CROSS^EIEERE CE TO RELATED APPLICATIONS

This application cites the riority of US 62/050,564, filed eft 19 December 2019 (pending), which is incorporated herein by reference in its entirety,

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support trader;: Grant No. 2016-31100 06001 awarded by the Knifed States Depart ent of A ricul ures Award DE-EE00O8483 awarded by the Unite States Department of Energy- Competitive Grant m, 2918-67021 27715 by the Unite States Department of Agriculture; and Match project ALA914-10T702S by the United States Department of Agriculture, The government has certain rights in the Invention,

In this context 'pvernnienB refers to th government of the United States of America,

BACKGROUND

FI ELD OF THE D ISCLOSU RE

The present disclosure relates generally to industrial biotechnology.

BACKS ROUND

Although tremendous efforts have been invested for Mofnef and biochemical research, It is still challenging to generate robust microbial strains that can produce target products at desirable levels. Fatty acid esters, or mono-alkyl esters, can be used as valuable fuels such as diesel components or specialty chemicals for food flavoring, cosmetic and pharmaceutical industries. The US mar ket deman for fatty acid esters could reach $4,99 billion by 2025.

Conventionally, esters ar produced through Fischer esterification which Involves high temperature and inorganic catalysts. The reaction consumes a large amount of energy and generates: tremendous wastes, and thus is not environmentally friendly. On the other hand, ester production through biological routes is renewable and environmentally benign. Using current techniques, bioproduction of most of the esters Is low, and is not economicall competitive with environmentally damaging abiotic approaches.

There is therefore a need in the art for ester bioproduction approaches with: higher production levels.

SUMMARY

I. MBS of microorganis ha e b e e el ed having vastly Improved pr duction of organic esters, by genetically modifying the microorganisms to introduce or enhance the activity of one or both of an alcohol acyl transferase and a lipase. This has resulted in some cases in 1-3 orders of magnit ude increase in ester production, with specific examples being m butyl acetate and n- utyl butyrate,

A first general embodiment is a modified microorganism capable of n-bntyJ acetate (BA) production, the microorganism capable of expressing; an alcohol acyl transferase (AAT); and comprising an n~butanol synthesis pathway,

A second general embodiment is a modified microorganism capable of mhulyl butyrate (BS ^' J production, the microorganis capable of expressing an AAT; an comp ising^ butyryl coenxyme A synthesis pathway and a butanol synthesis path ay.

A third general embodiment Is a genetically modified CkstrMium mcchamperhutykicetomcum having improved BA production and designated YM028F, having MC A designation number 20201.2114 deposited on lb December 2020 at the National Center for Marine Algae and Mierobiota at Bigelow Laboraloty for Ocean Sciences, 60 Bigelo Drive, bast Boothbay, Mi 04S4411SA,

A fourth general embodiment is a genetically modified Ciosiridium sotxhoroperhuiy cetaoiam· having improved BS production and designated YM016P8, having ftCMA designation number 202012115, deposited on l.b December 2020 at the National Center for Marine Algae and Microbiota at Bigelow Laboratory for Ocean Sciences, 60 Bigelow Drive, East Boothbay, ME 04544 USA.

A fifth general embodiment is a genetically modified Clostridium sacckaroper t^ to fe m having Improve BA production and designated B|430I, having NCMA designation number 202012114, deposited on 16 December 2020 at the National Center for Marine Algae and Mierobiota at Bigelow Laboratory lor Ocean Sciences, 60 Bigelow Drive, Bast Boothbay, MB 04544 USA,

A sixth genera! embodiment: is a method of ester production, comprising culturing a microorganism of the first through fifth general embodiments under conditions suitable to produce an ester.

A seventh general embodiment is an ester-containing composition comprising m ester compound tha is the product: of the method of the sixth embodiment.

The foregoing presents a simplified summery in order to provide a basic understanding of some aspects of the claimed subject matter. This summary is not a extensive overv ew. It s not ntended to Identify key or critical elements or to delineate the scope of the claimed subject matter. Its sole purpose i to present some concepts In a simplified form as a prelude to the more detailed description that is presented later.

BRIEF DESCRIPTION OF THE DRAWINGS FID, 1 Engineering of solventegefoe Clostridia for fatty acid ester production. Top; Fives rains out of four representative clostridial species were selected and evaluated as the host to be engineered for ester production in this study. We hypothesised that the different metabolic fluxes within different strains would make a big difference lor the desi rable ester production. The metabolic pathways of the four different species were represented in four different colors. Bottom: fatly add ester could be synthesized through two primary biological pathways; ms is through toe esterification of fatty acid an alcohol catalyzed by lipases, and the other Is through the condensation of acyhCoA and alcohol catalyzed by alcohol acyl transferases (AATs). Key genes in the pathway: pta, phosphofoansacetyiase; oc c acetate kinase; i¾ thiolase; hhd _> beta-hydroxyhutyry!-CoA dehydrogenase; erf, crotonase; be , butyryf-CoA dehydrogenase; p k, alcohol dehydrogenase; «dM, Aldehyde- alcohol dehydrogenase;: ode, acetoacetate decarboxylase; cfAB CoA transferase; ptk _? phosphotramshutyrylase; buk, butyrate kinase; oM _t aldehyde dehydrogenase.

FIG. 2 Screening of strains and enzy mes for ester synthesis (a) Schematic representation of devices and procedures for ester feroiehiatlon, (b) Ifeatmap results showed the ester production in various C stritUim strains with the overexpresslou of different enzymes. Empty plasmid: overexpression of blTL82lSi (for C, sa charaperbutyia etonicum N1-4-C, C pmteuriamm SD-l, C tyrobu rteum tl;;adh£t and cedxodbEsf] or pT|i (for C, Im ermckii 80S 2} as the con tool; EA: ethyl acetate; BE ethyl butyrate; BA; butyl acetate; 08; butyl butyrate, Scale bar on the right is In g/L FID. 3 Enhancing BA production through increasing the availability of acetyl -CoA (a & h) and dynamically expressing the άίb gene (c Bt d) (a) Introductio of Isopropanof synthesis pathwa (shaded in purple) and deletion of thiolase genes. The pathways in purple arrows represent the 'regeneration' of actyi -CoA. There are five annotated genes encoding thiolase in C saechem srbutytm ionimm _t only two (in red] of which could be delete (see Supplementar materials), (b) The fermentation results for BA production with various mutant strains corresponding to the genetic manipulations In (a), The reporte value is mean t: SD, fc) Four promoters associated with the biosynthesis of acetyi-CM or alcohols in the pathway were selected to drive the expression of nt l. (d) The fermentation results for BA production w th various mutant s rains in which ifferen promotors were m@d to dri e the expression of tfl as illustrated In (cl The reported value is mean ± SO. BA: butyl acetate, Key genes in the pathway; adh, alcohol dehydrogenase- boh, butanol dehydrogenase; ctfoiA CoA transferase; $& h _f secondary alcohol dehydrogenase; hy , 5 putative electron transfer protein; pyruvate form te lyase; n¾ aldehyde dehydrogenase

Big. 4. Enhancing BA production through rational organisation of the enzymes associated with BA synthesis, (a) Biological components evaluated In this study for rational organisation of enzymes; (I) CC-DI- A and CG-DfeB tags, (if) FduA* scaffold, M ni) C-iag.

III (h| Schematic representation of assembling two of the three enzymes for A synthesis with the CC-!M-A and CC-Ds-B tugs (c) Schematic representation of organizing the three enzymes for BA synthesis onto the PduA* formed scaffold, (d) Schematic representation of using the Mini) C tag: to draw ATF1 onto the ceil membrane. (e| The fermentation results for BA production with various mutant Strains corresponding to the genetic manipulations from 15 (¾Hd) ¾ ^e sporte value is mean ± SB, BA; butyl acetate, fig. S The deletion of the prophage genomes, (a) The gene cluster orpnizutlon of the :BM T prophage [PI) and another four putative prophages (P2-P5), {¾) Comparison of the cell growth of prophage deleted mutants and the control Nf-4-C strain (c) Comparison of the butanol production In prophage deleted mutants and the control N ifo-C strain, (dj 20 Comparison of the cell growth between AP1234 and API2345. (e) Comparison of butanol prodnefion between AP1234 and &P12345; (I) Transmission election microsco image of clostocin 0; (g) Transmission election microscop Image of the fife T prophage particles; (h, i| Ceil growth profiles of AP1234 and API2345 with the induction (at various ODssyl using mitomycin C at 2 or 4 pg/ i indicates that there was no ceil lysis; ^{* J} indicates 25 that most of the cells were lysed. The value at the right side of the cell growth profile figure represents the actual ODux value at which mitomycin C (with the applied concentration included in the parentheses) was added for the induction

FIG. 6 Fermentation results of the engineered strains for fatty acid ester production (a) BA production In serum hottles using glucose (F|-1291 and Ff-lBOi] or biomass hydrolysates 30 (FfolSdl (B)} as the substrate, (b) BA fermentation kinetics in FI420I In SOO-mL bioreactor using glucose as the substrate, (c) B fermentation kinetics in FJ-1201 in $00- L bioreactor using biomass hydrolysates as the substrate. {<!} BB production In seru bottles using glucose (F)4202 _S Bj42B3, and F)4204j or biomass hydrolysates (FJ-1202 (B)) as the substrate, (ej BB fermentation kinetics in B|Ί202 la SOO-miL blereactor using glucose as the substrate, (f BB fermentation kinetics In F| :i 202 In 500 - L btoreactor using biomass hydrolysates as the substrate. BA; butyl acetate; BB; butyl butyrate- Bth: ethanol; But: butanol

BIB, 7. Techno-economic analysis of buty l acetate production from com stover, a} process overview; b) total installed equipment cost; c} chemical production from each metric tonne (MT) of corn stover; d) butyl acetate production cost,

FIB. B Fermentation results in serum bottles with Cfastrkikm ccharoperbu l& toft um N1-4-C and; the mutant strains, a-h); ABB fermentation results for PJ-fCJO as compared to the control N1-4-C, The reporte value is menu SB,

Nf-A-C and the mutant strains with prophage deleted, (a-hj: ABB fermentation results for API, AP2, AP3, DR4, AP1234 as compared to the control Nl~4-C, (i-p): ABB fermentation results for ANPi, ANP2, L R3, AMP4, PI234 as compared to the: control 1- -C. The reposted value is mean & SB.

FIB, 10. Cell lysis in various prophage deletion mutants upon induction with different concentrations of mitomycin C, T indicates growth Inhibition; " * indicates that there was no cel! lysis fo” indicates that most of the cells were lysed; fo-k indicates that all th cells were lysed,

FIB. :i 1 TEM picture of eiostodn 0. The magnllkatfon of (a) was ii)0x: _s while the magnification of (b) was 200x.

FIB, 12, Ceil lysis In various prophage deletion mutant upon induction with different concentrations of mito ycin C. Indicates that there was no cell lysis; ^* 4” indicates that most of the ceils were lysed,

FIB, 13, HM T phage observed in the DR2345 strain upon induction. The magnification of (a, b} was IDOx, while the magnification of {c _* d) was 200x.

FIB, 14 ABB Fermentation results m serum bottles with Clostridium s cchawperbutyhce nicum DR1234 and API 2345, The reported value is mean ±. SB.

FIB, 15, ABE fermentation results in 500-mh b!oreactors with Clostridium cch&ropurbntyleeetonicum AF1234 and AFI2H4S, The reported value is mean ± SB.

FIB, 1&, Sensitivity of butyl acetate production cost to different parameters. The numbers In brackets in Y-axls are the potential low;, base and high values of each parameter. DETAILED DESCRIPTIO

Unless otherwise defined, ail terras (Including technical and scientific terras) used herein have the same meaning as commonly understood by one of ordinary skill in the art of this disclosure. It will he further understood that terms, such as those defined in commonly used dictionaries, should he interpreted as having a meaning that is consistent with their meaning in the context of the specification and should not be interpreted in an idealised or overly formal sense unless expressly so defined herein. Well known functions or constructions may not foe described in detail for brevity or clarity.

With reference to the use of the wordfs) "comprise ^® or "comprises" or "comprising" in the foregoing description and/or in the following claims, unless the contest: requires otherwise, those words are used on the basis and clear understanding that they era to be interpreted inclusively, rather titan exclusively, and that each of those words is to he so Interpreted in construing the foregoing description and/or the following claims.

The term "consisting essentially of means that, In addition to the recited elements, what Is claimed may also contain other elements (steps, structures, Ingredients, components, etc,) that do not adversely affect the operability of what is claimed for its intended purpose. Such addition of other elements that do not adversely affect the operability of what is claimed for ts intended purpose would not constitute a material change In the basic a nd novel characteristics of what Is claimed _*

Terms such as "at least one of A and 8” should be understood to mean "only A, only 8, or both A and 8," The same construction shoul he applied to longer list (e.g.. "at least one of A, B, and ),

The terms "about" and "approximately" shall generally mean an acceptable degree of error or variation for the quantity measured given the nature or precision of the measurements. Typical, exemplary degrees of error or variation are within 20 percent {%}, preferably within 10%, and mare preferably within 5% of a given value or range of values. For biologies! systems, the ter _: "about ^* refers to an acceptable standard deviation of error, preferably not more than 2 -fold of a given value. Numerical quantities given herein are approximate unless stated otherwise, meaning that the term "about" or "approximately" ca he Inferred when not expressly stated.

The terminology used herein is for the purpose of describing particular embodiments only and is not Intended to be limiting. As used herein, the singular form "a". “an" and "the" are intended to foehuie t e plural forms as well:, unless the contest clearly indicates otherwise.

The terms "first" "second" and the like are used herein to describe various features or ele ents, but these features or elements should hot be limited by these terms. These term are only used to distinguish one feature or element from another feature or element. Thus, a first feature or element discussed below could be terme a second feature or element, and similarly _* a second feature or element: discussed below could be termed a first feature or element without de arting f o the teachings of the present disclosure.

The term "nucleotide" as used herein refer to any such known groups, natural or synthetic, ft Include conventional DMA or RMA bases (A, G _* C, T, 0), base analogs (eg,, !nosine, S-niteohufasm!e and others), imidaxofe-d-earboxamlde, pyrimidine or purine derivatives (e.g. _s modified pyrimidin base bllBH-Srt-dlhydrop rs ldoffeS-eJii^Joxaxin ?~o (sometimes designated "IP" base that binds A or C»}) and; modifie purine base N6- methoxy-Hfo-diam opuriae (sometimes designated ^* K" base that binds C or T) _* hypoxanthine, -d-methyi deoxyguanosine, 4-ethyl d'-deoxycytidine, 46- dlihtorobenxjmidaaole and 2,4 dli1uoroben¾eoe nucleoside analogues, pyrene-· iunct!onaliaed LNA nucleoside analogues, deaza- or asa-modified purines and pyrimidines pyrimidines with substituents at the S or 6 position and purines with substituents at foe 2, 6 or 8 positions, 2-ammoaden!ne (uA), 2-tfiionradf (sU), H-amlno-b-methyla iuo urine, 0- 6-methyignanlne, d-tliio-pyrlmldiues _* d-amlno-pyrimldines, 4-d Imethyih raame- pyrimidines, O-d-aikybpyrimldmes an hydrophobic nucfeobases that form du lex DMA without hydrogen banding. Nucleobuses can be Joined together b a variety of linkages or conformations, including phosphodfester, phosphorothioafe or metbylpbosphonate linkages, peptide-nucleic add linkages,

The term "poiy nucleotide" as use d herein refers to a multi merle comp ound comprising nucleotides linked together to form a polymer, including conventional RNA, DMA, LNA, BMA, copolymers of any of the foregoing, and analogs thereof

The term "nucleic acid” as used herein refers to a single stranded paly nucleotide or a duplex of two polynucleotides. Such duplexes need not be annealed at all locations, and may contain gaps o overhangs,

The term "genetically modified" means that genetic material has been altered by human intervention, in the context of a self-replicating entity such os a ceil ora vims, such alteration may have been performed on the seif-repiieating entity In question, or on anancestor of the sell-replicating entity from who it acquired the alteration.

PENETICALLY MODIFIED MICROORGANIS

Strains: of microo ganism have been developed having vastly improved production of organic esters, by genetically modifying the microorganisms to introduce Or enhance the activity of one or both of an alcohol acyl transferase and a lipase. This has resulted in someembodiments of the microorganism in 1-3 orders of magnitude increase in ester production, with specific examples being butyl acetate and butyl butyrate. AATs catalyze the addition of an acyl moiety fro m m acyl-coonzyme A facybCoA) donor onto m alcohol accepter. The efficiencies of various AAT vary widely depending on the alcohol end the aeyi-CoA involved in the reaction. The work described herein discovered that some heterologous AATs in microorganisms: catalyse the formation of butyl Osiers at extremely high efficiency, an that even higher efficiencies can be realize by further genetic modifications.

A first general embodiment of the microorganisms is capable of highly efficient BA production. This microorganis is capable of expressing the AAT, and has a functioning butanol synthesis: pathway. Without wishing to be bound to « hypothetical model, it: is believed that the AAT catalyzes transacety!atkni from aceiy!-CoA to butanol to form the ester. The nucroor nlsin may also bo cha acterised as having an aceiyl-CoA synthesis pathway.

Although in theory any AAT could function to produce BA in this context, in preferred embodiments the AAT is one or more oh Vast Seat, Atfl, Ehti, anti a functional homolog of any of the foregoing, The strawberry AAT Vaaf (also found is banana] wa$ elucidated by Beekwlider et al. {Plant Physmi VOL 135, 2004), having a reported amino add sequence of SEQ 1 NO: 1 (see also ClenBank Accession No, AX025504 i) _> The strawberr AAT Seat was elucidated b Aharonl et ai, (The Plant Cell, Vet 12 _» 647-661, 2000 --· incorporated herein by reference to teach the amino aci sequence), having a reportedamino acid sequence of SEQ IP NO: 2 as reported in GeaBanfe Accession No. AAS13130/L The Saccharamyces cere i AAT Atfl was elucidated by Fuji et at (Appl Environ, Microbiol, VoL 66, 2786-2792, 1994 - incorporated herein by reference to teach the amino acid sequence), having a reported amino acid sequence of SEQ _. IP NO: 3 as reported in NCB1 Reference Sequence NT 015022.3. feast ethanol hexanoyi transferase IE Ini) wa elucidated by Saerens et aL (I Boll Cheur, Vol 281, 2086- incorporated herein by reference to teach the amino acid sequence), .having a reported amino acid sequence of SEC) 10 MOt 4 as reported in OmProt Accession Mo. P403S3,

Single or multiple copies of the AAT genes may be included. Various embodiments of the microorganism compris a nucleic acid encoding one or more AATs, which may include any of the ATI's discusse above or their functional variants. A preferred embodiment of the microorganism comprises multiple nucleic acid sequences each encoding Atil or a functional homolog of Atfl, Another preferred embodiment of the microorganism comprises multiple genomic nudaic acid sequences each encoding Atil or a functional homolog of Atil .

A second general embodiment Is a microorganism capable of butyl butyrate (BB) production, the microorganism capable of expressing an AAT and comprising a butanol synthesis pathway and a butyryi eoenxyme A synthesis pathway; It has been found that AATs can catalyze the condensation of hutyryi eoen¾yme A and butanol to produce SB at high efficiency, Btrtyryheoeozyme A is a four- carbon fatty acid that 1$ the coeoxyme A- activated fer of butyric acid, The pathway may bo endogenous or exogenous to the microorganism. One pathway was elucidated by Bennett and Rudolph (F£M5 Mkrobioi Rev, 17, 241-249, 1995} as including pffiydroxylmtyryhCoA dehydrogenase, crofona e, butyryl €oA dehydrogenase and the a and p subunits of an electron transfer !iavoprotein. In a preferred embodiment: of the mlerourganlsm the AAT Is Ehtl or a functional ho ofog of Ehtl, In a further preferre embodiment of the microorganism the AAT is Seat or a functional homolog of Seat

The microorganism may fee constructed to contain a nucleic add that encodes the AAT, Such nucleic acid may he genomic (part of the organisms genome, such a a chromosome of a bacterium) or extra-genomic (such as part of an episome, fur example a bacterial plasmid). The nucleic acid encoding the AAT may he a "canonical” sequence encoding the AAT, or it ma he a degenerate variant of a canonical sequence. Degenerate variants can he constructed based on an understanding of the standard thre nucleotide code and Its corresponding amino acids. This correspondence is understood both for RNA and for DMA. Some organisms ^* protein synthesis machinery employs atypical codons for certain amino acids, and this can he taken Into account when designing nucleic acids that encode the AAT or its functional homologs.

Various embodiments of the microorganism may he eukaryotic or prokaryotic, such as bacteria, archaea, fungi, and proifsts, A preferred embodiment of the microorganism is a prokaryote. Prokaryotes haw the advantage of rapid growth, easy cultivation, and simple genetics. The prokaryote may be a bacterium or an arehaean, Bacteria have the advantage of being well understood model organisms with a wide diversity of metabolic niches. Archaea have the a vantage of some specialized metabolic pathways and the ability in many cases to withstand extreme Industrial conditions. An embodiment of the microorganism is a fermentative bacterium. A specific preferred embodiment of the microorganism is a bacterium of genus €o$tri ium _f a well characterized group of fermentative anaerobic bacteria, capable of fermenta tion of a wide variet of substrates. In such embodiments the bacterium may be one of Ci&stoidium cchm~eperbirlyiacekmkam _f Clostridium beijefincM, Q trMkim pasimmmiim and Clostridium iy biiiyrkum In a particularly preferred embodiment, the bacterium: is Clostridium saeckdmperbutylQmtomeum,

Lipase B (CALB) from Can ida antarctim {Pmtdosymn Antarctica} catalyzes the formation of esters from fatty acids (including CoA fatt acids) and alcohols, and is commercially available for biochemical applications, Lipase B Increases ester production inso e embodiments of the microorganism. It has a reported amino acid sequence of SE ID MG: S a Uolprot Accession Mo. P4I3&5, Some embodiments of the microorganis ate capable of expressing Lipase B or a functional homolog of Lipase B. Such embodiments of the mioroorgunis may he capable of such expression owing to the presenc of a nucleic acid encoding Lipase B or a functional boroofog of Lipase B, The nucleic acid may take various forms, including RNA (fo example, as introduced mBNAJ or DMA (for example, genomic DMA, episome DMA, or plasmid DMA), Some embodiments of the microorganism capable of expressing Lipase B have one or both of an acetic acid synthesis pathway and a butyric acid synthesis pathway. Butyric add ca form butyl esters wife alcohols, and acetic acid can form acetyl esters with alcohols. Examples of suitable fermentativel produced alcohols Include ethano and butanol.

Enhancement of ester production can be achieved by reducing or eliminating the activity of the NADf-i-quinone oxidoreductase subunit G, which Is a subunit of the electron transport chain complex L The moG gene encode the MADLbqu!none exldoreduetase subunit G, Without wishing te be bound by any hypothetical model, it is believed that the availability of MAPfi is increase by reducing nr eliminating th activity of the NADH- qnlnone oxldoroductase subunit G, leading to Improved aster production. The nuoG in G saechwvperb&tytdcetonlmm is at locus tag Cspa _w e4?S60 Some embodiments of the ill microorganism have partial or complete deletions of toQ The partial deletion may he sufficient to either completely inhi it expression, reduce the activity of the resulting polypeptide _* or eliminate the activit of the resulting polypeptide _* In a preferred embodiment the mttiG gene Is deleted. Ester production can also be increased by the expression of secondary alcohol dehydrogenase alone or in combination with the expression of the putative electron transfer protein hydG. One such suitable secondar alcohol dehydrogenase, that cun convert acetone into isopropanol, is fee one encoded by the mdff gene in C beijsrinckii 8593 {Uniprof no AOALSSii&KS). The hyd& gene in the same operrm as $adh encodes a putative electron transfe protein. Some embodiments of fee microorganism are capabl of expressing one or both of hydG and sad (or fenctinnal homologs thereof). Further embodiments of fee microorganism comprise a sa h-hydG gone cluster _* One or more of the foregoing may be heterologous genes. One example of a suitable m tfdtydC gene cluster is of clostridial origin, such as from & bmjerinekit 8593, The AAT may be expressed using various promoters and other regulatory elements.

In this context the AAT is "operatively linked" to a promoter if the promoter controls the expression of the AAT, The promoter may be adfaeent to the AAT gene, or it may he remote. Some embodiments of the mkfoorganis comprise a constitutive promoter operatively United to the AAT. l¾w Is a promoter in Gmtmimm Sdcrhar&psrhutyhcetonkum^ Which controls expression of add, NADPH- epeudem butanol dehydrogenase at locus lag C pa _j dUBhil 1¼ is a promoter in Clostridium saceharoperfeufefeeefomcaM, which can sense the acidic state an switch cel metabolism from acidogenesis to solvenfugenes s. Operatively Unking fte* or to the AAT can Increase ester production In some embodi ents of the microorganism,. Some embodiments of the promoter are native to the microorganism

Additional advantages cab he realise by locaiixing the AAT at the cell membrane, Without wishing to fee bound fey any hypothetical model, it is believed feat the cell can be protected from toxic properties of the ester If the AAT is localized at the membrane. One such approach is to fuse fee AAT with a membrane-associated molecule, such as Mte O, Mini) is a me brane-associated protein and the localisation of Mini) Is mediated by an 8- 12 residue e-terminal niembrane-tergeiing sequence. It is an ubiquitous ATPase feat plays a crucial rote In selection of fee division site in eubaeterla and ch!orop!asts. The proteins wife MluD € terminal sequence are believed to he drawn to the ceil membrane. Thus, fee

SI a lica ion of MioP C-tag can led! irate the secretion of target product and enhance its production by mitigatin the intracellular toxicity as wall us promoting the catalysing process (FIG 4a). In so e embodiments of the microorganism the ΆAT is fuse to a C- terminal rtwmLrane-iargeting sequence of M The T may be fused at any positio on the MT; in a preferred embodiment the AAT Is fused at Its Oteroiinai end to the C-te inal membrane-targeting sequence of Mini). The C-termsnal membrane-targeting sequence of MmD may be part of a larger molecule _» such as a whole MinD molecule Some such embodiments of the microorganism may contain a nucleic acid encoding the AAT concatenated to a nucleic acid encoding mD. An example of a suitable Mini.) tag is Ba lhm suhl is Mini), having a canonical DMA sequence of SE ID MO; 6.

'^Functional homologs" of a polypeptide will have some degree of identity with the wild type polypeptide _» For example, it would be expected that most hmetionai homoiogs having from 95-100% identity with the native polypeptide woul retain at least some function. The likelihood that functionality would he retaine by a ho oiog to the polypeptide with at least any of the following levels of sequence identity coaid be predicted: 70, 80 _» 90 _» PS, 99, and 99.5%. it is understood that the minimum desirable Identity can he determined in some cases by identifying a known non-funefional homoiog to the polypeptide, and establishing that the minimum desirable identit must be above the Identify between the polypeptide and the known non-functional Identity. Persons having ordinary skill in the art will also understand that the minimum desirable Identity can he determined in some cases by identifying a known functional homolog to the polypeptide, and establishing that the range of desirable identity must encompass th percent identity between the polypeptide and the known nomionetiena! level of Identity,

Deletions, additions and substitutions can he selected to generate a desired polypeptide functional homolog. For example. It is not expected that deletions, additions and substitutions In a non-fbnetio b region of a polypeptide would alter the polypeptide activity. Likewise conservative substitutions or substitutions of amino acids with similar properties Is expected to he tolerated in a conserved region. Of course non- conservative substitutions in these regions would be expected to decrease or eliminate the polypeptide activity.

For example, a "conservative amino acid substitution ^' ' may involve a substitution of a native amino acid residue with a nonnative residue such that there is iitde or no

11 effect m fee polarity or charge of fee amino acid residue at that position, Furthermore, any native residue in fee polypeptide may also be su stituted wife alanine. Conservative amino acid substitutions also encompass non -naturally occurring a mu acid residues winch are typically incorporated by chemical peptide synthesis rather than by synthesis i biological systems. These include pepfidomirneties, and other reversed or inverted forms of amino a d moieties. It will he appreciated by those of skill in the art that nucleic acid and polypeptide molecules described herein may be chemically s nthesize as well as produced by recombinant means.

Naturally occurring residues may be divided into classes base on common si e chain properties;: 1) hydrophobic: norleucme, feet Ala, Va!, Leu, Iky 2} neutral hydrophilic' Cys, Ser, Thr, Asn, Gin; 3} acidic:; Asp, Glu; 4) bask; His. _» lys, Arg; S) residues that Influence chain orientation; Gly, Fro; and 6} aromatic: Trp, Tyr, Fhe,

For example, conservative substitutions may involve the exchange of a member of one of these class es for a member o f the same class, In making such changes, the hydropathic index of amino acids ma be considered. Each amino acid has been assigned a hydropathic index on the basis of their hydrophoblcit and charge characteristics, these are: isoieuclne (*4.$); valine (+4,2); leucine (-fe d); phenylalanine (42.8); cysteio e/cystine {425}; methionine {419}; alanine (rl.B); glycine (Ό.4 }; threonine {-0,7b serine (-9,8}; tryptophan (-0.9); tyrosine (1.3); proflne (1.6); histidine (-32); glutamate (-3,5}; glutamine (-3,5); aspartate [-3,5); asparagine (-3,5); lysine (-3,0); and arginine (-4.S), The importance of the hydropathic amino acid Index in conferring interactive biological functio on a protein Is understood In the art (byte efc ai„ / _< Mol Biol , IS dfeiS!, 1982). It is known that certain amino acids may be substituted for other amino acids having a similar hydropathic Index or score and still retain a similar biological activity. In making conservative substitutions based upon the hydropathic index, the substitution of amino acids whose hydropathic indices are within */- 2 ma be used; hi an alternate embodiment, the hydropathic Indices are with */- 1; in yet another alternate embodiment, the hydropathic indices are within fe 0.5 it i also understood in the art that tbs substitution of like amino adds can be made effectively on the basis of hydrophiii ty, The greatest local average hydrophi!icity of a polypeptide m governed by the hydrophillclty of Its adjacent amino acids, correlates with a biological property of the protein. The following hydropbilicity values have been assigned to amino acid residues: arginine lysine (+3.0); aspartate

(4-:3 _> 0,Kl}; glutamate (+ 3,0, tel); serine (+0.¾ asparagine (+0,2); glutamine (+0,2): glycine (0); threonine (-04); proline {-1> S, -,ί}; alanine {-ilS}; histidine { _* 0.¾ cysteine (-1 ); methlonme (-1,3); valine (4.S); leucine (4,8); isoleocine (-U); tyrosine (-2,3); phenylalanine (-2,5); tryptophan {- S,4j _< in making changes: based upon similar hydropbihdty values, the substitution of amino acids whose hydropfubdty values are withi +/· 2: may be used; in an alternate embodiment, the hydrophilidty values are wit +/- 1; in yet another alternate embodiment the hydrophilicity values are within

+/-as Desired amino add substitutions (whether conservative or non-conservative) can he determined by those skilled in the art at the ti e such substitutions are desired For exampl , amino add substitutions can be used to identify important residues of the polypeptide, or to Increase or decrease the affioli of the polypeptide with a particular binding target in order to Increase or decrease the polypeptide activity, Exemplary amino add substitutions are set forth in Table 1, For Identifying suitable areas of the molecule that may be changed without destroying ctivit ^y , me skilled in the art may target areas not believed to fee important for activity _: . For example, when similar polypeptides with similar activities from the same species or from other species are known, one skilled m the art may compare the amine add sequence of a given polypeptide to such similar polypeptides. With such a comparison, one can Identify resi dues and portio ns of the molecules that are conserved among similar polypeptides. It will be appreciated that changes in areas of the polypeptide that are not conserved relative to such similar polypeptides would be less likely to adversely affect the biological: activity and/or structure of the polypeptide One skilled in the art would also know that even in relatively conserve regions, one may substitute chemically similar amino acids for the naturally occurring residues while retaining activity {conservative amino acid residue substitutions). Therefore, even areas that may be Important for biological activity or for structure may fee su.b|eet to conservative amine acid substitutions without destroying the biological activity or without adversely affecting the polypeptide structu e.

Further advantages can be realised by curing the microorganism of viruses in its genome. In this contest 'Tore" means to introduce a mutation that prevents the virus from replicating or otherwise expressing viral genes. An example of curing is entirely deleting the viral genome from the microorganism. Another example is partially eleting the viral genome form the microorganism, to such a exten that viral replication Is not possible. Another example is mutating a bacteriophage integrase gene, preventing functioning phage intograse from befog expressed. The mutation may be an entire deletion, a partial deletion, a substitution, or an insertion so long as unctioning bacteriophage Integrase In net expressed. Some embodiments of the microorganism are a bacterium that has been cured of one, some, or all prophages, A preferred embodiment of th bacterium is a Ci tridium spp that has been cured of all native prophages. In a further preferred embodiment, the microorganism Is a bacterium of genus CfastrMmm which has beets cured of one or more of prophages PI, P2, P3, P4, and PS (described further below in the examples).

The rex gene was Identified as a redox-sensing transcriptional repressor, which modulates transcription i response to changes in cellular NAPK/MAP-r redox state, and represses the transcription of genes related to butanol synthesis. It wa originally Identified by Wietake and Bah! (Appl Miemhiol Bmt hml, 06(33: 749 61, 2012} (C mccheroperb ieceteni ui Cspa _;„ cO4320), Some embodiments of the microorganis do not express a functional redox-senslog transcriptional repressor. This lack of expression may be na ive to the organism, or mduced through genetic modification, Such genetic modification may take any suitable form, such as a complete deletion, a partial deletion, an insertion, or a substitution, Further advantages can be realised in embodiments of the microorganism capable of expressing soluble pyridine nucleotide transhydrogenase (StM), or a functional ho oiog thereof for converting MABPM to HA One exampl of a suitable SthA is fro £ wΐίBIZI fSEQID NO: 7),

Further advantages can he realized in embodiments of the mic oorganism that do not express a functional A1~ Bί gene duster {encoding acetoaeetyb

CoA:acetafa/butyrate:CoA transferase}. Acetoacetyl-CoAmeeiats/hatyrateiCoA transferase transfers CoA group from aeetoacetyl-CoA to either acetate or butyrate, generating acetyl CoA or hutyryi-CoA respectively, The cffol-cgfBi gene cluster catalyzes the fol Sowing two reactions: acetoacetyi-CoA + acetate acetoacetate + acetyhCoA, aeetoacetybCoA * butyrate acetoacetate * bntyryi-CoA, The ctfiii (at.oDl) Gene IP in £ eclrnmperbt cstomam is Cspa _.„ o2i00B csr:Cspa :2tO00 K01034 acetate

CoA/acetoaeetafe CoA-traosfecuse alpha subunit |1C:2, 8,3,8 2.8,3,9J | (Genianh) atoDl; acetate CoA-transferase subunit alpha {14); and ctfBl : Gene ID In £ saccha perbut^hcei icum is Cspa eEl B csnCspajcEiOlO EM03S acetate GoA/acetoacetate CoA-transferase beta subunit fi€ 2,8 .8 2,83,9] j (GenSank} ctfBl; butyrate- acetoaceiate CoA-transferase subi it B (N), This lack of expression may be native to the organism, or induced through genetic modification. Such genetic modification may fake any suitable form, suc as a complete deletion, a partial deletion, an insertion, or a substitution, A specific embodiment of the microorganism is a genetically modified

Cfastfi lum Mcck&ropetbtityhcetomcum having improved BA production and designated YM028P, having bICMA designation number 2020:12116, deposited on 16 December 2028 at the National Center for Marine Alpe and Microhiota at Bigelow Laboratory for Ocean Sciences, 60 Bigelow Drive, Bast Boothha , MB 04544 USA, A specific embodiment of the microorganism Is a genetically modified Clostridium

MicchQmperbu kceiQmam having: Improved 88 production and designated YM016FB,. having bICMA designation number 202012115, deposited on 16 December 2028 at the National Center for Marina Algae an Microbiota at Bigelow Laboratory for Ocean Sciences _» 60 Bigelow Drive, East Bootbbay, ME 04544: USA,

A further specific embodiment of the microorganism is a genet cally modified Omi idium sac harope intfyiaceUuuaiin having Improved BA production and designated l' ^r ) - 1 01 , having HCMA designation number 202012114 deposited on 16 December 2020 at the National Center for arine Algae and Mforob!ota at Bigelow Laboratory for Ocean Sciences, 60 Bigelow Drive _* East Booth bay. Ml 04544 USA,

METHOD OF ESTER PRODUCTION

A method of ester production Is disclosed, comprising culturing any one of the microorganisms above under conditions suitable to produce an ester. In a preferred embodiment of the method the ester produced is one or both of BA and BB. it has been observed that organisms that have been mod Bled as described above can generate extremely large amount of BA and BB at unprecedented levels of effi ciency.

Culturing may occur in the presence of a suitable carbo source an suitable energy source, depending on the organism. Some embodiments of the culture medium contain glucose, which Is widely used by heterotrophlc organisms as both a source of carbon and energy. The glucose can be part of a defined medium or part of a complex medium. Numerous suc glucose media are known in the art, some of which are cataloged in ft Atlas, mdkook of Micrdhloiogimi Media, Fourth Edition, CRC Press (2010) In a preferred embodiment the medium comprises a biomass hydrolysate, Defined me ia have the advantage of being of controlled composition, whereas a complex medium such as biomass hydrolysate are loss expensive to produce and require no supplementation, One suitable example of hydrolysate Is a corn stover hydrolysate, such as one prepared according to the method of 0, llumMrd et al, “Process design and economics for biochemical conversion of ii nocelluiosic biomass to etbanokdiiniMcid pretreatment and enzymatic hydrolysis of corn stover” (No flREL/TP-5tOO-4?764} National Renewable Energy laixfWRBL}, Golden, CD (United States} (2011) - which is incorporated by reference to the extent necessary to describe and enable such hydrolysates.

Culturing shoul he conducted at a temperature conducive to microbial metabolism, which will vary depending on the microorganism involved. Often mesop.hiIic organisms are used in ester fermentations, and in such cases culturing occurs a esophllic temperatures,. Higher temperatures can accelerate metabolic processes, and it is contemplated that

P thermophilic organisms could fee use as well, in which case culturing would preferably be at thermophilic temperatures.

Ester production is generally a fermentative process. To encourage fermentation the culturing ma be conducted under low oxygen conditions. Conditions may be hypoxic or anoxic, as necessary to create anaerobic conditions conducive to fermentation, it is possible that the microorganis could fee engineered to ferment under aerobic conditions as well

Sued fermentation processes have been observed to generate extremely high concentrations of HA in culture, in excess of 25 g/L Some embodiments of the culture method produce at least 1,5 g: BA/L of culture, further embodiments have been observed to produce at least 5, 7.5, 10, 12.5, 13, 14, IS, 16, 17, 18, 19, 20, or 25 g BA/L of culture. Higher concentrations of BB production have been observed than previous known methods. Some embodiments of the culture method produce at least 0.1 g HB/i, of culture. Further embodiments of the culture method produce at least 0,2, 0.3, 0.4, CIS, 6,6, 0,7, 0.8, 6,9, 1.0, .1, 12, ,3, 1,4, 1,5. or l,6g; BB/i of culture,

WORKING EXAMPLE; Renewable Fatty Acid Ester Production in Clostridium

An earlier version of this work was published on bioRxiv on 20 August 2020 (available at |H;b s:feyewwfefel2iy2fl2?SAuh¾nj|/l|tlllil _. il¾Cl¾0 _; d _; e9,ill. 74dv1,feilj. and is Incorporafe herein by reference Ί» It entirety. The terms "we” and ^< ouf" used below refer to anthers of fee manuscript and persons who contributed to the work, some of whom might not b considered ^' inventors” of what is claimed under th Saws of certain countries; the use of such terms is neither a representation nor an admission that an person Is legally an inventor of what Is cla imed.

Abstract

Production of renewable chemicals through biological routes Is considered a an urgent solution for fossil energy crisis. However en product toxicity inhibits ah erohiai performance and is a key bottleneck for biochemical production. To address this challenge, her we report an example of biosynthesis of high-value and easy-recoverable derivatives to alleviate endprodoct toxicity and enhance hloprodnetfon efficiency, By leveraging the natural pathways in so!ventogenlc Clostridia for co-producing acyl-Co&s, acids and alcohols as precursors, through rational screening lor host strains and enzymes, systematic metabolic engineering — including rational organization of ester-synthesizing enzymes Inside of the cell, and elimination of putative prophages, we developed strains that can

13 produce 253 g/ butyl acetate an 1.6 g/L butyl butyrate respectively, which were both the unprecedented: levels fit microbial hosts. Techno economi analysis Indicated a production cost of $086 per metric tonne for butyl acetate production from corn stover compared: to the market price of $1, 200-1,400 per metric tonne of butyl acetate, suggesting the economic competitiveness of the bioprocess.

Main

Although tremendous efforts have been invested for Mofoei and biochemical research, it is still challenging: to generate robust microbial strains that can produce target products at desirable ieveish One key bottlenec is the intrinsic tonicity of end products to host eeilsk Therefore;, the production of high-value bioproducts which can be easily recovered from fermentation might be a solution to tackle the bottleneck in bioproduction. Fatty acid esters, or mono-alkyl esters, can be used as valuable fuels such as diesel components or specialty chemicals for food flavoring, cosmetic and pharmaceutical Industries/ St is profeeted that the US market demand for fatty acid esters could reach. $4,99 billion by 2025/ In addition, esters, with fatt acid and alcohol moieties, are generally hydrophobic end can easily separate from fermentation; thus the production of ester can help mi igate end product toxici y to host cells and ell dent bioproducuon can he achieved.

Esters are produced: through Fischer esterification hich involves high temperature and inorganic catalysts*/ The reaction consumes a targe amoun of energy and generates tremendous wastes, and thus i not environmentally friendly ⁵ . On the other hand, ester production through biological routes is becoming more an more attractive because it is renewable and environmentally benign. There are at least two known biological pathways for ester production: one is through esterification of fatty acid and alcohol catalyse by lipases ⁵ and the other is based on condensation of acyi-CoA and alcohol catalyzed by alcohol acyl transferases (MTs) ⁵ , Previously, lipases from bacteria or fungi have been e ployed for catalysing esterification ^® ,

I» this study, we report highly efficient fatty acid ester production to unprecedented levels using engineered Clostridia. W selected soiveetogenic Clostridia to take advantage of their natural pathways for co-producing aeyi-CoAs (aeetyl-€oA and butyry!-€oA], fetty acids (acetate and butyrate), and alcohols (ethanol: and butanol), either as Intermediates or end products; we hypothesized that Clostridia can be excellent microbial platforms to he engineered for ellcfent ester production by Introducing heterologous Mi's and/or lipase genes, in eed, through screening for ost strains (from four well-known clostridial species] a ! enzymes (alcohol acyl tr nsferases and lipase), systematic metabolic engineering— including organization of estowsyntheslzing enzymes inside of the cell, and elimination of putative prophages, we ultimately obtained two strains which can produce 25.3 g/L butyl acetate (BA) and 1,6 g/h B8 respectively In extractive batch fermentations. These production levels were both highest in record to the best of our knowledge.

Results

Screening of host Strains and genes for osier production

We considered efostridfe as model platforms fo ester production thanks to their intrinsic Intermediates (fatty acids, acyl-CoAs, and alcohols) serving as precursors for este biosynthesis (FIG. 1). We hypothesised that different .tux levels of these precursors within various clostridial strains would make a big difference for the specific typefs) of ester production, Therefore, we selected five strains (from four representative species) including C tyrobutyrkum c tta dhE (, € _> tpmbufwkmn Acutf vod¾E2 _f C, pastemim m SD fo C. cchttmperbutyiac&tanimm Nl- -C ^{s :i} and betj rtnckii NCIMB 80$2 ^:W to evaluate their capabilities for ester production through metabolic engineering (Table SI), We included both (2 tyrobufyflctiin &c&tP; hEl and &mtl:: dhB2 here because they produce different levels (and thus ratios} of butanol and eihanoFMh Esters can be synthesized either through esterification of acid and alcohol catalyzed by lipase, or through condensation of aeyi-CoA and alcohol catalyzed by AATs (FIG, 1), Previously, lipase B (€ALB) from Candida n retim has been employed fo efficient: ester production through esterification ³ · ¹³ , in addition, four AATs including VAAT««, S Tfo ATFT ^{1 S3} _.. EN1£F. _. 29 EHTfe ³ have been recruited for ester production In various hosts ¹ - ^3·31 herefore, here, we evaluated all these genes in our clostridial hosts for ester production,

Six plasmids (pMTL-i wraf, pMTL-F«K-sooG p TLdhsi-ntfl, pMTfe! ebfe, pMTbdha ffposeB as well as pMTL82i5i as the control) were individually transformed: into C, stcc mperbulyMcektkciim Ni-4-G, C pastmria m SD-1, tymbutyrk&m c otivorfAhi and tl adhEi respectively. While pTfl-P^rVOUf, pTJl -F ernoG pTfl-i ofel, T|:ί Rΐ« _ί'Ό ΐ?6 and T|t-ftw*4fpa$a0 as: well a pip were transformed into C beqermckb 6052. Fermentations were performed (FIG. 2a), and results were shown i FIG, 2b, Four types of esters wore detected: FA, BA, ethyl butyrate (EB) and SB. Interestingly, control strains with the empty plasmid (pMT!,S2:iS 1 or pTJ i) also produced noticeable EA, BA and BE. This could be because; 1) the endogenous lipase in Clostridia can catalyze ester production* ³ ; 2] tP on pfdTL82!5i encoding a chloramphenicol acetyl transferase (belonging to the same class of e yrnes as Mi's) has AAf activities ⁵ ^ ³ ,.

Based on the results, It could be concluded that ATPt Is more favorable for SA production. All strains with &0 produced higher levels of BA compared to the same strain but with the overexpression of other genes (FIB, 2b). While VAAT, SAAT and EflTI seemed to have better activities for SB production. Among all the strains, C, ${ieehawperbu0&ee&miciim Ff 004 produced the highest titer of 5,5 g/L 8A, This Is the highes BA production that has been reported through microbial fermentation to the best of our knowledge C ac tobuvyiicum CaSAAT | wit the overexpressk of saat from Pmgatm ximmmssa} was reported to produce 837 mg/L BA ³³ The highest BB production of (13 g/L was observed in C pmtstiriamim |-5 with the overexpressfon of shlL This is also the highest BB production reported so far directly from glucose with engineered microorganisms to the best of our knowledge, which is significantly higher than the recently reported 50.07 mg/L In an engineered fe acetabutyhoim-. Some of our engineered _: strains could also produce small amount of E 8 with the highest of 0,02 g/t been observed In C Qs iriantim f-S _< With the UpeseB ove reap cession, C tymhutyrietm fXfo could generate 0,3 g/L EA, This was significantly higher than other strains tested in this work (mostly < 001 g/L) The mother strain Li tymhiliyrkam AcaiixmihEl could produce 208 g/l acetate an 5.3 g/l ethanol {precursors for BA synthesis) during a hatch fermentation, which might hav enabled high-level BA production In C iyrobu rimm JZfofe

The production levels of 8A and BB achieved above are bot significantly higher than the previously reported In microbial hosts to the best of our knowledge, in comparison, the BA level Is much higher than BB level, and thus has greater potentials towards economic viability. Therefore, in the following steps, we primaril focused on systematic metabolic engineering of the strain for further enhanced BA production.

Deletion of m € increased BA productio

Enhancement of the pool of precursors is one common strategy to Improve the production of targeted bioproduct. Butanol and acetylCoA are the two precursors for BA production. Therefore, we set out to increase butanol synthesis: to improve BA production, The ui od gene encodes the ADI f-quinone oxidoreduetase subunit G _f which is a subunit of the electron transport chain complex PA ADfiwpinone axldoreducfase can oxidise ADR to NAD* and transfer protons from cytoplasm to periplasm to form a proton gradient between periplasm and cytoplasm, which can then contribute to the energy conversion-^

2! In this stud , we hypothesized hat by deleting oG _> BA production would be boosted because of the potentially Incre se butanol production. Thus, we deleted moG (€spe _^ c4?5€&) m N I 4-C and generated FJ406, Further, Fj 101 was constructed based OH Fj-100 for BA production. Results demonstrated that although butanol production in F|- 100 was only slightly improved (16.5 g/L vs 15.8 g/L in Nt -4-C; FIB. 8], EA productio in Ff-lOl was remarkably enhanced compared to FI-004 (78 g/L vs. 5.5 g/L). Based on our experiences _* because C sadchamperkutykcetoniemri M14 (or b ^: i4-€} mother strai can naturally produce very high level butanol, it was generally very difficult to further Improve butanol production in. C saOcharop&thuitytacetaaicmn through simple meCaholic engineering strategies^. This is likely the case Ssi Fj- 100 (comparing to Ml -€)„ However, the increased NAIM! availability with m&C deletion in FI-101 would enable an enhanced InstonF flux/ availability of butanol which would serve as a precursor for BA production and thus enhance BA production in FI- 101. In this sense, the total butanol generated during the process (including the fraction serving as the precursor for BA production and the other fraction as the end product} in FJ - 16:1 would he actually much higher than in Fj-004, Enhancement of acetyl-CoA availabilit to Improve BA production

At the end of fermentation with FJ46I _* there was still 7.6 g/L of butanol remaining. This suggested that limited availability of Intracellular acetyi-CoA was likely the bottleneck for the further Improvement of BA production, To enhance the availability of acetyFCoA, we firstly introduced Isopropanoi synthesis (from acetone} pathway (FIG. 3A), We hypothesized that this could pull tlux from acetone to isopropanoi and thus boost the transferring of CoA from acetoacelyl-CoA to acetate driven by the CoA transferase, resulting in increased instant availability of acetyl· CoA, The sadh gene in C Mjerimkit 8593 encoding a secondary alcohol dehydrogenase can convert acetone into isoprupanol ²⁸ . The hy G gene In th same operon as sadh encodes a putative electron transfer protein. In this study, either sadh alone or the sadh iydG gene cluster was integrated into the chromosome of Fj-100, generating FI-260 an F] 400, respectively. Further, by introducing pMTL-catO , FI-201 and Fj-301 were obtained. Compared to J -101, about 50% of the acetone could be converted into Isopropanoi In FfrZOl _* while --95% of the acetone in Ffr 301 could be converted into isopiopanoh The total ti ters of acetone plus isopropanoi in Ffr

301 and FI-261 were 6.6 and 5,1 g/L respectively, both of which were higher than 4,8 g/L acetone in Ff-iO!, More significantly, BA productio in FJ~20:t and Ffr30i has bee ? remarkably Increased compared to FJRIOJ, and reache |0 . and 12.9 g/L, respectively, likely due to enhanced 'regeneration' of acetyi-CoA described above (FIG, 3 b),

Dynamic expression of atfl enhanced BA production in our engineered strain, BA Is synthesized through condensation of butanol and acetyl -CeA catalyzed by ATFL The constitutiveiy high expression of AT lwoa not necessarily lead to high BA production. For example, BA production in Pi-008 In which atfl was driven by the constitutive strong promoter ? from C tyrobufyricum was actually much lower (3,5 g/L ys 5,5 g/L) than In Ff-004 In which atfl was expressed under the promoter P from C tyrobtityrieam We hypothesised that, in order to obtain more efficient BA production, the synthesis of ATP 1 should be dynamically controlled and thu synchronous with the synthesis of precursors {butanol or acetyl-CoA) Therefore, fo the next step, we attempted to evaluate various native promoters for atfl expression, and identify the onefs) that can enable an appropriately dynamic expression ofATFi and lead to enhanced BA production, Four promoters associated with the synthesis of BA precursors were selecte to drive the atfl expression, and four strains were cons ructe correspondingly for BA production (FIG 3C). Fermentation results were shown in FIG, 3D. indeed, distinct results for BA production were observed i these strains. The BA level was only 1.0 and 1 1 g/L to F|-30S and J-302 with P * and ¾ br atfl expression, respectively, j¾« is m Importont promoter in C saccha perhutyi& tmicum, which can sense the acidic state and switch cell metabolism from acidogenesis to solvaotogenesifoL Ff-303 with the atfl expression driven by Bus produced 93g/L BA, which was still 20% lower than In Fj~30i, Interestingly, FI- 304 In which atfl was expressed under Y _<«® produced 1 7 g/L BA, which was about :10 _> S% higher than in F|-301. Based on the results, we speculated that the promoter of add (which encodes the key enzyme catalyzing butanol and ethanol production} might have resulted in the more appropriate dynamic expression of u¾fl In line with the dux of the precursors and thus led to enhanced BA production. On the other hand, ethanol production in FI-384 was also lower than in Ff-301 (0.3 g/L vs 0.6 g/L), The enhanced BA production in P)-304 consumed more acetyi-CoA, and thus dec ased production of ethanol, which also needed acetyi-CoA as the precursor.

OrgasitatioB of AsyMitim eszytsds t enhan e BA prodseti os in this study, we evaluated several reorganization approaches to increase BA production (FIG, 4), tc PduA protein, derived from Clt b cter fr mdii Pdu bacteria! microcompertment could form filaments in bacteria life £ cofofo The CC--DFA a CC-Di-B are des gne parallel heterodimerk coiled coils and two proteins with each of these self-assembling tags could combine an shorten the catalytic distance _* The enzyme (from the seine metabolic pathway), tagged with one of the coiled cods (CC-Df-A or CC-Ot-B) would attach onto the formed intracellular filaments (its Pd«A* was tagged by the other coiled coil); thus, the organize enzymes on the filaments would improve the catalytic efficiency of foe target metabolic pathway. Mini) is a membrane-associated protein and the localization of MinO is mediated by an 8-12 residue C-terminal membrane-targeting sequence. The proteins with iuD C-termina! sequence were able to he d awn to the cell membrane^ fo Thus, the application of MiuO C-fog can facilitate the secretion of target product and enhance Its production by mitigating the intracellular tozicity as well as promoting the catalyzing proces (FIG, 4A}

To evaluate whether the organization of enz es could improve HA production in our strain, three strategies were recruited; 1) assembling two of the three enzymes (enzymes associated with BA synthesis; Miff (related to acetyl-CoA synthesis), !MhA (related to butanol synthesis) and ATFI) with the CC-Bi-A and CC-DLh tags; 2) organizing the three enzymes onto PduA formed scaffold; or 3} introducing Mini) C-tag to draw ATfil onto the ceil mem brane, Firstly we assembled enzymes for BA synthesis by adding the CC-Bl-A tag to ATFI and the CC-Oi-8 tag to Nil) and BdhA (FIG, 48), Fermentatior; results showed that the addition of CC-Di-A tag to the C -terminus of ATFI in ) 306 had significantly negative effects on BA synthesis with only 2.6 g/L BA was produced (FIG, 4E), The assembly of AT FI together wit Mil) or BdhA had even severer negative effects on BA synthesis and BA productio was onl 0.86 and 0,03 g/L in Pf-308 and Ff-SlO, respectively. Further, we organized ATFi, Mil) and BdhA onto the PduA* scaffold, PduA* was fogged with CC-DLB, while the other thre enzymes were fagged with €C~0i-A (FIG. 4C), The generated FJ-311 (harhoring fee scaffold and ATFi-CC-DI- A) produced 3,1 g/L BA, while Fj--3i2 (harboring fee scaffold, ATFl-CC-DLA and Miff-Ce-Di-A} and B|-3I3 Charhor!ng the scaffold, ATFl-CC- Di-A, Mhl-BC-DI-A and BdhA-Cfofh-A) produced slightly higher amount of BA both at 3,5 g/L The scaffold seemed to have som positive effects on BA synthesis but didn't work as efficient as It was reported in other studies*-, We speculate that the colled coils fogs (CC-DF A or CC-TH-'B) _: might severel impair the catalytic activity of ATFI, Furthermore, th assembly of ATFl together w th Niff or BdhA could further inhibit the acti vity of ATFl., thus resulting In significant decrease i BA production in the corresponding strains.

Moreover, we evaluated the effect of the introduction of Mint) C-tag ft» draw ATFl onto the cel! membrane) on BA production (FIG. 4P), Fermentation results showed that the corresponding R|-·308 strain could produce _: 164 g/L B , which was 2fi% higher than i-1 304

(FIG 4E), The PI-36? strata (with GC-DhA-Atil- /nB) could also produce higher BA of 3.9 g/L compared to Ff~3§6 (26 g/L). AU these results indicated that the addition of the ceil membrane associated motif to draw' ATFl onto the ceil membrane coul facilitate the BA excretion and mitigate the Intracellular toxicity and therefore enhance BA production However, the assembly of BA synthetic enxy es or the organisation of relevant enayrnes onto the scaHd!d would significantly decrease BA production.

Elimination if f prophages increased BA (and SB) production

During our fermentations, we noticed that the ester production of the strains was not stable and could fee varied from hatch to hatch, ft has been reported that the Mi-4 (ESMT) strain contains a temperate phage named HM T which could release from the chromosome even without mdnctlon¾ In addition, the Nt-4 (flMT) strain can produce a phage-!ike particle dostocin 0 upon the Induction with mitomycin C-te yy¾ hypothesized that the instability oFier entatkms with C sa charoperb lyb e it c might be related to the prophages, and the deletion of prophages would enable more stable and enhanced production of desired endprodncts. We Identified five pro hage-like genomes (referred here a P1-P5 respectively wlthl» the chromosome of Ml -4 (BMT) (Fig. 5a}¾ Based on systematic evaluation through individual and combinatory deletion of the prophages, we demonstrate that PS is responsible for the clostocm 0 synthesi (Figs Sf, h A :l2)w, and further confirmed that Pi was the HM T phage genome (Figs, Sg & 14). However, the phage image wa different fro what was described feefortefe I was more like HM 7 (a hea with a long tail), rather than HM 1 (a bead with multiple short tails). Both AP1234 (with the deletion of Pi~P4) and AP1234S (with the deletion of Pi -PS} exhibited improve cell growth and enhanced butanol production (Figs, Sb-Se, Figs. 10, IS ¾ 16). &P12345 should be a more stable platform as there i no cell lysis at any Induction conditions with mitomycin C or norfloxacin (FIG Si), White DR1234 showed similar growth and even slightly higher fentanoi production compared to A Pi 2345 (Pip, 3d & So)

Thus, in a farther step, we used API 234 and API 2345 as the platform to be engineered for enhanced and more stable BA production. We deleted nouG and Integrate sadh-hydG duster in both DR1234 and API 2345, and obtaine F|-!2Q0 and FJ-1300 correspondingly, The plasmid pMTL-fL*-©L¾ -MfnD was transformed into Ff-1200 and FL 1300, generating: P|-120i and F| 301, respectively. Fermentation results showed that FI- 1201 and FI-1.301 produced 19,7 g/L and 19,4 g/L BA, respectively, which were both higher than Ff-308 (FIS, 6a & Table S3) BA production In both Ff-I2:0i and FJ4301 could be completed within 48 b, resulting in a productivity of 8.41 g/L/h, which was significantly higher than 0,23 g/L/h in F)-38B BA yield i F|-I20i reached 0.26 g/g, which was also higher than Fj -308 (0,24 g/g), We further determined BA concentrations In the fermentation broth as 06 g/L and 05 g/L respectively for the fermentation with FJ-120I and I-1303. Taken together, total BA production in Ff- 20! was 20,3 g/L which was the highest level that has ever been reported in a microbial host It Is 2400-fal higher than th highest level that lias been previously reported ²² and also 14,8 -fol higher than that by the very recently reported «1 diolis strain ²⁴

Besides BA production, BB production in Ff-1201 also reached 0,9 g/L, which was significantly higher than in F) 398 (0,81 g/L) and In £ p stemimwffi J-S (0,3 g/L) (FIG, 2i¾ FIG, 6a, & Table S3). This level (09 g ¾ was 1B,6-I»ld higher than the highest BB production that has been previously reported (0,85 g/L in C. etmtobutyKmm} ² *. All these results confirmed our hypothesis that the elimination of prophages would make .more robus host strain for enhanced and more stable ester production,

Expression of SAAT in F|-120ii further enhanced BB proefoetlon

As demonstrate In FIG, 2b, SAAT and LHTl were more relevant for BB production. Therefore, to achieve higher BB production, we expressed seat and efitl in FJ-120Q and obtained FI-1202 and FJ-1203, respectively. Fermentation showed that Ff-1282 and FI- 1203 produced 13 and 0.2 g/L BB, respectively (FIGikl). Further., we added Mini) C-tag to the SAAT and Introduced the reco binam gene into Ff- 1200 and: obtained Ff-1204 lor an attempt to further Improve SB production as observed for BA production in Fj-308. However, BB production i F)-1204 was only 10 g/L notwithstanding, 1.3 g/L BB obtained In FI-1202 is 25,8-foid higher than the highest level that has been previously reported ²² . Deletion of rex increased BA/BB production

The rex gene was identified as a redox-sensing transcriptionai repressor, which represses the transcription of genes related to butanol synthesis. The rex gene was deleted In Fi-1200, generating the strain VM016. Then the plasmid: pMTL-Pod not/l-MlnD was transformed into YMOlb, obtain ng the strain YM026F. The ferme«t»tSo« results Indicated that 22.0 g/L BA could be produced in a batch fermentation with YMOleP.

Further systematic genome engineering to enhance BA production Based on YM016, three copie of 'Padk-at -MmD' cassette were integrated into different loci (ktu, l cZl and kicZ2) Further, the cttl-cfiEi gene cluster (encoding aeetoacetyLCoA-aeetete/botyrateiCoA transferase} was deleted and the obtained strain was named TM028. Then, the plasmid pMTfeFodh-otfl - lnP was transformed into YMBEB, generating YM028F for BA production. Fer entation results showed that YM028P produced 2S3 g/L BA, which was the highest BA titer that we obtained by now.

Ester production with biomass hydrolysates as substrate

Fermentations were carried out using biomass hydrolysates as the substrate. In the hydrolysates, besides sugars (57,4 g/L glucose and 27,2 g/L xylose) as carbon source, there were also nutrients converted from biomass (corn stover). Therefore, we tested the effect of organic nitrogen (yeast and tryptone) of various levels on ester production. Interestingly, results showed that the highest BA production of 173 g/L was achieved in Ff ~ 1201 (la the extractant phase) without any exogenous nitrogen source supplemented (Table $4), in addition, 0,3 g/L BA was detected in aqueous phase, making a total BA production of 17 g/L in 103201 (FIG 6A & Table S4) Althoug this was slightl lower than when glucose was used as substrate (203 g/L), fer eniat!on with hydrolysates did not need any snpplementatfon of nutrients, which could significantly reduce production cost We further performed fermentation in SO0-mL bioreactor with pH controlled >3,0, BA productio reached 16,0 g/L with hydrolysates and 180 g/L wife glucose as substrate, both of which were lower than results from fermentation under the same conditions hut with serum bottles (Figs, 6S8 & 6C), During bioreactor fermentation, we noticed very strong smell of BA around the reactor. We suspected that significant evaporation during fermentation with hioreaetor resulted in the lower level of final BA titers as compared to fermentation with serum bottle, which was securely sealed with only minimu outlet for releasing gases. An improved bioprocess needs to be carefully designed for larger scale fermentation to minimlxe BA evaporation and enhance BA production and recoveiy.

Furthermore, we performed fermentation with Ff-1202 for BB production using hydrolysates in both serum bottle and SOB-raL hioreaetor. Results demonstrated that BS production in serum bottle from hydrolysates was 0.0 g/L {compared to 1,3 g/L when glucose used as substrate; FIG, ® }, BB production in bioreactor from hydrolysates reached

7? 0,9 g/L compared to 1,6 g/ ^' L w e glucose was used as substrate (Figs, BE & BE), The results were consistent with the case for BA production that tower-level 88 was obtained when hydrolysates (compared to glucose) was used as substrate. However, Interestingly, larger scale fermentation with bioreactor produced slightly higher level of 88 than the fermentation under the same conditions with serum bottle, which was different from the case for BA production. This might be because 88 is less evaporative (in bioreactor) than BA.

Techno-econo ic analysis (TEA) for 8.4 roiluction from biomass h lrolysafos

We performed a techno-economic analysis (TEA) to evaluate the economic comp titiveness of BA production from corn stover at a process capacity of 2,509 MT wet corn stover (20 moisture) per day. The whole process was developed based on the previous process using the deaeetylatfon and dish refining (DPR) pretreatment to produce com stover hydrolysate ^® *) which was the substrate used for our fermentation experiments to produce BA. The detailed process information is summarised in the supplementary materials. The process is composed of eight sections including feedstock handling, DDR pretreatment and hydrolysis, B fermentation, product recovery (distillation), wastewater treatment, steam and electricity generation, utilities:, and chemical and product storage (FID. 7 A). FIG 7B shows the equipment cost distribution of each process sections, with a total Installed equipmen cost of $263 million. The fermentation, steam & electricity cogeneration, and wastewater treatment contribute significant percentage to the total mstalied equipment cost, which aligns well with previou TEA models for chemical production from biomass via fermentation ^®® · ^4® . The total capital Investment (TO) Is $472 million by taking consideration of additional direct cost, indirect cost as well as working capitals (Table SS) From the process model., 95,2 kg of 8:4 can b produced from 1 MT of corn stover, meanwhile significant amounts of butane! (11,1 kg), isopropanoi (15.5 kg) and surplus electricity (209 kWh) are produced as coproducts (FIG. 7C) The BA production cost wa estimated to be S986/MT (FIG. ?d), which is much lower than the current

BA market price ranging between $1,200 and $1,490 per MT in year 2019 (based on the quotes from the industry**}, showing the highly economic competitiveness of BA production using our engineered strain. B looking into the cost breakdown, the corn stover feedstock cost contributes the most (38,2%) to the BA production cosh followed by other chemicals (223%) and capita! deprecation (180%) and utilities (143%). Sensitivity analysis shows that corn stover price, BA yiel , and BA liter are the most sensitive input parameters to the BA production cost (BIG, 16}

Discussion

Although tremendous efforts have been invested on blofaej/biochenneal research 5 worldwide, very linn ted success has been achieved. A key bottleneck is that the microbial host is subject to endproduet toxicity and thus desirable production efficiency cannot be obtained ^® . Our central hypothesis was that rnetaboheafiy engineering of microorganisms for high-value and easy-recoverable bioproduct production ca help alleviate en product toxicity and thus hig titer and productivity can he achieved, with which economicall 10 viable hiofnel/hioche ical production can he ultimately established. Here we tested this hypothesis by engineering solventogeoic dostr! is for highly efficient eater (high-value and. easy-recoverable) production. Based on the systematic screening of host strains and eusymes as well as multiple rounds of metabolic engineering: enriching precursors (alcohols and acefy!-CoAJ for ester production, dynamically expressing heterologous ester- 15 production pathways, orpniwing ester-synthesis enxymes, and improving strain robustness by eliminating putative prophages, we ultimately obtained strains: for efficient production of esters in both synthetic fermentation .medium and biomass hydrolysates, To the best of our knowledge, the production levels of BA and BS e achieved set up the new records.

BO ethods

Microorganisms and cultivation conditions

All the strains mid plasmids used In this study are listed in Table SI, C. pmteiifimmm ATCC 6613 and C sttcehkropetbu!tyiQ&stonicmn l-4 (H T) (DSM X4923) were requested from American Type Culture Collection (ATCC) and Deutsche Sammhmg von 25 Mikroergamsmea und lkulturen (OS Z), respectively, (2 hetfertnekii NCfMfi 80S2 was provided by Dr Hans P iiaschek® C fy b&tyticum Arc Amth:adhE2 are byper-bntanoi producing mutants constructed in our lab ^® , All the clostridial strains were grown in an anaerobic chamber ( s-COa-Ms with a volume ratio of 8S:10vS) at 3 S ^s €. Strains of C mbutytimm _* C mcch mperbutyiacet&ntettm and C 30 b germekii were cultivated using Oyptooe-gSueose-yaast extract (TGY) ium· ⁵³ , hile strains of C p i wkm m were cultivated using 2x¥T(j medium ^® . When required, clarithromycin fOa] or thiamphenicei (Tm) was supplemented into the medium at a final concentration of 39 gg/mL and IS pg/mh, respectively, £ljco/i.DHS« was used for routine plasmid propagation nd ma ie mce, E, calf CA434 was used as the doner strain for plasmi conjugation lot C iymhulyrimm. Strains of £ mil wore grown aerobically at 37 ^S C in Lurfo-Bertam (LB) medium supplemented with ISO pg/mL ampidliin (Amp) _» 50 pg/«iL kanauiycfn (Kan) or 34 pg/rnl chlorampheoica! (Cm) as needed.

Plasmi construction

All the plasmids used in this study are listed In Table Si _., and all the primers used In this study ate listed in Table S2 _<

The plasmids pMTL82lSi and pTjt wore use as mother vectors for heterogeneous gene expression* ^*, The promoter of the catt gene (GTIC_€:0652O) (f J and the promoter of the thl gene €TK C0l4$0j (Pte) from (2 iy bufyriaum ATC€ 2S755 were amplified and inserted fofo pMT.I 82i5l at the I&ORI site, and the generated plasmids were named us pMTSJIS iSt-P wi and p TL821$l-i¾¾ respectively, Promoters of the following gens, pflA f€spajrl3?103 (Piph aid (Csps*_c56880} (F,w} _> adh (Cspa c94380) {0 &) and Mb (€spa _. _c56?90j (Fw*) _: all from C. sacthawperbittyietdebomcim HI- 4 HMT) were amplifie and insetted Into pMTL8215X at the GcoRi site, and the generated plasmids were named as pMTL82f51“Pg _¾ pi e215H pMTl HtS tefteo and pMT:L82XSi~F¾ _<® ,respectively.

The mol gene from Fragaria vexeo, the sued gene from E mumas and the atf i gene from £ cer&vMde were amplified from plasmids pOLCKM, pDLQOl and pD.1,004, respectivel ³⁰⁵ JShlREF 26, The atfV (the codon optimized o f gene), ehtl fro £ c reebm T and lipaseB from Candida mtaretk ⁴⁷ were all syothesiKed by GenScript (Fiscatawa , Nj, USA], The obtained gene fragments of vent, seat, tfi, e hl^ and BpmeB wer insetted between the BtpZi an i&oRi sites i plvtTLeSlSl-lfow generating

E &dipa eB, respectively. The tfi gene was inserte between the BtgZ I and Bco sites i pMTL821SI-Pm, generating pMTtePwmfrl.

The I promoter and the gene fragments of vent, soot, ot/ 1, ethl, and lipase wer a pis ilea and ligated Into the ffeoRf site of pT.fi, generating pTJi-f voaf pTjtdfowsoaC T|I-Pi«i-ofr3, pTjl-f -e and Tli-P^-fipnseB, respectively. The atfi gen was Inserted Into the Beam site of pMT! 21Si-lfrg, pMTL82I,5l P,w, MTIJ S teFws and p T182lSl Paa, generating rMTI,-R,n iί/ί. pMTL-F, wat/2, p TfoPwi a¾fI and pMTLd¾,wi l, respectively,

DMA sequences of CC Di A CC-DEB, MmD and pduA · were synthesize by GenScript (Fiscataway, Nj, USA), The MinD-tag was fused to die end of atfi with PCR anti ligated Into the addition, the MmD-tag was fused to the end of soot w th PCR and inserte between the Big l ami BcoM sites of p Tl- generating Th^i-sant-MlnB,

The syn esized CC-ffiM fragment was llpted into the ffooRI site of pMTL-¾ _>¾ , generating p TL¾*-CC- t A. Th DMA fragments of atfi and bίb-MmD were amplified irons pMTfoPwi-uifi and MTLWwwot/WMmi) and then inserted into the EeoRI site of p TL-Ras-CC-Di-A, obtaining p TL-f A-Ot l and pM LdMirA -atfi-MmlX The CC-Bi-B sequence with the niff gene and C&BBB with the MM gene weird subsequently Inserted into the Kpn\ site of pM L-P^-A-uf ?, generatipg yHTI,-R _® %~ ~aίb~ ~hί$ and TL-S - A-agQ-8-nijff-S-hdM, The D A fragments of C&B B~pduA €€ BA~h$ CC-BbA-MHA were inserted into the MoR! site of pTfl-iM;, generating pT| i P _< » 8-p yA% jYTJiMa B-pbioWA- n¾J and T l-Psmt- -piuA^A- iff-A-MhA, respectively.

For the gene deletion or integration n 12 soeduov er M^Wonfoum, all the relevant plasmids were constructed based on pYW34, which carries the customized. €RISPS¾-€as9 system for genome editing in C echareperbutylac tmiatm * ⁷ . The promoter tfos and the gilMA (with 20-nt guide sequence targeting on the specific gene] were amplified fey two rounds of PCR with primers M-20nt/YW1342 and YW1330/VW1342 as described previously (M represents the targeted gene} ² ?. The obtained fragment as then insetted into p¥W34 (digested with B bI and Moil) through Gibson Assembly, generating the Intermediate vectors. For gene deletion, tire fragment containing the two corresponding homology arms {5004000 bp for each) for deleting the specific gene through home!ogoos recombination was amplified and inserted into the Moil site of the obtained intertnediate vector as described above, generating pYW34-&H (M represents the targeted gene), For gene integration, the fragment containing lire two corresponding homology arms ( - ) 000· b for each), the promoter an the gene fragment to he Integrated, was amplified and inserted into the MoM si te of the obtained intermediate vector as described above, generating the final plasmid for gene integration purpose. Fermentation with glucose as the substrate

For the fermentation for ester production, the C. pmi nr mm strain was cultivated: in Siehi ediu ⁴⁸ ith 50 g/L glycerol as the carbon source at 35 % ^' in the anaerobic chamber. When the OCRw reached ~0,8, the seed culture was inoculated at a ratio of tt% Into 100 ml of the same medium in a 250- b serum bottle and then cultivated a an agitation of ISO rpm and 30 *€ (on a shaker Incubator) for 72 h. The G beijerinckti strai was eufrfvated in TOY medium until the OBm* reached -41,8. Thee the seed culture was inoculated at a ratio of $% into 100 mL F2 e i along with 60 g/L glucose and I g/L yeast extract in a 250- L serum bottle. The fermentation was carried out at an agitation of 150 pm and 37 °C for 72 bfe The C. tyrobtityrkmi stsxbn was cultivated in RCM medium at 35 ^<: C until the ODosy reached --4,5. Then the seed culture was Inoculated at a ratio of 5% into 208 mL fermentation medium {contalnmg; 50 g/L glucose, 5 g/L yeast extract, 5 g/L try tene., 3 g/L (NIH ίH, 1,5 g/L IfeHPCfe 0,6 g/L MgSO^HsG, QM g/L FeSfe^lLO, and i g/L L-eysteine) hi a 500-mi hloreacfor (GS-MFC, Shanghai Gu Xin biological technology Co.. Shanghai, China} and the fermentation was carrie out at an agitation of 158 rpm and 37 tor 120 h with pH controlled >6,04 The C, $e£eher&perbuiytoe nicim strain was cultivated in TGV medium at 35 ^5> G in the anaerobic chamber until the ODme reached ~0,8. Then the seed culture was inoculated at a ratio of 5% into .100 mL F2 medium along with 60 g/L glucose and 1 g/L yeast extract In a 250-mL serum bottle. The fermentation was carried out at an agitation of 150 rpm and 30 for 120 life for the fermentation at larger scales in hioreactors, it was carried out in a :>ø()- i, fermenter (GS-MFC, Shanghai Gu Kin biological technology Co., Shanghai, China) with a working volume of 258 mL wit pH controlled >5.0, at SO rpm and 30 °C for 120 h. Samples were taken every 24 h for analysis.

For all fermentations In the serum bottle, a needle and hosepipe were connected to the top of bottle for releasing the gases produced durin the iernieutetion, For all the fermentations for ester production, the extractant pfeexadecane was added into the fermentation with a ratio of 14 (volume of the extractant vs, volume of fermentation broth) for in situ ester extraction. The reported ester concentrations were the determined values in the extractant phase. All the fermentations were carried out in triplicate. Fermentation with bio ass hydrolysates as the substrate

The biomass hydrolysates was kindly provided hy Or. Daniel Schell from Maternal Renewable Energy Laboratory f MREL) which was generated from corn stover through the innovative 'deacety!ation and mechanical refining in a disc refiner (DDR j approach" ⁵ ·/ For the fermentation, the biomass hydrolysate was diluted and supplemented info th P2 medium as the carbon source (wife fund sugar concentrat ons of 57.4 g/L glucose and 272 g/L xylose). In addition, various concentration of yeast extract (Y, g/L) and frypfrme (T, g L) were also added as the nitrogen source to evaluate their effects on the fomentation performance: OY-rdT; IY43T an 2YY6T, The fermentatio wa carried out under the same con iti ns as described above at 100 mb working volume in a 2S0~mL sen.it» botle, Ail the fermenta ions were carried out in triplicate.

Analytical methods

Concentrations of acetone, ethanol butanol acetic acid, butyric acid and glucose were measured using a fdgh-parfofmauce liquid chromatography (HPtC, Agilent Technologies 1260 infinity series, Santa Clara, €A) with a refractive index Detector (RID), equipped with an Amines: HP I7H column (Bfo-Ead Laboratories, Hercules, C:A), The column was eluted with S mM ¾SCb at a flow rate of 0.6 mh/m at 25 C The concentration of the ester in the n-haxadeeana phase was quantified using a gas ehro atography-mass spectrometry (GC- S, Agilent Technologies 680OM Santa Clara, CA) equipped with an HP-5 column (60mx0.25 mm, 0,25 film thickness) Helium was used as the carrier gas. The initial temperature of the oven was set at 30 °C for 2 mi», followed by a ramp of TO ^s €/min to reach 300 °C, and a ramp of 2 ^:> C/min to reach the final temperature of 320 °€, which was then held for 2 min. Th detector was kepi at 22SA References

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35 Ogata, S _* , Mlhara, O... EReda, ¥, & Kongo, M, Inducible phage tal!-fke particles of Clostridium mcchoraperlwtyldcetonimm and Its ate strains. Agriculture} ami Bioiogicai;Chemistry M, 14134421 (1972},

36 Zhou, Y,, yang, Y., Lynch, R, H,. Dennis, }, L & Wishart, !>, S Pf!AST: a fast phage search tool, hludetc acids research 3% W347-W3S2 (2011) _»

37 Ogata, S,, Rapp, fi, flidaka, 2, & Mongo, M. Bacteriophages of Clostridium saccharoperhu tyla ei a a iam Pari XVII, The structure of phage MM 2, Agrfctikurel and Biological Chemistry 3 15414552 (1969],

38 D. finmhlrd, R, P„ L, Tao, €, Kinchin,, D, Hsn, A, A., P, Seboers, ], Lukas, B, Oithof, M. Worley, & 0, Sexton, a, 1), IX Process design and economics lor biochemical conversion of flgnocelltdosic biomass to ethanobdifotemdd pretreatinent and enzymatic hydrolysis of corn stover. {Mo. NREL/TP-S100-47764}, Rational Renewabl Energy Lab (MREL), Golden, CO (I ted States), (2911},

39 Tao, L el aL Techno-economic analysis and !lfe-cyele assessment of cellulosle Isobiitanol and comparison with celinlosic ethanol and n-butanol. Biofuefs, Bioproducts and Bia fining 8, 3948 (2914],

40 Huang, ., long, S. & Singh, V, Techno-economic analysis of biodiesel and ethanol coproduction from lipi -producing sugarcane. Bipfmis, Bypro ucts imd Biorefining 19, 299- 315 (2016), 41 btips;//www,allbaha,c0m/pr0duct-detail/Po:riiy-99-S~8uty! Aceta:te·' Cas„62416lSS038Atffil?spm=a270O: _> ISe^olferIist.0.O,:6?372e613q _: q32W ^: &$«:p

42 Gong, 2,, Nielsen, J. & Zhuu, Y f Engineering robustness of microbial cel! fa clones. Biotech n ohgy _j ournal 12, 1700014 (2017),

43 Wang, Y at a Development of a gene knockout system using mobile group 1! iotrons (Targetrou) and genetic disruption ofadd production pathways in Clostridium begermckii. Applied mid environmental microbiology 79, S8S3-S863 (2013),

44 Sch arz, Si, M. et al Towards improved butanol: production through targeted genetic modification of Clostridium pasttntrianim. Metabolic engineering 40, 124-137 (20:17)

45 Heap, ), T., Pennington, H, Curtman, S T. & Minton, 14, 8, A modular system for OosO'i d/M shuttle plosm is. jaarnoi of micrabia!og al methods 78, 70-83 (2009)

40 Wang, Y, et of Development of a gene knockout system using mobile group II Introns (Targetron) and genetic disruption of acid production pa thways in Clostridium beijemickiL Applied and eimnmmental microbiology, 79, 3053-5863 (2:013).

47 Tama lam pndi, S et al Development of recombinant Aspergillus aryme whole-cell biocatalyst expressing ll pose- encoding gene from Candida antaretka. Applied microbiology and MatadmoIogy 387 (2907),

48 Bicbi, H Fermentation of glycerol: by Clostridium pas inrkmton-h&tch and conttniions culture studies. journal of industrial microbiology & biotechnology 27, 18-26 (2001 ).

49 Chen, X, et at A highly efficient dilute alkali deacetylation and mechanical (disc) reining: process for the conversion of renewable biomass to lower cost sugars. Biotechnology b ^h bίo oI$ 7, 98 (2014).

SUPPLEMENTAL MATERIALS TO EXAMPLE Methods

Plasmid transformation and mutant verification

Competent cells of C pastmriam were prepared fallowing the protocol as reported by Pyne ef alh pasteuriamm SD-1, an equivalent to C pastmrianum AcpoMR in which the cpaAiR gene (encoding the CpaAi Type II restriction endonuclease) was deleted and thus more efficient transformation was enahledh was used as the host strain. The overn!gbt-grown seed culture was Inoculated Into 20 ml 2*T¥G medium. When the OPwe reached 0 _> 3·-Ό,4> sucrose and glycine were added to a final concentration of 84 M an 1 S%, respectively. When the ODws further reached 0, 6—0.8, toe ceils were harvested by centrifugation at 4,200 gaud 4 *C for 10 mi». The cell pellets were resuspended In 5 mh of SfoF buffer (270 mM emse, i MgC¾ and 7 bi sodium phosphate, pH 0.5} and sploned down under the same conditions, The ob ained coll pellets were then resuspended in 0.6 l of SMF halier i pg of plasmid DBA was suspended with 20 pi, f 2 m Tris-Hd (pH 8.0) and then mixed with 530 ul competent cells and 30 pi, 96% cold ethanol. The mixture was transferred to a pre-ehiiied 4 m electroporation cuvette and incubated on ice for 5 min, Electroporation was then applied with a voltage of 1,800 V, capacitance of 25 pF and resistance of w «slug a Gone Pulset Xcell electroporation syste fBfo-Ra Laboratories, Hercules, CA), Afterwards, the culture was transferred Into 2 l of 2W'?G medium and recovered at 35 ^¾ C for 4 h. The culture was then spread onto the 2 xYTGT agar plates (PxYTCi agar plates containing 15 pg/mb of this phe nice!) for the selection of the transformants.

Competent cells of C. be^ermckii were p pa ed following the procedure as described by Wang et aid. Briefly, the overnight cel! culture was inoculated into f GY medium with an inoculation ratio of 1%. When the Ot « reached - 0.8. the cells were harvested by centrifugation at 4,200 and 4 for id min. The cell pellets were washed with the same volume (as the original cel! culture] of ice-cold 15% glycerol and ce trifugated under the same condition for 10 nun. The ceil pellets were resuspended with 5% volume of foe-cold 15% glycerol. Then 400 pi competent ceil and ~1.0 pg of plasmid DBA were mixe and transferred info a 2 nun pre-chilled electroporation cuvette and incubated on ice for 1.0 min, Electroporation was carried out at 2,000 V of voltage, 25 pf of capacitance and 200 tl of resistance Afterwards, the cells were transferred into 1,6 ml, of TOY ^' and incubated at 35 °C for 6-8 h for recovery. The culture was then spread onto TGYC agar plates (TOY agar plates containing 30 pg/mL of clarithromycin) for the selection of transformants.

The plasmid transformation through conjugation for C iymbniyrimm was performed following the procedure as deserihed by Zhang et aid, The donor strain £ cod CA434 carrying the desired plasmid as cultivated In LB medium supplemented with Cm and K . 3 mi of overnight-cultured £ red CA434 ceils were centrifuged and washed for twice (with fresh LB medium) to remove the antibiotics. The obtained donor ceils were then mixed with 0.4 ml of the ovcrnlghi-eu!tored £ rabutyrteum (grown In TOY medium}. The cell mixture was spotted onto the TGY agar plate and incubated in the anaerobic chamber at 37 ^¾ C for conjugation. After 24 h of cultivation, the cel! lawn on the plate was washed off «slag lml of TGY od rum and then spread onto the TOY plate containing IS pg/raLTm an 250 pg/mL B-cyeioserme (for eff inatfrtg the ldnal £ c&ti CA434 donor cells). The transformant colonies coaid bn observed after 48-72 h of meubatten in the anaerobic chamber.

5 Competent cells of C mccharoperinttytacetoniemi were prepare following the procedure as described by H rman et at, with slight modifications·* Briefly, the overnight- cultured ceils were inoculate into fresh TOY medium. W n the ©¾w of the cell culture reached -CbB, ceils were collected by centrifugation at 4,200 g and 22 · ^' € (room temperature) for 10 min. Cell pellets were then washed with the same volume (as the 10 original cell culture) of SMB buffer, The resuspen ion was centrifuged again under the same conditions as described above. The col! pellets were resuspended in 5% volume of S P holler. After that, the plasmid (~4.0 pg) was mixed with 400 pt of competen t cells and transferred into a 2 mm electroporation cuvette and incubated on ice for 3d min. Electroporation was teen applied with a voltage of 1,000 V, capacitance of 25 pF and 15 resistance of 3001! Subsequently, the culture was transferred into 2 mi, pre-warme TGY medium and incubated at 35 °C for 2-3 h. After that, the culture was spread onto TGYC or TGYT (TGY agar plates containing IS pg/mt of thiamphenicol) agar plates.

The positive mutants of £ secc eim erbutyiaeetmi m with desired gene deletion or integration were identified following our previously described procedures^ Briefly, th Iff C sacchamperhutyi cet fcum transforma ts harboring th plasmid designed for the gene deletion or integration were incubated in TGYC liquid medium at 3S *€ in the anaerobic chamber for about 24 h. The cell culture was then spread onto TGYLC plate (TGYC supplemented with 40 n\M lactose), When colonies appeared on the plates, colony PCB (cPCfi) was then carried out with the pair of primers of -U/H-B (N represents the targeted 25 gene name, U represent the upstream primer flanking the target locus, and I.) represents the downstream primer Hanking the target locus) to verify the gene deletion or gene integration. The selected mutants were then suhcultured In TGY medium for 3 to 5 generations to cure the plasmid for the gene deletion/integrationh The obtained plasmid- free and marker-free mutant strains wore used for the following steps

3ff Prophage Induction and phage harvesting

The strain was grown in TGY medium for overnight The cells were then transferred into fr sh TGY prio to he expose to the inducing reagent mitomycin C or noritexacm. Mitomycin € was added into the culture when the cel! growth reached th desired ©Owe (0,t-0,¾ 0,2-02, 0.3-b _> 4 or ii4-0.S), The cells of soma of the cultures grew too fast at the early stage for the induction purpose, and thus they were not Induced under every above mentioned ODsss conditions In this or For the induction, generally mitomycin € at final concentrations of 2 gg/mL and 4 pg/mL was usedT Besides, 1 pg/mt and 3 gg/ml of mitomycin C were also tried for the induction in AP234, After 30 min of treatment at 35 *C, the cells were harvested via centrifugation at 4,000 g for 5 nun and resuspended at the same volume of T6Y fresh medium. Then the ODsm was monitored carefully during the following 3· a h.

The seed culture with the CH is of around 0,2 _* 114 and 0,$ was treated using norfloxacin at the final concentrations of 0,3, 1 _* 3, 6, 93 gg/mL at 35 *€ for 24 , The final Q m was measured alter t treatment

The cells were collected via centrifugation at 4,000 g for 20 min, and then filtered through the 02 p fitter to obtain the supernatants. The supernatants were centrifugated at 20,000 rp for 3h, and the obtained precipitation (containing the phages) was then resuspended by 1/30 volume of ddf O,

Transmission election microscopy (TIM)

20 gb prepared phage sample was applied to a mesh copper grid and settled for 2 in, Then the lipoid was blotted off with a filter paper. The negative stain of 2% phosphotongsito acid (FTA) was applied to a esh copper grid and settled fo 30 s. Then the liquid was blotted oft with a liter paper followed by air dry- The prepared samples were then used for 7EM observation under a Zeiss B 10 transmission electron microscope (Carl Zeiss AS, Oherkochen, Germany) at an accelerating oltage of 60 fcV.

T eehm> -eeonmni c anal sis (TEA)

A comprehensive TEA model was developed fa evaluate the economic feasibility of BA production from corn stover using the eteacetylatlon and disk refining EffiE) pretreatmenf, The model originally developed to produce ethanoP was modifie to produce BA by mainly substituting the fermentation and distillation unit operations. The processing capacity was set at 2,500 wet metric tonnes {MT, 20% moisture) of corn stover per day. The process was assumed to run 350 days (8,41.0 hours) per year; thus the annual corn stover consumption is 075,000 wet MT per year- The composition of corn stover was 35,05% cellulose, 10-53% henuceflniosw 15,76% lignin, 4.03% ash, 3 _> 10% of protein, and 21.63% other solids on a dry basisfo Ah process was simulated using the software SuperPro Designer (inte!!igen foe, NT). The whole process can he divided into eight sections, including feedstock han ling, DOR pretreataerrt and hydrolysis, fermentation, product recovery (distillation), wastewater treatment, steam and electricit cogeneration, utilities, and chemical and product storage, in the process, corn stover Is milled at the pre-processing plant and 5 delivered to toe Seed handling section front a uniform corn stover supply sysfentfe Received com stover Is added with water to obtain a 25% solid slurry. The slurry is added with sodium hydroxide of a loading of 40 kg/MT dry co stover, heated to 80 % and hel for 2 hours to remove acetyl groups from earn stovers The alkali-treated corn stover is then washed by using the same amount of added water, followed by dewatering using screw- lit type presses to remove excess water to attain 40% solids content for the subsequent disk refining and enzymatic hydrolysis. The parameters of disk refining are adapted from a previous optimisation study using wet disk mils! The electricity consumption of disk milling is 212 kWh per dry MT of corn stover. The detail of the DDR process are described In Chen et al 20151 The p re treated corn stover Is then coole and sent to hydrolysis tanks 15 where it i hydrolysed into monomeric sugars by cellulose at 48 % for 84 hr. The enzyme loading for the en¾ymaMc hydrolysis Is 1.9 g protein/g cellulose according to a previous study! After the enzymatic hydrolysis, 10% of the hydrolysate i split off to seed fermenters for production of seed culture, and the rest 90% of the hydrolysates is fermented into BA an coproducts in large fermenters at 32 % where hexadeeane Is added 20 to the fermenter at 1:1 ratio v/vf to extract BA from the fermentation broth. The fermentation lakes 96 hours to convert all hydrolyze sugars to 8A and coproducts butanol and isopropanoi The key parameter for the conversion and fermentation yields are summarized In Table $6. The fermentation beer is then sent to the decanter to separate the extractant phase and the aqueous phase, which are separately pumped to the dis illation 25 sectio to recover BA and coproducts butanol and Isopropanoi. The distillation stillage is press filtered to separate insoluble solids for combustion to produce steam and electricity; whereas the pressed filtrate is sent ip the waste treatment section. The remaining sections of the model, including wastewater treatment, steam and electricity cogeneration, and utilities, inherited most of the original designs from the NEEL process models- %

39 The energy and mass balance and Bow rate information for the process were generated te determine the capital and operating costs. The purchased equipment costs were determined based on previous literature, particularl from fhimhlrd et al. (2011)% and Chen et ai. (20I5)A The cost of the product recovery (distillation) section was mainly

4| determined by she embedded cost esti a or of SuperPro Designer. The purcha d equipment costs were scaled «slag the exponential scaling equation with exponents ranged between 05 and 0.8 depending on th type of equipment^. The equipment cost obtained in previous year is adjusted to the year of 2019 using the plan cos index from chemical 5 engineering magazine Th total capital investment (TCI) was calculated: as a su of direct and indirect costs, which were determined based on the installed equipment costs. Direct costs included installed equipment cost, site development (9% of Inside· batterydimits (ISBL) equipment cost), warehouse (4.0% of 1S8L), and: additional piping (4.5% of 1881,). Indi rect cost is the sum of prorateah!e costs (16% of total direct cost (T0C¾, field expenses(1 (10% of TDQ, home office and construction (20% of TDC), project contingency (10% of

TOC), and other costs (10% of TDC), Working capital was assumed to he 5% of the fixed: capital investment A su ary of the total operating cost is listed in Table $5, The total operating cost included both fixed an variable operating costs. Fixed operating costs Include labor and various overhead items and variable operating costs in dude raw5 material costs, utility costs, and en-produe credits. The detailed variable and fixed operating costs are summarized in Table ST

The BA production cost was calculated based on the methods described in prior studies^.44, For the process model, BA was assigned as the main product and butanol, isopropanol, and electricity were assigned as the coproduc s _* which were sold to generateO coproduct credits. Sensitivity analyses were also performed at ± 20% variation range to evaluate the most inilnenfiai: variables on th BA production cost Results ivaluatlop of cod o-opti ixafio» of atft lor BA synthesis

Different microorganisms have different codon usage preferences. Th gene5 was originall from 5, cerevlsine and Its genetic codon usage might not he preferable for the C secihemper^ufyi& tonicmn host. Thus, a codon optinuxed aif! gene (designated m at ) was synthesized and evaluated for potentially i p oved expression and BA production in the €, s cch reperbutylocetonicmn host strain. However, fermentation results demonstrated tha FJOB? (carrying o 'rather tha atfi) actually generated slightly lower0 concentration of BA than F|:~0O4 (50 g/L vs S5 g/L, Table SB). The result was unexpected, but not totally surprising. Similar cases have been reported previously where the original natural gene showed better efficiency than the codon-optlnhaed counterpart for desirable biochemical production^, We speculate that the natural gene might be able to transcribe nto more stable mRMA structu e, and thus lead to higher translation level than the codon- op imised genefo fo addition, It has been recently reported that codon-optlni aed genes could bring abou toxicity to the host eeflsto

Enhancement of acetyl-CoA avnilahlilty to tapwe BA production 5 in the pathway;, thiolase is the enzyme that coverts acetyhCoA into scetoaeetyl-CoA

(Fig, 3a), and the attenuation of the tfdolase activity might be able to repress the acetyl -CoA ftax towards butanol (and other products) and lead to the accumulation of Intracellular acetyhCoA, which could be beneficial for enhanced BA production. Therefore, we attempted to delete the thiolase gen to further Increase the acetyhCoA availability. There id ar five annotated genes encoding thiolase in C saic ropi-rhuiyia e o kiim: Cspa cddi¾ GspsipiTSfQ, C$pa 20S20 Cspa^0B90 _t €$ paj:S€3M. After numerous attempts, we were only able to delete Csp& t20B®& and €$ra $ΰ3M ⁾ individually In FI-300, generating the mutant strains Ff-406 and F|-S0O, respectively. By Introducing the vector pMTL-cat-mp Into these two strains for BA production, ff-401 and Fj-SOl were obtained, respectively, 15 Fermentation results Indicated that BA prnduction was actually slightly lower in FJ-401 (108 g/i.) and Ff-Sdl (9.8 g/l) titan tha In Ff-301 (Fig, 3b), The result was unexpected, AcetoacetybCaA Is one of the ke metabolites for the cell; the repression for Its biosynthesis might impair the ceil metabolism an thus lead to decreased BA production. The ©f tainaimn of prophages Increased cell growth and butanol roeSoctfon b During our fermentation: process, we noticed that the performance for ester production of the strains was not vary stable and could he varied from batch to batch. Our industrial collaborator also observed that C. mschar&perbutyi&cetonfciim often had instable performance tor ABE production in the continuous fermentation process (data not shewn) it has been previously reported that th KU-4 (H T) strain contains a temperate phage 5 named Bid T which could release from the chromosome even without induction^ In addition, the Ml -4 (t-IMT) strain can produce a phage-like particle dostoeto 0 with the induction of mitomycin C. We hypothesize that the instability of the fermentation with C cehdmperbit facetxmicum might be related to the existence of prophages, and the deletion of these prophages and clostocin 0 encoding sequences would improve the 0 stability of the strain and thus enable more stable and enhanced production of the desired endproduct (8 A here). The online program FiiAST was used to predict the prophage sequences in N I -4 (B T)-W with lour possible prophage genomes were identified: the HM T prophage (renamed as PI) as well as three other putative prophages which were named as F2, P3 and P4 here {f g. 5a). Besides, »ne additional incomplete prophage g no e (without Infograse gene) was foun and name as PS,

We firstly constructed the mutant with single deletion of each prophage genome an generated the API, DR2, AP3 and DR4 strains. Because there are genes within the prophage genome possibly responsible for the normal ceil metabolism, we also constructed the mutant with foe deletion of only the integrate gene {without the integrate, the prophage cannot release from the chromosome}, obtaining the P ^' i, DNR2, DNR3 and DNR4 strains. In addition, we also constructed the mutant DRI234 (with foe deletion of all four prophage genomes) an DNR1234 (with the deletion of all four fntegrase genes). Fermentations were first conducted in foe serum bottle to investigate the effects of the elimination of prophages on butanol production in the mutant strains. As shown in Pigs Sb, Sc & S3, AF and DR1234 showed Increased cell growth and butanol production, while all the other eight mutants did not demonstrate significance difference in terms of the ceil growth and solve t production compare to the mother strain. DR4 and DR1234 reached the maximum 0&¼»> of 17, 9 and 17,6, which were and 13 5% higher than that of the control 1-4-C strain, respectively, The butanol production in DR4 and &F1234 reached 17.1 and 16.8 g/L respectively, which were also higher than that of the control N 1-442 strain (1.6.0 g/L}.

To further stud the individual prophage, we eonstmeted the trlplefoeletion mutants DR234, 4FI34, DR124, DRί23, Phage induction experiments of DR234, DR134, DRI24, DR223 and DR1234 with mitomycin € revealed that ail the mutants exhibited cell lysis (FIG, 10). Transmission election microscopy (TF ) results indicated that all the mutants produced the faiPIike particles, which showed likely the same appearance as eiostoem 0 as reported previously^ (FIG, 11), We were not able to observe the EM T phage in the supernatant of DR234. it might he because there were too many dostocin O particles in the view which made it difficult to observe the HM T phage.

Base on the above results, we tentatively concluded tha PS might he fes ooslhkv for the production of ctestocln 0 To verily this hypothesis and obtain a more robust strain for bloprednctlon, PS was deleted in DR234, AP134, AP124, AP123 and DR1234, obtaining AP234S, DR1345, DR2243, 4P1.23S and DR12345, The induction e perime ts indicated that the cell iysis was detected in AP2343 with the addition of 4 pg/mh of mitomycin C at the of O 2-0,S (PIG 12), while no cell lysis was defected in any other mutants at any conditions with the treatment using mitomycin C or norfloxacin (data not shown). Furthermore, alter in uction, phage-like particles were only observed In the supernatant of AF234S (f g· Sg & FIG. 13] an hey were likely the H t phages. However, the phage linage was different from what was described befo e^ It was more like HM 7 (a head with a long tali), rather than H t (a ead with multiple short tails). It Is worthwhile to mention 5 that this Is the first time that an image of the MM T phage has been reported

After the deletion of PS, no clestodn 0 particle was observed in the supernatant of DRI2345, which confirmed that F5 indeed encoded clostocin 0, In addition, as mentione above, no ceil lysis was observed I» 4812345 with im!ueifon (Pig, Si & FIG, 12), suggesting that DR!2345 could be a more stable platform to be engineered for enhanced ester id production. On the other and, we showed above that AB1234 grew fester and produced more butanol than the control N1-4-C strain. Therefore, we further compared the fermentation performance of AF1234 vs APi234S in both serum bottles and bioreactors (Figs. 54, 5e,S8, Si S9), Sesulis showed that ire further deletion of P5 In AP1234S did not result i significant: difference i cell growth or butanol production when compared to 15 DR1234; actually the butanol production In AP1234S was slightly lower than in AP1234,

Discussion

We firstl set out to screen the host strains and ester synthesis genes for specific ester production. With the combination of five clostridial strains (C saccha perhutyla ixmimm ffXM-G, C pm ewi mm este SD-i heijemc n 8052, C 0 tymhufyr tm mth: dk£l and mtl:;adh£2} an five ester synthesis genes (vnnt sent, atfl , ehtl and Pp eB} we obtained very promising results. Most of the engineered strains could produce BA, BA and BB at the same time (Fig, 2), and son» of the strains could also produce small amount of BB. C sac hm-vperbu iamteni n FI-004 produced S.S g/l BA, which was the highest BA production level that has ever been reported^ c, p rkmmn 5 ]-5 produced 8 g/L BB, which was also significantly higher than the previousl reported level of 0,05 g/L in an engineered C aegtohutykeum strain-c. is The results confirmed our hypothesis that solventogenic Clostridia are outstanding platforms to be engineered for ester production. Because the BA production in FJ--O04 was significantly higher than the production levels of other esters, we decided to focus on further improving BA production 0 In s< ~hamperfyiit lacet(imcu through systematic metabolic engineering.

Butanol and acetylCoA are the two precursors for BA synthesis. The enhancement of th intracellular pool of these two precursors in the host could help improve BA production, We thus firstly deleted moG to save HADM and improve butanol and thus BA production, 0»r fermentation results showed that Ff-IOt with the deletion of moG hod increased BA production to ? _> 8 g/L However, there was still 7.6 g/L butanol remaining at t e end of fermentation with Fj - 101 ; it was thus reasonable to speculate that the availability? of aeeiyhCoA was the bottleneck for further improving BA production. We tried two strategies to improve Intracellular acetybCoA availability, On was for the enhanced ‘regeneration:' of aeetyLCoA, and the other was for ‘blocking ⁵ the pathway that consumes cetyl -CoA _* Comparatively, the former seemed a better strategy. By introducing a heterologous isopropanol synthesis pathway to promote the 'regeneration' of initeeelluiar aceiyS-CoA, the FI -301 strain could produce up to 129 g/L BA (Fig 3b}

The dynamic expression of the heterologous pathway to he synchronous with the production of the precursors could be highly beneficial for foe production of the target bioproduct On the other hand, the Imbalance of intracellular metabolism and the accumulation of toxic precursors would harm the cells and lead to decreased production of the forget product We hypothesized that the appropriate regulation of the BA synthesis enapue using the native promoter of the host strain could achieve the similar effect: a OSRS. In this work, four native promoters associated with BA precursors formation were selected and evaluated to control the to gene expression (Fig, 3c}. Results indicated that the otp gene controlled by the ! ¾ promoter showed 10.5% increase in BA production compared with the control F| 301 steam (Fig, 3d}, The promoter is esponsibl for the ethanol and butanol synthesis. The synchronous expression of alcohol dehydrogenase and ATF remarkably increased BA production,

Spatial organisation of the enz es associated with BA synthesis is another strategy that we employed to enhance BA production. The cross-link of the enzy mes associated with 8A synthesis or anchoring these enzymes onto a synthetic scaffold iPduA*} was- nut able to improve the BA production; while anchoring the AXFi enzyme to the cell membrane by adding a MmD C-teg to the C-torminns of the enzyme led to significantl increased BA production. The obtained Ff~388 produced 164 g/L BA, which was 29% mote than that In Ff-384 (Fig, 4}. The attachment of ATFl to th ceil membrane facilitated the excretion of BA from the cells, which could mitigate the intracellular toxicity caused by BA and meanwhile boost the BA synthesis.

During the fermentation, the performance of the strain lor BA production was not stable, and remarkable cell lysis was also observed at the end of the fermentation. We speculated that the testabilit of the strain could be because of the prophages existing in the chromosome of C sacchewperbutyfaceixmteum _* Based on analysis, we identified four putative prophages Fi P4 and one incomplete prophage genome PS In the genome of €. saccha psrhuty cetoniam Hl-4 (II T), PS was demonstrated to he responsible for the synthesis of lostoein Q (Figs, SF & SS). Ultimately, we obtained two mutant strains API 234 and DR12345, both of which grew faster and produced more butanol than the wild type «train (Figs. SS e, S3, SB, & SB) Thus, we further constructed the BA-pmducing strains FT 1201 and 8/1301 respectively base on DR1234 and DR12343, Fermentation results emons irate! that PH 201 could produce 20.3 g/l BA (Fig. FA & Table S3), which was the highest pro uction level of B A known to us in any microbial biocatalyst fiosH The BA yield in !'j-120i reached 026 g/g, which was also significantly higher than the initial !!A- pr duefog strain Bf-004 (0.07 g/g). Thus the deletion of prophages from C $ chximperbtit i®miimmm co«M not only increase the cell growth (and stability} hot also the production of the desired end products (butanol or BA).

In addition, we also noticed that the 811 production in ) T 201 reache 0.9 g/l, which was significantly higher than that in C p tmmmm }-S (BJ g/l, the highest .88 productio level base on onr initial screening of the strains and en mes for ester production) (Pig, 6A & Table S3). We further overexpressed: stint (instead of atfi} in the strain, and obtained the Fj-1202 strain in which the BB production _: readied unprecedented i g/h CflgHB). Both the BA-prodnemg € mcekiiroperkutytecetmiemn F)-T201 and the B8~ producing C saccha perb tyiucetom m PH 202 performed well when biomass hydrolysates was used as the substrate for the fermentation R)· 2d! could produce 178 g/l BA and PJ-12B2 could produce 0.9 g/L BB from biomass hydrolysates (with no need to supplement any exogenous nitrogen source). Although these levels were slightly lower than when glucose was used as the substrate for the fermentation with the same strain, the operation eliminated the requirement of yeast extract and tryptone and thus would significantly decrease the cost of the hioprocess for fatty acid ester production.

36

54

00

Table SO. ¾ operation parameters for toe. co o stover eooversloa and butyl acetate fermentation considered m the techno mconsruk: analysts (TBA) fAetato eni and hydrolysis So iu hy ride loading 40 kg/MT dry com stever Deaectyladon eyfeaik temperature 80 ^V Disk milling energy consumption 212 kWkMT dry co stover EngY atk hydrolysis solids loading 2078 M&zfms loading 19 a¾g ssfeia g celMose Cellulose hydrolysis efficiency MenticelMose hydrolysis eifieieucy 74%

6\ FermefUaiutu

Mexadecaue load 1:1 by volume Fe jcmofkm time 96 hours BA yield 0.25 g BA/g consumed sugar Butanol yield 0,63 g butauol/g consumed sugar Isopropaooi yield 0.04 g Isopropaool/g consumed sagar Addif I o«al nutrients None (based on verified expert meals) _

Table ¾?, Summary of key raw materi al costs item Cost _.§}

Haw maicriaix a»d ntlBtles

Cunt stdvcr (gOSS mossi rr) SFAMT* Sudio IgM te t S 6/ T ^!> CeHu!asa 4240311 ' t xadeeaae 0031 ^::

Wastewater Ixeatsueat cheat ieats 45 FAIT ^¾

Bcd r ChesnkMs 45 '3 Ml *

CiKsOog sewer dirstiesk 466331 k

Sube ic acid 4L0iMT ^¾

Freshwater 0.2231 i ^: ' Fixed Ogeraisag Casts Labor cm is 3306400 ^i: LMsm feiai&!B 0055 of labor cost Maint n nce 53 «PABί, Property imsara x; 0,7% af iked capital investment

¾e rice of com stover end o-lua ctfcsaksds «¾ fiats* the ssv s tsieumac including HuiBtbrd ct at, (201 ! 14 C¾eu et at (2015 ) _% and Dalle Ave amt Ada s (201 Fy4 :¾ts to dibcteBt sources, hietudiog the JOS cknuksd price tcgart and dusi ! quotes;

"Assxustag SO employees wall* ats ver ge sternal misty of 550.000 per esupldyee.

Table S8 Butyl acetate roduci ai »» (be esigineeted sirams; tor the evaiaatbo of die effect of gene c

BA 5,540.5 5,04(1,2 Lactate 0.00 0.7*0,!

Acetate i asD.00 ./mi}.]

LflumOi .0 i 0 _. 0 IMO

Acetone 5.0- .2 3,6e0/2

Butyrate , f.KMt {>,00 tfetenol 7,fe . .5- ) ?

* PA: ethyl acetate; BA: ftf! scciai ; 08: t l t ra(«- All values * In Cl,

Supplemental References References

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Mseherichiu coll Metabolic engineering 36, 77 -88 (2014), 2 Dalle Ave, , & Adams, TA _· Techno-eeonoohe comparison of Acetone-Butanol- Ethanol fermentation using various extractants. Energy Conversion and Management: IS 6. The following are nomliraiting examples of specific embodiments of the subject matter disclosed above. This disclosure specifically hut noomxcfusively supports claims to these embodiments;

Emb. 1, A modified microorganism capable of butyl acetate (BA) production, the microorganism capable of expressing an alcohol acyl transferase (AAT); and comprising a butanol synthesis pathway.

Em 2. Any one of the microorganisms above, wherein the AAT Is selected from one or more oh Vaat, East, Atfl, Bhtl, and a fimetioual homoiog of an of the foregoing.

Emh, Any one of th microorganisms above, whereto the AAT is Vaat having at least ?ø% sequence identit with SEQ; ID NO;: 1, Emb, 4, Any one of the microorganisms above, whereto the AA Is Seat having at least 70% sequence iden ity with SEQ ID NO; 2,

Emh, 5, Any one of the microorganisms above, wherein the AAT is Atfl having at feast 70% se uence identity with $BQ ID O; 3,

Emh, 6, Any one of the mscroor sms above, wherein the AAT is E.htl having at least 70 sequence identity with SEQ ID MOr 4.

Emh, 7. Any one of the microorganisms above, comprising an acetyl CoA synthesis pathway,

Emb, 8- Any one of the microorganisms s ove, comprising multiple nucleic acid sequences each encoding Atfl or a functional homolog of Atfl, Emh, 9. Any one of the microorganisms above, comprising multiple genomic nucleic acid sequences each encoding Atfl or a Amctional homolog of Atfl,

Emh, 10, A modi hod microorganism capable of butyl butyrate (BB) production, the microorganism capable of expressing an alcohol acyl transferase (MT); and comprising a butanol synthesis pathway and a botyryi eoenayme A synthesis pathway. Emb. tl. The microorganism of embodiment 0, wherein the AAT is Bhtl or a ft ct!ona! homoiog of Ehtl .

Emb, 12, The microorganism of any one of embodiments 0-0 _» wherein the AAT is Saat or a functional homolog of Saab Emb, 1.3, Arty one of the noeroorga sms above, comprising the AAT en y e,

Emb, 1.4 Any one of the mierobrpmsms above, comprising a nucleic acid encodlngthe

AAT.

Emb, IS, An one of the microorganisms above, comprising a genomic nucleic acid encoding the AAT,

Emb lb. Any one of the microorganisms above, comprisin a plasmid encoding the AAT.

Emb. 17, Any one of the microorganisms above, wherein the microorganism is a prokaryote.

Emb. IS. Any one of the microorganisms above, wherein the microorganism is fermentative,

Emb, 19, Any one of the microorganisms above, wherein the microorpnlsm is a bacterium of goons Clostridium,

Emb. 29, Any one of the mieroorpnis s above, where in the microorganism Is selected from Clostridium Mix mperbutytoce omcunc Clostridium beijmmkii Clostridium pa$ ur mm and Chs ridium ty hutyn m ,

Emb 21, Any one of the microorganisms above, capable of expressing Lipase B or a fanctional homoiog of Lipase B.

Emb, 22. An one of the microorganisms above, capable of expressing a functional homoiog of Lipase B having at least 79% sesfuence identity with SEQ ID NO: 5,

Emb, 23, Any one of the microorganisms above, capable of expressing Lipase 8 or a itmctlona! homoiog of Lipase B, and comprising an noetic acid synthesis pathway

Emb, 29. Any one of the microorganisms above, capabl of expressing Lipase B or a functional homoiog of Lipase B, and comprising a butyric acid synthesis pathway.

Emb, 25. Any one of the microorganisms above, comprising a nucleic acid encoding Lipase B or a functional homoiog of Lipase B

Emb 26. An one of the microorganisms above, comprising a nucleic acid encoding Lipase B or a functional homoiog of Lipase B having at least 70% sequence identity with SEQ ID O: S.

Emb. 27. Any one of the microorganisms above, having reduced or eliminated MuoG activity.

Emb. 28. Any one of the microorganisms above, capable of expressing a h eterologous Sadh ora functional homoiog thereof. Emb, 29. Any one of the microorganisms above, comprising a heterologous nude»: add encoding a Sadh or a ilmctfonai ho mo log of a Sadh.

Emb 3§. Any one of the microorganisms above, comprising a sodh-ftyrfO gene dasher, Emb, 31 An one of the icroo ganis s above, comprising an exogenous AAT gene operatively linked to promoter

Emb 32. Any one of the microorganisms above, comprising an exogenous .AAT gene operatively linked to a promoter native to the microorganism,

Emb. 33, Any one of the microorganisms above, comp ising an exogenous AAT gene operatively linked to promoter

Emb. 34 Any one of the microorganisms above, comprising an exogenou AAT gene operatively linked to promoter ¾ native to the mieroorganlsm.

Emb, 35, Any one of the microorganisms above, comprising an AAT that is focalized at the cell membrane,

Emb, 36, Any one of the microorganisms above, wherein the AAT is fused to a C- ter inal membrane· targeting sequence of Min P _<

Emb, 37. Any one of the microorganisms above, wherein the AAT Is fused to a C~ terminal membrane-targeting sequence of Min D encoded by SSQ ID MCfc 6.

Emb 38. Any one of the microorganisms above, wherein the AAT Is fused to an 84.2 residue C-terminal memhrane-targeting sequence of Mint ⁾ ,

Emb 39, Any one of the microorganisms above, wherein the AAT is fused at Its €~ terminal end to the (.-terminal mbran -targeting sequence of MinO.

Emb. 40, An one of the mieroorpnisnis above, comprising a nucleic a id encodi g a polypeptide comprising an alcohol acyltranslerase and a C-formlnal membrane-targeting sequence of MinD,

Emb, 41. An one of the microorganisms above, wherein the microorganis has been cure of a prophage,

Emb 42, Any one of the icroorgmus s above, wherein the microorganism has been cured of all native prophages,

Emb. 43. Any one of the mlcroorp s s above, wherein the microorganism ho® been cured of one or mors prophages b inactivation of an integrate gene of the one or more prophages. £mb _< 44 Any om of the m cr organ sms above, wherein line microorganism Ires been cured of one or more prophages by inactivation of an tntegrase gene of the one or more prophages through the partial or entire deletion of the Integrase gene

Erah, 45. Any one of the microorganisms above, wherein the microorganism has been cured of one or more prophages hy deletion of the one or more prophages,

Emb, 46. Any one of the microorganisms above, wherein the microorganism Is a bacterium of genus Cimtri ium _* an wherein the microorganism has been cured of one or more of prophages Pi, P2, P3. F4, and PS,

Emb. 47, Any one of the microorganisms above, wherein the microorganism is a bacterium of genus Clostridium an wherein the microorganism has been cured of all of prophages PI, P2, F3, and P4.

Emb, 48, Any one of the microorganisms above, wherein said microorganism does not express a functional redox-sensing transcriptional repressor Res,

Emb, 4% Any one of the microorganisms above, capabl of expressing soluble pyridine nucleotide transhydrogenase (StM) or functional homolog thereof,

Emb. 50, Any one of the microorganisms above, capable of expressing solubl pyridine nucleotide traiwhydrogenase (SthA) having at least 70% sequence identity with SEQ ID 140: 7,

Emb, 51 Any one of the microorganisms above, wherein said microorganism does not expres a functional c/ i-egfSi gene cluster,

Emb, 52, Any one of the microorganisms above, wherein the functional homolog has only exemplar substitutions from Table i compared to the AAT, Lipase B, SthA, or Sadh, Emb, S3. An one of the microorganisms above, wherein the functional homolug has only preferred substitutions from Table l compared to the AAT, Lipase B, SthA, or Sadh, Emb, 54. An one of the microorganisms above, wherein the functional homoleg has a amino add sequence Identity level to the AAT, Li ase B, SthA, or Sadh of at least 7(1, 75, 70, 85, 90, 01, 92, 93, 94, 95, 96 ,97, 8, 99, 99.S, 99.9, or 109%.

Emb, 55, A genetically modified Qostri iim mcchamperhutyiacetonimm having unproved BA production and designated YM028F, having NChiA designation number 30201211.6, deposited on 1.6 December 2020 at the National Center for Marine Algae and Microbiota at Bigelow Laboratory for Ocean Sciences, 90 Bigelow Drive, East Bootbbay, ME 04544 USA, Emh, S6, A genetically modified CimtrMmm mcchmxrperbsMy e tQmcum havinmprove BB production ami designated YMO!bPS, ha ng NCMA esignation number 202012115, deposited on 16 December 2020 at the National Center for Marine Algae and Mlerobiota at Bigelow Laboratory tor Ocean Sciences, 60 Bigelow Drive, East Boothhay, ME 04544 USA,

Emb. S7, A genetically modified Cl&strii m mcdmmpetimt laait icim having

Improved BA production and designated Ef-iSOl, having NCMA designation ntsmher 202012114, deposited on 16 December 20 0 at the National: Center for Marine Algae and Mlecobiota at Bigelow Laboratory for Ocean Sciences, 60 Bigelow Drive, lastBoothhay, ME 04544 USA. b 58. A method of ester production, comprising eoliuring an one of the microorganisms above under conditions suitab e to produce an ester,

Emb; 59. Any one of the methods above, comprising culturing any one of the microorganisms above in a medium containing glucose. E b. 60, Any one of the methods above, comprising culturing any one of the microorganisms above in a medium containing a biomass: hydrolysate,

Emb, 61, Any one of the methods above, comprising culturing any one of the microorganisms above in a medium containing a corn stover hydrolysate.

Emh, 62, Any one of the methods above, wherein culturing occurs at mesophllle temperatures,

Emh, 63, Any one of th methods aboye, wherein culturing occurs under anaerobic conditions.

Emh, 64. Any one of the methods above, wherein the ester Is at least one of BA and BB,

Emb, 65. Any one of the methods above, producing at least about 1,5 g BA/L of culture, Emb, 66. An one of the methods above, producing at least 1 ,5 g BA/L of culture.

Emb, 67, Any one of th methods above, producing at least about 5, 7.5, 10, 12.5, 13, 14. 15, 16, 17, 18, 9, 26, or 25 g BA/L of culture,

Emb, 68. Any one of the methods above, producing at least 5, 7,5, 10, 12.5, 13, 14, 15, 16, 17, 18 20, or 25 g BA/L of culture, Emh, 69, Any one of the methods above, producing at least about 0.1 g BB/L of culture,

Emb, 76, Any one of the methods above, producing at least 0,1 g BB/L of ailture,

Emb, 71, Any one of the methods above, producing at least about 0,2, 6,3, 0,4, 0,5, 6.6, 0.7, 0.8, 0,9, 1 , 1,1, 1,2, 1.3, 1.4 1,5, or 1.6 g BB/L of culture. Emh, 72, An one of the methods above, producing at least 0.2, 0,3, 04, 0,5, 0,6, 07, Oil.. 0.9, _. 1.0, 1.1. 1,2, 1.3, 1 A LS. or 1 6 g: i!B/L of culture. It is to fee understood that any given ele ent of the disclosed embodiments of the invention ma he embodied in a single structure, a single step., a single substance, or the like, Similarly, a given element of the disclosed embodiment may beembodied in multiple structures, steps, substances, or the like. The foregoing description and accompanying drawings illustrate and describe certain processes, machines, manufactures, and composition of matter, some of which embody the inveniionfs), Such descriptions or Illustrations are not intended to limit the scope of what can be claimed, and are provided as aids in understanding the claims, enabling the making and use of what is claimed, and: teaching the best mode of use of the inveaiiou(s}. If this description and accompanying drawings are interpreted to disclose only a certain embodiment or embodiments, it shall not be construed to limit what can he claimed to that embodiment or embodiments, Any examples or embodiments of the invention described herein are not intended to Indicate that what is claimed must be coextensive with such examples or embodiments. Where it is stated that the inventkm(s) or embodiments thereof achiev one or snore objectives, it is not I ten e to limit what can he claimed to versions capable of achieving all such objectives. Any statements In this description criticising the prior art are not intended to limit what is claimed to exclude any aspects of the prior art Additionally, ho disclosure shows and describes certain embodiments of the processes, machines, manufactures, compositions of matter, and other teachings disclosed, but It is to be understood that the teachings of the present disclosure are capable of use In various other combinations, modifications, and environments and is capable of changes or modifications wltMn the scope of the teachings as expressed herein. Any section headings herein are provide only for consistency with the suggestions of 37 C.ER, § 1 7 or otherwise to provide organizational queues. These headings shall not limit or characterize the invention(s) set forth herein.