1^mg^of^calcium^pantoth^nat^,^0.5^mg^of^biotin,^100^mL^of^2^M^potassium^phosphat^^buff^r^ (pH^ 7.8),^ 10^ mL^ of^ a^ trac^^ ^l^m^nt^ solution^ (200^ mg^ L^1 of^ F^^l ^1 3·6H2O,^ 200 mg^ L of^ MnS04·H O,^ 50^ mg^ L^1 ,^ of^ ZnSO ·7H O,^ 20^ m ^1 ^1 24 2g^ L of^ ^u^l2·2H2O,^ 20^ mg^ L of^ ^^ ^a B O ·10H O,^10^mg^L^1 of^(^H ) Mo O ·4H O ^ 24 7 2 4 6 7 24 2,^adjust^d^to^pH^1.0^^ith^H^l),^and^30 µg^L 1 of^3,4^dihydroxyb^nzoic^acid.^^o^maintain^^pisomal^plasmids,^ th^^m^dium^^as^am^nd^d^ ^ith^ 50^ µg^ mL^1^ kanamycin.^ For^ th^^ pr^paration^ of^ solid^ m^dium,^ 20 g L^1 of^ Difco^ agar^ (B^cton^Dickinson)^^as^add^d^to^th^^corr^sponding^liquid^formulation. ^^ 1.4.^Ba^c^^cul^iva^i^n^in^^^ak^^fla^k^ Singl^^coloni^s^from^BHI^agar^plat^s,^pr^^incubat^d^at^30^°^^for^48^h,^^^r^^us^d^to^inoculat^^ th^^ first^pr^^cultur^^ (10^mL^BHI^m^dium^ in^100^mL^baffl^d^shak^^ flasks)^^hich^^as^gro^n^ ov^rnight^ on^ a^ rotary^ shak^r^ (230^ rpm,^ 85^ %^ humidity,^ Multitron,^ Infors^ AG,^ Bottming^n,^ S^itz^rland).^ ^h^n,^c^lls^^^r^^harv^st^d^ (3^min,^ 8800^ xg,^ room^ t^mp^ratur^)^ and^us^d^ to^^^ inoculat^^ a^ s^cond^ pr^^cultur^^ in^minimal^m^dium^ (25^mL^ in^ 250^mL^baffl^d^ shak^^ flask).^ ^^lls^^^r^^harv^st^d^(3^min,^8800^xg,^room^t^mp^ratur^)^during^mid^^xpon^ntial^gro^th^and^ us^d^to^ inoculat^^th^^main^cultur^s^in^minimal^m^dium.^^h^s^^^^r^^ inoculat^d^in^biological^ triplicat^^(50^mL^m^dium^in^500^mL^baffl^d^shak^^flasks)^and^incubat^d^on^an^orbital^shak^r^ as^giv^n^abov^.^^h^^cultivation^ t^mp^ratur^^^as^s^t^ to^d^fin^d^valu^s^b^t^^^n^30^and^37^^^ °^,^as^giv^n^b^lo^. ^^ 1.5.^Ba^c^^cul^iva^i^n^in^a^minia^uriz^^^micr^^i^^r^pla^^^^y^^^m Scr^^ning^ of^C^^ g^u^am^^um strains^ ^as^ conduct^d^ in^ 48^^^ll^ flo^^r^ plat^s^ in^ a^ Biol^ctor^ bior^actor^ syst^m^ (1,300^ rpm,^30^ °^,^85%^humidity,^B^ckman^^oult^r^GmbH,^Ba^s^^il^r,^ G^rmany)^^ith^ onlin^^ s^nsing^of^ c^ll^ gro^th^at^ 620^nm^ (OD620)^ (B^ck^r^ ^t^ al.,^Microb^^^ll^ ^ Fact^(2018),^17:^115).^Each^^^ll^^as^fill^d^^ith^1^mL^minimal^glucos^^m^dium.^^r^^cultur^s^ ^^r^^pr^par^d^as^d^scrib^d^abov^^and^us^^to^inoculat^^th^^main^cultur^s^to^an^initial^OD620of^0.2.^^h^^cultur^s^^^r^^p^rform^d^as^biological^triplicat^. 1.6.^F^^-ba^c^^pr^^uc^i^n^^f L-pip^c^lic^aci^ ^^ ^h^^p^rformanc^^of^L^pip^colic acid^producing^strains^^as^^valuat^d^in^a^f^d^batch^proc^ss,^ p^rform^d^ in^ duplicat^^ in^ 1^ L^ lab^ scal^^ bior^actors,^ monitor^d^ and^ controll^d^ using^ th^^ DASGI^^ control^ soft^ar^^ (SR0700ODLS,^ Epp^ndorf,^ Hamburg,^ G^rmany).^ ^h^^ batch^ m^dium^ (300^ mL)^ contain^d^ p^r^ lit^r:^ 90^ g^ of^ glucos^,^ 25^ g^ of^ (^H4)2SO4,^ 15 g^ of^ y^ast^ ^xtract^(B^cton^Dickinson),^2^g^of^citric^acid,^1.25^g^of^KH2^O4,^1.25^g^of^^a2H^O4,^1.25^g^of^^^ MgSO4·^ 7^H2O,^ 168^mg^of^ ^aSO4·^ 2^H2O,^ 70^mg^of^ F^SO4·^ 7^H2O,^30^mg^of^ ZnSO4·^ 7 H O,^9.1^mg^of^MnSO ·^H O,^15^mL^of^vitam ^1 ^1 24 2in^solution^(300^mg^L of^biotin,^500 mg^L of^ thiamin^·^H^l,^600 mg^L^1 of^nicotinamid^,^2 g L^1 of^calcium^pantoth^nat^),^and^1.43^mL^of^a^ trac^^^l^m^nt^solution^(2.1^g L^1 of^citric^acid,^228^mg^L^1 of^^uSO ^1 4 · 5 H2O,^214^^mg^L ^^of^ ^oSO ·^7^H O,^169^mg^L^1 ^iSO ·^6^H O,^150^mg^L^1 of^^ ^1 42 4 2a2B4O7·10H2O,^and^29.5^mg^L^^ ^a MoO ·^ 2^H O).^Additionally,^50 µg mL^1^of^ kanamycin,^ 13 mg ^1 2 4 2 L pyridoxal^5^phosphat^^ monohydrat^,^ and^ 1^ mL^ of^ antifoam^ 204^ (Sigma^ Aldrich,^ St.^ Louis,^ MI,^ USA)^ ^as^ suppl^m^nt^d^to^th^^batch^m^dium. ^rior^to^th^^proc^ss,^a^first^pr^^cultur^,^inoculat^d^from^an^agar^plat^^cultur^^(s^^^abov^)^^as^^^ gro^n^in^50^mL^BHI^m^dium^ov^rnight^at^34^°^.^^h^n,^c^lls^^^r^^harv^st^d^(3^min,^8800^xg,^ room^ t^mp^ratur^)^and^ r^^susp^nd^d^ in^200^mL^batch^m^dium^ in^ a^2^L^ shak^^ flask^ to^an^ initial^optical^d^nsity^OD660of^0.2.^Aft^r^ 20^h,^ c^lls^^^r^^harv^st^d^again^ (3^min,^8800^xg,^ room^t^mp^ratur^),^r^^susp^nd^d^in^50^mL^batch^m^dium,^^hich^^as^th^n^fill^d^into^th^^pr^^ st^riliz^d^bior^actor^ that^alr^ady^contain^d^250^mL^batch^m^dium.^During^ th^^proc^ss,^ th^^^^ t^mp^ratur^^^as^k^pt^at^34^°^^±^0.1^(^WD4^Bioblock,^Epp^ndorf),^^hil^^th^^pH^valu^^^as^ controll^d^at^7.0 ± 0.2^using^a^pH^prob^^ (405^D^AS^S^^K8S / 225,^M^ttl^r^^ol^do,^Gi^ss^n,^ G^rmany)^ and^ automatic^ addition^ of^ 6^ M^ ^aOH^ (M^8^ pump^ syst^m,^ Epp^ndorf).^ Onlin^^ monitoring^of^th^^dissolv^d^oxyg^n^(DO)^l^v^l^^as^don^^by^an^optical^s^nsor^ (VisiF^rm^DO^ 225,^Hamilton,^Höchst,^G^rmany).^ Initially,^ th^^stirring^rat^^^as^s^t^ to^800^rpm.^During^ lat^r^^^ proc^ss^stag^s,^it^^as^automatically^adjust^d^to^k^^p^th^^DO^l^v^l^abov^^30^%^of^saturation.^ ^h^^ a^ration^ rat^^^as^maintain^d^at^ 1 vvm.^ ^h^^ l^v^ls^ of^^O2and^O2in^ th^^^xhaust^ gas^ ^^r^^r^cord^d^throughout^th^^proc^ss^(GA4,^Epp^ndorf). ^^ At^ th^^^nd^of^ th^^batch^phas^,^ th^^f^^d^phas^^^as^start^d. Ev^ry^ tim^^^h^n^glucos^^^as^ consum^d,^th^^r^sulting^sudd^n^incr^as^^in^th^^DO^signal^ trigg^r^d^th^^automat^d^addition^ of^ a^ 20^mL^ f^^d^ puls^.^^h^^ f^^d^ solution^ contain^d^ p^r^ lit^r:^600 g^of^glucos^,^ pr^f^rably^ ^ 500 g^of^glucos^,^200 g^of^(^H4)2SO4,^15 g^of^y^ast^^xtract^(B^cton^Dickinson),^14 g^of^ur^a^ (Grüssing^ Analytica,^ Filsum,^ G^rmany),^ 2^ g^ of^ citric^ acid,^ 1.25^ g^ of^ KH2^O4,^ 1.25^ g^ of^ ^a2H^O4,^1.25^g^of^MgSO4·^7^H2O,^168^mg^of^^aSO4·^2^H2O,^70^mg^of^F^SO4·^7^H2O,^30^ mg^ of^ ZnSO ^1 4 ·^ 7 H2O,^9.1^mg^ of^ MnSO4·^ H2O,^ 15^mL^of^ vitamin^ solution^ (300^mg^ L of^ biotin,^ 500^ mg^ L^1 of^ thiamin^ ·^ H^l,^ 600 mg^ L^1 of^ nicotinamid^,^ 2 g L^1 of^ calcium^^^ pantoth^nat^),^and^1.43^mL^of^a^trac^^^l^m^nt^solution^(2.1^g L^1 of^citric^acid,^228^mg^L^1 of^ ^uSO · 5 H O,^214^ m ^1 ^1 ^1 42g^L ^^of^^oSO4·^7^H2O,^169 mg L of^^iSO4·^6^H2O,^150^mg^L of^ ^a2B4O7·10H2O,^29.5^mg^L^1 of^^a2MoO4·^2^H2O).^ 1.7.^Quan^ifica^i^n^^f^c^ll^c^nc^n^ra^i^n ^^ ^h^^c^ll^ conc^ntration^^as^quantifi^d^as^optical^d^nsity^at^660^nm^(OD660).^ ^h^^corr^lation^ b^t^^^n^c^ll^dry^^^ight^(^DW)^and^optical^d^nsity^(^DW^[g^L^1]^=^0.32 x OD660)^^as^tak^n^ from^pr^vious^^ork^(Rohl^s^^t^al.,^Microb^^^ll^Fact^(2016),^15:^154). 1.8.^Quan^ifica^i^n^^f^^x^rac^llular^^ub^^ra^^^^an^^pr^^uc^^ ^^ Glucos^^ ^as^ quantifi^d^ by^ H^L^^ (1260^ Infinity^ S^ri^s,^ Agil^nt^ ^^chnologi^s)^ using^ an^ Amin^x^H^X^87H^column^(7.8^mm^x^300^mm^x^9^µm,^55 °^,^Bio^Rad^Laboratori^s,^H^rcul^s,^ ^A,^USA)^as^stationary^phas^^and^3.5^mM^H ^1 2SO4as^mobil^^phas^^(0.5 mL min )^(Rohl^s^^t^ al.,^ Gr^^n^ ^h^m^ (2018),^ 20:^ 4662^4674).^ Glucos^^ ^as^ d^t^ct^d^ using^ r^fractiv^^ ind^x^ analysis^ (1260^ RID,^ G1362A,^ Agil^nt^ ^^chnologi^s),^ and^ its^ quantification^ ^as^ bas^d^ on^^^ ^xt^rnal^ standards.^ ^h^^ conc^ntration^ of^L^pip^colic^ acid^ ^as^ d^t^rmin^d^ by^H^L^^ (1290^ Infinity^S^ri^s,^Agil^nt^^^chnologi^s)^using^an^Eclips^^^lus^^18^column^(4.6^mm^x^100^mm^x^ 3.5^µm,^Agil^nt^^^chnologi^s),^k^pt^at^10^°^,^and^a^gradi^nt^of^^at^r^(A)^and^ac^tonitril^^(B)^ at^a^flo^^rat^^of^0.7^mL^min^1 as^^lu^nt^(0 min:^100^%^A,^0^^ 3 min:^90^%^A,^3^^ 3.5^min:^0^%^ A,^3.5^^ 4^min:^100^%^A,^4^– 7 min:^100 %^A).^For^d^t^ction,^a^diod^^array^d^t^ctor^^as^us^d^^^ (194^ nm,^ 1290^ Infinity^ II^ Diod^^ Array^ D^t^ctor,^ Agil^nt^ ^^chnologi^s).^ Ext^rnal^ standards^ ^^r^^us^d^ for^ quantification.^ ^h^^quantification^of^L^lysin^^^as^ conduct^d^ by^H^L^^ (1200^ S^ri^s,^Agil^nt^^^chnologi^s)^as^d^scrib^d^b^for^^(Kröm^r^^t^al.,^Anal^Bioch^m^(2005),^340:^ 171^173).^ ^rior^ to^ analysis,^ sampl^s^ ^^r^^ dilut^d^ ^ith^ α^aminobutyric^ acid^ (220 µM)^ as^ int^rnal^ standard.^ ^r^halos^,^ lactat^,^ and^ ac^tat^^ ^^r^^ analyz^d^ via^ H^L^^ (1260^ Infinity^^^ S^ri^s,^ Agil^nt^ ^^chnologi^s)^ Hoffmann^ ^t^ al.,^ 2018,^ ^^^^^ ^^^.).^ ^h^^ phosphat^^ l^v^l^ ^as^ d^t^rmin^d^by^ion^chromatography^(^h^rmo^Sci^ntific,^Karlsruh^,^G^rmany)^using^an^anion^ ^xchang^^column^(Ion^ac^AS9^H^,^2^mm^x^250^mm^x^9^µm,^^h^rmo^Sci^ntific)^at^30^°^ as^ ^7 th^^stationary^phas^^and^12^mM^^a ^O as^ th^^m ^1 23obil^^phas^^ (0.25 mL^min )^ (Kuhl^^t^al.,^ Biot^chnol^Bio^ng^(2020),^117:^3858^3875). 1.9.^Qu^nc^ing,^^x^rac^i^n,^an^^quan^ifica^i^n^^f^in^rac^llular^amin^^aci^^ ^ Intrac^llular^ m^tabolit^s^ ^^r^^ coll^ct^d^ using^ fast^ filtration^ and^ ^xtraction^ in^ boiling^ ^at^r^ (Bolt^n^^t^al.,J^Microbiol^Biot^chnol^(2009),^19:^23^26).^^h^^obtain^d^c^ll^^xtract^^as^dilut^d^ 1:10^^ith^α^aminobutyric^acid^as^int^rnal^standard^(Kröm^r^^t^al.,^Microbiol^(R^ading)^(2008),^ 154:^ 3917^3930).^ Subs^qu^ntly,^ L^pip^colic^ ^as^ quantifi^d^ in^ th^^ obtain^d^ c^ll^ ^xtract^ by^ H^L^^ (1260^ Infinity^ S^ri^s,^ Agil^nt),^ using^ a^ ^18^ column^ (4.6^ mm^ x^ 100^ mm^ x^ 3.5^ µm,^^^ G^mini^ ^h^nom^n^x,^ ^h^nom^n^x,^ ^orranc^,^ ^A,^USA)^ at^ 40 °^,^ a^ gradi^nt^ of^ ^lu^nt^ A^ (10 mM^^aH2^O4,^0.125^g^L^1 sodium^azid^,^pH^7.8)^and^^lu^nt^B^ (70^%^ac^tonitril^,^20^%^ m^thanol,^10^%^^at^r)^as^mobil^^phas^^ (1 mL min^1 ,^0 min:^56^%^A,^0^^ 1^min:^56 %^A,^1^^ 7.5^min:^36.5^%^A,^7.5^^ 8^min:^0^%^A,^8^^ 10^min:^0^%^A,^10^^ 13^min: 56^%^A,^13^^ 15^min:^ 56 % A)^and^fluor^sc^nc^^d^t^ction^(266^ / ^305^nm,^G1321^A,^Agil^nt^^^chnologi^s).^^rior^to^^^ analysis,^ th^^ analyt^s^ ^^r^^ d^rivatiz^d^ ^ith^ fluor^nylm^thoxycarbonyl^ (FMO^)^ und^r^ r^ducing^conditions^(0.5^%^2^m^rcaptopropionat^^in^500 mM^bicin^,^pH 9.0)^using^th^^H^L^^ autosampl^r^ and^ a^ pr^d^fin^d^ sampl^^ s^qu^nc^.^ Ext^rnal^ standards^ ^^r^^ us^d^ for^ quantification.^ Intrac^llular^ conc^ntrations^ ^^r^^ ^stimat^d^ bas^d^ on^ a^ corr^lation^ factor^ of^ 1.95 (µL^cytoplasm)^mg ^1 ^DW(Kröm^r^^t^al.,^J^Bact^riol (2004),^186:^1769^1784). ^^ 1.10.^S^ruc^ural^v^rifica^i^n^^f^L-pip^c^lic^aci^^u^ing^GC-^S First,^ cultur^^ sup^rnatant^ (20^ µL)^^as^ dri^d^ und^r^ nitrog^n.^ ^h^n,^ th^^obtain^d^ solid^ ^as^ dissolv^d^ in^ 50^ µL^ dim^thyl^ formamid^^ (0.1%^ pyridin^)^ and^ 50 µL^ ^^m^thyl^^^^^ butyldim^thylsilyl^trifluoroac^tamid^^ (Mach^r^y^^ag^l,^ Dür^n,^ G^rmany),^ follo^^d^ by^^^ incubation^for^30^min^at^80^ °^,^clarification^ from^d^bris^ (Sch^^chh^im^r^ ^t^al.,^M^tab^Eng^ (2018),^47:^357^373) and^G^^MS^analysis^ (Ki^f^r^^t^al.,^Appl^Environ^Microbiol^ (2004),^ 70:^ 229^239).^^h^^s^tup^compris^d^a^gas^chromatograph^ (G^^7890B,^Agil^nt^^^chnologi^s),^a^ H^^5MS^ column^ ((5%^ ph^nyl)^m^thylpolysiloxan^,^ 30^ m^ x^ 250^ µm^ x^ 0.25^ µm,^ Agil^nt^ ^^chnologi^s)^ as^ stationary^ phas^,^ H^lium^ 5.0^ as^ carri^r^ gas^ (1.2 mL min^1),^ and a^^^ quadrupol^^d^t^ctor^(MSD^5977A,^Agil^nt^^^chnologi^s)^(Kröm^r^^t^al.,^2008,^ ^^^^^^^^.).^For^ analysis,^ 2^ µL^ sampl^^ ^as^ inj^ct^d^ at^ a^ split^ ratio^ of^ 1:10.^ ^h^^ column^ t^mp^ratur^^ ^as^ initially^k^pt^at^120 °^^(0^^ 2 min)^and^th^n^ incr^as^d^up^to^300^°^^at^a rat^^of^10^°^^min^1.^ ^h^^instrum^nt^^as^op^rat^d^at^th^^follo^ing^t^mp^ratur^^s^ttings:^inl^t:^300^°^,^ion^sourc^:^ 230 °^,^ int^rfac^:^ 300 °^,^ quadrupol^:^ 150 °^.^Using^ this^ protocol,^ L^pip^colic^ acid^ ^lut^d^^^ aft^r^ 10.6^ minut^s.^ Structural^ id^ntification^ ^as^ bas^d^ on^ a^ library^ s^arch^ (MassHunt^r^ Library^v^rsion^^IS^08.L,^Agil^nt^^^chnologi^s)^and^analysis^of^a^comm^rcial^standard. ^8 1.11.^Quan^ifica^i^n^^f^^nzyma^ic^ac^ivi^i^^ 1.11.1.L-ly^in^^ 6-^^^y^r^g^na^^.^ Expon^ntially^ gro^ing^ c^lls^ ^^r^^ harv^st^d^ (5 min,^ 8000 x^ g,^ 4^ °^)^ and^ ^ash^d^ t^ic^^ ^ith^ 100^ mM^ glycin^^KOH^ buff^r^ (pH^ 10.0,^ 0.5^ mM^ dithiothr^itol)^ or^ 100^ mM^ potassium^ phosphat^^ buff^r^ (pH^ 7.8,^ 0.5^ mM^ dithiothr^itol)^ ^ d^p^nding^on^th^^d^sir^d^pH^for^th^^lat^r^assay.^^h^n,^c^lls^^^r^^r^susp^nd^d^in^th^^sam^^ buff^r^ to^ a^ final^ conc^ntration^of^ 1^ g^ (c^ll^ ^^t^mass)^mL^1 and^aliquot^d^ in^ lysing^matrix^B^ tub^s^ (M^^ Biom^dicals,^ Esch^^g^,^ G^rmany).^ ^^ll^ disruption^ ^as^ th^n^ p^rform^d^ in^ a^ ribolys^r^(^r^c^llys^24,^B^rtin^^^chnologi^s,^Il^^d^^Franc^,^Franc^)^^ith^a^2^min^cooling^st^p^ on^ ic^^ in^ b^t^^^n^ t^o^ disruption^ cycl^s^ (30^ s,^ 5500 xg).^ Subs^qu^ntly,^ c^ll^ d^bris^ ^as^^^ r^mov^d^by^c^ntrifugation^(17,000 xg,^4 °^,^20 min).^^h^^mast^r^mix^for^ th^^^nzym^^assay^ ^as^bas^d^^ith^r^ on^glycin^^KOH^ (pH 10.0)^ or^ on^ potassium^phosphat^^ (pH^ 7.8).^ In^ ^ach^ cas^,^it^contain^d^10 mM^ ^lysin^ ^1 L ^H^l,^2 mM^^AD,^and^50^µL^mL of^dilut^d^c^ll^^xtract.^For^ th^^ inv^stigation^of^ ^nzym^^ kin^tics,^ th^^ conc^ntration^of^ L^lysin^^ and^^AD^^as^ vari^d^ as^ sp^cifi^d^b^lo^.^Unl^ss^not^stat^d^oth^r^is^,^all^assays^^^r^^p^rform^d^at^30 °^.^^^gativ^^^^ controls^^ithout^^AD,^L^lysin^,^and^c^ll^^xtract^^^r^^ includ^d^ in^all^ ^xp^rim^nts.^Enzymatic^ activity^^as^assay^d^as^chang^^in^absorbanc^^at^340 nm^ov^r^tim^. 1.11.2.L-ly^in^^ 6-amin^^ran^f^ra^^.^ Expon^ntially^ gro^ing^ c^lls^ ^^r^^ harv^st^d^ (5 min,^ 8000 x^g,^4^°^)^and^^ash^d^t^ic^^^ith^100^mM^^ris^H^l^buff^r^(pH^8.5,^0.5^mM^dithiothr^itol,^^^ 0.5^ mM^ pyridoxal^ phosphat^)^ or^ 100^ mM^ potassium^ phosphat^^ buff^r^ (pH^ 7.8,^ 0.5^ mM^ dithiothr^itol,^0.5^mM^pyridoxal^phosphat^),^d^p^nding^on^th^^d^sir^d^pH^for^th^^subs^qu^nt^ analysis.^^h^n,^c^lls^^^r^^r^susp^nd^d^in^th^^sam^^buff^r^to^a^final^conc^ntration^of^1^g^(c^ll^ ^^t^ mass)^ mL^1 and^ aliquot^d^ in^ lysing^ matrix^ B^ tub^s^ (M^^ Biom^dicals,^ Esch^^g^,^ G^rmany).^M^chanical^c^ll^disruption^^as^p^rform^d^as^d^scrib^d^abov^.^Subs^qu^ntly,^c^ll^^^ d^bris^ ^as^ r^mov^d^ by^ c^ntrifugation^ (17,000^ x^ g,^ 4 °^,^ 20 min). ^h^^ mast^r^ mix^ for^ ^nzymatic^ analysis^ compris^d^ ^ith^r^ 100^ mM^ ^ris^H^l^ (pH^ 8.5)^ or^ 100 mM^ potassium^ phosphat^^ (pH^ 7.8)^ buff^r^ plus^ L^lysin^^ H^l^ (0^ ^ 30 mM),^ 10^ mM^ α^k^toglutarat^,^ 0.5 mM^ pyridoxal^ phosphat^,^ and^50^ µL^mL^1 c^ll^ ^xtract.^A^ volum^^ of^ 10^mL^of^ r^action^mix^^as^ fill^d^ in^a^baffl^d^shak^^flask^ (100^mL)^and^ incubat^d^for^1^h^at^30 °^^on^an^orbital^shak^r^^^ (230^ rpm,^ 5^ cm,^ H^^ Infors^ Multitron).^ Sampl^s^ ^^r^^ tak^n^ ^v^ry^ 5^ minut^s,^ follo^^d^ by^ imm^diat^^inactivation^(100^°^,^15^min,^^h^rmoMix^r ^,^Epp^ndorf^D^utschland,^W^ss^ling^ B^rzdorf,^G^rmany).^^rot^in^d^bris^^as^r^mov^d^(17,000 xg,^4^°^,^3 min).^Enzymatic^activity^ ^as^th^n^inf^rr^d^from^th^^lin^ar^d^cr^as^^of^th^^L^lysin^^conc^ntration^^hich^^as^quantifi^d^ via^H^L^^as^d^scrib^d^abov^. ^^ In^all^cas^s,^th^^total^prot^in^conc^ntration^of^c^ll^^xtract^^as^^stimat^d^using^th^^Bradford^ m^thod^(^rot^in^Assay^Dy^^R^ag^nt^^onc^ntrat^,^Bio^Rad^Laboratori^s)^and^bovin^^s^rum^ albumin^as^a^standard (B^ck^r^^t^al.,^Microb^^^ll^Fact^(2008),^7:^8). ^9 1.12.^Ex^rac^i^n^an^^quan^ifica^i^n^^f^r^^^x^m^^ab^li^^^ ^ultur^^sampl^s^^^r^^qu^nch^d^using^a^protocol^from^pr^vious^^ork^(Hoffmann^^t^al.,^2021,^ ^^^^^^^^.).^5^mL^cultur^^broth^^as^mix^d^^ith^10^mL^pr^^cool^d^m^thanol^(^58^°^,^60^%^v / v),^ ^ c^ntrifug^d^ (9,400 x^ g,^ ^10 °^,^ 5^ min)^ and^ r^susp^nd^d^ in^ 1.1 mL^ ^BS^ buff^r.^ ^o^ obtain^ s^parat^^sampl^s^for^^AD+ and^^ADH^analysis,^r^sp^ctiv^ly,^th^^susp^nsion^^as^split^in^t^o^ fractions^ of^ 500^ µL^ ^ach.^ Each^ fraction^ ^as^ c^ntrifug^d^ (10,000^ x^ g,^ 4°^,^ 5^ min).^ ^h^^ intrac^llular^^AD+ and^^ADH^ l^v^ls^^^r^^ th^n^d^t^rmin^d^using^an^assay^ kit^ (Enzy^hrom^ ^AD+ / ^ADH^Assay^Kit,^BioAssay^Syst^m,^Hay^ard,^^A,^USA)^follo^ing^th^^manufactur^r’s^^^ protocol.^Finally,^this^yi^ld^d^th^^^AD+ to^^ADH^ratio. 1.13.^Gl^bal^g^n^^analy^i^ A^customiz^d^microarray^ (Sur^^rint^G3^^ustom^GE^8x60^K,^part^ numb^r^G4863A,^Agil^nt^ ^^chnologi^s)^ ^as^ d^sign^d^ using^ th^^ ^Array^ ^orkspac^^ (Agil^nt^ ^^chnologi^s).^ ^hr^^^^^ diff^r^nt^prob^s^(45^to^60 bp)^cov^r^d^^ach^g^n^^of^th^^C^ g^u^am^^um A^^^^13032^g^nom^^ (^ntry^numb^r:^#^^00102,^KEGG^databas^)^plus^h^t^rologous^g^n^s^of^int^r^st:^ ^ysDH from^ R^ p^mer^y^ in^ nativ^^ and^ codon^optimiz^d^ form,^ nativ^^ ^ysDH from^ Agr^ba^^er^um^ ^umefa^^ens and^Ge^ba^^^^us s^ear^^herm^ph^^us,^nativ^^ ^a^ from^F^av^ba^^er^um^ ^u^es^ens,^ and^th^^codon^optimiz^d^pr^C from^C^^g^u^am^^um,^r^sp^ctiv^ly.^Each^prob^^^as^appli^d^as^^^ 6^r^plicat^s^and^randomiz^d^to^f^atur^^locations^on^th^^slid^^using^th^^Sur^^rint^t^chnology^ (Agil^nt^ ^^chnologi^s).^ First,^ R^A^ ^as^ isolat^d,^ cl^an^d^ up^ (R^^asy^ Mini^ Kit,^ Qiag^n,^ Hild^n,^ G^rmany)^ and^ quantifi^d^ (^anoDrop^ 1000,^ ^EQLAB^ Biot^chnology,^ Erlang^n,^ G^rmany).^ ^h^n,^ th^^ R^A^ quality^ ^as^ ^valuat^d^ (R^A^ 6000^ ^ano^Kit,^ 2100^Bioanalyz^r^ Syst^m,^Agil^nt^^^chnologi^s).^All^sampl^s^^xhibit^d^an^R^A^int^grity^numb^r^(RI^)^>^9.9.^^^ In^ a^n^xt^st^p,^ fluor^sc^nt^ cR^A^^as^g^n^rat^d^ (Lo^^ Input^Quick^Amp^W^^Lab^ling^On^^ ^olor^ Kit,^ Agil^nt^ ^^chnologi^s,^ R^A^ Spik^^In^ On^^^olor^ Kit,^ Agil^nt^ ^^chnologi^s,^ and^ R^^asy^ Mini^ Kit,^ Qiag^n).^ Fifty^ nano^ grams^ of^ total^ R^A^ s^rv^d^ as^ starting^ mat^rial^ and^ yi^ld^d^>^825^ng^of^lab^l^d^cR^A^^ith^a^sp^cific^^y3^activity^of^15^pmol^^y3^p^r^µg^cR^A.^ An^amount^of^600^ng^ lab^l^d^ cR^A^^as^hybridiz^d^onto^ th^^microarray^ (G^n^^Expr^ssion^^^ Hybridization^ Kit,^ hybridization^ gask^t^ slid^s,^ Sur^Hyb^ chamb^r,^ hybridization^ ov^n,^ all^ Agil^nt^ ^^chnologi^s).^ Subs^qu^ntly,^ th^^ slid^^ sand^ich^s^ ^^r^^ disass^mbl^d^ (G^n^^ Expr^ssion^Wash^ Buff^r^ Kit,^ Agil^nt^ ^^chnologi^s).^ Scanning^ ^as^ th^n^ conduct^d^ by^ th^^ Sur^Scan^Microarray^Scann^r^ (G4900DA,^Sur^Scan^Microarray^Scann^r^^ontrol^Soft^ar^,^ V^rsion^ 9.1.11.13, Agil^nt^ ^^chnologi^s).^ ^ranscriptom^^ analysis^ includ^d^ thr^^^ biological^^^ r^plicat^s.^ ^ranscriptomic^ data^ ^xtraction^ ^as^ p^rform^d^ ^ith^ th^^ microarray^ and^ f^atur^^ ^xtraction^soft^ar^^(V^rsion^12.1.1.1,^Agil^nt^^^chnologi^s).^^h^^r^sults^^^r^^visualiz^d^and^ furth^r^^valuat^d^ using^ th^^ soft^ar^^G^n^Spring^ (V^rsion^14.9,^ Agil^nt^ ^^chnologi^s).^ For^ ^^ statistical^analysis,^a^mod^rat^d^t^t^st^^as^appli^d,^consid^ring^asymptotic^computation^of^p^ valu^s^adjust^d^ for^multipl^^ t^sting^ according^ to^ th^^B^njamini^Hochb^rg m^thod^ and^ a^q^ valu^^cut^off^of^0.05 (Kohlst^dt^^t^al.,^M^tab^Eng^(2022),^72:^337^352).^^h^^data^^^r^^th^n^ filt^r^d^for^g^n^s^^ith^a^ log2^fold^chang^^≥^1^(p^valu^^≤^0.05).^R^A^^xtraction^and^analysis^ ^ ^^r^^conduct^d^in^biological^triplicat^^for^^ach^condition.^^h^^^ntir^^transcriptom^^data^s^t^is^ availabl^^at^GEO^(GSESE216736). 2.^R^^ul^^ 2.1. Expr^^^i^n^ ^f^ L-ly^in^^ 6-^^^y^r^g^na^^^ an^^ Δ1-pyrr^li^in^-5-carb^xyla^^^^^ r^^uc^a^^^in ^.^glutamicum LYS-12 C^^ g^u^am^^um LYS^12^ ov^rproduc^sL^lysin^,^ th^^ pr^cursor^ of^L^pip^colic^ acid^ and^ ^as^ th^r^for^^ tak^n^ as^ a^ starting^ point^ for^ strain^ ^ngin^^ring.^ ^o^ ^nabl^^L^pip^colic^ acid^ production,^diff^r^nt^g^n^tic^modul^s^^^r^^d^sign^d^and^constitutiv^ly^^xpr^ss^d^that^^ach^ ^ncod^d^for^a^h^t^rologous^L^lysin^^6^d^hydrog^nas^^(LysDH)^and^ th^^nativ^^Δ1^pyrrolin^^^^ 5^carboxylat^^r^ductas^^(^ro^)^(Fig.^1).^ ^h^^^xpr^ssion^of^L^lysin^^6^d^hydrog^nas^^^as^kno^n^to^^nabl^^th^^conv^rsion^of^L^lysin^^ into^ α^amino^ adipic^ acid^ s^miald^hyd^^ ^hich^ ^ould^ spontan^ously^ transform^ into^ 1^pip^ridin^^ 6^carboxylic^ acid^ (^^r^z^Garcia^ ^t^ al.,^ Appl^ Microbiol^ Biot^chnol^ (2016),^ 100:^^^ 8075^8090).^ ^h^^ latt^r^ int^rm^diat^^ ^ould^ b^^ conv^rt^d^ by^ ^ro^,^ nativ^ly^ part^ of^ th^^ L^prolin^^biosynth^sis,^into^L^pip^colic^acid^(Ankri^^t^al.,^J Bact^riol^(1996),^178:^4412^4419).^ 4L^lysin^^6^d^hydrog^nas^^variants^from^thr^^^diff^r^nt^donors ^^r^^ impl^m^nt^d,^nam^ly^ R^ p^mer^y^,^ a^ gram^n^gativ^^ bact^rium^ from^ th^^ marin^^ ^nvironm^nt,^ that^ b^n^fits^ from L^lysin^^d^gradation^via^LysDH^by^incr^as^d^salt^tol^ranc^^(^^shich^^t^al.,^Ism^^J^(2013),^7:^^^ 2400^2410) G^ s^ear^^herm^ph^^us, a^gram^positiv^^th^rmophilic^Ba^^^^us^(H^ydari^^t^al., Appl^ Environ^Microbiol^(2004),^70:^937^943),^and^A^^^umefa^^ens,^a^gram^n^gativ^^phytopathog^n (^agasaki^^t^al.,^J^Bioch^m^(1989),^105:^1002^1008).^Each^^ysDH g^n^^^as^^xpr^ss^d^^ith^ its^ nativ^^ codon^ usag^^ und^r^ th^^ constitutiv^ tuf^promot^r^ (B^ck^r^ ^t^ al.,^ Appl^ Environ^ Microbiol^ (2005),^71:^8587^8596) on^an^^pisomal^v^ctor^ tog^th^r^^ith^pr^C,^ r^sulting^ in^ th^^^^ strains^^IA^1A,^^IA^1B,^and^^IA^1^. In^ shak^^ flask^ cultivations,^ th^^ n^^ly^ d^riv^d^ strains^ C^ g^u^am^^um ^IA^1A^ and^ ^IA^1^^ form^d^ L^pip^colic^ acid,^ as^ confirm^d^by^G^^MS^analysis^ (Fig.^ 8).^ In^ contrast,^ th^^^IA^1B^ mutant^did^not^^xhibit^th^^d^sir^d^production,^indicating^that^th^^^nzym^^from^A^^^umefa^^ens^^ ^as^not^functionally^^xpr^ss^d.^C^ g^u^am^^um ^IA^1A^p^rform^d^b^st^and^all^s^cr^t^d^about^ 6^mM^L^pip^colic^acid^into^th^^m^dium,^tog^th^r^^ith^20 mM^L^lysin^^(Fig.^2A).^In^r^lation^to th^^ utiliz^d^ glucos^,^ th^^ ^1 L^pip^colic^ acid^ yi^ld^ (88 mmol mol )^ and^ th^^ sp^cific^ production^ ^^ rat^^ (0.31 mmol^ g^ h^1)^ ^^r^^ n^v^rth^l^ss^ lo^^ (^abl^s 3^ and^4).^ Lo^^l^v^l production^ of^L^ pip^colic^ acid^ in^ a^ mixtur^^ ^ith^ major^ shar^s^ of^ L^lysin^^ match^d^ pr^vious^ findings^ from^ ov^r^xpr^ssion^of^a^similar^ construct^ in^anoth^r^ L^lysin^^ov^rproduc^r^ (^^r^z^Garcia^ ^t^al.,^ 2016,^^^^^^^^^.).^^h^^p^rformanc^^of^strain^^IA^1A^^as^consid^r^d^poor. ^ Tabl^^ 3:^ In^rac^llular^ amin^^ aci^^ l^v^l^^ in^ ^^^^ L-pip^c^lic^ aci^^ pr^^ucing^ ^^rain^^ ^. glutamicum PIA-1A^an^^ PIA-7.^^h^^ sampl^s^^^r^^ tak^n^ from^ glucos^^bas^d^ cultur^s^ during^ mid^^xpon^ntial^ gro^th.^ ^h^^ non^producing^ r^f^r^nc^^ strain^ ^IA^0^ is^ sho^n^ for^ comparison.^n=3.^ PIA-1A PIA-7 PIA-0 Amin^^aci^ [m^] [m^] [m^] L^Lysin^ 35.3^±^05.8 22.4^±^03.8 63.5^±^06.9 L^Aspartic^acid 16.6^±^00.8 13.5^±^01.5 12.1^±^01.3 L^Glutamic^acid 227.4^±^19.8 186.3^±^21.3 238.2^±^19.6 L^Asparagin^ 7.7^±^00.5 6.5^±^00.6 4.1^±^00.4 L^S^rin^ 7.7^±^00.5 6.9^±^00.6 4.9^±^00.5 L^Glutamin^ 22.0^±^01.0 16.6^±^01.8 42.3^±^05.8 L^Histidin^ 0.0^±^00.0 0.0^±^00.0 0.0^±^00.0 Glycin^ 11.6^±^01.2 10.9^±^01.1 9.8^±^01.1 L^^hr^onin^ 6.2^±^00.5 6.1^±^00.5 3.2^±^00.4 L^Arginin^ 8.0^±^00.5 6.9^±^00.6 5.0^±^05.0 L^Alanin^ 14.3^±^00.9 11.4^±^01.2 12.1^±^01.1 L^^yrosin^ 0.0^±^00.0 0.0^±^00.0 0.0^±^00.0 L^Valin^ 32.3^±^02.8 22.7^±^02.4 28.4^±^02.9 L^M^thionin^ 4.5^±^00.4 4.2^±^00.4 1.7^±^00.1 L^^h^nylalanin^ 8.9^±^00.7 7.5^±^00.8 5.1^±^00.5 L^Isol^ucin^ 16.7^±^01.3 12.5^±^01.1 14.3^±^01.5 L^L^ucin^ 11.9^±^00.8 10.0^±^01.0 6.9^±^00.6 L^^ryptophan 21.7^±^01.4 18.0^±^01.6 11.8^±^01.0 L^^rolin^ 49.9^±^05.8 35.0^±^04.3 59.1^±^04.4 L^^ip^colic^acid 266.9^±^48.1 222.3^±^26.2 0.0^±^00.0 ^^ Tabl^^ 4:^ Kin^^ic^^ an^^ ^^^ic^i^m^^ry^ ^f^ gr^w^^^ an^^ pr^^uc^^ f^rma^i^n^ in^ ba^ic^ L- pip^c^lic^ aci^^ pr^^ucing^ ^.^ glutamicum^ ^^rain^.^ ^h^^ produc^rs^ ^^r^^ bas^d^ on^ ^xpr^ssion^ of^ lysin^^ 6^d^hydrog^nas^^ from^ diff^r^nt^ donors^ and^ combin^d^ ^ith^ diff^r^nt^ supporting^ modifications^ (^abl^^ 1).^ All^ strains^ ^^r^^ gro^n^ on^ glucos^^ minimal^ m^dium^ in^^^ shak^^ flasks^ at^ 30^ °^^ and^ 34 °^.^ ^h^^ data^ compris^^ rat^s^ for^ gro^th^ (µ),^ substrat^^ consumption^ and^ product^ formation^ (q),^ as^ ^^ll^ as^ yi^lds^ (Y).^ GL^^ =^ glucos^;^ ^IA =^ L^ pip^colic^acid,^LYS^=^L^lysin^,^X^=^biomass.^n=3.^ PIA- PIA- PIA- PIA-4 PIA-5 PIA-6 PIA- PIA-4 PIA-5 1A 2A 2C (30^ (30^ (30^ 1A (34^ (34^ (30^ (30^ (30 °C) °C) °C) (34^ °C) °C) °C) °C) °C) °C) Ra^^^ µ^[h^1] 0.18^±^ 0.19^±^ 0.18^±^ 0.14^±^ 0.19^±^ 0.18^±^ 0.13^±^ 0.07^±^ 0.10^±^ 0.00 0.00 0.00 0.00 0.00 0.01 0.00 0.00 0.00 qGL^3.58^±^ 4.08^±^ 4.27^±^ 3.25^±^ 4.00^±^ 3.86^±^ 3.42^±^ 2.59^±^ 3.65^±^ [mmol^g^1 0.08 0.17 0.09 0.11 0.08 0.08 0.27 0.10 0.11 h^1] q^IA 0.31^±^ 0.05^±^ n.d. * 0.38^±^ 0.15^±^ 0.05^±^ 0.63^±^ 0.44^±^ 0.32^±^ [mmol^g^1 0.00 0.01 0.00 0.00 0.00 0.07 0.01 0.02 ^^ h^1] qLYS0.68^±^ 1.20^±^ 0.88^±^ 1.09^±^ 1.71^±^ 1.03^±^ 0.53^±^ 0.76^±^ 1.47^±^ [mmol^g^1 0.06 0.00 0.09 0.02 0.03 0.02 0.34 0.03 0.04 h^1] Yi^l^^ YX / GL^48.9^±^ 45.8^±^ 41.0^±^ 42.7^±^ 46.8^±^ 45.5^±^ 37.4^±^ 28.8^±^ 28.8^±^ [g^mol^1] 0.7 1.0 0.4 0.9 1.3 2.3 0.9 0.5 0.8 Y^IA / GL^88.1^±^ 13.3^±^ n.^d. 118.0 37.0^±^ 12.5^±^ 182.3^ 169.8^ 87.6^±^ [mmol^ 2.8 1.5 4^±^ 1.3 0.2 ±^7.0 ±^2.9 7.5 mol^1] 1.8 YLYS / GL^191.8^ 293.3^ 204.9^ 335.6^ 427.0^ 266.5^ 155.4^ 294.5^ 403.8^ [mmol^ ±^20.4 ±^17.3 ±^1.8 ±^9.2 ±^7.6 ±^5.7 ±^1.2 ±^21.5 ±^13.9 mol^1] *^n.d.^=^not^d^t^ct^d. 2.2.^ ^^n^ci^^r^nic^ lysDH^pr^^ m^^ul^^ wi^^^ na^iv^^ c^^^n^ u^ag^^ f^r^ L-pip^c^lic^ aci^^ pr^^uc^i^n ^ An^ alt^rnativ^^ pyrrolin^^ 5^carboxylat^^ r^ductas^ ^as^ us^d.^ Diff^r^nt^ to^C^^ g^u^am^^um,^B^^ sub^^^^s poss^ss^s^t^o^activ^^pyrrolin^^5^carboxylat^^r^ductas^s:^^roG^and^^roH^(B^litsky^^t^ al.,^J^Bact^riol^(2001),^183:^4389^4392;^Forlani^^t^al.,^Front^Microbiol^(2017),^8:^1442).^^roG^ ^as^ kno^n^ to^ play^ a^ constitutiv^^ rol^^ and^ ^as^ r^gard^d^ similar^ to^ th^^ ^ro^^ from^ C^^ g^u^am^^um,^^h^r^as^^roH^had^b^^n^sho^n^to^b^^activat^d^und^r^str^ss^for^incr^as^d^l^v^ls^^^ of^L^prolin^^(Forlani^^t^al.,^2017,^ ^^^^^^^^.).^^h^^prot^in^s^qu^nc^s^of^^ro^^and^^roH^shar^d^ only^about^35^%^of^cons^rv^d^r^gions,^sugg^sting^^roH^as^promising^alt^rnativ^.^^his^l^d^to^ th^^construction^of^a^fourth^path^ay^mutant,^nam^ly^^IA^3^(LYS^12^P Rp^m Bsub ^uf^ysDH pr^H ).^ ^h^^n^^^strain^achi^v^d^a^product^ tit^r^of^ 3.8^mM,^significantly^high^r^ than^ that^of^ th^^ t^o^ oth^r^variants^but^still^b^lo^^that^of^^IA^1A^(Fig.^3). ^^ 2.3.^T^mp^ra^ur^^impac^ Wh^n^int^grating^^nzym^^data^^ith^flux^data,^^hil^^consid^ring^that^no^oth^r^limitations^had^ ^m^rg^d^ from^ m^tabolit^^ and^ g^n^^ ^xpr^ssion^ profiling,^ th^^ h^t^rologous^ L^lysin^^ 6^ d^hydrog^nas^^ from^ R^^ p^mer^y^ turn^d^ out^ to^ b^^ a^ k^y^ ^nzym^^ that^ controll^d^ ^^ p^rformanc^.^Und^r^assay^conditions,^its^maximum^^n^v^^r^ activity^^as^900^mU^(mg^prot^in)^ 1.^ ^his^ valu^^ confirm^d that a^ high^ amount^ of^ activ^^ LysDH^ ^as^ ^xpr^ss^d.^ Assuming^ a^ c^llular^prot^in^cont^nt^of^50%,^th^^^n^v^^r^ activity^corr^spond^d^to^a^maximum^flux^capacity^ of^ th^^^nzym^^ for^L^pip^colic^acid^production^of^27^mmol^g^1 h^1 und^r^optimum^conditions.^ ^h^^^ff^ctiv^^ ^pip^ ^1 ^1 L colic^acid^flux^of^^IA1^A ^as,^ho^^v^r,^only^0.31^mmol^g h .^Obviously,^^^L^lysin^ 6^d^hydrog^nas^^ op^rat^d^ only^ at^ approximat^ly^ 1%^ of^ its^ th^or^tical^ ability^ although^th^^^nzym^^^as^suffici^ntly^suppli^d^^ith^L^lysin^. ^^ R^garding^ t^mp^ratur^,^LysDH^ ^xhibit^d^optimum^ activity^ at^55^ °^^ (Fig.^ 4A),^much^high^r than^ th^^ us^d^ gro^th^ t^mp^ratur^^ of^ C^^ g^u^am^^um (30 °^).^ Inspir^d^ by^ ^ctoin^^ and^ hydroxy^ctoin^^producing^C^^g^u^am^^um strains^ that^ had^ b^^n^ cultivat^d^ up^ to^42^ °^^ and^ ^xhibit^d^maximum^production^of^ th^^^xtr^molyt^s^at^35 °^^ (B^ck^r^^t^al.,^Microb^^^ll^Fact^ ^ (2013),^12:^10),^production^^as^t^st^d^at^incr^as^d^t^mp^ratur^^h^r^.^Ind^^d,^^h^n^gro^n^at^ 34^°^^(Fig.^4B),^^IA^1A^produc^d^L^pip^colic^acid^at^doubl^d^yi^ld^(182 mmol^mol^1)^and^rat^^ (0.62 g mol^1 h^1 ,^ ^abl^^ 4), ^h^r^as^ no^ gro^th^ ^as^ obs^rv^d^ at^ 37^ °^.^ In^ addition^ to^ th^^ significant^ improv^m^nt in^production,^ th^^outcom^^und^rlin^d^th^^ importanc^^of^LysDH^as^ major^pac^mak^r^for^L^pip^colic^acid^biosynth^sis. ^^ 2.4.^Al^^rna^iv^^^f^r^L-pip^c^lic^aci^^pr^^uc^i^n L^lysin^^6^aminotransf^ras^^(LA^)^from^th^^L^lysin^^d^gradation^path^ay^of^F^av^ba^^er^um^ ^u^es^ens^^as^us^d.^LA^ transaminat^s^L^lysin^^into^α^ aminoadipic^acid^s^miald^hyd^^using^ th^^ cofactor^ α^k^toglutarat^^ (Fuji^ ^t^ al.,^ J^ Bioch^m^ (2000),^ 128),^ follo^^d^ by^ spontan^ous^^^ r^action^ into^ 1^pip^rid^in^^ 6^carboxylat^,^ and^ pr^C-m^diat^d r^duction^ to^ L^pip^colic^ acid (Fuji^^t^al.,^Biosci^Biot^chnol^Bioch^m^(2002),^66:^622^627).^ ^h^^ ^a^^g^n^^^as^clon^d^^ith^its^nativ^^codon^usag^^tog^th^r^^ith^pr^C into^a^P^uf^controll^d^ hybrid^modul^^(P^uf^a^^pr^C),^yi^lding^C^^g^u^am^^um ^IA^7.^^h^^n^^^produc^r^C^ g^u^am^^um^^ ^IA^7^ form^d^ an incr^as^d^ amount^ of^ 7.5^ mM^L^pip^colic^ acid^ and^ ^xhibit^d^ improv^d^ s^l^ctivity,^b^tt^r^than^that^of^all^oth^r^strains^(Fig.^3).^In^shak^^flask^at^30^°^,^^IA^7^s^cr^t^d^ 8^ mM^L^pip^colic^ acid^ at^ 60%^ incr^as^d^ yi^ld^ and^ ^v^n^ doubl^d^ production^ rat^,^ as^ compar^d^to^^IA^1A^(Fig.^5A,^^abl^^5).^^his^discov^ry^^as^a^surpris^^giv^n^th^^fact^that^th^^ L^lysin^^6^aminotransf^ras^^bas^d^rout^^is^not^consid^r^d^a^promising^m^tabolic^path^ay^for^^^ L^pip^colic^acid synth^sis^(Wang^^t^al.,^Adv^appl^Micriobiol^(2018),^115:^1^33). ^IA^7,^sampl^d^during^th^^mid^^xpon^ntial^gro^th^phas^,^^xhibit^d^an^^n^v^^r^ activity^of^170^ mU^mg^1 at^10^mM^ ^lysin^, ^hich^^as^inc ^1 L r^as^d^to^230^mU^mg ,^^h^n^assay^d^at^30^mM^L^ lysin^^and^pH^7.8,^and^to^^v^n^500^mU^mg^1 at^30^mM^L^lysin^^and^pH^8.5^(Fig.^6). ^^ ^h^^ intrac^llular^ l^v^l^ of^ L^lysin^^ in^ th^^ n^^^ strain^ ^as^ 22^ mM^ (^abl^^ 3).^ ^h^ valu^^ ^as^ significantly^lo^^r^than^in strain^^IA^1A^(30^mM^L^lysin^),^indicating^fast^r^conv^rsion^into^L^ pip^colic^ acid.^ ^h^^ data^ translat^d^ into^ a^ L^lysin^^corr^ct^d^ flux^ capacity^ of^ approximat^ly^ 200^mU^mg^1 ,^ i.^ ^.^ 2.6^mmol^ g^1 h^1 ,^ off^ring^ high^^ffici^ncy^production.^ ^omparativ^^ g^n^^^^ ^xpr^ssion^ profiling^ of^ ^IA^7^ and^ ^IA^0^ r^v^al^d^ strong^ upr^gulation^ of^L^lysin^^ 6^aminotransf^ras^,^ as^ d^sir^d^ (^abl^^ 6),^ plus^ a^ f^^^ oth^r^ chang^s^ of^ appar^ntly^ minor^ r^l^vanc^^ that^ ^^r^^ also^ visibl^ in^ ^IA^1A.^ Accordingly,^ th^^ transcriptom^s^ of^ th^^ t^o^ ^^ produc^rs^^IA^1A^ and^^IA^7^ sho^^d^ no^ significant^ diff^r^nc^,^ ^xc^pt^ for^ th^^ h^t^rologous^ g^n^s^(data^not^sho^n).^ Tabl^^ 5:^ Kin^^ic^^ an^^ ^^^ic^i^m^^ry^ ^f^ gr^w^^^ an^^ pr^^uc^^ f^rma^i^n^ in^ ^up^ri^r^ L- ^ pip^c^lic^ aci^^ pr^^ucing^ ^.^ glutamicum^ ^^rain^.^ ^IA^7^ ^as^ bas^d^ on^ LYS^12^ and^ ^pisomally^^xpr^ss^d^^a^ from^A^^^umefa^^ens and^pr^C^from^C^^g^u^am^^um^und^r^control^of^ Puf^(^ab fbr ^ l^^ 1).^ ^IA^8^ ^as^ bas^d^ on^C^^g^u^am^^um^ ^ysC D^ysE^ and^ ^xpr^ss^d^ th^^ sam^^ plasmid.^Both^strains^^^r^^gro^n^on^glucos^^minimal^m^dium^in^shak^^flasks^at^30^°^^and^ 34 °^,^ partially^ suppl^m^nt^d^ ^ith^ pyridoxal^ phosphat^^ (12.5^ mg^ L^1).^ ^h^^ data^ compris^^^^ rat^s^for^gro^th^(µ),^substrat^^consumption^and^product^formation^(q),^as^^^ll^as^yi^lds^(Y).^ GL^^=^glucos^;^^IA =^L^pip^colic^acid,^LYS^=^L^lysin^,^X^=^biomass.^n=3. PIA-7 PIA-7 PIA-7 PIA-7 PIA-7 PIA-8 30^°C 34^°C 30^°C+^ 34^°C^+^ 37^°C 30^°C PP PP Rat^s µ^[h^1] 0.23^±^ 0.16^±^ 0.24^±^0^ 0.16^±^ 0.10^±^ 0.40^±^ 0.01 0.01 01 0.01 0.01 0.01 qGL^4.14^±^ 3.63^±^ 3.74^±^ 3.59^±^ 3.39^±^ 4.56^±^ [mmol^g^1 h^1] 0.08 0.13 0.33 0.04 0.14 0.06 q^IA0.58^±^ 1.10^±^ 0.73^±^ 1.09^±^ 1.91^±^ 0.12^±^ [mmol^g^1 h^1] 0.01 0.05 0.01 0.12 0.09 0.01 qLYS 0.32^±^ 0.31^±^ 0.34^±^ 0.44^±^ 0.09^±^ n.d.* [mmol^g^1 h^1] 0.01 0.02 0.03 0.04 0.00 Yi^lds Y ^ X / GL^[g^mol 56.0^±^ 42.9^±^ 63.3^±^ 41.4^±^ 29.9^±^0.6 87.0^±^1.1 1] 03.2 03.0 06.5 01.6 Y^IA / GL^139.9^±^^ 304.3^±^ 196.1^±^ 304.1^±^ 562.1^±^ 26.0^±^1.6 [mmol^mol^1] 0.5 08.4 12.4 36.2 2.8 YLYS / GL^76.3^±^ 84.8^±^ 89.6^±^ 123.0^±^ 27.4^±^1.0 n.d. [mmol^mol^1] 01.3 02.1 00.2 12.9 *^n.d.^=^not^d^t^ct^d. ^^ Tabl^^6:^Tran^crip^i^n^pr^filing^^f^L-pip^c^lic^aci^^pr^^ucing^^. glutamicum PIA-7.^^h^^ data^ compris^^ significantly^ up^ and^ do^n^r^gulat^d^ g^n^s^ as^ compar^d^ to^C^ g^u^am^^um ^IA^0^^xpr^ssing^th^^^mpty^plasmid^(log2^fold^chang^^≥^1^and^≤^^1,^p^≤^0.05).^^h^^sampl^s^ ^^r^^ tak^n^ from^ glucos^^bas^d^ cultur^s^ during^ mid^^xpon^ntial^ gro^th.^ ^h^^ statistical^ quality^of^th^^data^^as^v^rifi^d^by^^^A. n=3. L^cu^^^ag G^n^ G^n^^^^^crip^i^n L^g2f^l^^ c^ang^ ^a^L^lysin^^6^aminotransf^ras^ +^9.6 CGL_R^02095 pr^C Δ1^pyrrolin^^5^carboxylat^^r^ductas^ +^4.6 CGL_R^15485 -Hypoth^tical^prot^in +^2.5 CGL_R^03280 prpD2 MmgE / ^rpD^family^prot^in +^2.4 CGL_R^14845 NCg^2877 ^adR^family^transcriptional^r^gulator +^2.4 CGL_R^03285 prpB2 M^thylisocitrat^^lyas^ +^2.3 CGL_R^00835 NCg^0156 Hypoth^tical^prot^in +^2.3 CGL_R^12550 f^n F^rritin +^2.3 CGL_R^00840 msmA ^oA^acylating^m^thylmalonat^^ +^2.2 s^miald^hyd^^d^hydrog^nas^ CGL_R^05910 narI R^spiratory^nitrat^^r^ductas^^subunit^ +^1.6 ^^ gamma CGL_R^02010 m^rAHtaA^domain^containing^prot^in ^ 4.8 CGL_R^02015 m^rA HtaA^domain^containing^prot^in ^ 4.4 CGL_R^06520 NCg^1254 Hypoth^tical^prot^in ^ 3.5 CGL_R^01750 NCg^0329 Iron^sid^rophor^^AB^^transport^r^ ^ 3.4 substrat^^binding^prot^in CGL_R^04925 r^pA H^lix^turn^h^lix^transcriptional^r^gulator ^ 3.3 CGL_R^02555 NCg^0483 Iron^ch^lat^^uptak^^AB^^transport^r ^ 3.0 family^p^rm^as^^subunit CGL_R^02550 NCg^0482 AB^^transport^r^A^^^binding^prot^in ^ 2.9 CGL_R^04045 NCg^0773Sid^rophor^^int^racting^prot^in ^ 2.9CGL_R^10120 NCg^1959 Iron^sid^rophor^^AB^^transport^r^ ^ 2.8 substrat^^binding^prot^in CGL_R^03335 Irp1B Iron^AB^^transport^r^p^rm^as^ ^ 2.7 2.5.^A^^i^i^n^^f^Pyri^^xal^p^^^p^a^^ ^roduction^ ^as^ boost^d^ by^ th^^ addition^ of^ pyridoxal^ phosphat^^ (13 mg L^1),^ a^ stabilizing^ co^nzym^^of^L^lysin^^6^aminotransf^ras^^(^oqu^^^t^al.,^J^Bact^riol^(1991),^173:^6258^1664).^ ^ At^30 °^,^th^^supply^of^pyridoxal^phosphat^^(10^mg^L^1)^incr^as^d^th^^productivity^of^^IA^7^by^ 25%^to^0.73^mmol^g^1 h^1 (Fig. 5B,^^abl^^5),^^hil^^ th^^yi^ld^^as^^v^n^ incr^as^d^by^40%^ to^ 140^mmol^ mol^1 (^abl^^ 5).^ ^h^^ cultivation^ t^mp^ratur^^ b^n^ficially^ stimulat^d^ strain^ ^IA^7^ (Fig. 5^).^At^34^°^,^th^^produc^r^yi^ld^d^L^pip^colic^acid^at^30^%^yi^ld^and^a^production^rat^^ of^1.1 mmol g^1 h^1 (^abl^^5).^Diff^r^nt^to^th^^L^lysin^^6^d^hydrog^nas^^bas^d^strains,^^IA^7^^^ ^as^abl^^to^gro^^at^37 °^^ (^abl^^5).^Und^r th^s^^conditions,^for^th^^first^ tim^,^strain^^IA^7^ achi^v^d^n^arly^s^l^ctiv^^production^of^L^pip^colic^acid^(96%)^(Fig.^3).^ In^ batch^mod^^ at^ 37^ °^,^ ^IA^7 ^xhibit^d^ an^ ^xtraordinary^L^pip^colic^ acid^ yi^ld^ of^ 562 mmol mol^1 ,^mor^^than^six^fold^mor^^than^th^^basic^produc^r^^IA^1A^at^ th^^start^(^abl^^^^ 5).^ ^h^^ yi^ld^ corr^spond^d^ to^ 74%^ of^ th^^ th^or^tical^ maximum^ (B^ck^r^ ^t^ al.,^ M^tab^ Eng^ (2011),^13:^159^168),^d^monstrating^high^synth^tic^capability^of^ th^^n^^^c^ll^ factory.^^h^^L^ lysin^^yi^ld^of^th^^par^nt^L^lysin^^produc^r^LYS^12,^us^d^to^cr^at^^^IA^7,^^as^only^0.26^mol^ mol^1 ,^ ^h^n^ d^t^rmin^d^ und^r^ similar^ conditions^ (B^ck^r^ ^t^ al.,^ 2011,^ ^^^^^ ^^^.).^ ^IA^7^ surpass^d^this^valu^^by^216%.^ ^^ 2.6.^Fin^-^un^^^^xpr^^^i^n^^f^lat^u^ing^^yn^^^^ic^pr^m^^^r^varian^^^ ^o^^liminat^^ th^^ formation^ of^L^lysin^^as^a^by^product^ in^ th^^ cr^at^d^ strains (^abl^^5),^^^^ aim^d^ to^ fin^^tun^^ th^^ activity^ of^L^lysin^^ amino^ transf^ras^ and focus^d on^ optimiz^d^ ^xpr^ssion^of^ th^^ ^a^ g^n^.^ As^an^alt^rnativ^^ to^P^uf,^^^^ s^l^ct^d^ th^^promot^r^Ps^d,^^hich^^^ m^diat^s th^^^xpr^ssion^of^ th^^s^d (sup^roxid^^dismutas^)^g^n^^in^C^^g^u^am^^um and^^as^ pr^viously^ succ^ssfully^ us^d^ for^ constitutiv^^ ov^r^xpr^ssion^ in^ th^^microb^^ (B^ck^r^ ^t^ al.,^ 2007,^ loc.^ cit.) (SEQ^^o.^ 2,^ ^IA^9). Mor^ov^r,^ ^^^ cr^at^d^ a^ synth^tic^ variant^ of^Ps^d^ith^ ^^ improv^d^ str^ngth^ (SEQ^^o.^3,^^IA^10).^^o^sho^^ th^^ importanc^^of^promot^r^ s^l^ction,^^^^ additionally^ impl^m^nt^d^ th^^ alt^rnativ^^ promot^r^PdapB(SEQ^ ^o.^ 44),^ nativ^ly^ controlling^ ^xpr^ssion^ of^ th^^ dapB g^n^^ ^ncoding^ 4^hydroxy^t^trahydrodipicolinat^^ r^ductas^^ in^ C^^ g^u^am^^um ^ith^r^alon^^(^IA^11)^or^in^combination^^ith^P^uf (^IA^12). ^IA^9^form^d^th^^sam^^ ^ amount^of^L^pip^colic^acid^ as^^IA^7^but^significantly^ l^ss^ L^lysin^^ (Tabl^^7).^^IA^10^ form^d^ significantly^mor^^L^pip^colic^acid^than^^IA^7 and^almost^no^L^lysin^.^^h^^strain^achi^v^d^at^ high^ yi^ld^ of^ 525^ mmol^ mol^1 at^ 34^ °^. ^IA^11^ and^ ^IA^12^ s^cr^t^d^ significantly^ l^ss^ L^ pip^colic^ acid^ than^ th^^pr^vious^ r^port^d^ strains,^^h^r^by^ th^^d^coupl^d^^xpr^ssion^ of^ ^a^ and^pr^C slightly^incr^as^d^th^^tit^r^to^3.7^mM^in^^IA^12 (Figur^^8). ^^ Obviously,^ th^^ fin^^tun^d^ ^a^^ ^xpr^ssion^ und^r^ control^ of^ Ps^d^and Ps^d,^ m^dstimulat^d^ th^^ conv^rsion^ of^L^lysin^^ into^L^pip^colic^ acid^ insid^^ th^^ c^ll^ to^ obtain^L^pip^colic^ acid^ at^ incr^as^d^ s^l^ctivity.^ Using^ ^IA^10^ ^nabl^d^ to^ produc^^L^pip^colic^ acid^ ^v^n^ as^ ^xclusiv^^ product.^ ^h^^ improv^m^nt^ off^r^d^attractiv^^^conomics:^ production^at^L^pip^colic^ acid^ high^^^ yi^ld^and^simplifi^d,^lo^^cost^do^nstr^am^proc^ssing.^ Tabl^^ 7:^L-pip^c^lic^ pr^^uc^i^n^ in^ C. glu^amicum^ PIA-7,^ PIA-9,^ an^^ PIA-10.^ ^IA^7^ ^pisomally^^xpr^ss^d^th^^ ^a^^pr^C op^ron^und^r^control^of^ th^^^uf-promot^r^s^qu^nc^^(SEQ^ ID^^O.^1),^^IA^9^^pisomally^^xpr^ss^d^th^^ ^a^^pr^C op^ron^und^r^control^of^ th^^nativ^^sod^^^ promot^r^s^qu^nc^^ (SEQ^ID^^O.^2),^and^^IA^10:^^pisomally^^xpr^ss^d^th^^ ^a^^pr^C op^ron^ und^r^control^of^ th^^modifi^d^sod^promot^r^s^qu^nc^^ (SEQ^ID^^O.^3).^^^h^^data^r^pr^s^nt^ batch^cultivations in^a^miniaturiz^d^microtit^r^plat^^syst^m (conditions:^1,300^rpm,^34^°^,^85^ %^humidity,^minimal^glucos^^m^dium) and^sho^^th^^final^tit^rs^aft^r^48^h.^n=3. S^rain L-pip^c^lic^aci^^[m^] L-ly^in^^[m^] ^IA^7 20.8^±^0.0 4.1^±^0.0 ^IA^9 21.1^±^0.1 2.3^±^0.1 ^IA^10 29.0^±^0.0 0.1^±^0.0 ^IA^11 1.9^±^0.0 17.1^±^0.1 ^IA^12 3.7^±^0.3 16.6^±^0.3 ^^ 2.7.^F^^-ba^c^^pr^c^^^ ^o^ ass^ss^ p^rformanc^^ und^r^ industrially^ r^l^vant^ conditions,^ th^^ ^IA^7^ strain^ ^as^ b^nchmark^d^in^a^f^d^batch^proc^ss^on^a^glucos^^m^dium^at^34^°^^(Fig.^7).^L^pip^colic^acid production^ ^as^ significant^ from^ th^^ start.^ During^ th^^ batch^ phas^,^ ^IA^7^ accumulat^d^ ^7 L^pip^colic^acid at^a^yi^ld^of^0.24 mol^mol^1 (0.17 g g^1)^and^r^ach^d^a^conc^ntration^of^12.9^g^ L^1.^ H^r^by,^ th^^ strain^ gr^^^ ^xpon^ntially^ to^ a^ c^ll^ conc^ntration^ of^ 29.5^ g^ L^1 ,^ ^hil^^ it^ ^ffici^ntly^utiliz^d th^^high^start^l^v^l^of^th^^sugar^(90^g^L^1)^(Rohl^s^^t^al.,^M^tab^Eng^(2022),^ 73:^ 168^181;^ Rohl^s^ ^t^ al.,^ Gr^^n^ ^h^m^ (2018),^ 20:^ 4662^4674).^ Aft^r^ 16^ h,^ th^^ initially^ ^ suppli^d^glucos^^^as^d^pl^t^d,^and^th^^f^^d^phas^^^as^start^d.^^rigg^r^d^by^a^sudd^n^ris^^ of^th^^DO^signal,^puls^s^of^th^^conc^ntrat^d^f^^d^^^r^^automatically^add^d^^h^n^th^^sugar^ ^as^^xhaust^d. ^his^DO^bas^d^ control^^ork^d^highly^ robustly and^ allo^^d^ to^op^rat^^ th^^ proc^ss^^ithout^^xt^rnal^monitoring^(Rohl^s^^t^al.,^2022,^ ^^^^^^^^.).^Aft^r^28^h,^ th^^maximum^ biomass^ conc^ntration^ (32.9^ g^ L^1)^ ^as^ r^ach^d.^ ^h^^ conc^ntration^ of^ L^pip^colic^ acid^^ continuously^incr^as^d^to^a^final^tit^r^of^82^g^L^1 (636^mM)^aft^r^110^hours,^th^^high^st^valu^^ r^port^d^so^far^for^de-n^v^ production^of^this^int^r^sting^ch^mical^(Xu^^t^al.,^A^S^Synth^Biol^ (2022),^11:^760^769) and^^v^n^approaching^th^^^ffici^ncy^of^bio^catalytic^proc^ss^s^from^L^ lysin^^as^a^substrat^^(Han^^t^al.,^Enzym^^Microb^^^chnol^(2020),^140:^109643).^ ^^ ^8

Claims

Claim^ 1. A^ m^thod for^ th^^ production^ of^ L^pip^colic^ acid^ by^ a^ bact^rial^ host^ c^ll in^ cultur^,^ comprising^conv^rting^L^lysin^^by^th^^us^^of^lysin^^6^aminotransf^ras^ (LA^)^to^d^lta^ 1^pip^rid^in^^6^carboxylic^acid;^and^ r^ducing^ d^lta^1^pip^rid^in^^6^carboxylic^ acid^ by^ th^^ us^^ of^ pyrrolin^^5^carboxylat^^ r^ductas^^(^5^R)^to^L^pip^colic^acid, ^h^r^in^said^c^ll^cultur^^is^carri^d^out^by^a^t^mp^ratur^^of^mor^^than^32°^.

2. ^h^^m^thod of^claim^1,^^h^r^in^pyridoxal phosphat^^is^add^d^to^cultur^.

3. ^h^^ m^thod of^ any^ on^^ of^ th^^ pr^c^ding^ claims,^ ^h^r^in^ said^ cultur^^ is^ f^d^batch^ cultur^.

4. ^h^^m^thod^of^ any^ on^^of^ th^^pr^c^ding^ claims,^^h^r^in^ said^bact^rial^ host^ c^ll^ is^ charact^riz^d^ by^ having^ pyrrolin^^5^carboxylat^^ r^ductas^^ activity^ and^ b^ing^ g^n^tically^^ngin^^r^d^to^^xpr^ss^lysin^^6^aminotransf^ras^.

5. ^h^^m^thod^of^ any^ on^^of^ th^^pr^c^ding^ claims,^^h^r^in^ said^bact^rial^ host^ c^ll^ is^ charact^riz^d^ by^ having^ lysin^^ 6^aminotransf^ras^^ activity^ and^ b^ing^ g^n^tically^ ^ngin^^r^d^to^^xpr^ss^pyrrolin^^5^carboxylat^^r^ductas^.

6. ^h^^m^thod^of^any^on^^of^ th^^pr^c^ding^claims,^^h^r^in^said^host^c^ll^ is^g^n^tically^ ^ngin^^r^d^ to^ ^xpr^ss^ lysin^^ 6^aminotransf^ras^ and^ pyrrolin^^5^carboxylat^^ r^ductas^.

7. ^h^^ m^thod^ of^ claim^ 6,^ ^h^r^in^ said^ lysin^^ 6^aminotransf^ras^ and^ pyrrolin^^5^ carboxylat^^r^ductas^ ar^^^xpr^ss^d^in on^^op^ron.

8. ^h^^ m^thod^ of^ any^ on^^ of^ claims^ 4^ to^ 7,^ ^h^r^in^ ^xpr^ssion^ of^ said^ lysin^^ 6^ aminotransf^ras^^ and / or^ said^ pyrrolin^^5^carboxylat^^ r^ductas^^ is^ und^r^ th^^ control^ of^ th^^tuf^promot^r,^pr^f^rably^ th^^tuf^promot^r^sho^n^in^SEQ^ID^^O:^1 or^und^r^th^^ control^of^th^^sod^promot^r,^pr^f^rably^th^^sod^promot^r sho^n^SEQ^ID^^os:^2^or^3.

9. ^h^^ m^thod^ of^ any^ on^^ of^ th^^ pr^c^ding^ claims,^ ^h^r^in^ said lysin^^ 6^ aminotransf^ras^^is^d^riv^d^from^F^av^ba^^er^um^^u^es^ens^ ^910. ^h^^m^thod^of^any^on^^of^th^^pr^c^ding^claims,^^h^r^in^said pyrrolin^^5^carboxylat^^ r^ductas^^is^d^riv^d^from Es^her^^h^a^^^^^ or^a^coryn^form^bact^rium.

11. ^h^^ m^thod^ of^ any^ on^^ of^ th^^ pr^c^ding^ claims,^ ^h^r^in^ said^ m^thod^ furth^r^ compris^s^conv^rting^L^pip^colic^acid to^L^hydroxy^pip^colic^acid.

12. ^h^^m^thod^of^any^on^^of^ th^^pr^c^ding^claims,^^h^r^in^said^host^c^ll^ is^g^n^tically^ ^ngin^^r^d^to^^xpr^ss^a^Flavin^monooxyg^nas^.

13. ^h^^m^thod^of^ any^ on^^of^ th^^pr^c^ding^ claims,^^h^r^in^ said^bact^rial^ host^ c^ll^ is^ charact^riz^d^by^lysin^^ov^rproduction.

14. ^h^^m^thod^of^any^on^^of^ th^^pr^c^ding^claims,^^h^r^in^said^bact^rial^host^c^ll^ is^a^ ^oryn^bact^rium^sp^ci^s,^pr^f^rably^C^ryneba^^er^um^g^u^am^^um.

15. A^bact^rial^host^c^ll^b^ing^g^n^tically^^ngin^^r^d^to^^xpr^ss^ in^on^^op^ron^ lysin^^6^ aminotransf^ras^ and^ pyrrolin^^5^carboxylat^^ r^ductas^^ is^ und^r^ th^^ control^ of^ th^^ tuf^promot^r,^pr^f^rably^th^^tuf^promot^r^sho^n^in^SEQ^ID^^O:^1^or^und^r^th^^control^ of^ th^^ sod^promot^r,^ pr^f^rably^ th^^ sod^promot^r sho^n^ SEQ^ ID^^os:^ 2^or^ 3, and^ b^ing furth^r^charact^riz^d^by^ov^rproduction^of^lysin^. ^^

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