Biocatalytic C-C Bond Formation for One Carbon Resource Utilization

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International Journal of Molecular Sciences

Review Biocatalytic C-C Bond Formation for One Carbon Resource Utilization

Qiaoyu Yang 1,2,3, Xiaoxian Guo 1,2, Yuwan Liu 1,2,* and Huifeng Jiang 1,2,*

1 Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China; [email protected] (Q.Y.); [email protected] (X.G.) 2 National Technology Innovation Center of Synthetic Biology, Tianjin 300308, China 3 University of Chinese Academy of Sciences, Beijing 100049, China * Correspondence: [email protected] (Y.L.); [email protected] (H.J.)

Abstract: The carbon-carbon bond formation has always been one of the most important reactions in C1 resource utilization. Compared to traditional organic synthesis methods, biocatalytic C- C bond formation offers a green and potent alternative for C1 transformation. In recent years, with the development of synthetic biology, more and more carboxylases and C-C ligases have been mined and designed for the C1 transformation in vitro and C1 assimilation in vivo. This article presents an overview of C-C bond formation in biocatalytic C1 resource utilization is ﬁrst provided. Sets of newly mined and designed carboxylases and ligases capable of catalyzing C-C bond

formation for the transformation of CO2, formaldehyde, CO, and formate are then reviewed, and their catalytic mechanisms are discussed. Finally, the current advances and the future perspectives for the development of catalysts for C1 resource utilization are provided.

Keywords: C1 resource utilization; carboxylases; C-C ligases; designed pathway

Citation: Yang, Q.; Guo, X.; Liu, Y.; Jiang, H. Biocatalytic C-C Bond Formation for One Carbon Resource 1. Introduction Utilization. Int. J. Mol. Sci. 2021, 22, It has been estimated that more than 35% of industrial chemicals will be produced by 1890. https://doi.org/10.3390/ bio-manufacturing until 2030 [1]. C1 resources, including methane, methanol, formalde- ijms22041890 hyde, formic acid, and carbon dioxide, are ideal raw materials for bio-manufacturing, due to their low cost and easy availability. In nature, six CO2 fixation pathways have been Academic Editor: Gianfranco Gilardi identified in photoautotrophic and chemoautotrophic microorganisms [2]. In addition to Calvin–Benson–Bassham (CBB) cycle, there are three major pathways that operate in Received: 22 December 2020 methanotrophs or methylotrophic yeast for assimilation of methane or methanol, which Accepted: 5 February 2021 are first oxidized to formaldehyde, and then is assimilated via the ribulose monophosphate Published: 14 February 2021 (RuMP) cycle, serine cycle, or xylulose monophosphate pathway (XuMP) [3]. Engineer- ing natural C1 assimilation pathways to achieve the production of biofuels or industrial Publisher’s Note: MDPI stays neutral chemicals has always been a research hotspot in synthetic biology [4–16]. However, the with regard to jurisdictional claims in biotransformation of C1 resources is restricted in the industry by low production efficiency, published maps and institutional affil- limited product types, and high production costs. iations. With the development of synthetic biology, more and more C1 utilization pathways have been exploited in recent years. These pathways can be broadly divided into two categories. One refers to C1 transformation in vitro. Carboxylases and C-C ligases from naturally occurring C1 assimilation pathways are highly specialized, and thus, restricted Copyright: © 2021 by the authors. their use for the C1 biotransformation in vitro. It has been shown that (de)carboxylases in- Licensee MDPI, Basel, Switzerland. volved in the secondary metabolism are generally reversible and promiscuous [17], several This article is an open access article C-C ligases, such as aldolases and thiamine diphosphate (ThDP)-dependent enzymes [18], distributed under the terms and can receive formaldehyde as a receptor. These newly mined (de)carboxylases and C-C conditions of the Creative Commons Attribution (CC BY) license (https:// ligases greatly expand the scope of C1 resource utilization in vitro. The other refers to C1 creativecommons.org/licenses/by/ assimilation in vivo. Based on highly active carboxylases or designed C-C ligases, several 4.0/). artificial C1 assimilation pathways have been constructed, such as malyl-CoA-glycerate

Int. J. Mol. Sci. 2021, 22, 1890. https://doi.org/10.3390/ijms22041890 https://www.mdpi.com/journal/ijms Int.Int. J.J. Mol.Mol. Sci.Sci. 20212021,, 2222,, xx FORFOR PEERPEER REVIEWREVIEW 22 ofof 2323 Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 2 of 23

Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 2 of 23 involvedinvolved inin thethe secondarysecondary metabolismmetabolism areare generallygenerally reversiblereversible andand promiscuouspromiscuous [17],[17], involved in the secondary metabolism are generally reversible and promiscuous [17], severalseveral C-CC-C ligases,ligases, suchsuch asas aldolasesaldolases anandd thiaminethiamine diphosphatediphosphate (ThDP)-dependent(ThDP)-dependent several C-C ligases, such as aldolases and thiamine diphosphate (ThDP)-dependent enzymesenzymes [18],[18], cancan receivereceive formaldehydeformaldehyde asas aa receptor.receptor. TheseThese newlynewly minedmined enzymes [18], can receive formaldehyde as a receptor. These newly mined (de)carboxylases(de)carboxylasesinvolved in the secondaryandand C-CC-C ligaseligase metabolismss greatlygreatly are expandexpand generally thethe reversiblescopescope ofof C1C1and resourceresource promiscuous utilizationutilization [17], inin (de)carboxylases and C-C ligases greatly expand the scope of C1 resource utilization in vitro.vitro.several TheThe C-C otherother ligases, refersrefers such toto C1C1 as assimilationassimilation aldolases an inind vivo. vivo.thiamine BasedBased diphosphate onon highlyhighly active active(ThDP)-dependent carboxylasescarboxylases oror vitro. The other refers to C1 assimilation in vivo. Based on highly active carboxylases or designeddesignedenzymes C-CC-C[18], ligases,ligases, can severalseveralreceive artificialartificialformaldehyde C1C1 asassimilationsimilation as a receptor. pathwayspathways These havehave beenbeennewly constructed,constructed, mined designed C-C ligases, several artificial C1 assimilation pathways have been constructed, suchsuch(de)carboxylases asas malyl-CoA-glyceratemalyl-CoA-glycerate and C-C ligase s greatlypathwaypathway expand (MCG)(MCG) the scope[19],[19], ofcrotonyl-CoA/ethylmalonyl- crotonyl-CoA/ethylmalonyl-C1 resource utilization in Int. J. Mol. Sci. 2021, 22, 1890 suchvitro. asThe malyl-CoA-glycerateother refers to C1 assimilation pathway in vivo.(MCG) Based [19], on highly crotonyl-CoA/ethylmalonyl- active carboxylases or2 of 22 CoA/hydroxybutyryl-CoACoA/hydroxybutyryl-CoA cyclecycle pathwaypathway (CETCH)(CETCH) [20,21],[20,21], formolaseformolase pathwaypathway (FLS)(FLS) [22],[22], CoA/hydroxybutyryl-CoAdesigned C-C ligases, several cycle artificial pathway C1 (CETCH)assimilation [20,21], pathways formolase have beenpathway constructed, (FLS) [22], andand syntheticsynthetic acetyl-CoAacetyl-CoA pathwapathwayy (SACA)(SACA) [23].[23]. ComparedCompared toto thethe naturalnatural C1C1 assimilationassimilation andsuch synthetic as malyl-CoA-glycerate acetyl-CoA pathwa ypathway (SACA) [23].(MCG) Compared [19], crotonyl-CoA/ethylmalonyl- to the natural C1 assimilation pathways,pathways, thesethese artificialartificial pathwayspathways havehave obviousobvious advantagesadvantages inin catalyticcatalytic efficiency,efficiency, pathways,CoA/hydroxybutyryl-CoA these artificial cyclepathways pathway have (CETCH) obvious [20,21], advantages formolase in pathway catalytic (FLS) efficiency, [22], biomassbiomasspathway yield,yield, (MCG) andand [ driving19driving], crotonyl-CoA/ethylmalonyl-CoA/hydroxybutyryl-CoA force,force, andand areare expectexpecteded toto improveimprove thethe efficiencyefficiency ofof C1C1 cycle resourceresource path- biomassand synthetic yield, andacetyl-CoA driving pathwa force, andy (SACA) are expect [23]. edCompared to improve to the the natural efficiency C1 assimilationof C1 resource utilizationutilizationway (CETCH) inin thethe [ future.20future.,21], formolase pathway (FLS) [22], and synthetic acetyl-CoA pathway utilizationpathways, in these the future. artificial pathways have obvious advantages in catalytic efficiency, (SACA)TheThe overall [overall23]. Compared efficiencyefficiency to ofof the C1C1 natural transformationtransformation C1 assimilation inin vitrovitro pathways, andand C1C1 theseassimilationassimilation artificial inin pathways vivovivo isis biomassThe overallyield, and efficiency driving force,of C1 and transformation are expected to in improve vitro and the C1efficiency assimilation of C1 resource in vivo is generallygenerallyhave obvious determineddetermined advantages byby thethe in biochemical catalyticbiochemical efficiency, proppropertieserties biomass ofof carboxylasescarboxylases yield, and driving andand C-CC-C force, ligases.ligases. and areAA generallyutilization determined in the future. by the biochemical properties of carboxylases and C-C ligases. A betterbetterexpected understandingunderstanding to improve theofof the efficiencythe catalyticcatalytic of C1mechanmechan resourceismsisms utilization ofof thesethese inenzymesenzymes the future. isis necessarynecessary forfor better Theunderstanding overall efficiency of the of catalyticC1 transformation mechanisms in vitro of theseand C1 enzymes assimilation is necessary in vivo is for miningminingThe oror overalldesigndesign efficiency moremore efficientefficient of C1 transformationcarboxylcarboxylasesases andandin vitroC-CC-C andligases.ligases. C1 assimilationHerein,Herein, wewe in mainlymainly vivo is mininggenerally or determineddesign more by theefficient biochemical carboxyl propaseserties and of carboxylasesC-C ligases. andHerein, C-C ligases.we mainly A summarizedsummarizedgenerally determined carboxylases,carboxylases, by the andand biochemical C-CC-C ligasesligases properties forfor formaldehyde,formaldehyde, of carboxylases CO,CO, andandand C-Cformicformic ligases. acid.acid. A summarizedbetter understanding carboxylases, of the and catalytic C-C ligasesmechan forisms formaldehyde, of these enzymes CO, isand necessary formic foracid. Furthermore,Furthermore,miningbetter understanding or design C1C1 transformationtransformation more of theefficient catalytic in incarboxyl vitrovitro mechanisms ananasesdd C1 C1and of assimilationassimilation theseC-C enzymesligases. inin Herein,vivovivo is necessary basedbased we mainlyonon for newlynewly mining Furthermore, C1 transformation in vitro and C1 assimilation in vivo based on newly minedminedsummarizedor design oror designeddesigned more carboxylases, efficient carboxylasescarboxylases carboxylases and C-C andand ligases C-CC-C and ligasesligases C-Cfor formaldehyde, ligases. areare highlighted.highlighted. Herein, CO, we Finally,Finally,and mainly formic a summarizeda summarysummary acid. mined or designed carboxylases and C-C ligases are highlighted. Finally, a summary aboutaboutFurthermore,carboxylases, thethe currentcurrent C1 and advancesadvancestransformation C-C ligases andand forthe thein formaldehyde,futurevitrofuture an perspectivesperspectivesd C1 assimilation CO, andofof thethe formic inC1C1 vivo resourceresource acid. based Furthermore, utilizationutilization on newly are are C1 about the current advances and the future perspectives of the C1 resource utilization are presented.presented.minedtransformation or designedin vitro carboxylasesand C1 assimilation and C-C ligasesin vivo arebased highlighted. on newly Finally, mined a or summary designed car- presented. aboutboxylases the current and C-C advances ligases areand highlighted. the future perspectives Finally, a summary of the C1 aboutresource the utilization current advances are and the future perspectives of the C1 resource utilization are presented. 2.2.presented. CarboxylasesCarboxylases forfor COCO22 BiotransformationBiotransformation 2. Carboxylases for CO2 Biotransformation COCO22 isis aa poorpoor electrophileelectrophile andand usuallyusually existsexists asas bicarbonatebicarbonate inin anan aqueousaqueous solution.solution. 2. Carboxylases CarboxylasesCO2 is a poor forfor electrophile CO CO2 Biotransformation2 Biotransformation and usually exists as bicarbonate in an aqueous solution. Therefore,Therefore, thethe carboxylationcarboxylation reactionreaction ofteoftenn requiresrequires energyenergy (adenosine(adenosine triphosphatetriphosphate Therefore,CO2 isthe a poorcarboxylation electrophile reaction and usually often existsrequires as bicarbonateenergy (adenosine in an aqueous triphosphate solution. (ATP),(ATP),CO nicotinamidenicotinamide2 is a poor electrophile adenineadenine dinucleotidedinucleotideand usually exists phosphatephosphate as bicarbonate (NADPH),(NADPH), in an oror aqueous ferredoxin)ferredoxin) solution. oror the the (ATP),Therefore, nicotinamide thethe carboxylationcarboxylation adenine reactiondinucleotidereaction oftenoften requiresphosphate requires energy energy (NADPH), (adenosine (adenosine or ferredoxin) triphosphate triphosphate or (ATP), the assistanceassistance ofof coenzymescoenzymes (metal(metal ion,ion, ThDPThDP,, andand prenylatedprenylated flavinflavin mononucleotidemononucleotide assistance(ATP),nicotinamide nicotinamide of coenzymes adenine adenine dinucleotide (metal dinucleotide ion, phosphate ThDP phosphate, and (NADPH), prenylated (NADPH), or ferredoxin) flavinor ferredoxin) mononucleotide or the or assistance the (prFMN),(prFMN), etc.)etc.) [24].[24]. WeWe dividedivide carboxylasescarboxylases inintoto sevenseven categories:categories: (1)(1) OnlyOnly divalentdivalent metal-metal- (prFMN),assistanceof coenzymes etc.) of [24].coenzymes (metal We ion, divide ThDP,(metal carboxylases andion, prenylated ThDP in,to and seven flavin prenylated categories: mononucleotide flavin (1) Only mononucleotide (prFMN), divalent etc.) metal- [24]. dependentdependent carboxylases,carboxylases, (2)(2) ATP-dependenATP-dependentt carboxylases,carboxylases, (3)(3) redoxredox equivalents-equivalents- dependentWe(prFMN), divide etc.) carboxylasescarboxylases, [24]. We divide into (2) seven carboxylasesATP-dependen categories: into seven (1)t carboxylases, Only categories: divalent (1) metal-dependent(3) Only redox divalent equivalents- metal- carboxy- dependentdependent carboxylases,carboxylases, (4)(4) substrate-actisubstrate-activatedvated carboxylases,carboxylases, (5)(5) ThDP-dependentThDP-dependent dependentdependentlases, (2) ATP-dependent carboxylases,carboxylases, (4)(2) carboxylases, substrate-actiATP-dependen (3)vated redoxt carboxylases, carboxylases, equivalents-dependent (3) redox(5) ThDP-dependent equivalents- carboxylases, carboxylases,carboxylases, (6)(6) multi-enzymemulti-enzyme complexcomplex constructedconstructed carboxcarboxylase,ylase, (7)(7) prFMN-dependentprFMN-dependent carboxylases,dependent(4) substrate-activated carboxylases,(6) multi-enzyme carboxylases, (4) substrate-acticomplex (5) constructed ThDP-dependentvated carboxylases, carbox carboxylases,ylase, (5) (7) ThDP-dependentprFMN-dependent (6) multi-enzyme carboxylases.carboxylases. RepresentativeRepresentative carbcarboxylasesoxylases areare shownshown inin TableTable 1.1. carboxylases.carboxylases,complex constructed Representative (6) multi-enzyme carboxylase, carb complexoxylases (7) prFMN-dependentconstructed are shown carbox in Tableylase, carboxylases. 1. (7) prFMN-dependent Representative carboxylases.carboxylases Representative are shown in Table carboxylases1. are shown in Table 1. TableTable 1.1. RepresentativeRepresentative carboxylasescarboxylases forfor COCO22 fixation.fixation. Table 1. Representative carboxylases for CO2 fixation. AliphaticAliphatic SubstratesSubstrates Table 1. 1. RepresentativeRepresentative Product Product carboxylases carboxylases for for CO CO Enzyme Enzyme2 fixation.2 fixation. andand CategoryCategory Pathway Pathway Aliphatic Substrates Product Enzyme and Category Pathway Aliphatic Substrates Product Enzyme and Category Pathway Aliphatic Substrates Product Enzyme and Category Pathway RubiscoRubisco Rubisco Rubisco RubiscoECEC 4.1.1.394.1.1.39 Calvin–Benson–Calvin–Benson– EC 4.1.1.39 Calvin–Benson– EC 4.1.1.39 Calvin–Benson–BasshamBassham (CBB)(CBB) EC 4.1.1.39 Calvin–Benson–BasshamBassham (CBB) OnlyOnlyOnly divalentdivalent divalent metal-dependentmetal-dependent Basshamcycle cycle(CBB) Only divalent metal-dependent (CBB) cyclecycle Onlymetal-dependent divalent metal-dependent cycle carboxylasecarboxylase carboxylasecarboxylase carboxylase

Int.Int. J.J. Mol.Mol. Sci.Sci. 20212021,, 2222,, xx FORFOR PEERPEER REVIEWREVIEW 33 ofof 2323 Int. Int. J.J. Mol.Mol. Sci.Sci. 20212021,, 2222,, xx FORFOR PEERPEER REVIEWREVIEW PCPC 33 ofof 2323 PC PC PC ECEC 6.4.1.16.4.1.1 // EC 6.4.1.1ECEC 6.4.1.1 6.4.1.1 // / ATP-dependentATP-dependentATP-dependent carboxylase carboxylasecarboxylase 3-3- ATP-dependent carboxylase ATP-dependent carboxylase hydroxypropionatehydroxypropionate 3-3- ACCACC cyclecycle (HP)(HP) hydroxypropionatehydroxypropionate ACC cycle (HP) ECECACC 6.4.1.26.4.1.2 3-hydroxypropionatecycleandand (HP) cycle ACC (HP) ECEC 6.4.1.26.4.1.2 andand ATP-dependentATP-dependentEC 6.4.1.2 carboxylasecarboxylase and3-HP/4-3-HP/4- hydroxybutyratehydroxybutyrate ATP-dependentATP-dependentATP-dependent carboxylase carboxylasecarboxylase 3-HP/4-hydroxybutyrate3-HP/4-3-HP/4- cyclecyclecycle (HB) (HB)(HB) hydroxybutyratehydroxybutyrate cyclecycle (HB)(HB)

PCCPCC 3-HP3-HP PCC PCC 3-HP3-HP ECECPCC 6.4.1.36.4.1.3 3-HPandand EC 6.4.1.3 and EC 6.4.1.3 and ATP-dependentATP-dependentATP-dependentEC carboxylase 6.4.1.3 carboxylasecarboxylase 3-HP/4-HB3-HP/4-HB3-HP/4-HBand

ATP-dependentATP-dependent carboxylasecarboxylase 3-HP/4-HB3-HP/4-HB

MCCMCC MCC ECECMCC 6.4.1.46.4.1.4 // EC 6.4.1.4 / ATP-dependentATP-dependentEC 6.4.1.4 carboxylasecarboxylase /

ATP-dependentATP-dependent carboxylasecarboxylase

GCCGCC // GCC ECECGCC 6.4.1.56.4.1.5 / / EC 6.4.1.5 ATP-dependentATP-dependentEC 6.4.1.5 carboxylasecarboxylase

ATP-dependentATP-dependent carboxylasecarboxylase

ACAC AC ECEC AC 6.4.1.66.4.1.6 // EC 6.4.1.6 / ATP-dependentATP-dependentEC 6.4.1.6 carboxylasecarboxylase /

ATP-dependentATP-dependent carboxylasecarboxylase

APCAPC APC ECECAPC 6.4.1.86.4.1.8 // EC 6.4.1.8 / ATP-dependentATP-dependentEC 6.4.1.8 carboxylasecarboxylase / ATP-dependentATP-dependent carboxylasecarboxylase

PSPS PS ECEC 1.2.7.1PS1.2.7.1 4-HB4-HB EC 1.2.7.1 4-HB RedoxRedox equivalents-dependentequivalents-dependentEC 1.2.7.1 carboxylasescarboxylases 4-HB

RedoxRedox equivalents-dependentequivalents-dependent carboxylasescarboxylases

OGSOGS OGS ECECOGS 1.2.7.31.2.7.3 rTCArTCA EC 1.2.7.3 rTCA RedoxRedox equivalents-dependentequivalents-dependentEC 1.2.7.3 carboxylasecarboxylase rTCA RedoxRedox equivalents-dependentequivalents-dependent carboxylasecarboxylase

Int.Int.Int.Int. J.J.J.J. Mol.Mol.Mol.Mol. Sci.Sci.Sci.Sci. 20212021,,,, 2222,,,, xxxx FORFORFORFOR PEERPEERPEERPEER REVIEWREVIEWREVIEWREVIEW 33 ofof 2323

3-3- hydroxypropionatehydroxypropionate ACCACC cyclecyclecycle (HP) (HP)(HP)

ECEC 6.4.1.26.4.1.2 andand

ATP-dependentATP-dependent carboxylasecarboxylase 3-HP/4-3-HP/4-

hydroxybutyratehydroxybutyrate cyclecyclecycle (HB) (HB)(HB)

Int. J. Mol. Sci. 2021, 22, 1890 3 of 22 PCCPCC 3-HP3-HP

ECEC 6.4.1.36.4.1.3 andand Cont. Table 1. ATP-dependentATP-dependent carboxylasecarboxylase 3-HP/4-HB3-HP/4-HB

Aliphatic Substrates Product Enzyme and Category Pathway MCCMCC MCC ECEC 6.4.1.46.4.1.4 //// EC 6.4.1.4 / ATP-dependentATP-dependentATP-dependent carboxylase carboxylasecarboxylase

GCCGCC GCC //// ECEC 6.4.1.56.4.1.5 EC 6.4.1.5EC 6.4.1.5 /

ATP-dependentATP-dependentATP-dependent carboxylase carboxylasecarboxylase

ACAC AC ECEC 6.4.1.66.4.1.6 // EC 6.4.1.6ECEC 6.4.1.66.4.1.6 / /// ATP-dependentATP-dependentATP-dependent carboxylase carboxylasecarboxylase

APCAPC APC ECEC 6.4.1.86.4.1.8 // EC 6.4.1.8ECEC 6.4.1.86.4.1.8 / /// ATP-dependentATP-dependentATP-dependent carboxylase carboxylasecarboxylase

PSPS PS EC 1.2.7.1ECEC 1.2.7.11.2.7.1 4-HB4-HB ECEC 1.2.7.11.2.7.1 4-HB4-HB4-HB Redox equivalents-dependent RedoxRedox equivalents-dependentequivalents-dependentcarboxylases carboxylasescarboxylases

OGSOGS OGS Int.Int. J.J. Mol.Mol. Sci.Sci. 20212021,, 2222,, xx FORFOR PEERPEER REVIEWREVIEW 44 ofof 2323 EC 1.2.7.3ECEC 1.2.7.31.2.7.3 rTCArTCA ECEC 1.2.7.31.2.7.3 rTCArTCArTCArTCA Redox equivalents-dependent RedoxRedox equivalents-dependentequivalents-dependentcarboxylase carboxylasecarboxylase

IDHIDH IDH rTCArTCA EC 1.1.1.41/42ECEC 1.1.1.41/421.1.1.41/42 EC 1.1.1.41/42 rTCA Redox equivalents-dependent RedoxRedox equivalents-dependentequivalents-dependentcarboxylase carboxylasecarboxylase

CCRCCR CCR EC 1.3.1.85ECEC 1.3.1.851.3.1.85 CETCHCETCH pathwaypathway ECEC 1.3.1.851.3.1.85 CETCHCETCHCETCH pathway pathwaypathway Redox equivalents-dependent RedoxRedox equivalents-dependentequivalents-dependentcarboxylase carboxylasecarboxylase

PEPCPEPC 4-HB4-HB andand

ECEC 4.1.1.314.1.1.31 MCGMCG

Substrate-activatedSubstrate-activated carboxylasecarboxylase pathwaypathway

PDCPDC

ECEC 4.1.1.14.1.1.1 //

ThDP-dependentThDP-dependent carboxylasecarboxylase

KdcAKdcA

fromfrom LactococcusLactococcus lactislactis //

ThDP-dependentThDP-dependent carboxylasecarboxylase

reductivereductive glycineglycine GCSGCS pathwaypathway Multi-enzymeMulti-enzyme complexcomplex constructedconstructed carboxylasecarboxylase

FerulicFerulic acidacid decarboxylasedecarboxylase (FDC1)(FDC1) // prFMN-dependentprFMN-dependent carboxylasecarboxylase

BenzeneBenzene carboxylasecarboxylase // prFMN-dependentprFMN-dependent carboxylasecarboxylase

3,4-dihydroxybenzoic3,4-dihydroxybenzoic acidacid decarboxylasedecarboxylase fromfrom E.E. cloacaecloacae (EcAroY)(EcAroY) // prFMN-dependentprFMN-dependent carboxylasecarboxylase

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IDH IDHIDHIDHIDH rTCA rTCArTCArTCArTCA Int.Int. J.J. Mol.Mol. Sci.Sci. 20212021,, 2222,, xx FORFOR PEERPEER REVIEWREVIEW EC 1.1.1.41/42 44 ofof 2323 ECECEC IDH 1.1.1.41/42 1.1.1.41/421.1.1.41/42 rTCA

Redox equivalents-dependentEC 1.1.1.41/42 carboxylase RedoxRedoxRedox equivalents-dependent equivalents-dependentequivalents-dependent carboxylase carboxylasecarboxylase

Redox equivalents-dependent carboxylase Int. J. Mol. Sci. 2021, 22, 1890 4 of 22

CCR CCRCCRCCR ECCCR 1.3.1.85 CETCH pathway ECECEC 1.3.1.85 1.3.1.851.3.1.85 CETCHCETCHCETCH pathway pathwaypathway Table 1. Cont.Redox equivalents-dependentEC 1.3.1.85 carboxylase CETCH pathway RedoxRedoxRedox equivalents-dependent equivalents-dependentequivalents-dependent carboxylase carboxylasecarboxylase

Redox equivalents-dependent carboxylase Aliphatic Substrates Product Enzyme and Category Pathway

PEPCPEPCPEPC 4-HB4-HB4-HB andandand PEPCPEPCPEPC 4-HB4-HB4-HB and andand PEPC 4-HB4-HB andand EC 4.1.1.31EC 4.1.1.31 MCG ECECEC 4.1.1.31 4.1.1.314.1.1.31 MCGMCGMCGMCG Substrate-activatedEC 4.1.1.31 MCG Substrate-activated carboxylase pathwaypathway Substrate-activatedSubstrate-activatedSubstrate-activatedSubstrate-activatedcarboxylase carboxylase carboxylasecarboxylasecarboxylase pathwaypathwaypathway Substrate-activated carboxylase pathway

PDC PDCPDCPDC PDCPDCPDC PDC ECECEC 4.1.1.1 4.1.1.14.1.1.1 ///// ECEC 4.1.1.1ECEC 4.1.1.1 4.1.1.14.1.1.1 // /// ThDP-dependentThDP-dependent carboxylase carboxylase ThDP-dependentThDP-dependentThDP-dependent carboxylase carboxylasecarboxylase ThDP-dependent carboxylase

KdcA KdcAKdcAKdcAKdcA KdcA fromfromfromfromfromfrom Lactococcus LactococcusLactococcus LactococcusLactococcusLactococcus lactis lactis lactislactis / ///// from Lactococcusfromfromfrom LactococcusLactococcus lactis lactislactis / /// ThDP-dependent carboxylase ThDP-dependentThDP-dependentThDP-dependentThDP-dependentThDP-dependent carboxylase carboxylase carboxylase carboxylasecarboxylase

reductivereductivereductivereductive glycine glycineglycineglycine GCSGCSGCS reductivereductivereductivereductive glycine glycineglycineglycine GCSGCSGCS pathwaypathway GCS pathwaypathwaypathway Multi-enzymeMulti-enzymeMulti-enzymeMulti-enzyme complex complexcomplex constructed complex constructedconstructed carboxylase carboxylasecarboxylasereductive glycine pathway Multi-enzymeMulti-enzymeMulti-enzyme complex complexcomplex constructed constructedconstructed carboxylase carboxylasecarboxylase constructed carboxylase

Ferulic acid decarboxylase (FDC1) FerulicFerulic acidacid decarboxylasedecarboxylase (FDC1)(FDC1) FerulicFerulicFerulicFerulic acid acid acidacid decarboxylase decarboxylase decarboxylasedecarboxylase (FDC1) (FDC1)(FDC1) / / prFMN-dependent(FDC1) carboxylase ///// prFMN-dependent carboxylase / prFMN-dependentprFMN-dependentprFMN-dependent carboxylase carboxylasecarboxylase

carboxylase

Benzene carboxylase Benzene carboxylase BenzeneBenzeneBenzene carboxylase carboxylasecarboxylase / Benzene carboxylase / prFMN-dependent carboxylase ///// prFMN-dependentprFMN-dependent carboxylase / prFMN-dependentprFMN-dependentprFMN-dependent carboxylase carboxylasecarboxylase carboxylase

3,4-dihydroxybenzoic acid decarboxylase from 3,4-dihydroxybenzoic3,4-dihydroxybenzoicE. cloacae (EcAroY) acid decarboxylase acid from 3,4-dihydroxybenzoic3,4-dihydroxybenzoic3,4-dihydroxybenzoic3,4-dihydroxybenzoic acid acidacidacid decarboxylase decarboxylasedecarboxylasedecarboxylase from fromfromfrom / decarboxylaseE.E. cloacae cloacae from (EcAroY) E.(EcAroY) cloacae E.E.E. cloacae cloacaecloacae (EcAroY) (EcAroY)(EcAroY) /// prFMN-dependent(EcAroY) carboxylase / //// prFMN-dependent carboxylase prFMN-dependentprFMN-dependentprFMN-dependent carboxylase carboxylasecarboxylase carboxylase

Pyrrole-2-carboxylicPyrrole-2-carboxylicPyrrole-2-carboxylic acidacid acid decarboxylasedecarboxylase decarboxylase / / / prFMN-dependentprFMN-dependent carboxylasecarboxylase

carboxylase

2.1.2.1.2.1. OnlyOnly Only DivalentDivalent Divalent Metal-DependentMetal-Dependent Carboxylases CarboxylasesCarboxylases

The CBB cycle is the most important CO2 assimilation pathway on earth, and is used by most photosynthetic organisms (such as plants, algae, cyanobacteria, and most

aerobic or facultative aerobic Eubacteria) to assimilate CO2 into biomass [25]. D-ribulose- Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 5 of 23

Pyrrole-2-carboxylic acid decarboxylase / prFMN-dependent carboxylase

Int. J. Mol. Sci. 2021, 22, 1890 2.1. Only Divalent Metal-Dependent Carboxylases 5 of 22 The CBB cycle is the most important CO2 assimilation pathway on earth, and is used by most photosynthetic organisms (such as plants, algae, cyanobacteria, and most aerobic or facultative aerobic Eubacteria) to assimilate CO2 into biomass [25]. D-ribulose-1, 5- 1,bisphosphate 5-bisphosphate carboxylase/oxygena carboxylase/oxygenasese (Rubisco, (Rubisco, EC 4.1.1.39) EC 4.1.1.39) is the key is thecarboxylase, key carboxylase, which whichconverts converts CO2 and CO ribulose-1,2 and ribulose-1, 5-bisphosphate 5-bisphosphate to 3-phosphoglycerate to 3-phosphoglycerate [26]. The [catalytic26]. The catalyticprocess processof Rubisco of Rubisco is that isthe that enolate the enolate form formof ribulose-1, of ribulose-1, 5-bisphosphate 5-bisphosphate launches launches a 2+ β anucleophilic nucleophilic attack attack onto onto CO CO2 2assistedassisted by by an an essential essential Mg Mg2+, to, toproduce produce a alabile labile C6- C6-β- - ketoacidketoacid intermediate,intermediate, which is hydrolytically hydrolytically cleaved cleaved into into 3-phosphoglycerate 3-phosphoglycerate (Figure (Figure 1).1 ). RubisCORubisCO showsshows twotwo major flaws. ﬂaws. One One is is the the low low catalytic catalytic activity, activity, which which has has an an average average −1 turnoverturnover numbernumber ofof 55 ss−1 [27,28].[27,28]. The The other other is is a aside side reaction reaction with with O O2, 2causing, causing carbon carbon loss. loss.

FigureFigure 1.1. TheThe catalyticcatalytic mechanism of Rubisco Rubisco [26]. [26].

DueDue toto thethe highhigh complexitycomplexity of the cataly catalytictic mechanism mechanism and and the the unique unique position position of of RubisCORubisCO inin biosynthesis,biosynthesis, it is unlikely that that this this enzyme enzyme can can accept accept non-natural non-natural substrate substrate analogsanalogs to to produce produce carboxylic carboxylic acids. acids. In addition In addition to engineering to engineering cyanobacteria cyanobacteria or microalgae or tomicroalgae produce variousto produce biofuels various and biofuels industrial and chemicals,industrial chemicals, the reconstruction the reconstruction of the CBB of cyclethe basedCBB cycle on RubisCO based on in RubisCO industrial in microbialindustrial model microbial strains model (Escherichia strains (Escherichia coli, etc.) has coli, made etc.) a serieshas made of progress a series [ 29of –progress31]. By co-expressing [29–31]. By co Rubisco,-expressing phosphoribulokinase, Rubisco, phosphoribulokinase, and formate dehydrogenase,and formate dehydrogenase, Shmuel Gleizer Shmuel et al. engineeredGleizer et al.E. engineered coli to produce E. coli all to its produce biomass all carbon its frombiomass CO 2carbonvia the from CBB CO cycle2 via [ 30the]. CBB By adding cycle [30]. eight By heterologous adding eight genesheterologous and deleting genes and three nativedeleting genes, three Thomas native genes, Gassler Thomas et al. Gassler engineered et al. theengineered peroxisomal the peroxisomal methanol-assimilation methanol- pathwayassimilation of P. pathway pastoris ofinto P. pastoris a CO2 -ﬁxationinto a CO pathway2-fixation resemblingpathway resembling the CBB the cycle CBB [31 cycle], the resulting[31], the resulting strain can strain grow can continuously grow continuously withCO with2 asCO a2 soleas a sole carbon carbon source source at at a µamax μmaxof 0.008of 0.008 h− 1h.−1.

2.2.2.2. ATP-DependentATP-Dependent CarboxylasesCarboxylases Biotin-dependentBiotin-dependent carboxylases include include pyruva pyruvatete carboxylase carboxylase (PC, (PC, EC EC 6.4.1.1), 6.4.1.1), acetyl- acetyl- CoACoA carboxylasecarboxylase (ACC,(ACC, EC 6.4.1.2), propionyl-CoA propionyl-CoA carboxylase carboxylase (PCC, (PCC, EC EC 6.4.1.3), 6.4.1.3), 3- 3- methylcrotonoyl-CoAmethylcrotonoyl-CoA carboxylasecarboxylase (MCC, (MCC, EC EC 6.4.1.4), 6.4.1.4), and and geranoyl-CoA geranoyl-CoA carboxylase carboxylase (GCC, EC(GCC, 6.4.1.5). EC 6.4.1.5). They are They widely are distributedwidely distributed in nature in and nature can beand found can be in archaea,found in bacteria, archaea, algae,bacteria, fungi, algae, plants, fungi, and plants, animals and [32 ].animals The catalytic [32]. The process catalytic of biotin-dependent process of biotin-dependent carboxylases cancarboxylases be divided can into be two divided steps (Figureinto two2). steps First, (F theigure biotin 2). carboxylaseFirst, the biotin (BC) carboxylase domain catalyzes (BC) thedomain ATP-dependent catalyzes the carboxylation ATP-dependent of the carboxylation N10 atom of of the the biotin N1′ atom cofactor, of the using biotin bicarbonate cofactor, asusing the CObicarbonate2 donor. Second,as the theCO2 carboxyltransferase donor. Second, the (CT) carboxyltransferase domain transfers (CT) the COdomain2 from carboxy-biotintransfers the CO to2 from the substrates carboxy-biotin [33– 35to]. the The substrat site fores [33–35]. carboxylation The site isfor on carboxylation the α-carbon ofis saturatedon the α-carbon substrates of saturated (pyruvate, substrates acetyl-CoA, (pyruvat ande, propionyl-CoA) acetyl-CoA, and orpropionyl-CoA) the γ-carbon oforα , βthe-unsaturated γ-carbon substratesof α, β-unsaturated (3-methylcrotonyl-CoA, substrates (3-methylcrotonyl-CoA, geranyl-CoA). Acetyl-CoA geranyl-CoA). carboxylase and propionyl-CoA carboxylase are two carboxylases of 3-hydroxypropionate/malyl-CoA cycle and 3-hydroxypropionate/4-hydroxybutyrate cycle [36,37]. Acetone carboxylases (AC, EC 6.4.1.6) are soluble cytoplasmic enzymes, and can be found in many species of aerobic, anaerobic phototrophic bacteria, and even microaerobic gastric human pathogenic species Helicobacter pylori. They catalyze the carboxylation of acetone to form acetoacetate at the expense of ATP [38]. There are two different types of acetone carboxylases. One requires 2 ATP equivalents as an energy supply for the carboxylation reaction, while another requires 4 ATP equivalents. The main difference in catalytic mechanism lies in the processes of substrate activation. The catalytic mechanism proposed for acetone carboxylase of Xanthobacter/Rhodobacter is that one ATP is sequentially hydrolyzed to ADP and AMP to activate acetone and bicarbonate, respectively. While the catalytic mechanism of acetone carboxylase from Aromatoleum is that 2 ATP are hydrolyzed Int. J. Mol. Sci. 2021, 22, 1890 6 of 22

Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 6 of 23 to 2 AMP to active two substrates [39]. Acetoacetate can be activated by a CoA ligase to

form acetoacetyl-CoA, which is cleaved to form 2 acetyl-CoA by thiolase. Therefore, a new pathway from isopropanol and CO2 to acetyl-CoA can be constructed. Acetophenone carboxylaseAcetyl-CoA (APC,carboxylase EC 6.4.1.8) and catalyzespropionyl-CoA the carboxylation carboxylase ofare acetophenone two carboxylases to benzoylac- of 3- etatehydroxypropionate/malyl-CoA [40]. Different from the abovecycle and two 3-hy activationdroxypropionate/4-hydroxybutyrate processes of acetone carboxylases, cycle acetophenone[36,37]. and bicarbonate are all activated by hydrolyzing ATP to ADP.

FigureFigure 2. 2.The The catalytic catalytic processprocess ofof biotin-dependentbiotin-dependent carboxylases carboxylases [33–35]. [33–35 ].

2.3. RedoxAcetone Equivalents-Dependent carboxylases (AC, EC Carboxylases 6.4.1.6) are soluble cytoplasmic enzymes, and can be foundPyruvate in many synthase species of (PS, aerobic, EC 1.2.7.1) anaerobic and phototrophic 2-oxoglutarate bacteria, synthase and (OGS,even microaerobic EC 1.2.7.3) are agastric class of human enzymes pathogenic sharing species a similar Helicobacter catalytic mechanismpylori. They [catalyze41,42]. Theythe carboxylation belong to strictly of anaerobicacetone to enzymes form acetoacetate and show at lowthe expense catalytic of activity. ATP [38]. Acetyl-CoA There are two can different be reductively types of car- boxylatedacetone carboxylases. by pyruvate synthaseOne requires at the 2 expense ATP equivalents of two equivalents as an energy of ferredoxin supply tofor generate the pyruvate.carboxylation Similarly, reaction, succinyl-CoA while another can requires be converted 4 ATP equivalents. to 2-oxoglutarate The main by difference 2-oxoglutarate in synthasecatalytic [mechanism43]. Different lies from in the the processes above two of substrate enzymes, activation. isocitrate The dehydrogenase catalytic mechanism (IDH, EC 1.1.1.41/42)proposed for converts acetone 2-oxoglutarate carboxylase toof isocitrateXanthobacter/Rhodobacter at the expense is of NAD(P)Hthat one ATP [44]. is The carboxylationsequentially processhydrolyzed of isocitrate to ADP dehydrogenaseand AMP to isactivate assumed acetone to proceed and viabicarbonate, the enolate intermediaterespectively. of While 2-oxoglutarate, the catalytic which mechanism is formed of acetone with the carboxylase assistance from of divalent Aromatoleum metal is ions that2+ 2 ATP2+ are hydrolyzed to 2 AMP to active two substrates [39]. Acetoacetate can be Mg or Mn . After the addition of CO2, the unstable keto-tricarboxylic acid intermediate isactivated immediately by a CoA reduced ligase by to NAD(P)H form acetoacetyl-CoA, to yield stable which isocitrate is cleaved (Figure to form3A). 2 2-oxoglutarate acetyl-CoA Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 7 of 23 synthaseby thiolase. and Therefore, isocitrate dehydrogenasea new pathway arefrom carboxylases isopropanol of and the CO reductive2 to acetyl-CoA tricarboxylic can be acid

(rTCA)constructed. cycle. Acetophenone carboxylase (APC, EC 6.4.1.8) catalyzes the carboxylation of acetophenone to benzoylacetate [40]. Different from the above two activation processes of acetone carboxylases, acetophenone and bicarbonate are all activated by hydrolyzing ATP to ADP.

2.3. Redox Equivalents-Dependent Carboxylases Pyruvate synthase (PS, EC 1.2.7.1) and 2-oxoglutarate synthase (OGS, EC 1.2.7.3) are a class of enzymes sharing a similar catalytic mechanism [41,42]. They belong to strictly anaerobic enzymes and show low catalytic activity. Acetyl-CoA can be reductively carboxylated by pyruvate synthase at the expense of two equivalents of ferredoxin to generate pyruvate. Similarly, succinyl-CoA can be converted to 2-oxoglutarate by 2- oxoglutarate synthase [43]. Different from the above two enzymes, isocitrate dehydrogenase (IDH, EC 1.1.1.41/42) converts 2-oxoglutarate to isocitrate at the expense of NAD(P)H [44]. The carboxylation process of isocitrate dehydrogenase is assumed to proceed via the enolate intermediate of 2-oxoglutarate, which is formed with the assistance of divalent metal ions Mg2+ or Mn2+. After the addition of CO2, the unstable FigureFigure 3. The 3. catalytic The catalytic mechanism mechanism of isocitrateof isocitrate dehydrogenase dehydrogenase and crotonyl-CoA carboxylase/reductase. carboxylase/reductase. (A) The (A) catalytic The catalytic keto-tricarboxylic acid intermediate is immediately reduced by NAD(P)H to yield stable mechanismmechanism of isocitrate of isocitrate dehydrogenase dehydrogenase [44 [44].]. (B ()B The) The catalytic catalytic mechanismmechanism of of croton crotonyl-CoAyl-CoA carboxylase/reductase carboxylase/reductase [45]. [45]. isocitrate (Figure 3A). 2-oxoglutarate synthase and isocitrate dehydrogenase are carboxylases of the reductive tricarboxylic acid (rTCA) cycle. Enoyl-CoAEnoyl-CoA carboxylases/reductases carboxylases/reductases (ECRs) (ECRs) are a are class a classof carboxylases of carboxylases that exist that in exist in secondary metabolism, metabolism, as well as well as in as central in central carbon carbon metabolism metabolism of α-proteobacteria of α-proteobacteria and Streptomycetes [45]. The best-studied ECR is crotonyl-CoA carboxylase/reductase (CCR, and Streptomycetes [45]. The best-studied ECR is crotonyl-CoA carboxylase/reductase EC 1.3.1.85) that catalyzes NADPH-dependent reductive carboxylation of crotonyl-CoA (CCR, EC 1.3.1.85) that catalyzes NADPH-dependent reductive carboxylation of crotonyl- into (2S)-ethylmalonyl-CoA. The mechanism of CCR is assumed to proceed via CoAnucleophilic into (2S)-ethylmalonyl-CoA. hydride attack at β-carbon The of mechanism the enoyl-CoA of CCR ester; is the assumed forming toenolate proceed is via β nucleophilictrapped by hydrideCO2 to generate attack at (2S)-ethylmalonyl-CoA-carbon of the enoyl-CoA (Figure 3B). ester; Recently, the forming combining enolate is experimental biochemistry, protein crystallography, and advanced computer simulations, Gabriele M. M. Stoffel et al. determined the CO2-binding residues at the active site of crotonyl-CoA carboxylase/reductase from Kitasatospora setae [46]. Propionyl-CoA synthase from Erythrobacter sp. NAP1, as well as an acrylyl-CoA reductase from Nitrosopumilus maritimus, have almost no carboxylation activity. Based on the determined CO2-binding residues, they used rational design to engineer two enzymes into carboxylases by increasing interactions of the proteins with CO2 and suppressing diffusion of water to the active site [47]. Relative to Rubisco, CCR is oxygen-insensitive, does not react with O2, requires only the NADPH, and catalyzes CO2 fixation with higher efficiency (Kcat/Km = 1642.6 s−1 mM−1) [48,49]. All of these characteristics make CCR a good candidate enzyme for the fixation of CO2. Based on CCR, Tobias J. Erb research group constructed a crotonyl- CoA/ethylmalonyl-CoA/hydroxybutyryl-CoA (CETCH) cycle in vitro [21], which consists of 17 enzymes and can convert CO2 into organic molecules at a rate of 5 nanomoles of CO2 per minute per milligram of protein (Figure 4). Recently, they successfully encapsulated thylakoids isolated from the spinach plant along with all enzymes of the CETCH pathway within water-in-oil droplets [20]. The encapsulated system could use light energy to produce glycolate from CO2, while also phosphorylating ADP to ATP.

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trapped by CO2 to generate (2S)-ethylmalonyl-CoA (Figure3B). Recently, combining experimental biochemistry, protein crystallography, and advanced computer simulations, Gabriele M. M. Stoffel et al. determined the CO2-binding residues at the active site of crotonyl-CoA carboxylase/reductase from Kitasatospora setae [46]. Propionyl-CoA synthase from Erythrobacter sp. NAP1, as well as an acrylyl-CoA reductase from Nitrosopumilus maritimus, have almost no carboxylation activity. Based on the determined CO2-binding residues, they used rational design to engineer two enzymes into carboxylases by increasing interactions of the proteins with CO2 and suppressing diffusion of water to the active site [47]. Relative to Rubisco, CCR is oxygen-insensitive, does not react with O2, requires only the −1 −1 NADPH, and catalyzes CO2 fixation with higher efficiency (Kcat/Km = 1642.6 s mM )[48,49]. All of these characteristics make CCR a good candidate enzyme for the ﬁxation of CO2. Based on CCR, Tobias J. Erb research group constructed a crotonyl-CoA/ethylmalonyl- CoA/hydroxybutyryl-CoA (CETCH) cycle in vitro [21], which consists of 17 enzymes and can convert CO2 into organic molecules at a rate of 5 nanomoles of CO2 per minute per milligram of protein (Figure4). Recently, they successfully encapsulated thylakoids Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW isolated from the spinach plant along with all enzymes of the CETCH8 pathwayof 23 within water-in-oil droplets [20]. The encapsulated system could use light energy to produce glycolate from CO2, while also phosphorylating ADP to ATP.

Figure 4. TheFigure crotonyl-CoA/ethylmalonyl-CoA/hydroxybutyryl-CoA 4. The crotonyl-CoA/ethylmalonyl-CoA/hydroxybutyryl-CoA cycle pathway cycle pathway (CETCH (CETCH pathway) [21]. CCR, crotonyl-CoA carboxylase/reductase;pathway) [21]. CCR, crotonyl-CoA EEM, ethylmalonyl-CoA carboxylase/re epimerase,ductase; EEM, and mutase; ethylmal MOX,onyl-CoA methylsuccinyl-CoA epimerase, oxidase; MMD, methylmalyl-CoAand mutase; dehydratase; MOX, methylsuccinyl-CoA MML, methylmalyl-CoA oxidase; lyase;MMD, POX, methylmalyl-CoA propionyl-CoA dehydratase; oxidase; MEM, MML, methylmalonyl- CoA epimerase,methylmalyl-CoA and mutase; SSDH, lyase; succinate POX, propionyl-CoA semialdehyde oxid dehydrogenase;ase; MEM, methylmalonyl-CoA HBDH, 4-hydroxybutyrate epimerase, dehydrogenase; and HBS, 4-hydroxybutyryl-CoAmutase; SSDH, synthetase;succinate semialdehyde HBD, 4-hydroxybutyryl-CoA dehydrogenase; dehydratase.HBDH, 4-hydroxybutyrate dehydrogenase; HBS, 4-hydroxybutyryl-CoA synthetase; HBD, 4-hydroxybutyryl-CoA dehydratase.

2.4. Substrate-Activated Carboxylases Phosphoenolpyruvate (PEP) carboxylase (PEPC, EC 4.1.1.31) catalyzes the irreversible carboxylation of PEP to form oxaloacetate (OAA) using Mg2+ or Mn2+ as a cofactor [50]. This kind of enzyme is present in most photosynthetic organisms [51]. PEPC is used to replenish intermediates of the TCA cycle for amino acid biosynthesis, or to shuttle CO2 between the mesophyll and bundle sheath cells in C4 plants. The catalytic mechanism of PEPC has been well studied (Figure 5). First, bicarbonate act as a nucleophile to attack phosphate groups in PEP, yielding carboxyphosphate and enolates of pyruvate, which is stabilized by metal ions Mn2+. Next, carboxyphosphate decomposes into inorganic phosphate and CO2, which is attacked by enolates of pyruvate to form OAA [52].

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Figure 4. The crotonyl-CoA/ethylmalonyl-CoA/hydroxybutyryl-CoA cycle pathway (CETCH pathway) [21]. CCR, crotonyl-CoA carboxylase/reductase; EEM, ethylmalonyl-CoA epimerase, and mutase; MOX, methylsuccinyl-CoA oxidase; MMD, methylmalyl-CoA dehydratase; MML, methylmalyl-CoA lyase; POX, propionyl-CoA oxidase; MEM, methylmalonyl-CoA epimerase, and mutase; SSDH, succinate semialdehyde dehydrogenase; HBDH, 4-hydroxybutyrate Int. J. Mol. Sci. 2021, 22, 1890 8 of 22 dehydrogenase; HBS, 4-hydroxybutyryl-CoA synthetase; HBD, 4-hydroxybutyryl-CoA dehydratase.

2.4. Substrate-Activated Carboxylases 2.4. Substrate-Activated Carboxylases Phosphoenolpyruvate (PEP) carboxylase (PEPC, EC 4.1.1.31) catalyzes the Phosphoenolpyruvate (PEP) carboxylase (PEPC, EC 4.1.1.31) catalyzes the irreversible irreversible carboxylation of PEP to form oxaloacetate (OAA) using Mg2+ or Mn2+ as a carboxylation of PEP to form oxaloacetate (OAA) using Mg2+ or Mn2+ as a cofactor [50]. cofactor [50]. This kind of enzyme is present in most photosynthetic organisms [51]. PEPC Thisis used kind to of replenish enzyme isintermediates present in mostof the photosynthetic TCA cycle for organismsamino acid [51biosynthesis,]. PEPC is usedor to to replenish intermediates of the TCA cycle for amino acid biosynthesis, or to shuttle CO shuttle CO2 between the mesophyll and bundle sheath cells in C4 plants. The catalytic 2 betweenmechanism the mesophyllof PEPC has and been bundle well sheath studied cells (Figure in C4 plants. 5). First, The bicarbonate catalytic mechanism act as a of PEPCnucleophile has been to attack well studied phosphate (Figure groups5). in First, PEP, bicarbonate yielding carboxyphosphate act as a nucleophile and enolates to attack phosphateof pyruvate, groups which in is PEP,stabilized yielding by metal carboxyphosphate ions Mn2+. Next,and carboxyphosphate enolates of pyruvate, decomposes which 2+ isinto stabilized inorganic by phosphate metal ions and Mn CO2., which Next, is carboxyphosphate attacked by enolates decomposes of pyruvate to into form inorganic OAA phosphate[52]. and CO2, which is attacked by enolates of pyruvate to form OAA [52].

Figure 5. The catalytic mechanism of phosphoenolpyruvate carboxylase [52].

−1 −1 PEPC is known to be one of the most active carboxylases (Kcat/Km = 23,792 s mM )[53]. Based on PEPC, Hong Yu et al. constructed a synthetic malyl-CoA-glycerate (MCG) pathway [19], which is capable of converting one C3 sugar to two acetyl-CoA via ﬁxation of one CO2 equivalent, or assimilating glyoxylate, a photorespiration intermediate, to produce acetyl-CoA without carbon loss (Figure6). Coupling the MCG pathway with the CBB cycle, photosynthetic organisms utilize only 5.5 ATP and 1.5 Rubisco turnovers to produce one acetyl-CoA from CO2 equivalents, while the native pathway requires 7 ATP and 3 Rubisco turnovers. When transferring the MCG pathway into a photosynthetic organism Synechococcus elongates PCC7942, the intracellular acetyl-CoA level increased, and bicarbonate assimilation was improved by roughly 2-fold.

2.5. ThDP-Dependent Carboxylases Pyruvate decarboxylase (PDC, EC 4.1.1.1) is a key enzyme of carbon metabolism at the branching point between aerobic respiration and anaerobic alcoholic fermentation, and can be found in some bacteria, yeasts, and plants [54]. PDC catalyzes the decarboxylation of pyruvate by using ThDP and Mg2+ as cofactors (Figure7). This enzyme has been successfully applied to yield pyruvic acid through the reverse carboxylation reaction. To favor the carboxylation, high pH and high bicarbonate concentration are needed [55]. In addition to using a high concentration of bicarbonate solution as a CO2 source, elevated CO2 pressure is also an effective way to drive the direction of carboxylation. Combining branched-chain α-keto acid decarboxylase (KdcA) from Lactococcus lactis with transaminase or amino acid dehydrogenase, Julia Martin et al. achieved the synthesis of L-methionine from the abundant industrial intermediate methional under a 2 bar CO2 atmosphere [56]. Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 9 of 23

Figure 5. The catalytic mechanism of phosphoenolpyruvate carboxylase [52].

PEPC is known to be one of the most active carboxylases (Kcat/Km = 23,792 s−1 mM−1) [53]. Based on PEPC, Hong Yu et al. constructed a synthetic malyl-CoA-glycerate (MCG) pathway [19], which is capable of converting one C3 sugar to two acetyl-CoA via fixation of one CO2 equivalent, or assimilating glyoxylate, a photorespiration intermediate, to produce acetyl-CoA without carbon loss (Figure 6). Coupling the MCG pathway with the CBB cycle, photosynthetic organisms utilize only 5.5 ATP and 1.5 Rubisco turnovers to produce one acetyl-CoA from CO2 equivalents, while the native pathway requires 7 ATP and 3 Rubisco turnovers. When transferring the MCG pathway into a photosynthetic Int. J. Mol. Sci. 2021, 22, 1890 9 of 22 organism Synechococcus elongates PCC7942, the intracellular acetyl-CoA level increased, and bicarbonate assimilation was improved by roughly 2-fold.

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Lactococcus lactis with transaminase or amino acid dehydrogenase, Julia Martin et al. FigureFigure 6.6. TheThe syntheticsynthetic malyl-CoA-glyceratemalyl-CoA-glycerate (MCG) pathway [19]. [19]. PEPC, PEPC, PEP PEP carboxylase; carboxylase; MDH, MDH, achieved the synthesis of L-methionine from the abundant industrial intermediate malatemalate dehydrogenase; dehydrogenase; MTK, MTK, malate malate thiokinase; thiokinase; MCL, MCL, malyl-CoA malyl-CoA lyase; lyase; GCL, GCL, glyoxylate glyoxylate carboligase; TSR,methionalcarboligase; tartronate under TSR, semialdehyde tartronate a 2 bar CO semialdehyde reductase;2 atmosphere GK, reductase; glycerate [56]. GK, kinase; glycerate ENO, kinase; enolase. ENO, enolase.

reaction. To favor the carboxylation, high pH and high bicarbonate concentration are Figure 7. The catalytic mechanism of pyruvate decarboxylase [54,55]. Figureneeded 7. The [55]. catalytic In addition mechanism to using of pyruvate a high decarboxylase concentration [54,55]. of bicarbonate solution as a CO2 2.6.source, Multi-Enzyme elevated ComplexCO2 pressure Constructed is also Carboxylase an effective way to drive the direction of 2.6.carboxylation. Multi-Enzyme Combining Complex Constructed branched-chain Carboxylase α-keto acid decarboxylase (KdcA) from The glycine cleavage system (GCS) is common among many organisms because of The glycine cleavage system (GCS) is common among many organisms because of its involvement in glycine and serine catabolism [57]. The GCS converts glycine to CO2, its involvement+ in glycine and serine catabolism [57]. The GCS converts glycine to CO2, NH4 , and methylene-THF. GCS is composed of four proteins, a carrier protein, and three NH4+, and methylene-THF. GCS is composed of four proteins, a carrier protein, and three enzymes. They are lipoic acid-containing protein (GcvH), glycine dehydrogenase (GcvP), enzymes. They are lipoic acid-containing protein (GcvH), glycine dehydrogenase (GcvP), aminomethyltransferase (GcvT), and lipoamide dehydrogenase (Lpd), respectively. The aminomethyltransferase (GcvT), and lipoamide dehydrogenase (Lpd), respectively. The glycine cleavage process can be divided into three steps. The ﬁrst step is the decarboxylation glycine cleavage process can be divided into three steps. The first step is the of glycine by the glycine dehydrogenase. The decarboxylated moiety is then further decarboxylation of glycine by the glycine dehydrogenase. The decarboxylated moiety is degraded by the aminomethyl transferase with the aid of tetrahydrofolate. The last step then further degraded by the aminomethyl transferase with the aid of tetrahydrofolate. is the reoxidation of the two sulfhydryl groups to form lipoic acid-generating NADH by The last step is the reoxidation of the two sulfhydryl groups to form lipoic acid-generating dihydrolipomide dehydrogenase. Two sulfhydryl groups or lipoate attached to the lipoic NADHacid-containing by dihydrolipomide protein act as dehydrogenase. intermediate shuttles. Two sulfhydryl groups or lipoate attached to theNow, lipoic GCS acid-containing is conﬁrmed protein to be reversibleact as intermediate (rGCS) and shuttles. can condense the C1 moiety Now, GCS is confirmed to be reversible (rGCS) and can condense the C1 moiety of of methylene-THF with CO2 and ammonia to produce glycine [58], which shows great methylene-THF with CO2 and ammonia to produce glycine [58], which shows great application potential for reductive glycine pathway (RGP). Arren Bar-Even’s research group has made a series of encouraging progress [59–61]. Especially, they redesigned the central carbon metabolism of the model bacterium E. coli for growth on one-carbon compounds (formate and methanol) using the RGP [61] (Figure 8). Recently, Irene Sánchez-Andrea et al. demonstrated that sulfate-reducing bacterium Desulfovibrio desulfuricans (strain G11) could grow autotrophically via the RGP using hydrogen and sulfate as energy substrates [62]. This work first demonstrates that autotrophic microbial growth can be fully supported by RGP, which is a highly ATP-efficient CO2 fixation pathway.

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application potential for reductive glycine pathway (RGP). Arren Bar-Even’s research group has made a series of encouraging progress [59–61]. Especially, they redesigned the central carbon metabolism of the model bacterium E. coli for growth on one-carbon compounds (formate and methanol) using the RGP [61] (Figure8). Recently, Irene S ánchez-Andrea et al. demonstrated that sulfate-reducing bacterium Desulfovibrio desulfuricans (strain G11) could Int. J. Mol. Sci. 2021, 22, x FOR PEERgrow REVIEW autotrophically via the RGP using hydrogen and sulfate as energy substrates11 of 23 [ 62]. This work first demonstrates that autotrophic microbial growth can be fully supported by RGP, which is a highly ATP-efficient CO2 fixation pathway.

FigureFigure 8.8. TheThe reductive glycine pathway pathway (RGP) (RGP) [61]. [61]. FTS, FTS, formyl-THF formyl-THF synthase; synthase; FTCH, FTCH, formyl- formyl-THF cyclohydrolase;THF cyclohydrolase; MTDH, MTDH, methylene-THF methylene-THF dehydrogenase; dehydrogenase; GLYA, GLYA, L-serine L-serine hydroxymethyltransferase; hydroxymethyltransferase; SDAA, L-serine dehydratase. SDAA, L-serine dehydratase.

2.7.2.7. prFMN-DependentprFMN-Dependent Carboxylases Carboxylases prFMN-dependentprFMN-dependent decarboxylasesdecarboxylases catalyze catalyze the non-oxidativethe non-oxidative reversible reversible decarboxyla- tiondecarboxylation of aromatic substrates,of aromatic andsubstrates, play a pivotaland play role a pivotal in bacterial role ubiquinonein bacterial ubiquinone (coenzyme Q) biosynthesis(coenzyme Q) and biosynthesis microbial and biodegradation microbial biod ofegradation aromatic of compounds aromatic compounds [63,64]. The [63,64]. prFMN cofactorThe prFMN is provided cofactor is by provided an associated by an associ prenyltransferaseated prenyltransferase (UbiX), (UbiX), which extendswhich extends the isoal- loxazinethe isoalloxazine FMN ring FMN system ring system through through prenylation prenylation with with a fourth a fourth non-aromatic non-aromatic ring. ring. The iminium catalyticallyThe catalytically active active iminium iminium species species of the cofactorof the cofactor (prFMN (prFMNiminium) is obtained) is obtained by oxidizing by oxidizing the reduced prFMN with O2. There are two different catalytic reaction the reduced prFMN with O2. There are two different catalytic reaction mechanisms for mechanisms for the prFMN-assisted (de)carboxylation reaction. For α, β-unsaturated carboxylic acids, the reaction proceeds through the intermolecular 1, 3-dipolar cycloaddition step. While for protocatechuic acid-type substrates, the electrophilic character of the iminium ion of prFMNiminium enables reversible (de)carboxylation via a mono-covalently bound quinoid–cofactor intermediate. prFMN-dependent decarboxylases encompass a wide range of substrates [17], including non-aromatic α, β- unsaturated (acrylic) acid derivatives, catechol, and 4-hydroxybenzoic acid derivatives,

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the prFMN-assisted (de)carboxylation reaction. For α, β-unsaturated carboxylic acids, the reaction proceeds through the intermolecular 1, 3-dipolar cycloaddition step. While for protocatechuic acid-type substrates, the electrophilic character of the iminium ion of prFMNiminium enables reversible (de)carboxylation via a mono-covalently bound quinoid– cofactor intermediate. prFMN-dependent decarboxylases encompass a wide range of substrates [17], including non-aromatic α, β-unsaturated (acrylic) acid derivatives, catechol, and 4-hydroxybenzoic acid derivatives, polycyclic aromatic hydrocarbons (PAHs), and heterocyclic substrates. Recently, combining ferulic acid decarboxylase (FDC, prFMN- Int. J. Mol. Sci. 2021, 22, x FOR PEER dependent)REVIEW with carboxylic acid reductase (CAR), alcohol dehydrogenase12 of 23 (ADH), or imine reductase (IRED), Godwin A. Aleku et al. designed cascade reactions to enable efficient functionalization of terminal alkenes to the corresponding aldehyde, alcohol, amide or aminepolycyclic derivatives aromatic hydrocarbons through ambient (PAHs), CO and2 fixation heterocyclic [65 ].substrates. Recently, combining ferulic acid decarboxylase (FDC, prFMN-dependent) with carboxylic acid 3.reductase C-C Ligases (CAR), alcohol for Formaldehyde dehydrogenase (ADH), Biotransformation or imine reductase (IRED), Godwin A. Aleku et al. designed cascade reactions to enable efficient functionalization of terminal alkenesFormaldehyde to the corresponding is a cytotoxic aldehyde, compoundalcohol, amide and or aamine central derivatives metabolic through intermediate in methy- lotrophsambient CO [266 fixation]. In addition[65]. to the naturally occurring formaldehyde condensing enzymes, such as dihydroxyacetone synthase (DAS), 3-hexulose-6-phosphate synthase (HPS), and 3. C-C Ligases for Formaldehyde Biotransformation serine hydroxymethyltransferase (SHMT), several promiscuous enzymes display catalytic Formaldehyde is a cytotoxic compound and a central metabolic intermediate in activitymethylotrophs towards [66]. In formaldehyde. addition to the na Basedturally onoccurring the catalytic formaldehyde mechanism, condensingthose enzymes can beenzymes, divided such intoas dihydroxyacetone 4 categories (Figuresynthase 9(DAS),). Class 3-hexulose-6-phosphate I aldolases activate synthase the donor substrate with(HPS), a and lysine serine residue. hydroxymethyltransferase Class II aldolases (SHMT), usea several metal promiscuous cofactor to enzymes facilitate the activation of donordisplay catalytic substrate activity to antowards enolate. formaldehyde. The third Based class on the is hydroxymethylcatalytic mechanism, pyridoxalthose 50-phosphate enzymes can be divided into 4 categories (Figure 9). Class I aldolases activate the donor (PLP)substrate dependent with a lysine enzymes. residue. Class The II aldo PLPlases coenzyme use a metal activates cofactor to the facilitate donor the substrate to form a quinoide-aldimineactivation of donor substrate intermediate. to an enolate. The third last class is ThDP-dependent is hydroxymethyl pyridoxal enzymes. 5′- The ThDP coen- zymephosphate activates (PLP) dependent the donor enzymes. substrate The PLP to coenzyme form an activates enamine/carbanion the donor substrate intermediate. to For the firstform a three quinoide-aldimine classes ofenzymes, intermediate. formaldehyde The last is ThDP-dependent cannot act enzymes. as a donor The ThDP substrate because it is coenzyme activates the donor substrate to form an enamine/carbanion intermediate. For notthe first enolizable. three classes For of enzymes, ThDP-dependent formaldehyde enzymes, cannot act as formaldehyde a donor substrate can because act either as a donor or anit is acceptor.not enolizable. C-C For ligases ThDP-dependent using formaldehyde enzymes, formaldehyde as a donor can act oreither acceptor as a donor were summarized in Tableor an acceptor.2. Furthermore, C-C ligases using the formaldehyde formaldehyde as a assimilation donor or acceptor pathways were summarized published in recent years arein Table highlighted. 2. Furthermore, the formaldehyde assimilation pathways published in recent years are highlighted.

FigureFigure 9. 9.FourFour types types of C-C of Ligases C-C Ligasesfor formaldehyde for formaldehyde biotransformation biotransformation [18]. [18].

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TableTable 2. 2. Summary Summary of of C-C C-C Ligases Ligases with with formaldehyde formaldehyde as as a a donor donor or or acceptor. acceptor. TableTableTableTable 2.2.2. 2. SummarySummarySummary Summary ofofof of C-CC-CC-C C-C LigasesLigasesLigases Ligases withwithwith with formaldehydeformaldehydeformaldehyde formaldehyde asasas as aaa a donordonordonor donor ororor or acceptor.acceptor.acceptor. acceptor. TableTableTable 2. 2. 2.Summary SummarySummary of of of C-C C-C C-C Ligases Ligases Ligases with with with formaldehyde formaldehyde formaldehyde as as as a a adonor donor donor or or or acceptor. acceptor. acceptor. SubstratesSubstrates Product Product Enzyme Enzyme and and Category Category Pathway Pathway SubstratesSubstratesSubstratesSubstrates Product Product Product Product Enzyme Enzyme Enzyme Enzyme andandand and CategoryCategoryCategory Category Pathway Pathway Pathway Pathway Substrates Product Enzyme and Category Pathway OO OOOO 4-hydroxy-2-oxogluta4-hydroxy-2-oxoglutaraterate aldolase aldolase (EC (EC 4-hydroxy-2-oxogluta4-hydroxy-2-oxogluta4-hydroxy-2-oxogluta4-hydroxy-2-oxoglutaraterateraterate aldolasealdolasealdolase aldolase (EC(EC(EC (EC OHOHOHOH OHOH 4-hydroxy-2-oxoglutarate4.1.3.16)4.1.3.16)4.1.3.16)4.1.3.16)4.1.3.16) aldolase (EC / / 4.1.3.16)4.1.3.16) /// // O 4.1.3.16) / OOOOO classclass I Ialdolase aldolase OO classclassclassclassclass I aldolase III aldolaseIaldolasealdolase aldolase pyruvatepyruvatepyruvate pyruvatepyruvatepyruvate

D-Fructose-1,6-bisphosphateD-Fructose-1,6-bisphosphate (FBP) (FBP) D-Fructose-1,6-bisphosphateD-Fructose-1,6-bisphosphateD-Fructose-1,6-bisphosphateD-Fructose-1,6-bisphosphate (FBP)(FBP)(FBP) (FBP) aldolasesaldolases (FruA, (FruA, EC EC 4.1.2.13), 4.1.2.13), and and D-Fructose-1,6-bisphosphatealdolasesaldolasesaldolasesaldolases (FruA,(FruA,(FruA, (FruA, ECECEC EC 4.1.2.13),4.1.2.13),4.1.2.13), 4.1.2.13), (FBP) andandand and aldolases (FruA, EC 4.1.2.13), and TagatoseTagatoseTagatoseTagatoseTagatose 1,6-diphosphate1,6-diphosphate1,6-diphosphate 1,6-diphosphate1,6-diphosphate aldolasealdolasealdolase aldolasealdolase (Tag(Tag(Tag (Tag(Tag / / TagatoseTagatoseTagatose 1,6-diphosphate 1,6-diphosphate 1,6-diphosphate aldolase aldolase aldolase (Tag (Tag (Tag //// // A,A,A, ECEC EC 4.1.2.40):4.1.2.40): 4.1.2.40): A,A,A,A, EC ECEC EC 4.1.2.40): 4.1.2.40):4.1.2.40): 4.1.2.40): class I aldolase classclassclassclassclass III IaldolaseIaldolasealdolase aldolasealdolase classclass I Ialdolase aldolase

D-Fructose-6-phosphateD-Fructose-6-phosphate aldolase aldolase (FSA) (FSA) D-Fructose-6-phosphateD-Fructose-6-phosphateD-Fructose-6-phosphateD-Fructose-6-phosphate aldolasealdolasealdolase aldolase (FSA)(FSA)(FSA) (FSA) D-Fructose-6-phosphate aldolase (FSA) / / //// // classclassclass I aldolase I Ialdolase aldolase classclassclassclass III aldolaseIaldolasealdolase aldolase

D-Fructose-6-phosphateD-Fructose-6-phosphate aldolase aldolase (FSA) (FSA) D-Fructose-6-phosphateD-Fructose-6-phosphateD-Fructose-6-phosphateD-Fructose-6-phosphate aldolasealdolasealdolase aldolase (FSA)(FSA)(FSA) (FSA) D-Fructose-6-phosphate aldolase (FSA) // / //// // classclass I Ialdolase aldolase classclassclassclassclass I aldolase III aldolaseIaldolasealdolase aldolase

HexuloseHexulose phosphate phosphate synthase synthase (HPS, (HPS, EC EC HexuloseHexuloseHexuloseHexulose phosphatephosphatephosphate phosphate synthasesynthasesynthase synthase (HPS,(HPS,(HPS, (HPS, ECECEC EC 4.1.2.43)4.1.2.43) Hexulose phosphate4.1.2.43)4.1.2.43)4.1.2.43)4.1.2.43) synthase (HPS, EC RuMPRuMPRuMP 4.1.2.43) RuMPRuMPRuMPRuMP classclass II II aldolase aldolase classclassclassclassclass II IIaldolaseIIII II aldolasealdolasealdolase aldolase

OOO OOO 2-keto-3-deoxy-L-rhamnonate2-keto-3-deoxy-L-rhamnonate2-keto-3-deoxy-L-rhamnonate aldolasealdolase aldolase 2-keto-3-deoxy-L-rhamnonate2-keto-3-deoxy-L-rhamnonate2-keto-3-deoxy-L-rhamnonate aldolasealdolase aldolase thethethethethe OHOHOHOH thethe OHOH 2-keto-3-deoxy-L-rhamnonate(YfaU,(YfaU,(YfaU,(YfaU,(YfaU, ECECEC ECEC 4.1.2.53)4.1.2.53)4.1.2.53) 4.1.2.53)4.1.2.53) aldolase homoserinehomoserine (YfaU,(YfaU, EC EC 4.1.2.53) 4.1.2.53) thehomoserinehomoserinehomoserinehomoserine O (YfaU, EC 4.1.2.53) OOOOO classclass II II aldolase aldolase cyclecyclecyclecyclecyclecycle OO classclassclassclassclass II IIaldolaseIIII II aldolasealdolasealdolase aldolase cyclecycle pyruvatepyruvatepyruvate pyruvatepyruvatepyruvate

SerineSerine hydroxymethyltransferase hydroxymethyltransferase serineserineserineserineserine cyclecyclecycle cyclecycle SerineSerineSerineSerine hydroxymethyltransferasehydroxymethyltransferasehydroxymethyltransferase hydroxymethyltransferase serineserine cycle cycle Serine hydroxymethyltransferase(SHMT,(SHMT,(SHMT,(SHMT, ECEC ECEC 2.1.2.1)2.1.2.1) 2.1.2.1)2.1.2.1) (SHMT,(SHMT,(SHMT,(SHMT, ECEC ECEC 2.1.2.1)2.1.2.1) 2.1.2.1)2.1.2.1) serine cycleandandandand and (SHMT, EC 2.1.2.1) andand PLP-dependentPLP-dependentPLP-dependent aldolasealdolase aldolase RGP PLP-dependentPLP-dependentPLP-dependentPLP-dependent aldolase aldolasealdolase aldolase RGPRGPRGPRGP RGPRGPRGP

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Table 2. Cont.

Substrates Product Enzyme and Category Pathway MSHMTMSHMT for for D-alanine. D-alanine. α α-Methylserine-Methylserine MSHMTMSHMT for for D-alanine. D-alanine. α α-Methylserine-Methylserine-Methylserine aldolasealdolase for for MSHMT for D-alanine.aldolasealdolaseα -Methylserinefor for aldolase for / / D-alanineD-alanine and and D-butanine D-butanine /// / D-alanineD-alanineD-alanine and and and D-butanine D-butanine D-butanine PLP-dependent aldolase PLP-dependentPLP-dependent aldolase aldolase PLP-dependentPLP-dependent aldolase aldolase

DihydroxyacetoneDihydroxyacetone synthase synthase (DAS, (DAS, EC EC DihydroxyacetoneDihydroxyacetone synthase synthase (DAS, (DAS, EC EC Dihydroxyacetone synthase (DAS, EC 2.2.1.3)2.2.1.3) XuMPXuMP 2.2.1.3)2.2.1.3) XuMPXuMPXuMP ThDP-dependentThDP-dependentThDP-dependent C-C C-C C-C Ligase Ligase Ligase ThDP-dependentThDP-dependent C-C C-C Ligase Ligase

FormolaseFormolase (FLS) (FLS) FLSFLS FormolaseFormolase (FLS) (FLS) FLSFLS Formolase (FLS) FLS ThDP-dependentThDP-dependentThDP-dependent C-C C-C C-C Ligase Ligase Ligase pathwaypathwaypathway ThDP-dependentThDP-dependent C-C C-C Ligase Ligase pathwaypathway

GlycolaldehydeGlycolaldehyde synthase synthase (GALS) (GALS) SACASACA GlycolaldehydeGlycolaldehyde synthase synthase (GALS) (GALS) SACASACA Glycolaldehyde synthase (GALS) SACA ThDP-dependentThDP-dependent C-C C-C Ligase Ligase pathwaypathway ThDP-dependentThDP-dependentThDP-dependent C-C C-C C-C Ligase Ligase Ligase pathwaypathwaypathway

2-hydroxyacyl2-hydroxyacyl CoA CoA lyase lyase (HACL) (HACL) 2-hydroxyacyl2-hydroxyacyl CoA CoA lyase lyase (HACL) (HACL) / / 2-hydroxyacyl CoA lyase (HACL) // / ThDP-dependentThDP-dependent C-C C-C Ligase Ligase / ThDP-dependentThDP-dependentThDP-dependent C-C C-C C-C Ligase Ligase Ligase

3.1.3.1. Class Class I IAldolases Aldolases for for Formaldehyde Formaldehyde Biotransformation Biotransformation 3.1.3.1.3.1. Class Class Class I IAldolases IAldolases Aldolases for for for Formaldehyde Formaldehyde Formaldehyde Biotransformation Biotransformation Biotransformation NaturalNatural class class I Ialdolases aldolases are are generally generally pr promiscuous.omiscuous. It It has has been been confirmed confirmed that that NaturalNaturalNatural class classclass I I I aldolasesaldolases aldolases areare are generally generally generally promiscuous. pr promiscuous.omiscuous. It hasIt It has beenhas been confirmedbeen confirmed confirmed that several that that severalseveral class class I Ialdolases aldolases can can tolerate tolerate formaldehyde formaldehyde as as an an acceptor acceptor substrate. substrate. From From the the severalseveralclass Iclass aldolasesclass I Ialdolases aldolases can tolerate can can tolerate tolerate formaldehyde formaldehyde formaldehyde as an acceptor as as an an acceptor acceptor substrate. substrate. substrate. Fromthe From From perspec- the the perspectiveperspective of of biocatalysis, biocatalysis, th the epromiscuity promiscuity of of class class I Ialdolases aldolases provides provides solutions solutions for for perspectiveperspectivetive of biocatalysis, of of biocatalysis, biocatalysis, the promiscuity th the epromiscuity promiscuity of class of Iof aldolases class class I Ialdolases providesaldolases provides solutionsprovides solutions forsolutions producing for for producingproducing valuable valuable chemicals chemicals from from formaldehyde. formaldehyde. The The following following are are examples examples of of class class I I producingproducingvaluable chemicalsvaluable valuable chemicals fromchemicals formaldehyde. from from formaldehyde. formaldehyde. The following The The arefollowing following examples are are of examples classexamples I aldolases of of class class cat- I I aldolasesaldolases catalyzing catalyzing formaldehyde formaldehyde as as a areceptor. receptor. 4-hydroxy-2-oxoglutarate 4-hydroxy-2-oxoglutarate aldolase aldolase (EC (EC aldolasesaldolasesalyzing formaldehydecatalyzing catalyzing formaldehyde formaldehyde as a receptor. as as 4-hydroxy-2-oxoglutarate a areceptor. receptor. 4-hydroxy-2-oxoglutarate 4-hydroxy-2-oxoglutarate aldolase (EC 4.1.3.16,aldolase aldolase class(EC (EC I 4.1.3.16,4.1.3.16, class class I Ialdolase) aldolase) can can catalyze catalyze the the aldol aldol addition addition of of pyruvic pyruvic acid acid or or phenylpyruvic phenylpyruvic 4.1.3.16,4.1.3.16,aldolase) class class can I I catalyzealdolase) aldolase) the can can aldol catalyze catalyze addition the the aldol of aldol pyruvic addition addition acid of orof pyruvic phenylpyruvicpyruvic acid acid or or phenylpyruvic acid phenylpyruvic to formalde- acidacid toto formaldehydeformaldehyde [18].[18]. DHAP-dependentDHAP-dependent aldolases,aldolases, includingincluding D-Fructose-1,D-Fructose-1, 6-6- acidacidhyde toto [18 formaldehydeformaldehyde]. DHAP-dependent [18].[18]. DHAP-dependentDHAP-dependent aldolases, including aldolases,aldolases, D-Fructose-1, includingincluding 6-bisphosphate D-Fructose-1,D-Fructose-1, (FBP) 6-6- bisphosphatebisphosphate (FBP) (FBP) aldolases aldolases (FruA, (FruA, EC EC 4.1.2.13), 4.1.2.13), and and tagatose tagatose 1, 1, 6-diphosphate 6-diphosphate aldolase aldolase bisphosphatebisphosphatealdolases (FruA, (FBP) (FBP) EC aldolases aldolases 4.1.2.13), (FruA, (FruA, and tagatose EC EC 4.1.2.13), 4.1.2.13), 1, 6-diphosphate and and tagatose tagatose aldolase 1, 1, 6-diphosphate 6-diphosphate (TagA, EC aldolase aldolase 4.1.2.40), (TagA,(TagA, EC EC 4.1.2.40), 4.1.2.40), can can catalyze catalyze the the aldol aldol addition addition of of dihydroxyacetone dihydroxyacetone phosphate phosphate (TagA,(TagA,(TagA,can catalyze EC ECEC 4.1.2.40), 4.1.2.40),4.1.2.40), the aldol can cancan addition catalyze catalyzecatalyze ofthe thethe dihydroxyacetone aldol aldolaldol addition additionaddition of of phosphateof dihydroxyacetone dihydroxyacetonedihydroxyacetone (DHAP) tophosphate phosphatephosphate formalde- (DHAP)(DHAP) to to formaldehyde formaldehyde [67,68]. [67,68]. D-Fructose-6-phosphate D-Fructose-6-phosphate aldolase aldolase (FSA, (FSA, EC EC 4.1.2.n) 4.1.2.n) can can (DHAP)(DHAP)(DHAP)hyde [ 67 toto to, 68formaldehydeformaldehyde formaldehyde]. D-Fructose-6-phosphate [67,68].[67,68]. [67,68]. D-Fructose-6-phosphateD-Fructose-6-phosphate D-Fructose-6-phosphate aldolase (FSA, EC aldolasealdolase aldolase 4.1.2.n) (FSA,(FSA, can (FSA, catalyze ECEC EC 4.1.2.n)4.1.2.n) 4.1.2.n) the aldolcancan can catalyzecatalyze the the aldol aldol addition addition of of dihydroxya dihydroxyacetonecetone (DHA), (DHA), hydrox hydroxyacetoneyacetone (HA), (HA), and and catalyzecatalyzeaddition the the of dihydroxyacetonealdol aldol addition addition of of (DHA),dihydroxya dihydroxya hydroxyacetonecetonecetone (DHA), (DHA), (HA), hydrox hydrox and glycolaldehydeyacetoneyacetone (HA), (HA), (GA) and and to glycolaldehydeglycolaldehyde (GA) (GA) to to formaldehyde formaldehyde [69]. [69]. glycolaldehydeglycolaldehydeformaldehyde (GA) [(GA)69]. to to formaldehyde formaldehyde [69]. [69]. 3.2.3.2. Class Class II II Aldolases Aldolases for for Formaldehyde Formaldehyde Biotransformation Biotransformation 3.2.3.2.3.2. Class Class Class II II IIAldolases Aldolases Aldolases for for for Formaldehyde Formaldehyde Formaldehyde Biotransformation Biotransformation Biotransformation HexuloseHexulose phosphate phosphate synthase synthase (HPS, (HPS, EC EC 4.1.2.43) 4.1.2.43) is is a aclass class II II aldolase aldolase found found in in aerobic aerobic HexuloseHexuloseHexulose phosphate phosphate phosphate synthase synthase synthase (HPS, (HPS, (HPS, EC EC EC4.1.2.43) 4.1.2.43) 4.1.2.43) is is a a isclass class a class II II aldolase aldolase II aldolase found found found in in aerobic aerobic in aer- methylotrophicmethylotrophic bacteria bacteria and and is is involved involved in in the the ribulose ribulose monophosphate monophosphate (RuMP) (RuMP) cycle. cycle. It It methylotrophicmethylotrophicobic methylotrophic bacteria bacteria bacteria and and is is involved andinvolved is involved in in the the ribulose ribulose in the monophosphate ribulose monophosphate monophosphate (RuMP) (RuMP) cycle. cycle. (RuMP) It It catalyzescatalyzes D-ribulose D-ribulose 5-phosphate 5-phosphate with with formaldehyde formaldehyde to to yield yield D-arabinose D-arabinose 3-hexulose 3-hexulose 6- 6- catalyzescatalyzescycle. It D-ribulose D-ribulose catalyzes 5-phosphate D-ribulose 5-phosphate 5-phosphate with with formaldehyde formaldehyde with formaldehyde to to yield yield D-arabinose D-arabinose to yield 3-hexulose D-arabinose 3-hexulose 6- 6- 3- phosphate.phosphate.hexulose 6-phosphate.Due Due to to strict strict Duesubstrate substrate to strict specificity, specificity, substrate it it specificity,is is difficult difficult itto to isuse use difficult hexulose hexulose to use phosphate phosphate hexulose phosphate.phosphate. Due Due to to strict strict substrate substrate specificity, specificity, it it is is difficult difficult to to use use hexulose hexulose phosphate phosphate synthasesynthasephosphate toto synthase synthesizesynthesize to synthesize value-addedvalue-added value-added chirchiralal products.products. chiral products. While,While, byby While, combiningcombining by combining the the non-non- the synthasesynthase to to synthesize synthesize value-added value-added chir chiralal products. products. While, While, by by combining combining the the non- non- oxidativeoxidativenon-oxidative glycolysis glycolysis glycolysis (NOG) (NOG) (NOG)with with the the with RuMP, RuMP, the Igor RuMP,Igor W. W. Bogorad IgorBogorad W. et Bogorad et al. al. constructed constructed et al. constructed a amethanol methanol a oxidativeoxidative glycolysis glycolysis (NOG) (NOG) with with the the RuMP, RuMP, Igor Igor W.W. W. BogoradBogorad Bogorad etet et al.al. al. constructedconstructed constructed aa a methanolmethanol methanol condensationcondensationmethanol condensation cycle cycle (MCC) (MCC) cycle [70], [70], (MCC)which which can [can70 ],convert convert which methanol can methanol convert to to methanolhigher-chain higher-chain to higher-chainalcohols alcohols or or condensationcondensation cycle cycle (MCC) (MCC) [70], [70], which which can can convert convert methanol methanol to to higher-chain higher-chain alcohols alcohols or or otherotheralcohols acetyl-CoA acetyl-CoA or other derivatives acetyl-CoAderivatives using derivativesusing enzymatic enzymatic using reactions reactions enzymatic in in a reactions acarbon-conserved carbon-conserved in a carbon-conserved and and ATP- ATP- otherother acetyl-CoA acetyl-CoA derivatives derivatives using using enzymatic enzymatic reactions reactions in in a acarbon-conserved carbon-conserved and and ATP- ATP- independentindependent system system (Figure (Figure 10). 10). It It is is generally generally believed believed that that the the RuMP RuMP cycle cycle is is the the most most independentindependentindependent systemsystem system (Figure(Figure (Figure 10).10). 10). ItIt It isis is generallygenerally generally believedbelieved believed thatthat that thethe the RuMPRuMP RuMP cyclecycle cycle isis is thethe the mostmost most efficientefficient naturally naturally occurring occurring route route for for meth methanolanol assimilation. assimilation. However, However, realizing realizing the the efficientefficient naturally naturally occurring occurring route route for for meth methanolanol assimilation. assimilation. However, However, realizing realizing the the

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and ATP-independent system (Figure 10). It is generally believed that the RuMP cycle is the most efﬁcient naturally occurring route for methanol assimilation. However, realizing heterogeneousthe heterogeneous construction construction of the of theRuMP RuMP cycle cycle is a is challenging a challenging task. task. Recently, Recently, a series a series of progressof progress has has been been made made [71,72]. [71,72 ].By By using using metabolic metabolic robustness robustness criteria criteria followed by laboratorylaboratory evolution,evolution, FredericFrederic Y.-H. Y.-H. Chen Chen et et al. al. enabled enabled the the growth growth of theof the engineered engineeredE. coli E. coliwith with methanol methanol as the as solethe sole carbon carbon source source [72]. [72].

Figure 10.10. The methanol condensation cycle (MCC)(MCC) [[70].70]. HPS, hexulose phosphate synthase;synthase; PHI,PHI, phosphohexulosephosphohexulose isomerase; TAL, transaldolase;transaldolase; TKT, transketolase;transketolase; RPE, D-ribuloseD-ribulose 5-phosphate5-phosphate epimerase;epimerase; RPI,RPI, Ribose-5-phosphateRibose-5-phosphate isomerase; XPK, phosphoketolase;phosphoketolase; PTA, phosphatephosphate acetyltransferase.acetyltransferase.

Another class II aldolase, 2-keto-3-deoxy-L-rhamnonate aldolase (YfaU, EC 4.1.2.53), can also catalyze pyruvate with formaldehydeformaldehyde to generate 4-hydroxy-2-oxobutanoate.4-hydroxy-2-oxobutanoate. Combining YfaU with (S)-or (R)-selective transaminases, Karel Hernandez et al. achieved thethe stereoselectivestereoselective synthesis synthesis of (S)-and of (S)-an (R)-homoserined (R)-homoserine with high with yield fromhigh formaldehydeyield from formaldehydeand alanine [73 ].and Relying alanine on the [73]. aldol Relying reaction on of pyruvatethe aldol with reaction formaldehyde of pyruvate as a superior with formaldehydesynthetic pathway as a design,superior Hai synthetic He et al. pathwa constructedy design, the Hai homoserine He et al. cycle constructed [74], which the homoserinecan assimilate cycle two [74], molecules which can of formaldehyde assimilate two or molecules methanol of to formaldehyde generate one or molecule methanol of toacetyl-CoA generate (Figureone molecule 11). Compared of acetyl-CoA to the RuMP (Figure cycle, 11). theCompared homoserine to the cycle RuMP support cycle, higher the homoserinetheoretical yields cycle of support products higher that are theoretical derived from yields acetyl-CoA, of products including that are ethanol, derived acetone, from acetyl-CoA,butyrate, butanol, including citrate, ethanol, itaconate, acetone, 2-ketoglutarate, butyrate, and butanol, levulinic acid.citrate, itaconate, 2- ketoglutarate, and levulinic acid.

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Figure 11.11. TheThe homoserine homoserine cycle cycle [74 [74].]. SAL, SAL, serine serine aldolase; aldolase; SDA, SDA, serine serine deaminase; deaminase; HAL, HAL, 4-hydroxy-2-oxobutanoate 4-hydroxy-2-oxobutanoate (HOB) aldolase;(HOB) aldolase; HAT, HOB HAT, aminotransferase; HOB aminotransferase; HSK, homoserine HSK, homoserine kinase; TS,kinase; threonine TS, threonine synthase; synthase; LTA, threonine LTA, threonine aldolase; aldolase; ACDH, acetylatingACDH, acetylating acetaldehyde acetaldehyde dehydrogenase. dehydrogenase. Both SAL Both and SAL LTA and are catalyzedLTA are ca bytalyzed the same by the LtaE same enzyme. LtaE enzyme.

3.3. PLP-Dependent Aldolases for Formaldehyde Biotransformation Serine hydroxymethyltransferase (SHMT, EC 2.1.2.1) belongs toto thethe PLP-dependentPLP-dependent aldolase familyfamily and and is is involved involved in serinein serine cycle cycle and reductiveand reductive glycine glycine pathway pathway (RGP) [75(RGP),76]. The[75,76]. natural The serinenatural cycle serine pathway cycle pathway found in Methylobacteriumfound in Methylobacterium extorquens extorquens AM1 can assimilate AM1 can oneassimilate molecule one of molecule formaldehyde of formaldehyde or methanol or and methanol one molecule and one of molecule bicarbonate of bicarbonate into acetyl- CoA.into acetyl-CoA. Hong Yu et Hong al. constructed Yu et al. aconstruct modiﬁeded serine a modified cycle [77 serine], which cycle uses [77], formaldehyde which uses dehydrogenaseformaldehyde dehydrogenase (Faldh) to simplify (Faldh) the oxidationto simplify of formaldehydethe oxidation toof formate,formaldehyde and also to utilizeformate, the and combination also utilize ofthe alanine-glyoxylate combination of alanine-glyoxylate transaminase and transaminase serine dehydratase and serine to avoiddehydratase hydroxypyruvate to avoid hydroxypyruvate as an intermediate as in an the intermediate conversion from in the glyoxylate conversion to PEP. from By utilizingglyoxylate the to modiﬁed PEP. By utilizing serine cycle, the theymodified achieved serine the cycle, conversion they achieved of methanol the conversion to ethanol inof anmethanol engineered to ethanolE. coli instrain. an engineered E. coli strain. Similar toto SHMT,SHMT, there there are are two two types types of of PLP-dependent PLP-dependent enzymes enzymes that that can can achieve achieve the α synthesisthe synthesis of unnatural of unnatural amino acids.amino -methylserineacids. α-methylserine hydroxymethyltransferase hydroxymethyltransferase (MSHMT, EC 2.1.2.7) catalyzes the formation of α-methyl-L-serine from D-alanine and formalde- (MSHMT, EC 2.1.2.7) catalyzes the formation of α-methyl-L-serine from D-alanine and hyde [78]. α-Methylserine aldolase can achieve the enantioselective formation of α-methyl- formaldehyde [78]. α-Methylserine aldolase can achieve the enantioselective formation of L-serine and α-ethyl-L-serine from D-alanine and D-butanine with formaldehyde [79]. α-methyl-L-serine and α-ethyl-L-serine from D-alanine and D-butanine with Different from SHMT and MSHMT, α-Methylserine aldolase is tetrahydrofolate (THF)- formaldehyde [79]. Different from SHMT and MSHMT, α-Methylserine aldolase is independent in vitro. tetrahydrofolate (THF)-independent in vitro. 3.4. ThDP-Dependent C-C Ligases for Formaldehyde Biotransformation 3.4. ThDP-Dependent C-C Ligases for Formaldehyde Biotransformation Dihydroxyacetone synthase (DAS, EC 2.2.1.3) belongs to the ThDP-dependent en- zymesDihydroxyacetone and is involved synthase in xylulose (DAS, monophosphate EC 2.2.1.3) pathwaybelongs to (XuMP). the ThDP-dependent It catalyzes D- xyluloseenzymes 5-phosphateand is involved with in formaldehyde xylulose mono to yieldphosphate D-glyceraldehyde pathway (XuMP). 3-phosphate It catalyzes and dihy- D- droxyacetonexylulose 5-phosphate [80]. The with catalytic formaldehyde process can to be yield described D-glyceraldehyde as follows: The 3-phosphate activated ThDP and (C2dihydroxyacetone of the ThDP is deprotonated)[80]. The catalytic attack process the carbonyl can be group described of D-xylulose as follows: 5-phosphate, The activated and thenThDP the (C2 bond of the between ThDP C-2 is anddeprotonated) C-3 of D-xylulose attack 5-phosphatethe carbonyl break group with of theD-xylulose assistance 5- ofphosphate, basic residues, and then generating the bond 2-betweenα, β-dihydroxyethylidene-THDP C-2 and C-3 of D-xylulose 5- (DHETHDP,phosphate break enamine with orthe carbanion assistance intermediate). of basic DHETHDPresidues, generating reacts with formaldehyde2-α, β-dihydroxyethylidene-THDP to gain dihydroxyacetone.(DHETHDP, Currently, enamine DAS is or not carbanion exploited intermediat as a biocatalyst.e). DHETHDP However, reactsPichia with pastoris formaldehyde, a kind of

Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 17 of 23

Int. J. Mol. Sci. 2021, 22, 1890 16 of 22 to gain dihydroxyacetone. Currently, DAS is not exploited as a biocatalyst. However, Pichia pastoris, a kind of methylotrophic yeast, have been engineered to produce chemicals from methanol using XuMP [81]. Similar to DAS, transketolase (TK, EC 2.2.1.1) can catalyzemethylotrophic the reaction yeast, of have D-fructose-6-pho been engineeredsphate to produce or L-sorbose chemicals with from formaldehyde methanol using to generateXuMP [dihydroxyacetone81]. Similar to DAS, [82]. transketolase (TK, EC 2.2.1.1) can catalyze the reaction of D- fructose-6-phosphateThe benzaldehyde or lyase L-sorbose (BAL, withEC 4.1.2.38) formaldehyde catalyzes to generatethe reversible dihydroxyacetone ligation of two [82 ]. benzaldehydeThe benzaldehyde to yield an lyase (R)-benzoin (BAL, EC [83] 4.1.2.38). When catalyzes formaldehyde the reversible acts as ligation a receptor, of two benzaldehydebenzaldehyde first to yieldreacts an with (R)-benzoin formaldehy [83].de. When However, formaldehyde wild-type acts BAL as was a receptor, shown ben-to catalyzezaldehyde the ﬁrstoligomerization reacts with formaldehyde.of formaldehyde However, into glycolaldehyde wild-type BAL and was dihydroxyacetone shown to catalyze withthe oligomerizationlow activity. Justin of formaldehyde B. Siegel et al into. engineered glycolaldehyde the BAL and dihydroxyacetonethrough computationally with low activity. Justin B. Siegel et al. engineered the BAL through computationally design and design and gained formolase (FLS) with high formaldehyde catalytic activity [22]. By gained formolase (FLS) with high formaldehyde catalytic activity [22]. By combining FLS combining FLS with acetyl-CoA synthase (ACS), acetaldehyde dehydrogenase (ACDH), with acetyl-CoA synthase (ACS), acetaldehyde dehydrogenase (ACDH), and dihydrox- and dihydroxyacetone kinase (DHAK), they created an FLS pathway, which assimilates yacetone kinase (DHAK), they created an FLS pathway, which assimilates formate into formate into dihydroxyacetone phosphate with four steps (Figure 12A). Compared with dihydroxyacetone phosphate with four steps (Figure 12A). Compared with the natural the natural carbon fixation pathway, the FLS pathway has obvious advantages in chemical carbon ﬁxation pathway, the FLS pathway has obvious advantages in chemical driving driving force, biomass yield, and reaction conditions (aerobic). force, biomass yield, and reaction conditions (aerobic).

FigureFigure 12. 12. TheThe formolase formolase (FLS) (FLS) pathway pathway [22] [22] and and synthetic synthetic acetyl-CoA (SACA) pathwaypathway [23[23].]. ((AA)) The Theformolase formolase (FLS) (FLS) pathway. pathway. ACS, ACS, acetyl-CoA acetyl-CoA synthase; synthase; ACDH, ACDH, acetaldehyde acetaldehyde dehydrogenase;dehydrogenase; FLS, FLS,formolase; formolase; DHAK, DHAK, dihydroxyacetone dihydroxyacetone kinase. kinase. (B) ( TheB) The synthetic synthetic acetyl-CoA acetyl-CoA (SACA) (SACA) pathway. pathway. GALS, GALS,glycolaldehyde glycolaldehyde synthase; synthase; ACPS, ACPS, acetyl-phosphate acetyl-phosphate synthase; synthase; PTA, phosphatePTA, phosphate acetyltransferase. acetyltransferase. Benzoylformate decarboxylase (BFD, EC 4.1.1.7) is also a ThDP-dependent enzyme thatBenzoylformate catalyzes the conversion decarboxylase of benzoylformate (BFD, EC 4.1.1.7) to benzaldehydeis also a ThDP-dependent and CO2 [84 enzyme]. When thatformaldehyde catalyzes the is theconversion only substrate, of benzoylformate trace amounts to of benzaldehyde glycolaldehyde and can CO be2detected. [84]. When Our research group engineered the BFD through directed evolution and gained glycolaldehyde synthase (GALS) with high catalytic activity for formaldehyde [23]. Different from FLS, GALS enables synthesis of glycolaldehyde even under high formaldehyde concentration Int. J. Mol. Sci. 2021, 22, 1890 17 of 22

conditions. By combining GALS with repurposed phosphoketolase (ACPS), and phosphate acetyltransferase (PTA), we constructed a synthetic acetyl-CoA (SACA) pathway, which assimilates formaldehyde into acetyl-CoA with only three steps (Figure 12B). Compared to the FLS pathway, SACA has obvious advantages in the synthesis of acetyl-CoA derivatives. When dihydroxyacetone phosphate (C3), the product of the FLS pathway, is converted to acetyl-CoA, one carbon is lost as CO2. Although both the FLS pathway and SACA pathway have shown great potential in assimilating C1 compounds, the low activity and afﬁnity of FLS and GALS towards formaldehyde restrict their further application in vivo. Recently, Alexander Chou et al. reported that 2-hydroxyacyl CoA lyase (HACL) could catalyze the ligation of formyl-CoA with formaldehyde to produce glycolyl-CoA [85]. Compared to FLS and GALS, HACL shows higher catalytic activity and afﬁnity towards formaldehyde. Although they mainly focus on the bioconversion of formaldehyde to glycolate, perhaps this alternative pathway will be available in vivo in the future.

4. C-C Ligases for CO and Formate Biotransformation CO and formate are relatively inert compounds, and few enzymes can catalyze the C-C forming with them. However, they are ideal electron donors [86], which can be easily oxidized to CO2 and reduce power. The reducing power can support CO2 ﬁxation and serves to provide the cell with energy. Several microbes can grow on formate or CO as the sole carbon source based on this strategy. CO is a toxic and ﬂammable gas with low solubility, while formic acid is readily soluble and of low toxicity. So formic acid is a preferred carbon source and mediator of electrons.

4.1. C-C Ligases for CO Biotransformation Diverse microbes can grow on CO as the sole carbon source, including anaerobes, such as Moorella thermoacetica, some purple sulfur bacteria akin to Rhodospirillum rubrum, and Carboxydothermus hydrogenoformans, as well as some aerobic carboxydobacteria like Oligotropha carboxidovorans [87]. For aerobes, CO is first oxidized to CO2 by Mo-Cu−CODH (CO dehydrogenase), and then CO2 is fixed by the CBB cycle. While for anaerobic microbes, CO is oxidized to CO2 by Ni−CODH, and the CO2 is then fixed by the WL pathway. Only CO dehydrogenase/acetyl-CoA-synthase complex existing in WL pathway can directly achieve the C-C extension [88]. CO dehydrogenase (CODH) reversibly catalyzes CO2 reduction into CO, and then acetyl-CoA synthase (ACS) catalyzes the condensation of in situ generated CO with CoA and a methyl group bound to the cobalt center in a B12- containing protein to generate acetyl-CoA. The WL pathway is not only a predominant CO2 sink under anaerobic conditions, but also used to synthesize desired products, such as ethanol and 2, 3-butanediol from industrial waste gases (mainly CO, CO2,H2)[89]. In addition to chemical energy, several anaerobic bacteria, such as Sporomusa ovata, and Moorella thermoacetica, can employ light energy to enable the photosynthesis of acetic acid from CO2 [90–92].

4.2. C-C Ligases for Formate Biotransformation Similar to CO assimilation, the CBB cycle and WL pathways are two carbon-fixation pathways known to support formatotrophic growth (i.e., growth on formate) by full oxidation of formate [93]. Different from CO, formate can not only be oxidized to CO2 to achieve carbon fixation, but also can be reduced to methylene-THF in vivo. The serine pathway in methylotrophic organisms is also known to support formatotrophic growth [94,95]. The metheylene-THF can spontaneously release formaldehyde, so RuMP, XuMP, RGP, FLS, and SACA pathways are, thus, highly promising for supporting formatotrophic growth. For- mate is a relatively strong acid (pKa = 3.75), and normally it is deprotonated. So formate is a much poorer electrophile than CO2 [88]. The direct formate-fixing reactions are rare. Only one formate-fixing reaction catalyzed by pyruvate formate-lyase (PFL) is confirmed. This enzyme is mostly known to support pyruvate cleavage, producing an extra ATP molecule during anaerobic sugar fermentation. Now the reversibility of PFL has been demonstrated. Int. J. Mol. Sci. 2021, 22, 1890 18 of 22

Especially, the enzyme was shown to achieve the condensation of acetyl-CoA and formate in vivo [96], supporting efﬁcient growth of E. coli on acetate and formate. The catalytic mechanism of PFL is a radical process, in which a single electron is extracted from formate by cysteine radical. The resulting formyl radical then attacks an enzyme-bound acetate moiety to generate a pyruvyl radical, which is released as pyruvate.

5. Conclusions Newly mined or designed carboxylases and C-C ligases not only greatly expand the scope of C1 transformation in vitro, but also are expected to improve the efficiency of C1 assimilation in vivo. For example, mined prFMN-dependent (de)carboxylases enable efficient functionalization of terminal alkenes to the corresponding aldehyde, alcohol, amide, or amine derivatives through ambient CO2 fixation [65]. The reversible glycine cleavage system (GCS) enables the growth of E. coli on one-carbon compounds (formate and methanol) using the reductive glycine pathway, which is theoretically the most efficient route for formate assimilation to date [61]. With the development of synthetic biology, we believe that more and more carboxylases and C-C ligases are being mined or designed. Compared to C1 transformation in vitro, constructing an efficient C1 assimilation pathway is more desirable. Since an efficient C1 assimilation pathway will be possible to greatly reduce industrial production cost and also alleviate the pressure of resource supplement for bio-manufacturing in the future. It is encouraging that the constructed CETCH pathway based on crotonyl-CoA carboxylase/reductase, can use light energy to produce multi-carbon molecule glycolate from CO2, while also phosphorylating ADP to ATP. Although several artificial pathways, such as the homoserine cycle, formolase pathway (FLS), and synthetic acetyl-CoA (SACA) pathway, show great application potential, model strains (E. coli) that integrate these artificial pathways have low growth efficiency when C1 compounds is used as carbon source or energy source. Of course, a lot of work needs to be done to make these artificial pathways truly applied to industrial C1 biotransformation. C1 compounds (CO2, HCOOH, HCHO, CH3OH, and CH4) can be interconverted. In methanotrophs, CH4 can be continuously oxidized to CO2 and then assimilated through the CBB cycle. In methanogens, CO2 can be continuously reduced to CH4 [97]. CO2 also can be continuously reduced to CH3OH in vitro [98]. Therefore, constructing a new assimilation pathway based on any of the C1 compounds can theoretically be used to assimilate other C1 compounds. However, among C1 compounds, only CO2 and formaldehyde are good candidate substrates for exploiting carboxylases and C-C ligases. For the CO2 assimilation pathway, the energy source is an issue that must be considered. Both electric energy and light energy may be ideal energy sources. Recently, the semiconductor–bacteria biohybrid photosynthetic system was reported to efficiently realize the synthesis of acetic acid from CO2 with the non-photosynthetic bacteria [90]. Formaldehyde exhibits high and versatile reactivity, in comparison to other C1 compounds. However, formaldehyde is toxic to the cell, therefore, the mined or designed C-C ligases must have high activity and affinity towards formaldehyde.

Author Contributions: Q.Y. and X.G. reviewed related studies and wrote the first draft; H.J. and Y.L. reviewed and edited the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the National Key R&D Program of China (Grant No. 2018YFA0901600), Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project (Grant No. TSBICIP-KJGG-007) and the Drug Innovation Major Project (2018ZX09711001-006-003). Conflicts of Interest: The authors declare no conflict of interest.

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