Strategy for the Biosynthesis of Short Oligopeptides: Green and Sustainable Chemistry

biomolecules

Review Strategy for the Biosynthesis of Short Oligopeptides: Green and Sustainable Chemistry

, Tao Wang † * , Yu-Ran Zhang †, Xiao-Huan Liu, Shun Ge and You-Shuang Zhu * School of Biological Science, Jining Medical University, Jining 272000, China; [email protected] (Y.-R.Z.); [email protected] (X.-H.L.); [email protected] (S.G.) * Correspondence: [email protected] (T.W.); [email protected] (Y.-S.Z.) These authors contributed equally to this manuscript. † Received: 1 October 2019; Accepted: 7 November 2019; Published: 13 November 2019

Abstract: Short oligopeptides are some of the most promising and functionally important amide bond-containing components, with widespread applications. Biosynthesis of these oligopeptides may potentially become the ultimate strategy because it has better cost efficiency and environmental-friendliness than conventional solid phase peptide synthesis and chemo-enzymatic synthesis. To successfully apply this strategy for the biosynthesis of structurally diverse amide bond-containing components, the identification and selection of specific biocatalysts is extremely important. Given that perspective, this review focuses on the current knowledge about the typical enzymes that might be potentially used for the synthesis of short oligopeptides. Moreover, novel enzymatic methods of producing desired peptides via metabolic engineering are highlighted. It is believed that this review will be helpful for technological innovation in the production of desired peptides.

Keywords: short oligopeptides; biosynthesis; non-ribosomal peptide synthesis; ATP-grasp enzyme; β-lactam; cyclic dipeptides; metabolic engineering

1. Introduction Short oligopeptides, especially l-α-dipeptides and their derivatives, are the simplest amide bond-containing components. However, they display various special and interesting biological activities, including taste-enhancing, antibacterial, nutritional, and anti-tumor activities [1] (Table1). These activities are mainly due to the special structures of dipeptides. A dipeptide can be seen either as a derivative of an amino acid or as the dipeptide itself. As a derivative of an amino acid, the dipeptide and the parent amino acid usually show different physicochemical properties but the same biological effects, because dipeptides, such as Ala-Gln and Gly-Tyr, can be degraded into individual amino acids via specific proteases in organisms. In contrast, various dipeptides, such as aspartame and carnosine, have unique bioactivities that cannot be found in the parent amino acids. Compared with the numerous studies on the function, application, and preparation of proteins or amino acids, the research progress on dipeptides has been relatively slow, and only a few dipeptides, such as Ala-Gln and aspartame, are available. One of the major reasons is the lack of cost-effective and efficient manufacturing processes.

Biomolecules 2019, 9, 733; doi:10.3390/biom9110733 www.mdpi.com/journal/biomolecules Biomolecules 2019, 9, 733 2 of 23

Table 1. The dipeptides with interesting biological activities.

Function Chemical Compound Reference Gly-Tyr [2] Parenteral nutrition Ala-Gln [3] Sweetener: Aspartame [4] Taste-enhancing Salt substance: Pro-Gly [5] Cytosolic buﬀering Carnosine [6] Ophthalmic drug N-Acetyl carnosine [7] Analgesic Kyotorphin (Arg-Tyr) [8] Anti-tumor Lys-Glu [9] Neuroprotective Leu-Ile [10] Bacilysin/Chlorotetaine [11] Anti-bacterial rhizocticin [12] tabtoxin [13]

Various methods have been reported for the synthesis or formation of peptide bonds, such as chemical synthesis, chemo-enzymatic synthesis, and enzymatic synthesis (biosynthesis). The chemical synthesis of dipeptides usually includes four principal procedures [14]: (1) protection of functional groups, (2) activation of the free carboxy group, (3) formation of a peptide bond, and (4) removal of the protecting groups. With chemical synthesis, almost all designed dipeptides can be synthesized with appropriate protecting groups, and the yield is usually high. However, the disadvantages of chemical synthesis, such as the high cost, possibility of racemization, and lack of environmental-friendliness, are also very clear. The chemo-enzymatic synthesis of dipeptides results from the reverse reaction catalyzed by peptide bond-hydrolyzing enzymes (proteases or esterases). This method includes two distinct types of reaction processes: the thermodynamically controlled process or the kinetically controlled process. The former is carried out to drive the equilibrium toward peptide synthesis with necessary interventions, such as the precipitation of synthesized dipeptides or reaction with a large excess of substrates. The latter is dependent on an acylated serine or cysteine protease, which will then undergo a competitive deacylation process with water and the other amino acid. This method leads to temporary accumulation of the formed dipeptide. Compared with the chemical synthesis process, these two methods usually involve stricter stereoselectivity and milder conditions. However, they are usually influenced by many complicated factors such as severe hydrolytic side-reactions, the racemization-free preparation of activated peptide esters, and the limited availability of efficient peptide coupling and enzymes with high catalytic performance. The synthesis of peptides by amide bond formation between specific (or partially protected) amino acid derivatives is, unfortunately, one of the most wasteful and least green chemical processes [15,16]. Due to the rapid development of DNA sequencing, tremendous progress has been made in the technologies of metagenomics, proteomics, and metabolomics, which lead to the identification of various enzymes that could be used to efficiently catalyze the synthesis of dipeptides. Given the great advantages of dipeptide biosynthesis, this review details strategies for dipeptide biosynthesis. Recent successful biosynthesis processes are also highlighted.

2. Biocatalysts Available for the Biosynthesis of Short Oligopeptides

2.1. Enzymes Used as Biocatalysts for the Biosynthesis of Dipeptides Various enzymatic machineries that can catalyze the synthesis of dipeptides, such as the ribosome, non-ribosomal peptide synthetases (NRPSs), ATP(Adenosine triphophate)-grasp enzymes, and α-amino acid ester acyltransferases, have been found in nature [17]. These naturally occurring peptide-synthesizing enzymes seem to be ideal catalysts for dipeptide synthesis, even though they are diverse in their speciﬁcity and physiological function. However, one common feature is the requirement for ATP to catalyze the peptide bond-forming reaction. Based on the intermediates Biomolecules 2019, 9, 733 3 of 23 formed in the catalytic process, either aminoacyl-AMP (Adenosine monophosphate) or aminoacyl phosphate, these enzymes are generally divided into two categories [18]. The representatives of the former category are the ribosome, tRNA-dependent ligases, adenylate-forming amide ligases, and the Biomolecules 2019, 9, x 3 of 23 NRPSs (Figure1). The latter category usually contains a variety of enzymes, which usually shares a characteristicenzymes. This ATP-grasp includes motifglutathione in their aminosynthetase, acid sequenceD-alanine- andD-alanine are known ligase, as the and ATP-grasp other ATP-grasp enzymes. Thisenzymes. includes glutathione synthetase, d-alanine-d-alanine ligase, and other ATP-grasp enzymes.

FigureFigure 1.1. The reaction mechanism of two distinct types of biocatalysts for the synthesis of dipeptides. ((11)) AcyladenylateAcyladenylate intermediatesintermediates areare formedformed inin thethe processprocess catalyzedcatalyzed byby NRPSs.NRPSs. ((22)) AcylphosphateAcylphosphate intermediatesintermediates areare formedformed inin thethe processprocess catalyzedcatalyzed byby ATP-graspATP-grasp enzymes. enzymes.

2.1.1.2.1.1. NRPSs Non-ribosomalNon-ribosomal peptide synthesis synthesis is is a auniversal universal and and critical critical biochemical biochemical process process catalyzed catalyzed by byNRPSs NRPSs in bacteria in bacteria and fungi, and through fungi, through which a wide which array a wide of therapeutically array of therapeutically important peptides important with peptideshighly diverse with highlystructures diverse and structuresbioactivities, and incl bioactivities,uding penicillin, including bleomycin, penicillin, and cyclosporine, bleomycin, and are cyclosporine,produced [19,20]. are producedBiocatalysts [19 ,capable20]. Biocatalysts of NRP synthesis capable could of NRP be synthesisdivided into could two be groups divided (ATP- into twoindependent groups (ATP-independent and ATP-dependent and ATP-dependentenzymes) [21] enzymes)based on [21the] baseddifference on the in di substratefference in activation substrate activation[22,23]. In [22the,23 ATP-dependent]. In the ATP-dependent process, process,enzymes enzymes such as such tRNA-dependent as tRNA-dependent ligase ligase can activate can activate the thesubstrate substrate through through aminoacyl-adenosine aminoacyl-adenosine monophosph monophosphate.ate. In In the the ATP-independent ATP-independent process, process, enzymes suchsuch asas transacylasetransacylase useuse aminoacylaminoacyl phosphate.phosphate. NRPSsNRPSs areare largelarge multi-functionalmulti-functional proteinsproteins organizedorganized intointo didifferentfferent modules,modules, wherewhere eacheach consistsconsists ofof thethe catalyticcatalytic domainsdomains responsibleresponsible forfor incorporatingincorporating oneone aminoamino acidacid intointo thethe growinggrowing peptidepeptide product.product. A A standard standard NRPS NRPS complex complex usually usually contains contains at atleast least four four enzymatic enzymatic domains domains (Figure (Figure 2): 2the): thecondensation condensation domain domain (C-domain), (C-domain), the theadenylation adenylation domain domain (A-domain), (A-domain), the thethiolation thiolation domain domain (T- (T-domain),domain), and and the the thioesterase thioesterase domain domain (Te-domain). (Te-domain). The The A-domain A-domain could could activate activate a specificspecific aminoamino acidacid substrate as an an aminoacyl aminoacyl adenylate. adenylate. In In the the PCP PCP domain, domain, a thioester, a thioester, aminoacyl-S-PCP, aminoacyl-S-PCP, could could be beformed. formed. The The C-domain C-domain then then catalyzes catalyzes the the amide amide bond bond formation formation by by releasing releasing a a dipeptide. The Te domaindomain isis usually usually located located at theat endthe ofend the of large the proteins large proteins and is capable and is of capable terminating of terminating the biosynthesis the ofbiosynthesis specific peptides. of specific The peptides. process of The NRPS-catalyzed process of NRPS-catalyzed peptide bond formation peptide bond canbe formation summarized can asbe followssummarized (Figure as3 follows). First, the(Figure A-domain 3). First, can the identify A-domain the substratecan identify amino the acidsubstrate and then amino activate acid and it as then an aminoacyl-AMP.activate it as an Then, aminoacyl-AMP. the activated amino Then, acid the is transferredactivated amino to the 40acid-phosphopantetheine is transferred to moiety the 4 of′- thephosphopantetheine T-domain, accompanied moiety with of thethe release T-domain, of adenosine accompanied monophosphate with the (AMP). release Next, of theadenosine peptide bondmonophosphate is formed in (AMP). the adjacent Next, condensationthe peptide bond domain is formed (C-domain). in the adjacent Lastly, the condensation synthesized domain peptide (C- is releaseddomain). from Lastly, the the NRPS synthesized complex bypeptide catalysis is released via the Te-domain.from the NRPS In addition, complex the by PCP catalysis is a small via the 8 kDa Te- domaindomain. thatIn addition, belongs the to the PCP non-overlapping is a small 8 kDa membersdomain that of abelongs superfamily to the ofnon-overlapping carrier protein domains,members whichof a superfamily play many of roles carrier in acyl protein group domains, transport. which play many roles in acyl group transport. Due to the superior catalytic activity and the unique reaction mechanism of NRPSs in dipeptide synthesis, modular manipulation of NRPS has been successfully applied to dipeptide synthesis. This approach is detailed in the following section. Biomolecules 2019, 9, 733 4 of 23

Due to the superior catalytic activity and the unique reaction mechanism of NRPSs in dipeptide synthesis, modular manipulation of NRPS has been successfully applied to dipeptide synthesis. This Biomolecules 2019, 9, x 4 of 23 approachBiomolecules is detailed 2019, 9, x in the following section. 4 of 23

FigureFigure 2. Structure 2. Structure of typicalof typical NRPS NRPS enzymes enzymes (PDB (PDB ID:ID: 2VSQ,2VSQ, in this this family, family, enzymes enzymes usually usually consist consist of Figure 2. Structure of typical NRPS enzymes (PDB ID: 2VSQ, in this family, enzymes usually consist the Aof domain, the A domain, the C the domain, C domain, the Tethe domain, Te domain, and and the the PCP PCP domain). domain). of the A domain, the C domain, the Te domain, and the PCP domain). PPi PPi+ + H2N R 1 R1 R1 O-AMP H2N R H N R O-AMP 1 2 2 R1 R1 O H2N R2 NH2 O O O NH NH2 NH2 S 2 NH NH O O O HN O 2 R1 2 S S R S O HN O HN 1 R2 HN O R2 R2 P T R T O 2 P T A S O T domain C +A domaindomainC S O OH + domain Te OH OH A domain domainTe O OH A domain A domain domain O NH2 A domain NH2

FigureFigure 3. The3. The process process of of amide amide bondbond format formationion catalyzed catalyzed by byNRPS NRPS enzymes. enzymes. Figure 3. The process of amide bond formation catalyzed by NRPS enzymes. 2.1.2.2.1.2. ATP-Grasp ATP-Grasp Enzymes Enzymes 2.1.2. ATP-Grasp Enzymes TheThe ATP-grasp ATP-grasp enzymes enzymes (or ATP-dependent(or ATP-dependent carboxylate-amine carboxylate-amine ligases) ligases) activate activate carboxylic carboxylic acids The ATP-grasp enzymes (or ATP-dependent carboxylate-amine ligases) activate carboxylic acids such as acylphosphate intermediates. ATP-dependent carboxylate-amine ligases are seen in suchacids as acylphosphate such as acylphosphate intermediates. intermediates. ATP-dependent ATP-dependent carboxylate-amine carboxylate-amine ligases ligases are are seen seen in in many many different biological systems, such as de novo purine biosynthesis, which is the assembly of the diﬀerentmany biological different biological systems, systems, such assuch de as novo de novo purine purine biosynthesis, biosynthesis, which which is isthe the assembly assembly of the of the pentapeptide chain of peptidoglycan. Another biologically important example is RimK, which pentapeptidepentapeptide chain chain of of peptidoglycan. peptidoglycan. Another biologically biologically important important example example is RimK, is RimK, which which catalyzes the tandem addition of L-glutamic acids to the carboxyl terminus of ribosomal proteins. catalyzes the tandem addition ofl L-glutamic acids to the carboxyl terminus of ribosomal proteins. catalyzesThis the tandemfamily was addition the first of of-glutamic the amide acidsbond-forming to the carboxyl enzymes terminus to be recognized of ribosomal and includes proteins. This family was the first of the amide bond-forming enzymes to be recognized and includes biotinThis family carboxylase, was the D-Ala- ﬁrstD of-Ala the ligase amide (Ddl), bond-forming and glutathione enzymes synthetase to be [24]. recognized ATP-grasp and enzymes includes are biotin biotin carboxylase, D-Ala-D-Ala ligase (Ddl), and glutathione synthetase [24]. ATP-grasp enzymes are carboxylase,usually maded-Ala- upd-Ala of three ligase conserved (Ddl), and domains glutathione (the N-terminal synthetase as [24 well]. ATP-grasp as central enzymesand C-terminal are usually usually made up of three conserved domains (the N-terminal as well as central and C-terminal madedomains), up of three which conserved is a structure domains unique (theto this N-terminal kind of enzyme as well (Figure as central4). As their and name C-terminal implies, these domains), domains), which is a structure unique to this kind of enzyme (Figure 4). As their name implies, these whichenzymes is a structure usually uniquehave a nonclassical to this kind ATP of enzymebinding fold (Figure comprising4). As their two α name + β domains implies, that these “grasp” enzymes enzymes usually have a nonclassical ATP binding fold comprising two α + β domains that “grasp” usually have a nonclassical ATP binding fold comprising two α + β domains that “grasp” an ATP Biomolecules 2019, 9, 733 5 of 23 Biomolecules 2019, 9, x 5 of 23 moleculean ATP molecule between between the central the and central C-terminal and C-termin domains.al domains. Most ATP-grasp Most ATP-grasp enzymes requireenzymes an require Mg2+ ion, an whichMg2+ ion, is coordinated which is coordinated by ATP in by the ATP active in site.the active site.

Figure 4. (1)A typical ATP-grasp enzyme: glycinamide ribonucleotide synthetase (PDB ID: 2IP4), and ((22)) thethe processprocess ofof ATP-graspATP-grasp enzyme-catalyzedenzyme-catalyzed peptidepeptide bondbond formation.formation. l-Amino Acid Ligase (Lal) L-Amino Acid Ligase (Lal) l-Amino acid ligase (Lal) is a special type of ATP-grasp enzyme that can catalyze only dipeptide L-Amino acid ligase (Lal) is a special type of ATP-grasp enzyme that can catalyze only dipeptide synthesis from unprotected amino acids in an ATP-dependent manner (Figure5). BacD (or YwfE, EC synthesis from unprotected amino acids in an ATP-dependent manner (Figure 5). BacD (or YwfE, EC 6.3.2.28), which is identified from Bacillus subtilis in 2005 by Tabata et al. [25], was the first identified 6.3.2.28), which is identified from Bacillus subtilis in 2005 by Tabata et al. [25], was the first identified Lal. To date, several Lals have been identified and investigated, including RizA, Rsp1486a, BL00235, Lal. To date, several Lals have been identified and investigated, including RizA, Rsp1486a, BL00235, PSPPH 4299, plu1440, TabS, and FtyB (details shown in Table2). PSPPH 4299, plu1440, TabS, and FtyB (details shown in Table 2). As shown in Table2, almost all Lals identified to date show di fferent substrate specificities, which leads to the production of different dipeptides. For example, the substrate specificity of BacD is restricted to smaller amino acids (e.g., l-Ala) at the N-terminal end of the dipeptide, whereas a wide range of hydrophobic amino acids (e.g., l-Phe) are recognized at the C-terminal end [26]. However, TabS can accept larger amino acids as its N-terminal substrate [27]. Plu1440 synthesizes dipeptides that contain l-asparagine at the N-terminus [28], and RSp1486a accepts bulkier amino acids as N-terminal substrates and less bulky amino acids as C-terminal substrates [29]. Based on these findings, it is reasonable to biosynthesize target dipeptides by modified Lals with improved substrate specificity [30]. This approach has provided novel methods for the production of useful dipeptides.

Biomolecules 2019, 9, x 6 of 23

Figure 5. Typical L-amino acid ligases. (1) BL00235 (PDB ID: 3VOT); (2) RizA (PDB ID: 4WD3); (3) BiomoleculesBacD 2019(PDB, 9 ID:, x 3VMM). In this family, members consist of three conserved domains (the N-terminal,6 of 23 central-terminal, and C-terminal domains) and a nonclassical ATP binding fold comprising two α + β domains.Figure 5. Typical L-amino acid ligases. (1) BL00235 (PDB ID: 3VOT); (2) RizA (PDB ID: 4WD3); (3) BiomoleculesBacD (PDB2019, 9 ID:, x 3VMM). In this family, members consist of three conserved domains (the N-terminal,6 of 23 Ascentral-terminal, shown in Table and C-terminal 2, almost domains) all Lals andidentified a nonclassical to date ATP show binding different fold comprising substrate two specificities, α + β Biomolecules 2019, 9, x 6 of 23 whichdomains.Figure leads 5. to Typical the production L-amino acid of differentligases. (1 dipeptid) BL00235es. (PDB For ID: example, 3VOT); the(2) RizAsubstrate (PDB specificityID: 4WD3); of (3 )BacD BacD (PDB ID: 3VMM). In this family, membersL consist of three conserved domains (the N-terminal, is restrictedFigure 5. to Typical smaller L-amino amino acid acids ligases. (e.g., (1 ) -Ala)BL00235 at the(PDB N-terminal ID: 3VOT); end(2) RizA of the (PDB dipeptide, ID: 4WD3); whereas (3) a Ascentral-terminal, shown in Table and C-terminal2, almost domains)all Lals andidentified a nonclassical to date ATP show binding different fold comprising substrate twospecificities, α + β wideBacD range (PDB of ID:hydrophobic 3VMM). In thisamino family, acids members (e.g., consL-Phe)ist of are three recognized conserved domainsat the C-terminal (the N-terminal, end [26]. domains. However,whichcentral-terminal, leads TabS to the can production andaccept C-terminal larger of different domains)amino acids dipeptidand aas nonclassical itses. N-terminal For example, ATP binding substrate the substratefold [27]. comprising Plu1440 specificity two synthesizes α of+ β BacD dipeptidesis restricteddomains. that to smaller contain amino L-asparagine acids (e.g., at the L-Ala) N-terminus at the N-terminal [28], and RSp1486aend of the accepts dipeptide, bulkier whereas amino a acidswideAs asrange shownN-terminal of hydrophobicin Table substrates 2, almost amino and all lessacids Lals bulky (e.g.,identified amino L-Phe) toacids are date recognizedas show C-terminal different at substratesthe substrate C-terminal [29]. specificities, Basedend [26]. on theseHowever,whichAs findings, leads shown TabS to theitin can isTable production reasonableaccept 2, almostlarger ofto different aminobiosynthesizeall Lals acids dipeptididentified as target itses. N-terminalto Fordipeptides date example, show substrate by differentthe modified substrate [27]. substrate LalsPlu1440 specificity with specificities, synthesizes improved of BacD whichsubstratedipeptidesis restricted leads specificity that toto the smallercontain production [30]. aminoL -asparagineThis ofacidsapproach different (e.g., at thehas dipeptidL-Ala) N-terminusprovided ates. the For novelN-terminal [28], example, methodsand RSp1486atheend substrate forof the accepts dipeptide,production specificity bulkier whereas of of amino usefulBacD a L isdipeptides.acidswide restricted asrange N-terminal toof smallerhydrophobic substrates amino aminoacids and less(e.g.,acids bulky L -Ala)(e.g., amino at-Phe) the acids N-terminal are asrecognized C-terminal end ofat thesubstratesthe dipeptide,C-terminal [29]. whereas Basedend [26]. on a widetheseHowever, rangefindings, TabS of hydrophobicit can is reasonableaccept larger amino to biosynthesizeamino acids acids (e.g., as Ltarget-Phe) its N-terminal dipeptidesare recognized substrate by modified at the[27]. C-terminal LalsPlu1440 with synthesizes improvedend [26]. However,substratedipeptides specificityTabS that cancontain accept[30]. L -asparagineThis larger approachTable amino 2. at Typical theacidshas N-terminusprovided Lalsas its and N-terminal theirnovel [28], functions. methodsand substrate RSp1486a for [27].the accepts productionPlu1440 bulkier synthesizes of aminouseful acids as N-terminal substrates and less bulky amino acids as C-terminal substrates [29]. Based on dipeptidesdipeptides. that containComponents L-asparagine Catalyzed at the N-terminus Availability [28], and ofRSp1486a accepts bulkier amino these findings, it is reasonable to biosynthesize target dipeptides by modified Lals with improved acidsEnzyme as N-terminal substrates and less bulkyUnnatural amino acidsthe asCrystal C-terminal substratesSource [29]. BasedRef. on Natural Product thesesubstrate findings, specificity it is reasonable [30]. This toapproachTable biosynthesize 2. Typical hasProduct provided Lals target and theirdipeptides novelStructure functions. methods by modified for the productionLals with improved of useful substratedipeptides. specificity [30].Components This approach Catalyzed has provided Availabilitynovel methods of for the production of useful dipeptides.Enzyme Unnatural the Crystal Source Ref. Biomolecules 2019, 9, 733 Natural Product 6 of 23 Table 2. TypicalProduct Lals and theirStructure functions. +, PDB ID: BacD Ala-Gln Bacillus subtilis [25] ComponentsTable Catalyzed 2. Typical Lals and their Availability3VMM functions. of Enzyme Unnatural the Crystal Source Ref. NaturalComponents Product Catalyzed Table Availability 2. Typical of Lals and their functions. Product Structure Enzyme Unnatural the+, PDB Crystal ID: Source Ref. BacD Natural Product Components CatalyzedAla-Gln AvailabilityBacillus subtilis of the [25] Enzyme Product Structure3VMM Source Ref. Natural Product Unnatural Product Crystal Structure

+, PDB ID: BacD Ala-Gln +, PDB ID: Bacillus subtilis [25] RizA / 3VMM [31] BacD Ala-Gln+, PDB4WD3 ID: +,NBRC PDB ID: 3134 3VMM Bacillus subtilis [25] BacD Ala-Gln Bacillus subtilis [25] 3VMM +, PDB ID: Bacillus subtilis RizA / [31] 4WD3 NBRC 3134 Ala-

HomoArg, RizA /+, PDB ID: +Bacillus, PDB ID:subtilis 4WD3 Bacillus subtilis NBRC 3134 [31] RizA Ala-Arg/ [31] 4WD3 NBRC 3134 Ala-LysAla- +, PDB ID: Bacillus subtilis RizA / [31] HomoArg,Ala-His 4WD3 NBRC 3134 PSPPH Ala-HomoArg,+, PDB ID: Pseudomonas syringae Ala-Gln [32] 4299 Ala-Arg Ala-Arg3VMM pv. phaseolicola 1448A Ala-Asn Ala-LysAla- Ala-Lys Ala-Met HomoArg,Ala-His Ala-His PSPPH Ala- +, PDB ID: Pseudomonas syringae PSPPH 4299 Ala-PheAla-GlnAla-Arg Ala-Gln +, PDB ID: 3VMM [32] Pseudomonas syringae pv. phaseolicola 1448A [32] 4299 HomoArg,Ala-Trp 3VMM pv. phaseolicola 1448A Ala-AsnAla-Lys Ala-Asn Ala-Arg Ala-Met Ala-MetAla-His PSPPH Ala-Lys Ala-Phe+, PDB ID: Pseudomonas syringae Ala-PheAla-Gln [32] 4299 Ala-HisArg-Phe Ala-Trp3VMM pv. phaseolicola 1448A PSPPH Ala-AsnAla-Trp +, PDB ID: Pseudomonas syringae Arg-Phe Ala-GlnGln-Trp [32] 4299 Ala-Met Gln-Trp3VMM pv. phaseolicola 1448A Ala-AsnLeu-Ser Ala-Phe Leu-Ser Pseudomonas syringae TabS TabS Ala-MetGlu-Thr - - [26] Pseudomonas syringae NBRC 14081 [26] Arg-PheAla-Trp Glu-Thr NBRC 14081 Ala-PheLeu-Ile Biomolecules 2019, 9, x Gln-Trp Leu-Ile 7 of 23 Ala-TrpPro-Gly Leu-Ser Pro-Gly Pseudomonas syringae TabS Glu-ThrArg-Phe - [26] NBRC 14081 Gln-TrpLeu-Ile FtyB Arg-Phe / Pseudoalteromonas- Pseudoalteromonas tunicata D2 [33] FtyB Pro-GlyLeu-Ser/ - [33] Gln-Trp Pseudomonastunicata D2syringae TabS Glu-Thr - [26] Leu-Ser NBRC 14081 Leu-Ile Pseudomonas syringae Rsp1486aTabS Phe-Cys, His-Ala, His-Val, His-Gly Glu-Thr / - - [26] Ralstonia solanacearum JCM 10486 [28] Pro-Gly NBRC 14081 BL00235 Met-Gly, Met-Met Leu-Ile / +, PDB ID: 3VOT Bacillus licheniformis NBRC 12200 [29,34] Phe-Cys, His-Ala, His-Val, Ralstonia Photorhabdus luminescens laumondii plu1440 Asn-Gly, Asn-Ala, Asn-Cys, Asn-Gln Pro-Gly / - subsp. TT01 [27] Rsp1486a His-Gly / - solanacearum JCM [28]

10486 +, PDB ID: Bacillus licheniformis BL00235 Met-Gly, Met-Met / [29,34] 3VOT NBRC 12200 Photorhabdus Asn-Gly, Asn-Ala, Asn-Cys, plu1440 / - luminescens subsp. [27] Asn-Gln laumondii TT01

D-Alanine: D-Alanine Ligase

Recently, it was reported that compared with L,L-dipeptides, D-amino acid-containing dipeptides have novel biological properties and are expected to be novel functional compounds for pharmaceuticals and food additives [35,36]. D-Alanine: D-alanine ligase (carboxylate-amine ligase, EC 6.3.2.4) is involved in the biosynthesis of the peptidoglycan component of the bacterial cell wall [37] and catalyzes the ATP-driven ligation of two D-alanine molecules, which results in the formation of D-alaninyl-D-alanine dipeptides (Figure 6). Given that Ddls do not have any homologue in humans, they have historically been considered promising targets for developing novel anti-bacterial components [38]. In addition to D-Ala-D-Ala, the formation of D-Ala-D-Ser dipeptides or D-Ala-D-Lac depsipeptides can also be catalyzed by Ddls [37].

Figure 6. Proposed mechanism of Ddls.

Figure 7. The reaction catalyzed is by poly-α-glutamic (αPGA) acid synthetase (RimK). Biomolecules 2019, 9, x 5 of 23

an ATP molecule between the central and C-terminal domains. Most ATP-grasp enzymes require an Mg2+ ion, which is coordinated by ATP in the active site.

Figure 4. (1)A typical ATP-grasp enzyme: glycinamide ribonucleotide synthetase (PDB ID: 2IP4), and (2) the process of ATP-grasp enzyme-catalyzed peptide bond formation.

L-Amino Acid Ligase (Lal)

BiomoleculesL-Amino 2019, 9 acid, x ligase (Lal) is a special type of ATP-grasp enzyme that can catalyze only dipeptide7 of 23 synthesis from unprotected amino acids in an ATP-dependent manner (Figure 5). BacD (or YwfE, EC 6.3.2.28), which is identified from Bacillus subtilis in 2005 by Tabata et al. [25], was the first identified BiomoleculesLal. To date,2019, several9, 733 Lals have been identified and investigated, includingPseudoalteromonas RizA, Rsp1486a, BL00235,7 of 23 FtyB / - [33] PSPPH 4299, plu1440, TabS, and FtyB (details shown in Table 2). tunicata D2

Biomolecules 2019, 9, x 7 of 23

Phe-Cys, His-Ala, His-Val, Ralstonia Rsp1486a His-Gly / - solanacearum JCM [28] Pseudoalteromonas10486 FtyB / - [33] +, PDB ID: Bacillustunicata licheniformis D2 BL00235 Met-Gly, Met-Met / [29,34] 3VOT NBRC 12200

Photorhabdus Asn-Gly, Asn-Ala, Asn-Cys, plu1440 / - luminescens subsp. [27] Phe-Cys, Asn-GlnHis-Ala, His-Val, Ralstonia laumondii TT01 Rsp1486a His-Gly / - solanacearum JCM [28] 10486 D-Alanine: D-Alanine Ligase +, PDB ID: Bacillus licheniformis BL00235 Met-Gly, Met-Met / [29,34] FigureRecently, 5. Typical it wasl-amino reported acid ligases.that compared (1) BL00235 with (PDB ID:L3VOT,L-dipeptides, 3VOT); (2) RizA DNBRC-amino (PDB 12200 ID: acid-containing 4WD3); (3) Photorhabdus dipeptidesBacD (PDB haveAsn-Gly, ID: novel 3VMM). Asn-Ala, biological In thisAsn-Cys, family,properties members and consistare expected of three to conserved be novel domains functional (the compounds N-terminal, for plu1440 / - luminescens subsp. [27] pharmaceuticalscentral-terminal, and and Asn-Glnfood C-terminal additives domains) [35,36]. and D-Alanine: a nonclassical D-alanine ATP binding ligase fold(carboxylate-amine comprising two α ligase,+ laumondii TT01 EC 6.3.2.4)β domains. is involved in the biosynthesis of the peptidoglycan component of the bacterial cell wall [37] and catalyzes the ATP-driven ligation of two D-alanine molecules, which results in the formation Dd-Alanine: Dd-Alanine Ligase of D-alaninyl-D-alanine dipeptides (Figure 6). Given that Ddls do not have any homologue in humans, l l d they Recently,have historically itit was was reported reportedbeen thatconsidered comparedthat compared promising with , with-dipeptides, targets L,L-dipeptides, for-amino developing acid-containingD-amino novel acid-containing anti-bacterial dipeptides dipeptideshavecomponents novel biologicalhave [38]. novel In addition properties biological to and D properties-Ala- areD expected-Ala, and the to areformation be expected novel functional of Dto-Ala- be novelD compounds-Ser functionaldipeptides for pharmaceuticalscompounds or D-Ala-D-Lac for d d pharmaceuticalsanddepsipeptides food additives can and [also35 ,food36 be]. catalyzed additives-Alanine: by [35,36].-alanine Ddls [37].D-Alanine: ligase (carboxylate-amine D-alanine ligase ligase, (carboxylate-amine EC 6.3.2.4) is involved ligase, ECin the 6.3.2.4) biosynthesis is involved of thein the peptidoglycan biosynthesis component of the peptidoglycan of the bacterial component cell wall of [the37] bacterial and catalyzes cell wall the ATP-driven[37] and catalyzes ligation the of ATP-driven two d-alanine ligation molecules, of two which D-alanine results molecules, in the formation which results of d-alaninyl- in the formationd-alanine ofdipeptides D-alaninyl- (FigureD-alanine6). Given dipeptides that Ddls (Figure do not 6). haveGiven any that homologue Ddls do not in have humans, any homologue they have historicallyin humans, theybeen consideredhave historically promising been targets considered for developing promising novel targets anti-bacterial for developing components novel [38 ].anti-bacterial In addition componentsto d-Ala-d-Ala, [38]. the In formationaddition to of D-Ala-d-Ala-D-Ala,d-Ser the dipeptides formation or ofd D-Ala--Ala-dD-Lac-Ser depsipeptidesdipeptides or D can-Ala- alsoD-Lac be depsipeptidescatalyzed by Ddls can [also37]. be catalyzed by Ddls [37]. Figure 6. Proposed mechanism of Ddls.

The Poly-α-Glutamic Acid (αPGA) Synthetase RimK RimK is a member of the ATP-dependent carboxylate-amine/thiol ligase superfamily, which is reported to catalyze the modification of ribosomal protein S6 (RPS6) from Escherichia coli (E. coli) K- 12 (Figure 7). In this biological process, Glu is added to the C-terminus of RPS6, which leads to the Figure 6. Proposed mechanism of Ddls. biosynthesis of RPS6-Glu, RPS6-Glu-Glu,Figure 6. Proposed RPS6-Glu-Glu-Glu, mechanism of and Ddls. RPS6-Glu-Glu-Glu-Glu. The Poly-In addition,α-Glutamic Kino AcidAcid recently ((αPGA)PGA) reported SynthetaseSynthetase that RimKRimK could catalyze the biosynthesis of αPGA from unprotected amino acids via ATP hydrolysis [39]. The results showed that the lengths of the resulting productsRimK changed is a member with pH, of the and, ATP-dependent at a pH of 9.0, carboxylate-aminecarbox a maximumylate-amine/thiol 46-mer/thiol of Gluligase was superfamily, obtained. RimK which has is reportedstrict substrate to to catalyze catalyze specificity the the modification modiﬁcation for Glu. ofTherefore, of ribosomal ribosomal it proteinis protein possible S6 S6 (RPS6) (RPS6)to produce from from Escherichia Escherichiavarious biological coli coli (E. (E.coli) Glu- coli) K- 12K-12containing (Figure (Figure 7).products,7 ).In In this this biological such biological as dipeptides process, process, Glu Glu (e.g., is is added addedL-glutamyl- to to the the LC-terminus C-terminus-glutamate and of RPS6, poly which-glutamic leadsleads acid) toto thethe or biosynthesistripeptides. of RPS6-Glu, RPS6-Glu-Glu, RPS6-Glu-Glu-Glu,RPS6-Glu-Glu-Glu, andand RPS6-Glu-Glu-Glu-Glu.RPS6-Glu-Glu-Glu-Glu. In addition, Kino recently reported that RimK could catalyze the biosynthesis of αPGA from unprotected amino acids via ATP hydrolysis [39]. The results showed that the lengths of the resulting products changed with pH, and, at a pH of 9.0, a maximum 46-mer of Glu was obtained. RimK has strict substrate specificity for Glu. Therefore, it is possible to produce various biological Glu- containing products, such as dipeptides (e.g., L-glutamyl-L-glutamate and poly-glutamic acid) or tripeptides.

Figure 7.7. The reaction catalyzed is by poly-α-glutamic-glutamic ((αPGA) acid synthetase (RimK).(RimK).

In addition, Kino recently reported that RimK could catalyze the biosynthesis of αPGA from unprotected amino acids via ATP hydrolysis [39]. The results showed that the lengths of the resulting

Figure 7. The reaction catalyzed is by poly-α-glutamic (αPGA) acid synthetase (RimK). Biomolecules 2019, 9, 733 8 of 23

Biomolecules 2019, 9, x 8 of 23 products changed with pH, and, at a pH of 9.0, a maximum 46-mer of Glu was obtained. RimK has 2.1.3.strict α-Amino substrate Acid specificity Ester Acyltransferase for Glu. Therefore, it is possible to produce various biological Glu-containing products, such as dipeptides (e.g., l-glutamyl-l-glutamate and poly-glutamic acid) or tripeptides. Kenzo et al. [40] reported an efficient enzymatic method for producing oligopeptides from unprotected2.1.3. α-Amino amino Acidacids Ester at a high Acyltransferase yield. In this study, Empedobacter brevis ATCC 14234 was found to produce L-alanyl-L-glutamine (Ala-Gln) much more efficiently than previous methods. Furthermore, Kenzo et al. [40] reported an efficient enzymatic method for producing oligopeptides from an enzyme catalyst (named carboxypeptidase Y) for the rapid production of Ala-Gln and other unprotected amino acids at a high yield. In this study, Empedobacter brevis ATCC 14234 was found to oligopeptides with unprotected substrates (L-alanine methyl ester hydrochloride, Gln, and more) was produce l-alanyl-l-glutamine (Ala-Gln) much more efficiently than previous methods. Furthermore, discovered in this strain and could be used to rapidly catalyze a reaction between L-alanine methyl esteran hydrochloride enzyme catalyst (AlaOMe) (named and carboxypeptidase Gln to synthesi Y)ze for Ala-Gln. the rapid However, production no additional of Ala-Gln detailed and other l information,oligopeptides including with unprotected the amino substratesacid sequence, ( -alanine the methylcoding ester gene hydrochloride, sequence, or Gln,the and3D more)crystal was l structure,discovered was inreported this strain in this and study. could beIsao used ABE to et rapidly al. [41] catalyze first reported a reaction the between cloning and-alanine expression methyl of ester α-aminohydrochloride acid ester (AlaOMe) acyl andtransferases Gln to synthesize (AETs) Ala-Gln. from However,Empedobacter no additionalbrevis ATCC14234 detailed information, and Sphingobacteriumincluding the aminosiyangensis acid AJ2458. sequence, The the proteins coding geneencoded sequence, are tw oro similar the 3D polypeptides crystal structure, composed was reported of α 616 inand this 619 study. amino Isao acid ABE residues, et al. [41] respectively. first reported Their the cloning amino and acid expression sequences of were-amino 35% acid and ester 36% acyl Empedobacter brevis Sphingobacterium siyangensis identicaltransferases to that of (AETs) the α-amino from acid ester hydrolaseATCC14234 from Acetobacter and pasteurianus, respectively. AETsAJ2458. wereThe believed proteins to encodeddisplay dipeptidyl are two similar peptidase polypeptides activity and composed transferase of 616 activity and 619simultaneously. amino acid residues, This respectively. Their amino acid sequences were 35% and 36% identical to that of the α-amino acid enzyme was reported to use L-alanine methyl ester hydrochloride and Gln to synthesize Ala-Gln in Acetobacter pasteurianus a highester yield hydrolase (Figure from 8) [42,43]. However, this enzyme, respectively. also shows AETs wide were substrate believed specificity to display for dipeptidyl both l acylpeptidase donors activityand nucleophiles, and transferase which activity leads simultaneously. to the synthesis This of enzyme not only was reporteddipeptides to usebut -alaninealso oligopeptidesmethyl ester from hydrochloride different accepted and Gln substrates to synthesize [40]. Ala-Gln To date, in there a high have yield been (Figure no studies8)[ 42, 43on]. the However, 3D structurethis enzyme of α-amino also shows acid ester wide acyltransferase substrate specificity and forthe bothdetailed acyl reaction donors and mechanisms nucleophiles, it catalyzes. which leads Theseto theaspects synthesis should of be not explored only dipeptides further. but also oligopeptides from different accepted substrates [40]. To date, there have been no studies on the 3D structure of α-amino acid ester acyltransferase and the detailed reaction mechanisms it catalyzes. These aspects should be explored further.

O OOH O O AET O CH3HCl O + H2N H2N OH N NH NH H 2 2 O FigureFigure 8. Reactions 8. Reactions catalyzed catalyzed by α by-aminoα-amino acid acid ester ester acyltransferase acyltransferase (AET). (AET). β β 2.1.4.2.1.4. Enzymes Enzymes Used Used for β for-Lactam-Lactam Biosynthesis Biosynthesis (β-Lactam ( -Lactam Acylases) Acylases) β-Lactamβ-Lactam antibiotics antibiotics are area large a large class class of antibiotics of antibiotics containing containing a β-lactam a β-lactam ring ring in their in their chemical chemical structurestructure (Figure (Figure 9) [44],9)[ 44such], such as peni ascillin, penicillin, cephalospori cephalosporins,ns, and thiamycins. and thiamycins. β-Lactamβ-Lactam antibiotics antibiotics are amongare amongthe most the widely most used widely clinical used anti-infective clinical anti-infective agents and agents occupy and an occupy important an important position in position the domesticin the domesticpharmaceutical pharmaceutical industry. industry. The enzymatic The enzymatic synthesis synthesis of β-lactam of β-lactam antibiotics antibiotics is more is more environmentally-friendlyenvironmentally-friendly and andeconomical economical than thantradit traditionalional chemical chemical methods, methods, with the with advantages the advantages of mildof reaction mild reaction conditions, conditions, a clean a and clean non-polluting and non-polluting nature, nature,and good and product good product quality. quality.Therefore, Therefore, this strategythis strategy has also has been also applied been applied successfully successfully in pilot-scale in pilot-scale production production in modern in modern pharmaceutical pharmaceutical enterprises.enterprises. Traditionally, Traditionally, the theβ-lactamβ-lactam acylases acylases are areused used for forthe thehydrolytic hydrolytic processing processing of β of-lactamβ-lactam antibioticsantibiotics (e.g., (e.g., penicillin penicillin G and G and cephalosporin cephalosporin C). C). However, However, some some other other acylases acylases can can also also be be used for for thethe synthesissynthesis of of semi-synthetic semi-syntheticβ-lactam β-lactam antibiotics antibiotics [45 ].[45]. To date, To date, several severalβ-lactam β-lactam acylases, acylases, including includingpenicillin penicillin acylase acylase (PA, EC (PA, 3.5.1.11), EC 3.5.1.11), glutaryl acylase glutaryl (GA, acylase EC 3.5.1.93), (GA, EC and 3.5.1.93),β-amino and acid β-amino ester hydrolase acid ester(AEH, hydrolase EC 3.1.1.43), (AEH, EC have 3.1.1.43), been widely have been investigated widely ininvestigated the biosynthesis in the ofbiosynthesisβ-lactam antibiotics. of β-lactam The antibiotics.biosynthesis The biosynthesis of nocardicin of is nocardicin performed is by performed an NRPS enzymeby an NRPS consisting enzyme of consisting two mega-enzymes of two mega- known enzymesas NocA known and NocBas NocA [46 ].and NocB [46]. Biomolecules 2019, 9, 733 9 of 23 Biomolecules 2019, 9, x 9 of 23

Penicillin G Thienamycin H OH H H NH2 N S S O N N O O O OH OH O N HO O O O O O N -Lactam O O O N O S N O NH H H H2N O Cephalexin Lactivicin OOH

HO O O N

N OH H NH2

FigureFigure 9.9. TheThe chemicalchemical structuresstructures ofof ββ-lactam-lactam andand severalseveral derivatives.derivatives.

PenicillinPenicillin acylaseacylase isis aa well-knownwell-known pharmaceuticallypharmaceutically importantimportant enzymeenzyme producedproduced byby variousvarious microorganisms.microorganisms. BasedBased on the substrate specificity,specificity, PAsPAs areare furtherfurther divideddivided intointo penicillinpenicillin GG acylasesacylases (PGAs)(PGAs) [[47]47] andand penicillinpenicillin VV acylasesacylases (PVAs).(PVAs). TheThe formerformer preferentiallypreferentially hydrolyzeshydrolyzes benzylpenicillinbenzylpenicillin (pen(pen G), G), and and the the latter latter preferentially preferentially hydrolyzes hydrolyzes phenoxymethyl phenoxymethyl penicillin penicillin (pen V).(pen These V). These enzymes enzymes could becould used be on used an industrial on an scaleindustrial for producing scale for the producing active pharmaceutical the active intermediatepharmaceutical 6-aminopenicillanic intermediate 6- acidaminopenicillanic (6-APA) by cleaving acid (6-APA) the side by chaincleaving from the natural side chain penicillins. from natural In addition, penicillins. they In couldaddition, be used they forcould the be potential used for synthesis the potential of newer synthesis semi-synthetic of newer antibioticssemi-synthetic by coupling antibiotics new by acylcoupling groups new to freeacyl βgroups-lactam to nuclei. free β-lactam On this nuclei. basis, On PAs this hold basis, great PAs potential hold great for potential application for in application the field of in novelthe field drug of development.novel drug development. For example, For these example, enzymes these could enzymes be used could directly be used to directly catalyze to the catalyze ligation the of ligation novel syntheticof novel fragmentssynthetic (novelfragment sides chains(novel andsideβ -lactamchains nuclei).and β-lactam Similarly, nuclei). the enzyme Similarly, engineering the enzyme (e.g., directedengineering evolution (e.g., directed or rational evolution design) or of rational PAs could design) be performed of PAs could for the be catalyticperformed synthesis for the ofcatalytic novel drugs.synthesis These of novel developments drugs. These will help developments to further expand will help and to increase further theexpand potential and ofincreaseβ-lactam the antibiotics potential forof β future-lactam biopharmaceutical antibiotics for future applications. biopharmaceutical In addition, applications. PAs are employed In addition, in peptide PAs are synthesis employed and inin thepeptide resolution synthesis of racemic and in mixturesthe resolu [48tion]. Dueof racemic to their mixtures enantioselectivity [48]. Due andto their promiscuity enantioselectivity [49], PAs and can alsopromiscuity be used for[49], producing PAs can achiral also andbe chiralused compoundsfor producing for theachiral preparation and chiral of synthons compounds and bioactive for the pharmaceuticalpreparation of synthons intermediates and bioactive on a laboratory pharmaceutical scale. intermediates on a laboratory scale. AlthoughAlthough PAsPAs havehave gainedgained aa uniqueunique positionposition amongamong thethe enzymesenzymes usedused byby thethe pharmaceuticalpharmaceutical industry,industry, theythey havehave seriousserious drawbacks,drawbacks, suchsuch asas thethe strongstrong inhibitoryinhibitory eeffectffect ofof thethe producedproduced phenylphenyl aceticacetic acidacid andand instabilityinstability atat alkalinealkaline pHpH values.values. GivenGiven thesethese considerations,considerations, αα-amino-amino acidacid esterester hydrolaseshydrolases (AEHs,(AEHs, ECEC 3.1.1.43)3.1.1.43) areare aa promisingpromising alternativealternative forfor thethe synthesissynthesis ofofα α-amino-containing-amino-containing cephalosporins.cephalosporins. Naturally,Naturally, AEHsAEHs areare capablecapable ofof thethe semi-synthesissemi-synthesis ofof ββ-lactam-lactam antibioticsantibiotics containingcontaining anan amino group, such such as as cephalexin, cephalexin, cefaclor, cefaclor, cefpro cefprozil,zil, and and cefadroxil cefadroxil [50,51]. [50,51 Since]. Since variations variations in the in theside side chain chain can canalter alter the biochemical the biochemical properties properties of a ofβ-lactam a β-lactam antibiotic, antibiotic, semisynthetic semisynthetic antibiotics antibiotics with withnovel novel side sidechains chains show show promise promise in the in the development development of ofnovel novel drugs drugs to to cope cope with with drug drug resistance. However,However, thethe presencepresence ofof aa hydroxylhydroxyl groupgroup atat thethe pp-position-position ofof thethe phenylglycinephenylglycine sideside chainchain hashas beenbeen reportedreported toto causecause aa drasticdrastic decreasedecrease inin specificityspecificity (Kcat(Kcat/Km)/Km) comparedcompared toto thatthat ofof thethe analogue without thisthis hydroxylhydroxyl group.group. ToTo addressaddress thethe issueissue ofof decreased activity toward componentscomponents withwith aa p-hydroxyl-hydroxyl group,group, YeYe etet al.al. [[52]52] exploredexplored thethe possibilitypossibility ofof improvingimproving thethe substratesubstrate specificityspecificity ofof AEHAEH towardtoward para-hydroxylpara-hydroxyl cephalosporincephalosporin synthesissynthesis byby site-directedsite-directed mutagenesis.mutagenesis. The results showed that Arg87, Ser131,Ser131, andand Y175Y175 playplay importantimportant rolesroles inin substratesubstrate recognitionrecognition andand thethe V131SV131S mutantmutant showed a 64% increaseincrease inin thethe maximummaximum accumulationaccumulation ofof thethe cefatrizinecefatrizine product.product. GlutarylGlutaryl acylasesacylases areare well-knownwell-known industrialindustrial biocatalystsbiocatalysts withwith widewide substratesubstrate specificityspecificity (cephalosporin(cephalosporin C (CPC) and/or and/or glutaryl glutaryl 7-aminoc 7-aminocephalosporanicephalosporanic acid acid (GL-7ACA)) (GL-7ACA)) for for producing producing 7- 7-aminocephalosporanicaminocephalosporanic acid acid (7-ACA) (7-ACA) [53]. [53]. These These enzymes enzymes have have further further been been classified classified into fivefive typestypes (class(class II toto classclass V)V) basedbased onon theirtheir genegene structuresstructures (sequence(sequence conservation),conservation), substratesubstrate specificity,specificity, andand enzyme properties [54]. All cephalosporin acylases are active toward GL-7ACA, but only members of classes I and III show appreciable activity toward cephalosporin C (CephC). Cephalosporin C Biomolecules 2019, 9, 733 10 of 23

enzymeBiomolecules properties 2019, 9, x [54]. All cephalosporin acylases are active toward GL-7ACA, but only members10 of of23 classes I and III show appreciable activity toward cephalosporin C (CephC). Cephalosporin C acylases (CAs)acylases [55 (CAs)] can speciﬁcally[55] can specifically use CephC use as CephC their substrate as their tosubstrate produce to 7-ACA. produce 7-ACA 7-ACA. is an7-ACA important is an βimportant-lactam nucleus β-lactam for nucleus preparing for manypreparing widely many used widely semisynthetic used semisyntheticβ-lactam antibiotics β-lactam [ 56antibiotics]. In contrast, [56]. glutaryl-7-ACAIn contrast, glutaryl-7-ACA acylases (GAs, acylases EC 3.5.1.93) (GAs, EC usually 3.5.1.93) preferentially usually preferentially use GL-7ACA use GL-7ACA as their substrate. as their Thesubstrate. most importantThe most important application application of GAs is theof GA expensives is the expensive and environmentally and environmentally hazardous hazardous two-step enzymatictwo-step enzymatic route for theroute synthesis for the of synthesis 7-ACA.As of an7-AC excellentA. As alternative,an excellent the alternative, single-step the production single-step of 7-ACAproduction can beof accomplished7-ACA can be using accomplished CephC acylase using (FigureCephC 10acylase)[57]. (Figure Unfortunately, 10) [57]. natural Unfortunately, CAs are usuallynatural CAs eﬃcient are usually in the deacylation efficient in ofthe GL-7ACA deacylation but of are GL-7ACA less active but toward are less adipyl-7-ADCA active toward adipyl-7- and are barelyADCA able and toare hydrolyze barely able CPC to hydrolyze for the industrial CPC for production the industrial of 7-ACA production [58]. of 7-ACA [58].

Figure 10. The reaction catalyzed by CephC acylase.

2.1.5. Cyanophycinases (CGPases) l Cyanophycin granule polypeptidepolypeptide (CGP, oror multi-multi-L-arginyl-poly)-arginyl-poly) isis anan intracellularintracellular storagestorage polymer foundfound inin mostmost cyanobacteria.cyanobacteria. Equimolar concentrations of arginine and aspartic acid are observed inin thethe aspartic aspartic acid acid backbone, backbone, where where the the arginine arginine moieties moieties are linked are linked to the toβ-carboxyl the β-carboxyl group ofgroup each of aspartic each aspartic acid through acid itsthroughα-amino its group.α-amino In mostgroup. genera In most of cyanobacteria, genera of cyanobacteria, the cyanophycin the synthetasecyanophycin gene synthetase (cphA) hasgene been (cphA identified) has been and identified verified forand the verified synthesis for the of CGP.synthesis In contrast, of CGP. the In intracellularcontrast, the and intracellular extracellular and degradation extracellular of CGPdegradation is catalyzed of CGP by cyanophycinases is catalyzed by (CphBcyanophycinases and CphE), which(CphB releasesand CphE), dipeptides which (releasesβ-Asp-Arg, dipeptides Table3, ( Figureβ-Asp-Arg, 11). In Table this respect, 3, Figureβ -Asp-Arg11). In this can respect, be effi βciently-Asp- synthesizedArg can be efficiently via the simultaneous synthesized productionvia the simultaneous of CGP and production CGPase, whichof CGP could and CGPase, be further which applied could in variousbe further fields applied requiring in various arginine fields (Arg) requiring content inarginine feed or (Arg) food. content However, in thefeed production or food. However, and efficient the isolationproduction of CGPand efficient from various isolation organisms of CGP from have vari beenous successfully organisms establishedhave been successfully in several recombinant established strains,in several including recombinantEscherichia strains, coli [including59], Nicotiana Escherichia tabacum [coli60], [59],Pseudomonas Nicotiana putida tabacum[61 ],[60], and Pseudomonas alcaligenesputida [61],DIP1 and Pseudomonas [62]. Therefore, alcaligenes it is very DIP1 feasible [62]. Therefore, to produce it is dipeptides very feasible (e.g., to βproduce-Asp-Arg) dipeptides via the metabolic(e.g., β-Asp-Arg) engineering via the of metabolic suitable engineering hosts [63] and of suitab chemo-enzymaticle hosts [63] and strategies chemo-enzymatic [64]. For strategies example, successful[64]. For example, co-expression successful of CGP co-expression and CGPase of in CGP the Nicotiana and CGPase tabacum in theplant Nicotiana was recently tabacum achieved plant [was65]. recently achieved [65]. A further study showed that it is possible to realize the goal of sufficient storage and efficient transport of arginine and β-Asp-Arg dipeptides in this synthetic model. Biomolecules 2019, 9, 733 11 of 23

A further study showed that it is possible to realize the goal of suﬃcient storage and eﬃcient transport of arginine and β-Asp-Arg dipeptides in this synthetic model.

Table 3. Enzymes used for the degradation of CGP.

Enzyme Source Product Reference Intracellular Cyanophycinase (CphB) Anabaena cylindrica β-Asp-Arg [66] CphEPa Pseudomonas anguilliseptica BI β-Asp-Arg [67] CphE Bacillus megaterium BAC19 Small molecules [68] Extracellular Bm CphE Sedimentibacter hongkongensis KI β-Asp-Arg, β-Asp-Lys [69] CphE Pseudomonas alcaligenes DIP1 Ethanol, acetic acid, succinic acid [70] Biomolecules 2019, 9, x 11 of 23

FigureFigure 11. 11. TheThe reaction reaction mechanism mechanism of of cyanophycinases. cyanophycinases.

2.1.6. Methods Used for theTable Biosynthesis 3. Enzymes of used Cyclic for Dipeptidesthe degradation of CGP.

Cyclic dipeptides,Enzyme or cyclodipeptides (CDPs), Source which are mainly produced Product by microorganismsReference as secondary metabolites,Cyanophycinase are the smallest cyclic peptides frequently found in nature and exhibit various Intracellular Anabaena cylindrica β-Asp-Arg [66] noteworthy biological(CphB) properties [71]. For example, cyclo(l-Phe-l-Pro), cyclo(l-Phe-trans-4-OH-l-Pro), clomycin, albonoursin,CphE pulcherrimin,Pa Pseudomonas mycocyclosin, anguilliseptica ambewelamides, BI β and-Asp-Arg phenylahistin are[67] several typical CDPs withCphE potentBm antibacterial,Bacillus antiviral, megaterium and BAC19 immunosuppressive Small molecules properties [72]. [68] From a Sedimentibacter hongkongensis chemicalExtracellular structural perspective,CphE CDPs are also called 2,5-diketopiperazines,β-Asp-Arg, and β-Asp-Lys they are characterized[69] KI by amide linkages formed to the two nitrogen atoms of a six-membered piperazine ring. Ethanol, acetic acid, CphE Pseudomonas alcaligenes DIP1 [70] In nature, the formation of the scaffold of CDPs is catalyzed bysuccinic two acid unrelated biosynthetic enzyme families (Figure 12): either CDP synthases (CDPSs) [72] or non-ribosomal peptide synthetases 2.1.6.(NRPSs) Methods [73]. Subsequently,Used for the Bios theynthesis resulting of cyclodipeptides Cyclic Dipeptides are usually further modified by tailoring enzymes, and the final CDPs are released [74–76]. CyclicCDPSs, dipeptides, first defined or incyclodipeptides 2002, can connect (CDPs), primary which metabolic are mainly pathways produced with by secondary microorganisms metabolic as secondarypathways bymetabolites, using aminoacyl-tRNAs are the smallest ascyclic substrates peptides to frequently catalyze the found formation in nature of fand diketopiperazines exhibit various noteworthy(DKPs) [75]. biological Based on properties the identity [71]. of For three example, essential cyclo( residues,L-Phe- CDPSsL-Pro), havecyclo( beenL-Phe-trans-4-OH- further dividedL- Pro),into twoclomycin, subfamilies: albonoursin, NYH (e.g., pulcherrimin, AlbC and mycocyclosin, YvmC) and XYP ambewelamides, (e.g., X40 and P203)and phenylahistin [77]. Enzymes are in severalthe former typical subfamily CDPs with are characterized potent antibacterial, by a typical antiviral, structural and architectureimmunosuppr (Rossmannessive properties fold) used [72]. for Fromsubstrate a chemical binding, structural which might perspective, lead to an CDPs exceptionally are also broadcalled tRNA2,5-diketopiperazines, substrate specificity, and producing they are characterizedvarious cyclodipeptides by amide linkages [78]. To formed date, the to structures the two nitrogen of six CDPSs atoms can of bea six-membered retrieved from piperazine the RCSB ring.PDB (https://www.rcsb.org/), including AlbC (PDB ID: 3OQV, Figure 13) from Streptomyces noursei [79], CDPSIn (PDBnature, ID: the 6EZ3) formation from Staphylococcus of the scaffold haemolyticus of CDPs [is80 ],catalyzed CDPS (PDB by two ID: 4Q24) unrelated from biosyntheticStreptomyces enzymenoursei [ 81families], CDPS (Figure from Fluoribacter 12): either dumo CDPffii [80synt](PDBhases ID: (CDPSs) 5OCD), CDPS[72] or from non-ribosomalRickettsiella gryllipeptide[80] synthetases(PDB ID: 5MLP), (NRPSs) and [73]. CDPS Subsequently, from Nocardia the brasiliensisresulting cyclodipeptides[80] (PDB ID: 5MLQ). are usually In addition, further modified with the byadvent tailoring of next-generation enzymes, and the sequencing, final CDPs manyare released more have[74–76]. been identified via BLAST (Basic Local Alignment Search Tool) searches and genome mining [82]. In addition to CDPSs, cyclic peptides could also be synthesized by NRPS modules [83]. In this study, BPSA prtein, which is a single NRPS module encoded by bpsA, was determined to be able to catalyze the synthesis of a blue pigment 5,50-diamino-4,40-dihydroxy-3,30-diazadiphenoquinone-(2,20). Biomolecules 2019, 9, 733 12 of 23 Biomolecules 2019, 9, x 12 of 23

Figure 12. TheThe reaction reaction mechanism mechanism of of typical typical cyclodipeptide cyclodipeptide synthases: synthases: (1) (Albc,1) Albc, (2) (YvmC,2) YvmC, and and (3) (Rv2275.3) Rv2275. Biomolecules 2019, 9, x 13 of 23 CDPSs, first defined in 2002, can connect primary metabolic pathways with secondary metabolic pathways by using aminoacyl-tRNAs as substrates to catalyze the formation of f diketopiperazines (DKPs) [75]. Based on the identity of three essential residues, CDPSs have been further divided into two subfamilies: NYH (e.g., AlbC and YvmC) and XYP (e.g., X40 and P203) [77]. Enzymes in the former subfamily are characterized by a typical structural architecture (Rossmann fold) used for substrate binding, which might lead to an exceptionally broad tRNA substrate specificity, producing various cyclodipeptides [78]. To date, the structures of six CDPSs can be retrieved from the RCSB PDB (https://www.rcsb.org/), including AlbC (PDB ID: 3OQV, Figure 13) from Streptomyces noursei [79], CDPS (PDB ID: 6EZ3) from Staphylococcus haemolyticus [80], CDPS (PDB ID: 4Q24) from Streptomyces noursei [81], CDPS from Fluoribacter dumoffii [80](PDB ID: 5OCD), CDPS from Rickettsiella grylli [80] (PDB ID: 5MLP), and CDPS from Nocardia brasiliensis [80] (PDB ID: 5MLQ). In addition, with the advent of next-generation sequencing, many more have been identified via BLAST (Basic Local Alignment Search Tool) searches and genome mining [82]. In addition to CDPSs, cyclic peptides could also be synthesized by NRPS modules [83]. In this study, BPSA prtein, which is a single NRPS module encoded by bpsA, was determined to be able to catalyze the synthesis of a blue pigment 5,5′-

diamino-4,4′-dihydroxy-3,3′-diazadiphenoquinone-(2,2′). FigureRecently,Figure 13. 13. The Thethe typical catalytic typical cyclodipeptide cyclodipeptide mechanism synthase synthase of CDPSs AlbC AlbC has (P (PDB DBbeen ID: ID: further 3OQV) 3OQV) andinvestigated and its its substrate substrate and binding binding reported mode. mode. to fit a ping-pong-type model [84] with two characteristic and conserved pockets known as P1 and P2 [77]. ConsideringRecently, the the catalytic great potential mechanism of CDPSs of CDPSs in the has biosynthesis been further of investigated CDPs, it is believed and reported that studies to fit a Catalysis starts with the binding of the first aa-tRNA (in the P1 pocket) and the subsequent transfer onping-pong-type the protein engineering model [84 ]of with CDPSs two will characteristic greatly faci andlitate conserved the production pockets of known a variety as P1of natural and P2 and [77]. of its aminoacyl moiety to a conserved serine, which leads to the formation of an aminoacyl enzyme unnaturalCatalysis startsbioactive with cyclodipeptides the binding of the[85,86]. first aa-tRNA (in the P1 pocket) and the subsequent transfer intermediate (in the P2 pocket). The aminoacyl moiety of a second aa-tRNA interacting with the pre- of its aminoacyl moiety to a conserved serine, which leads to the formation of an aminoacyl enzyme formed intermediate is then transferred to the aminoacyl enzyme, which leads to the formation of a 2.1.7.intermediate Biosynthesis (in the of Imidazole-Related P2 pocket). The aminoacyl Dipeptides moiety by Carnosine of a second Synthase aa-tRNA interacting with the dipeptidyl enzyme intermediate [81]. The final cyclodipeptide is released after intramolecular pre-formed intermediate is then transferred to the aminoacyl enzyme, which leads to the formation cyclizationHistidine of thedipeptides dipeptidyl (or moiety. imidazole-related dipeptides), such as carnosine, anserine, ophidine, of a dipeptidyl enzyme intermediate [81]. The final cyclodipeptide is released after intramolecular and homocarnosine, play a critical role in detoxifying cytotoxic reactive carbonyls and reversing cyclization of the dipeptidyl moiety. protein glycation (Figure 14) [87]. Structurally, all of these enzymes contain a non-α-amino acid (β- alanine or γ-aminobutyric acid) at the N-terminus and an imidazole-related amino acid (histidine) at the C-terminus. They are widely distributed in the skeletal muscle, heart, and central nervous system of most vertebrates and some invertebrates. Biomolecules 2019, 9, 733 13 of 23

Considering the great potential of CDPSs in the biosynthesis of CDPs, it is believed that studies on the protein engineering of CDPSs will greatly facilitate the production of a variety of natural and unnatural bioactive cyclodipeptides [85,86].

2.1.7. Biosynthesis of Imidazole-Related Dipeptides by Carnosine Synthase Histidine dipeptides (or imidazole-related dipeptides), such as carnosine, anserine, ophidine, and homocarnosine, play a critical role in detoxifying cytotoxic reactive carbonyls and reversing protein glycation (Figure 14)[87]. Structurally, all of these enzymes contain a non-α-amino acid (β-alanine or γ-aminobutyric acid) at the N-terminus and an imidazole-related amino acid (histidine) at the C-terminus. They are widely distributed in the skeletal muscle, heart, and central nervous system of Biomoleculesmost vertebrates 2019, 9, x and some invertebrates. 14 of 23

Figure 14. BiosynthesisBiosynthesis of imidazole-related dipeptides by carnosine synthase.

To date,date, three types of vertebrate enzymes havehave been identiﬁedidentified for the biosynthesis of imidazole-related dipeptides: carnosine carnosine synthase synthase (EC 6.3.2.11), carnosinecarnosine NN-methyltransferase (EC 2.1.1.22), and and histidine histidine NN-acetyltransferase-acetyltransferase (EC (EC 2.3.1.33). 2.3.1.33). Histidine Histidine N-acetyltransferaseN-acetyltransferase is a istype a type of Nα of- acetyl-histidineNα-acetyl-histidine (NAH) (NAH) synthesizi synthesizingng enzyme enzyme that that can can catalyze catalyze the the biosynthesis biosynthesis of of NAH NAH with with Ll-His and acetyl-CoA. Carnosine synthase is an ATP-grasp ligase that is one of the most important enzymes involved inin thethe biosynthesis biosynthesis of of anserine, anserine, homocarnosine, homocarnosine, and and carnosine carnosine (Figure (Figure 15)[ 15)88]. [88]. Similar Similar to Lals, to Lals,carnosine carnosine synthase synthase is a catalytically is a catalytically promiscuous promis enzyme.cuous enzyme. Therefore, Therefore, it can accept it can not accept only histidinenot only histidinebut also lysine, but also ornithine, lysine, and ornithine, arginine and as C-terminal arginine as substrates C-terminal to synthesize substrates various to synthesize dipeptides, various such dipeptides,as β-Ala-Lys such [89]. as This β-Ala-Lys promiscuity [89]. couldThis promiscuity also provide could an eﬃ alcientso provide approach an to efficient modify approach the catalytic to modifyfunction the of carnosinecatalytic function synthase of to carnosine form novel synthase natural to or form “unnatural” novel natural products. or “unnatural” products.

Figure 15. Crystal structure of carnosine synthase and the reaction catalyzed. Biomolecules 2019, 9, x 14 of 23

Figure 14. Biosynthesis of imidazole-related dipeptides by carnosine synthase.

To date, three types of vertebrate enzymes have been identified for the biosynthesis of imidazole-related dipeptides: carnosine synthase (EC 6.3.2.11), carnosine N-methyltransferase (EC 2.1.1.22), and histidine N-acetyltransferase (EC 2.3.1.33). Histidine N-acetyltransferase is a type of Nα- acetyl-histidine (NAH) synthesizing enzyme that can catalyze the biosynthesis of NAH with L-His and acetyl-CoA. Carnosine synthase is an ATP-grasp ligase that is one of the most important enzymes involved in the biosynthesis of anserine, homocarnosine, and carnosine (Figure 15) [88]. Similar to Lals, carnosine synthase is a catalytically promiscuous enzyme. Therefore, it can accept not only histidine but also lysine, ornithine, and arginine as C-terminal substrates to synthesize various dipeptides,Biomolecules 2019 such, 9, 733 as β-Ala-Lys [89]. This promiscuity could also provide an efficient approach14 of to 23 modify the catalytic function of carnosine synthase to form novel natural or “unnatural” products.

Figure 15. CrystalCrystal structure structure of carnosine synthase and the reaction catalyzed.

2.1.8. Proteases Although proteases are primarily used for the hydrolysis of proteins and peptides, they can also be used to catalyze the kinetically or thermodynamically controlled formation of peptide bonds with unprotected substrate amino acids [90–92]. Thermodynamically controlled (or equilibrium-controlled) peptide synthesis can be achieved with all types of proteases. In contrast, kinetically controlled peptide synthesis is usually conducted with serine and cysteine proteases [93] because the specific triads (Ser-His-Asp and Cys-His-Asn) in these two enzymes can catalyze the transfer of an acyl donor to the acceptor (nucleophile) via the formed acyl–enzyme intermediate [94]. Therefore, the kinetically controlled method is more widely applied in biosynthesing oligopeptides, and various proteases, including papain, thermolysin, trypsin, α-chymotrypsin, and ficin, have been thoroughly explored [90,95]. In a study by Wei Qi et al. [96], papain, which is a commercially available and low-cost protease, was used successfully for the biosynthesis of N-(benzyloxycarbonyl)-alanyl-glutamine (Z-Ala-Gln) through a kinetically controlled strategy. The results showed that the dipeptide yield was 35.5%, and the apparent maximum reaction rate was determined to be 6.09 mmol/(L min) under the optimized · conditions. Wen-Yong Lou et al. proposed a novel method for the more efficient synthesis of dipeptides with the same biocatalyst (papain) in deep eutectic solvents [97]. In this study, papain was successfully immobilized onto magnetic nanocrystalline cellulose, and the obtained nano-biocatalyst (PA@MNCC) showed improved stability, enhanced solvent tolerance, and increased enzyme-substrate affinity. When this method was used for the synthesis of Z-Ala-Gln, the yield of the dipeptide in deep eutectic solvent was approximately 71.5%, which was the highest reported yield. This strategy is a competitive method for the synthesis of Z-Ala-Gln. Moreover, this study provided a promising carrier (magnetic nanocrystalline cellulose) that might be widely applied for enzyme immobilization.

3. Emerging Approaches for the Eﬃcient Production of Short Oligopeptides: Rational Protein Engineering and Strain Development

3.1. Dipeptide Formation by Rational Engineering of NRPSs NRPSs are modular ‘mega-enzymes’ that can catalyze the assembly of many smaller units, which produces various bioactive molecules. NRPSs can synthesize and assemble peptides in-line from amino acid monomers, which are first activated by the A domains and then loaded onto the adjacent carrier domains. Lastly, the formation of peptide bonds and transfer of the growing chain are catalyzed by the C domains. Because each module of NRPSs performs specific reactions, such as substrate activation, modification, and condensation, the rational arrangement of these specific modules (domain assembly and module fusion) for the design of novel engineered NRPSs to produce interesting products is very promising [98,99]. Biomolecules 2019, 9, 733 15 of 23

In a pioneering study by Marahiel et al. (Figure 16)[98,100], different Asp-Phe synthetases were designed and constructed through fusion of the Asp and Phe activating modules and condensation domains. The product formation assay showed that two different forms of Asp-Phe were successfully bio-synthesized (α-Asp-Phe and β-Asp-Phe), while enzyme III 1 [A-PCP]SrfB2-[C-A]TycB2-[PCP-Te]TycC6 showed the best catalytic activity (Kcat = 0.7 min− , α:β = 1 100:0). The turnover rates (ranging from 0.01–0.7 min− ) and the purity of α-Asp-Phe (75–100% of the overall product) indicate that the rational engineering of NRPSs shows great potential for the design and efficient production of novel dipeptides. However, it should be noted that the different fusion sites mightBiomolecules play 2019 a critical, 9, x role in the resulting catalytic activities of the fused catalysts. 16 of 23

FigureFigure 16. 16.Dipeptide Dipeptide formation formation by by the the rational rational engineering engineering of NRPS. of NRPS. (1) Rational(1) Rational fusion fusion of the of Asp the and Asp Pheand activating Phe activating modules modules and condensation and condensation domains, domains, and (and2) the (2) biosynthesis the biosynthesis of Asp-Phe of Asp-Phe catalyzed catalyzed by theby rationallythe rationally constructed constructed enzyme. enzyme.

DueDue to to the the chemical chemical diversity diversity covered covered by by non-ribosomal non-ribosomal peptides, peptides, rational rational modification modification of of their their backbonesbackbones representsrepresents aa promisingpromising strategystrategy forforthe the development development ofofnovel novelproducts productswith with specific specific propertiesproperties [[101].101]. Therefore, Therefore, determining determining how how to toproduce produce the the designed designed component component via catalysis via catalysis by an byefficient an effi cientbiocatalyst biocatalyst might might display display great great potent potential.ial. From From this this perspective, engineeringengineering andand reprogrammingreprogramming modular modular NRPSs NRPSs to obtainto obtain novel novel catalysts catalysts with designedwith designed activities activities would makewould perfect make sense.perfect GrsA sense./GrsB1 GrsA/GrsB1 is a truncated is a truncated dipeptide dipeptid synthetasee synthetase excised excised from the from gramicidin the gramicidin S NRPS S [NRPS102], which[102], canwhich catalyze can catalyze the biosynthesis the biosynthesis of the of dthe-Phe- D-Phe-l-ProL-Pro diketopiperazine. diketopiperazine. Based Based on on this this finding, finding, DonaldDonald et et al. al. [ 103[103]] introduced introduced a a single single W239S W239S mutation mutation in in the the phenylalanine-specific phenylalanine-specific NRPS NRPS A-domain A-domain toto enlarge enlarge the the binding binding pocket. pocket. This This modification modification greatly greatly improved improved the the activation activation process process of of unnatural unnatural aromaticaromatic amino amino acids acids functionalized functionalized with with azide azide and and alkyne alkyne groups. groups. TheThe resultsresults showedshowed that that the the 5 5 substratesubstrate specificity specificity was was increased increased by by 10 10-fold-fold (for (forp -azido-p-azido-l-Phe,L-Phe, Kcat Kcat/K/KM M= =9000 9000 (25 (25 for for WT)) WT)) without without appreciableappreciable loss loss of of catalytic catalytic e ffiefficiency.ciency.

3.2.3.2. Engineering Engineering Modifications Modifications of of Cephalosporin Cephalosporin Acylase Acylase ConsideringConsidering the the nature nature of of the the similar similar chemical chemical structures structures of of glutaryl-7-ACA, glutaryl-7-ACA, adipyl-7-ADCA, adipyl-7-ADCA, andand cephalosporin cephalosporin C, C, attempts attempts have have been been made made to to create create mutants mutants of of cephalosporin cephalosporin acylases acylases with with improvedimproved activities activities toward toward adipyl-7-ADCA adipyl-7-ADCA and and cephalosporin cephalosporin C. C. From From this this perspective, perspective, engineering engineering modificationsmodifications of of cephalosporin cephalosporin acylase acylase would would be a feasiblebe a feasible strategy strategy to achieve to this achieve goal, andthis significant goal, and progresssignificant has progress been made has inbeen addressing made in theaddressing concerns the of lowconcerns substrate of low specificity, substratesubstrate specificity, inhibition, substrate andinhibition, product and inhibition product encountered inhibition encountered in practice [104 in practice,105]. [104,105]. TheThe study study by by Wim Wim J. J. Quax Quax [ 58[58,106],106] included included a a comprehensive comprehensive mutational mutational analysis analysis of of N266 N266 and and F375.F375. The The resulting resulting mutations mutations showed showed a a broad broad spectrum spectrum of of a ffiaffinitiesnities and and activities, activities, which which suggests suggests thethe flexibility flexibility ofof thethe glutarylglutaryl acylase from from Pseudo Pseudomonasmonas SY-77 SY-77 at at these these positions. positions. Moreover, Moreover, the the SY- N266Q N266H N266M SY-7777N266Q, SY-77, SY-77N266H, and, and SY-77 SY-77N266M mutantsmutants also also showed showed a modest a modest improvement improvement in in cephalosporin cephalosporin C hydrolysis. In a study carried out by Zhanglin Lin et al. [107], a positive mutation, H57βA/H70βY, of the CPC acylase acyII from Pseudomonas SE8 with no substrate inhibition was obtained via two rounds of combinatorial active site saturation testing. Further study with a quick pH indicator assay designed for real-time monitoring and screening libraries of site-directed saturation mutations led to the discovery of a new mutation, H57βA/H70βY/I176βN, which showed a Kcat 3.26-fold when compared to the wild type. In this study, it was suggested that a larger binding pocket might better accommodate CPC as the optimal substrate. However, the reason that this mutant abrogates substrate inhibition remains unclear. In addition to traditional molecular biology methods (such as random mutagenesis methods and directed evolution), rational protein design is a promising strategy in current enzyme engineering to improve enzymatic properties. In this field, several important studies carried out by Yu-shan Zhu et al. have demonstrated the importance of computational protein design [108–112]. In one study [111], Biomolecules 2019, 9, 733 16 of 23

C hydrolysis. In a study carried out by Zhanglin Lin et al. [107], a positive mutation, H57βA/H70βY, of the CPC acylase acyII from Pseudomonas SE8 with no substrate inhibition was obtained via two rounds of combinatorial active site saturation testing. Further study with a quick pH indicator assay designed for real-time monitoring and screening libraries of site-directed saturation mutations led to the discovery of a new mutation, H57βA/H70βY/I176βN, which showed a Kcat 3.26-fold when compared to the wild type. In this study, it was suggested that a larger binding pocket might better accommodate CPC as the optimal substrate. However, the reason that this mutant abrogates substrate inhibition remains unclear. In addition to traditional molecular biology methods (such as random mutagenesis methods and directed evolution), rational protein design is a promising strategy in current enzyme engineering to improve enzymatic properties. In this field, several important studies carried out by Yu-shan Zhu et al. have demonstrated the importance of computational protein design [108–112]. In one study [111], molecular dynamics (MD) simulations and molecular docking were applied to investigate the dynamic features of active site-transition state complex structures of cephalosporin acylase to potentially avoid an excess of false positives produced by high-throughput screening. Through this approach, the limiting step and well-maintained geometrical constraints in the hydrolysis reaction of cephalosporin C were determined and revealed, which could be further used to improve the activity of cephalosporin C acylase. In other studies [109,110], computational protein design strategies were successfully used for enzyme engineering to increase catalytic activities (thermostability or activity). The cephalosporin C acylase from the Pseudomonas strain N176 was reconstructed and analyzed via the PROtein Design Algorithmic (PRODA) package [113]. Through this method, rational protein design for the improvement of stability and activity could be achieved simultaneously by analyzing the functions of the hydrophobic core regions and the regions surrounding the active sites. This study [110] revealed that the instability caused by introduced mutations (V68βA) at the active site could be reversed by repacking the nearby hydrophobic core regions (L154βF and L180βF). One study [112] achieved the computational redesign of native penicillin acylase active sites for the condensation reaction between d-dihydrophenylglycine methyl ester (DHME) and 7-ADCA, which produces cephradine in fully aqueous medium. The great advantage of this method might be the development of a scoring function based on discounted folding energy instead of the single binding energy or the overall folding energy (∆Gfold). The results showed not only that the positive mutant (M142αF/F24βA/S67βA) displayed high substrate specificity but also that the catalytic activity was simultaneously increased by more than 10-fold. It is believed that this strategy provides a highly efficient and green approach to enzyme engineering to create novel biocatalysts for transforming a wide variety of substrates—both natural and unnatural compounds. It is known that the protein structure determines the function and the structural, chemical, and physical factors that play important roles in catalytic activity and inevitably affect substrate specificity or stability [114]. Therefore, computational protein design could provide a promising platform for the design of novel industrial biocatalysts and for the study of protein structure and function, which has also become a leading field in the biophysical sciences [115–117].

3.3. Metabolic Engineering of Microorganisms for the Biosynthesis of Desirable Dipeptides Ala-Gln is a very important compound from both the clinical and nutritional perspectives [118] and is the most suitable Gln-containing vector for the supply of l-glutamine (Gln). In addition to chemical synthesis and chemo-enzymatic synthesis, the metabolic engineering of E. coli for the biosynthesis of Ala-Gln has proven to be a promising strategy. Yoshinori Hirao et al. [42], Wenjie Yuan et al. [43], and Kino et al. [26] used α-amino acid ester acyltransferase and l-amino acid ligase as biocatalysts for the bio-catalysis of Ala-Gln. Wenjie Yuan engineered E. coli Origami 2 to produce Ala-Gln by overexpressing α-amino acid ester acyltransferase with the pET-29a(+) plasmid under the control of the T7 promoter. The engineered host could use l-alanine methyl ester hydrochloride (AlaOMe) and l-glutamine (Gln) as the substrates to synthesize Ala-Gln. The maximum molar yield Biomolecules 2019, 9, 733 17 of 23 and productivity were determined to be 94.7% and 1.89 g (L min) 1, respectively. Moreover, the high · · − SsAet activity of α-amino acid ester acyltransferase maintained during the repeated cycle experiments could guarantee a high Ala-Gln yield. An l-amino acid ligase, BL00235, was used and engineered for the selective synthesis of the salt taste enhancer Met-Gly [30]. Via site-directed mutagenesis of the P85 residue, the resulting P85F and P85Y mutants achieved selective Met-Gly synthesis without the synthesis of Met-Met. It was found that the key residues in the binding pockets (e.g., P85 of BL00235) play a critical role in substrate reorganization similar to that of BacD (Trp332). Therefore, rational modification of these sites would alter the substrate binding pockets, which leads to a restricted cavity for substrate binding. However, as seen from the abovementioned studies, a foreseeable result is that the obtained mutants showed lower yields but higher substrate specificities than the wild type. In our view, this difference arises because the catalytic performance of a specific catalyst is affected by various factors. Therefore, the simultaneous enhancement of multiple catalytic factors of L-amino acid ligase would be a feasible alternative for further studies.

4. Conclusions Due to their specific structures and functionality, polypeptides often show remarkable chemical and biological properties. Therefore, they have been widely employed in various fields, such as biomedicine. Compared with conventional solid-phase peptide synthesis, the widely used chemo-enzymatic synthesis might be advantageous, especially for the biosynthesis of dipeptides or tripeptides, due to its environmental-friendliness and increased yields. However, critical challenges might be posed by the lack of insight into the detailed enzymatic mechanisms and difficulties in determining the optical reaction conditions. In addition, the fermentative production of oligopeptides might potentially become the ultimate strategy. It is likely the most cost-efficient and environmentally-friendly approach. Thus, different types of key biocatalysts would be first used and engineered. Furthermore, the intracellular metabolic pathways of specific hosts would also be modified to redirect the metabolic flow of the substrate amino acids in order to suppress undesired pathways. As discussed above, the enzymes used (e.g., Lals) usually show broad substrate specificity. Therefore, the resulting spectrum of possible products would greatly affect the practical biosynthesis of oligopeptides. Protein engineering through directed evolution, rational design, and structure-based site-directed mutagenesis would help improve both the substrate specificity profiles and catalytic performance. Moreover, the use of engineered biocatalysts with improved catalytic performance would undoubtedly expand the scope of fermentative production of oligopeptides.

Funding: The authors are supported by Shandong Provincial Natural Science Foundation, China (no. ZR2019BB062), the project supported by Shandong Provincial Natural Science Foundation, China (no. ZR2017BC044), the National Natural Science Foundation cultivation project of Jining Medical University (grant no. JYP201704), the Supporting Fund for Teachers’ Research of Jining Medical University (grant no. JYFC2018KJ031), the Supporting Fund for Teachers’ Research of Jining Medical University (grant no. JYFC2018KJ020), and the Startup Fund of Jining Medical University. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from the one mentioned above. Conflicts of Interest: The authors declare no conflict of interest.

References

1. Santos, S.; Torcato, I.; Castanho, M.A. Biomedical applications of dipeptides and tripeptides. Biopolymers 2012, 98, 288–293. [CrossRef] 2. Bera, B.K.; Ray, S.; Mondal, S.; Karmakar, P.; Mandal, A.; Mallick, S.; Ghosh, A.K. Kinetic and mechanistic studies on the interaction of Glycyl-L-alanine, Glycyl-L-asparagine, and Glycyl-L-tyrosine with hydroxopentaaquarhodium(III) Ion. J. Chem. 2013, 2013.[CrossRef] Biomolecules 2019, 9, 733 18 of 23

3. Mccormack, W.P.; Hoffman, J.R.; Pruna, G.J.; Jajtner, A.R.; Townsend, J.R.; Stout, J.R.; Fragala, M.S.; Fukuda, D.H. Effects of l-Alanyl-l-Glutamine ingestion on one-hour run performance. J. Am. Coll. Nutr. 2015, 34, 488–496. [CrossRef] 4. Magnuson, B.A.; Burdock, G.A.; Doull, J.; Kroes, R.M.; Marsh, G.M.; Pariza, M.W.; Spencer, P.S.; Waddell, W.J.; Walker, R.; Williams, G.M. Aspartame: A safety evaluation based on current use levels, regulations, and toxicological and epidemiological studies. Crit. Rev. Toxicol. 2007, 37, 629–727. [CrossRef] 5. Kino, H.; Nakajima, S.; Arai, T.; Kino, K. Effective production of Pro-Gly by mutagenesis of l-amino acid ligase. J. Biosci. Bioeng. 2016, 122, 155–159. [CrossRef] 6. Davis, C.K.; Laud, P.J.; Zsanett, B.; Rajanikant, G.K.; Arshad, M. Systematic review and stratified meta-analysis of the efficacy of carnosine in animal models of ischemic stroke. J. Cereb. Blood Flow Metab. 2016, 36, 1686–1694. [CrossRef] 7. Babizhayev, M.A.; Deyev, A.I.; Yermakova, V.N.; Semiletov, Y.A.; Davydova, N.G.; Kurysheva, N.I.; Zhukotskii, A.V.; Goldman, I.M. N-Acetylcarnosine, a natural histidine-containing dipeptide, as a potent ophthalmic drug in treatment of human cataracts. Peptides 2001, 22, 979–994. [CrossRef] 8. Perazzo, J.; Castanho, M.A.; Sá, S.S. Pharmacological potential of the endogenous dipeptide kyotorphin and selected derivatives. Front. Pharm. 2016, 7, 530–540. [CrossRef] 9. Khavinson, V.K.; Morozov, V.G.; Malinin, V.V.; Kazakova, T.B.; Korneva, E.A. Effect of peptide Lys-Glu on interleukin-2 gene expression in lymphocytes. Bull. Exp. Biol. Med. 2000, 130, 898–899. [CrossRef] 10. Nakatani, M.; Shinohara, Y.; Takii, M.; Mori, H.; Asai, N.; Nishimura, S.; Furukawa-Hibi, Y.; Miyamoto, Y.; Nitta, A. Periocular injection of in situ hydrogels containing Leu-Ile, an inducer for neurotrophic factors, promotes retinal ganglion cell survival after optic nerve injury. Exp. Eye Res. 2011, 93, 873–879. [CrossRef] 11. Wang, T.; Wu, M.B.; Chen, Z.J.; Lin, J.P.; Yang, L.R. Separation, determination and antifungal activity test of the products from a new Bacillus amyloliquefaciens. Nat. Prod. Res. 2015, 30, 1215–1218. [CrossRef] 12. Gahungu, M.; Arguellesarias, A.; Fickers, P.; Zervosen, A.; Joris, B.; Damblon, C.; Luxen, A. Synthesis and biological evaluation of potential threonine synthase inhibitors: Rhizocticin A and Plumbemycin A. Bioorg. Med. Chem. 2013, 21, 4958–4967. [CrossRef] 13. Turner, J.G.; Taha, R.R.; Jill, D. Effects of tabtoxin on nitrogen metabolism. Physiol.Plant. 2010, 67, 649–653. [CrossRef] 14. Yagasaki, M.; Hashimoto, S. Synthesis and application of dipeptides; current status and perspectives. Appl. Microbiol. Biotechnol. 2008, 81, 13–22. [CrossRef] 15. Mandity, I.M.; Olasz, B.; Otvos, S.B.; Fulop, F. Continuous-flow solid-phase peptide synthesis: A revolutionary reduction of the amino acid excess. ChemSusChem 2014, 7, 3172–3176. [CrossRef] 16. Lawrenson, S.; Arav, R.; North, M. The peptide synthesis. Green Chem. 2017, 19, 1685–1691. [CrossRef] 17. Goswami, A.; Van Lanen, S.G. Enzymatic strategies and biocatalysts for amide bond formation: Tricks of the trade outside of the ribosome. Mol. Biosyst. 2015, 11, 338–353. [CrossRef] 18. Hashimoto, S.I. Occurrence, biosynthesis, and biotechnological production of dipeptides. In Amino Acid Biosynthesis ~ Pathways, Regulation and Metabolic Engineering; Wendisch, V.F., Ed.; Springer: Berlin/Heidelberg, Germany, 2007; Volume 5, pp. 327–348. 19. Süssmuth, R.D.; Mainz, A. Nonribosomal peptide synthesis-principles and prospects. Angew. Chem. Int. Ed. Engl. 2017, 56, 3770–3821. [CrossRef] 20. Wang, T.; Liang, Y.; Wu, M.; Chen, Z.; Lin, J.; Yang, L. Natural products from Bacillus subtilis with antimicrobial properties. Chinese J. Chem. Eng. 2015, 23, 744–754. [CrossRef] 21. Strieker, M.; Tanović,A.; Marahiel, M.A. Nonribosomal peptide synthetases: Structures and dynamics. Curr. Opin. Struc. Biol. 2010, 20, 234–240. [CrossRef] 22. Candela, T.; Fouet, A. Poly-gamma-glutamate in bacteria. Mol. Microbiol. Y 2006, 60, 1091–1098. [CrossRef] 23. Ogasawara, Y.; Dairi, T. Biosynthesis of Oligopeptides using ATP-grasp Enzymes. Chemistry 2017, 23, 10714–10724. [CrossRef] 24. Galperin, M.Y.; Koonin, E.V. A diverse superfamily of enzymes with ATP-dependent carboxylate-amine/thiol ligase activity. Protein Sci. 1997, 6, 2639–2643. [CrossRef] 25. Tsuda, T.; Suzuki, T.; Kojima, S. Crystallization and preliminary X-ray diffraction analysis of Bacillus subtilis YwfE, an l -amino-acid ligase. Acta Cryst. A 2012, 68, 203–206. 26. Tsuda, T.; Asami, M.; Koguchi, Y.; Kojima, S. Single mutation alters the substrate specificity of L-amino acid ligase. Biochemistry 2014, 53, 2650–2660. [CrossRef] Biomolecules 2019, 9, 733 19 of 23

27. Arai, T.; Arimura, Y.; Ishikura, S.; Kino, K. L-amino acid ligase from Pseudomonas syringae producing tabtoxin can be used for enzymatic synthesis of various functional peptides. Appl. Env. Microbiol. 2013, 79, 5023–5029. [CrossRef] 28. Kino, K.; Noguchi, A.; Arai, T.; Yagasaki, M. Identification and characterization of a novel L-amino acid ligase from Photorhabdus luminescens subsp. laumondii TT01. J. Biosci. Bioeng. 2010, 110, 39–41. [CrossRef] 29. Kino, K.; Nakazawa, Y.; Yagasaki, M. Dipeptide synthesis by L-amino acid ligase from Ralstonia solanacearum. Biochem. Biophys. Res. Commun. 2008, 371, 536–540. [CrossRef] 30. Kino, H.; Kino, K. Alteration of the substrate specificity of l-amino acid ligase and selective synthesis of Met-Gly as a salt taste enhancer. Biosci. Biotechnol. Biochem. 2015, 79, 1827–1832. [CrossRef] 31. Kagawa, W.; Arai, T.; Ishikura, S.; Kino, K.; Kurumizaka, H. Structure of RizA, an L-amino-acid ligase from Bacillus subtilis. Acta Cryst. A 2015, 71, 1125–1130. 32. Arai, T.; Kino, K. A novel -amino acid ligase is encoded by a gene in the Phaseolotoxin biosynthetic gene cluster from pv. 1448A. Biosci. E Biotechnol. Biochem. 2014, 72, 3048–3050. [CrossRef] 33. Blasiak, L.C.; Clardy, J. Discovery of 3-formyl-tyrosine metabolites from Pseudoalteromonas tunicata through heterologous expression. J. Am. Chem. Soc. 2010, 132, 926–927. [CrossRef] 34. Kino, K.; Noguchi, A.; Nakazawa, Y.; Yagasaki, M. A novel l -amino acid ligase from Bacillus licheniformis. J. Biosci. Bioeng. 2008, 106, 313–315. [CrossRef] 35. Nishanth Kumar, S.; Dileep, C.; Mohandas, C.; Nambisan, B.; Ca, J. Cyclo(D-Tyr-D-Phe): A new antibacterial, anticancer, and antioxidant cyclic dipeptide from Bacillus sp. N strain associated with a rhabditid entomopathogenic nematode. J. Pept. Sci. 2014, 20, 173–185. [CrossRef] 36. Dai, X.; Zhou, E.; Yang, W.; Zhang, X.; Zhang, W.; Rao, Y. D-Serine made by serine racemase in Drosophila intestine plays a physiological role in sleep. Nat. Commun. 2019, 10, 1986–1996. 37. Al-Bar, O.A.; O’Connor, C.D.; Giles, I.G.; Akhtar, M. D-alanine: D-alanine ligase of Escherichia coli. Expression, purification and inhibitory studies on the cloned enzyme. Biochem. J. 1992, 282, 747–752. [CrossRef] 38. Prosser, G.A.; de Carvalho, L.P.Metabolomics reveal D-Alanine: D-Alanine ligase as the target of D-cycloserine in Mycobacterium tuberculosis. ACS Med. Chem. Lett. 2013, 4, 1233–1237. [CrossRef] 39. Kino, K.; Arai, T.; Arimura, Y. Poly-alpha-glutamic acid synthesis using a novel catalytic activity of RimK from Escherichia coli K-12. Appl. Env. Microbiol. 2011, 77, 2019–2025. [CrossRef] 40. Yokozeki, K.; Hara, S. A novel and efficient enzymatic method for the production of peptides from unprotected starting materials. J. Biotechnol. 2005, 115, 211–220. [CrossRef] 41. Abe, I.; Hara, S.; Yokozeki, K. Gene cloning and characterization of α-amino acid ester acyl transferase in Empedobacter brevis ATCC14234 and Sphingobacterium siyangensis AJ2458. Biosci. Biotechnol. Biochem. 2011, 75, 2087–2892. [CrossRef] 42. Hirao, Y.; Mihara, Y.; Kira, I.; Abe, I.; Yokozeki, K. Enzymatic production of L-alanyl-L-glutamine by recombinant E. coli expressing alpha-amino acid ester acyltransferase from Sphingobacterium siyangensis. Biosci. Biotechnol. Biochem. 2013, 77, 618–623. [CrossRef] 43. Li, Y.; Yuan, W.; Gao, J.; Fan, C.; Wu, W.; Bai, F. Production of l-alanyl-l-glutamine by recycling E. coli expressing α-amino acid ester acyltransferase. Bioresour. Technol. 2017, 245, 1603–1609. [CrossRef] 44. Srirangan, K.; Orr, V.; Akawi, L.; Westbrook, A.; Moo-Young, M.; Chou, C.P. Biotechnological advances on Penicillin G acylase: Pharmaceutical implications, unique expression mechanism and production strategies. Biotechnol. Adv. 2013, 31, 1319–1332. [CrossRef] 45. Sklyarenko, A.V.; El’Darov, M.A.; Kurochkina, V.B.; Yarotsky, S.V. Enzymatic synthesis of β-lactam acids (review). App. Biochem. Micro. 2015, 51, 627–640. [CrossRef] 46. Gaudelli, N.M.; Long, D.H.; Townsend, C.A. β-Lactam formation by a non-ribosomal peptide synthetase during antibiotic biosynthesis. Nature 2015, 520, 383–387. [CrossRef] 47. Helena, M.O.; Martina, P.K.; Michal, G.; Pavel, K. Current state and perspectives of penicillin G acylase-based biocatalyses. Appl. Microbiol. Biotechnol. 2014, 98, 2867–2879. 48. Arroyo, M.; De la Mata, I.; Acebal, C.; Castillón, M.P. Biotechnological applications of penicillin acylases: State-of-the-art. Appl. Microbiol. Biotechnol. 2003, 60, 507–514. [CrossRef] 49. Grulich, M.; Štˇepánek, V.; Kyslík, P. Perspectives and industrial potential of PGA selectivity and promiscuity. Biotechnol. Adv. 2013, 31, 1458–1472. [CrossRef] 50. Lai, L.; Xu, Z.; Zhou, J.; Lee, K.D.; Amidon, G.L. Molecular basis of prodrug activation by human valacyclovirase, an alpha-amino acid ester hydrolase. J. Biol. Chem. 2008, 283, 9318–9327. [CrossRef] Biomolecules 2019, 9, 733 20 of 23

51. Polderman-Tijmes, J.J.; Jekel, P.A.; de Vries, E.J.; van Merode, A.E.; Floris, R.; van der Laan, J.M.; Sonke, T.; Janssen, D.B. Cloning, sequence analysis, and expression in Escherichia coli of the gene encoding an alpha-amino acid ester hydrolase from Acetobacter turbidans. Appl. Environ. Microbiol. 2002, 68, 211–218. [CrossRef] 52. Ye, L.J.; Wang, L.; Pan, Y.; Cao, Y. Changing the specificity of α-amino acid ester hydrolase toward -hydroxyl cephalosporins synthesis by site-directed saturation mutagenesis. Biotechnol. Let. 2012, 34, 1719–1724. [CrossRef] 53. Rosini, E.; Monelli, C.S.; Pollegioni, L.; Riva, S.; Monti, D. On the substrate preference of glutaryl acylases. J. Mol. Catal. B 2012, 76, 52–58. [CrossRef] 54. Bora, O.; Myungsook, K.; Jongchul, Y.; Kyungwha, C.; Yongchul, S.; Dongsoon, L.; Youngsoo, K. Deacylation activity of cephalosporin acylase to cephalosporin C is improved by changing the side-chain conformations of active-site residues. Biochem. Biophys. Res. Commun. 2003, 310, 19–27. 55. Loredano, P.; Elena, R.; Gianluca, M. Cephalosporin C acylase: Dream and(/or) reality. Appl. Microbiol. Biotechnol. 2013, 97, 2341–2355. 56. Volpato, G.; Rodrigues, R.C.; Fernandez-Lafuente, R. Use of enzymes in the production of semi-synthetic penicillins and cephalosporins: Drawbacks and perspectives. Curr. Med. Chem. 2010, 17, 3855–3873. [CrossRef] 57. Pollegioni, L.; Lorenzi, S.E.; Marcone, G.L.; Molla, G.; Verga, R.; Cabri, W.; Pilone, M.S. Evolution of an acylase active on cephalosporin C. Protein Sci. 2010, 14, 3064–3076. [CrossRef] 58. Otten, L.G.; Sio, C.F.; van der Sloot, A.M.; Cool, R.H.; Quax, W.J. Mutational analysis of a key residue in the substrate specificity of a cephalosporin acylase. Chembiochem 2004, 5, 820–825. [CrossRef] 59. Tseng, W.C.; Fang, T.Y.; Hsieh, Y.C.; Chen, C.Y.; Li, M.C. Solubility and thermal response of fractionated cyanophycin prepared with recombinant Escherichia coli. J. Biotechnol. 2017, 249, 59–65. [CrossRef] 60. Hühns, M.; Neumann, K.; Hausmann, T.; Ziegler, K.; Klemke, F.; Kahmann, U.; Staiger, D.; Lockau, W.; Pistorius, E.K.; Broer, I. Plastid targeting strategies for cyanophycin synthetase to achieve high-level polymer accumulation in Nicotiana tabacum. Plant Biotechnol. J. 2010, 6, 321–336. [CrossRef] 61. Simone Cardoso, D.; Ingo, V.; Alexander, S. Optimization of cyanophycin production in recombinant strains of Pseudomonas putida and Ralstonia eutropha employing elementary mode analysis and statistical experimental design. Biotechnol. Bioeng. 2010, 93, 698–717. 62. Sallam, A.; Kalkandzhiev, D.; Steinbüchel, A. Production optimization of cyanophycinase ChpE al from Pseudomonas alcaligenes DIP1. AMB Express 2011, 1, 38. [CrossRef] 63. Nausch, H.; Broer, I. Cyanophycinase CphE from P. alcaligenes produced in different compartments of N. benthamiana degrades high amounts of cyanophycin in plant extracts. Appl. Microbiol. Biotechnol. 2017, 101, 1–17. [CrossRef] 64. Sallam, A.; Kast, A.; Przybilla, S.; Meiswinkel, T.; Steinbuchel, A. Biotechnological process for production of beta-dipeptides from cyanophycin on a technical scale and its optimization. Appl. Env. Microbiol. 2009, 75, 29–38. [CrossRef] 65. Ponndorf, D.; Broer, I.; Nausch, H. Expression of CphB- and CphE-type cyanophycinases in cyanophycin-producing tobacco and comparison of their ability to degrade cyanophycin in plant and plant extracts. Transgenic Res. 2017, 26, 491–499. [CrossRef] 66. Ernst, A.; Liu, Y.D.; Reich, S.; Böger, P. Diurnal nitrogenase modification in the Cyanobacterium Anabaena variabilis. Plant Biol. 2015, 103, 183–189. 67. Martin, O.; Fred Bernd, O.S.; Heinrich, L.; Alexander, S. Isolation of cyanophycin-degrading bacteria, cloning and characterization of an extracellular cyanophycinase gene (cphE) from Pseudomonas anguilliseptica strain BI. The cphE gene from P. anguilliseptica BI encodes a cyanophycinhydrolyzing enzyme. J. Biol. Chem. 2002, 277, 25096–25105. 68. Obst, M.; Sallam, A.; Luftmann, H.; Steinbuchel, A. Isolation and characterization of gram-positive cyanophycin-degrading bacteria-kinetic studies on cyanophycin depolymerase activity in aerobic bacteria. Biomacromolecules 2004, 5, 153–161. [CrossRef] 69. Martin, O.; Andreas, K.; Heinrich, L.; Alexander, S. Degradation of cyanophycin by Sedimentibacter hongkongensis strain KI and Citrobacter amalonaticus strain G isolated from an anaerobic bacterial consortium. Appl. Env. Microbiol. 2005, 71, 3642–3652. Biomolecules 2019, 9, 733 21 of 23

70. Ahmed, S.; Alexander, S. Anaerobic and aerobic degradation of cyanophycin by the denitrifying bacterium Pseudomonas alcaligenes strain DIP1 and role of three other coisolates in a mixed bacterial consortium. Appl. Env. Microbiol. 2008, 74, 3434–3443. 71. Mishra, A.K.; Choi, J.; Choi, S.J.; Baek, K.H. Cyclodipeptides: An overview of their biosynthesis and biological activity. Molecules 2017, 22, 1796–1808. [CrossRef] 72. Gondry, M.; Sauguet, L.; Belin, P.; Thai, R.; Amouroux, R.; Tellier, C.; Tuphile, K.; Jacquet, M.; Braud, S.; Courcon, M.; et al. Cyclodipeptide synthases are a family of tRNA-dependent peptide bond-forming enzymes. Nat. Chem. Biol. 2009, 5, 414–420. [CrossRef] 73. García-Estrada, C.; Ullán, R.; Albillos, S.; Fernández-Bodega, M.; Durek, P.; Vondöhren, H.; Martín, J. A single cluster of coregulated genes encodes the biosynthesis of the mycotoxins roquefortine C and meleagrin in Penicillium chrysogenum. Chem. Biol. 2011, 18, 1499–1512. [CrossRef] 74. Giessen, T.W.; Marahiel, M.A. The tRNA-dependent biosynthesis of modified cyclic dipeptides. Int. J. Mol. R Sci. 2014, 15, 14610–14631. [CrossRef] 75. Belin, P.; Moutiez, M.; Lautru, S.; Seguin, J.; Pernodet, J.L.; Gondry, M. The nonribosomal synthesis of diketopiperazines in tRNA-dependent cyclodipeptide synthase pathways. Nat. Prod. Rep. 2012, 29, 961–979. [CrossRef] 76. Seguin, J.; Moutiez, M.; Li, Y.; Belin, P.; Lecoq, A.; Fonvielle, M.; Charbonnier, J.B.; Pernodet, J.L.; Gondry, M. Nonribosomal peptide synthesis in animals: The cyclodipeptide synthase of Nematostella. Chem. Biol. 2011, 18, 1362–1368. [CrossRef] 77. Jacques, I.B.; Moutiez, M.; Witwinowski, J.; Darbon, E.; Martel, C.; Seguin, J.; Favry, E.; Thai, R.; Lecoq, A.; Dubois, S. Analysis of 51 cyclodipeptide synthases reveals the basis for substrate specificity. Nat. Chem. Biol. 2015, 11, 721–727. [CrossRef] 78. Moutiez, M.; Seguin, J.; Fonvielle, M.; Belin, P.; Jacques, I.B.; Favry, E.; Arthur, M.; Gondry, M. Specificity determinants for the two tRNA substrates of the cyclodipeptide synthase AlbC from Streptomyces noursei. Nucleic Acids Res. 2014, 42, 7247–7258. [CrossRef] 79. Sauguet, L.; Moutiez, M.; Li, Y.; Belin, P.; Seguin, J.; Le Du, M.H.; Thai, R.; Masson, C.; Fonvielle, M.; Pernodet, J.L.; et al. Cyclodipeptide synthases, a family of class-I aminoacyl-tRNA synthetase-like enzymes involved in non-ribosomal peptide synthesis. Nucleic Acids Res. 2011, 39, 4475–4489. [CrossRef] 80. Bourgeois, G.; Seguin, J.; Babin, M.; Belin, P.; Moutiez, M.; Mechulam, Y.; Gondry, M.; Schmitt, E. Structural basis for partition of the cyclodipeptide synthases into two subfamilies. J. Struct. Biol. 2018, 203, 17–26. [CrossRef] 81. Moutiez, M.; Schmitt, E.; Seguin, J.; Thai, R.; Favry, E.; Belin, P.; Mechulam, Y.; Gondry, M. Unravelling the mechanism of non-ribosomal peptide synthesis by cyclodipeptide synthases. Nat. Commun. 2014, 5, 5141–5147. [CrossRef] 82. Skinnider, M.A.; Johnston, C.W.; Merwin, N.J.; Dejong, C.A.; Magarvey, N.A. Global analysis of prokaryotic tRNA-derived cyclodipeptide biosynthesis. BMC Genom. 2018, 19, 45–55. [CrossRef] 83. Takahashi, H.; Kumagai, T.; Kitani, K.; Mori, M.; Matoba, Y.; Sugiyama, M. Cloning and characterization of a Streptomyces single module type non-ribosomal peptide synthetase catalyzing a blue pigment synthesis. J. Biol. Chem. 2007, 282, 9073–9081. [CrossRef] 84. Bonnefond, L.; Arai, T.; Sakaguchi, Y.; Suzuki, T.; Ishitani, R.; Nureki, O. Structural basis for nonribosomal peptide synthesis by an aminoacyl-tRNA synthetase paralog. Proc. Nati. Acad. Sci. USA 2011, 108, 3912–3917. [CrossRef] 85. Dubois, P.; Correia, I.; Le Chevalier, F.; Dubois, S.; Jacques, I.; Canu, N.; Moutiez, M.; Thai, R.; Gondry, M.; Lequin, O.; et al. Reprogramming Escherichia coli for the production of prenylated indole diketopiperazine alkaloids. Sci. Rep. 2019, 9, 9208–9220. [CrossRef] 86. Liu, J.; Yu, H.; Li, S.M. Expanding tryptophan-containing cyclodipeptide synthase spectrum by identification of nine members from Streptomyces strains. Appl. Microbiol. Biotechnol. 2018, 102, 4435–4444. [CrossRef] 87. Chun, S.B.; Nam-Seok, J.; Giancarlo, A.; Yeum, K.J. Biological functions of histidine-dipeptides and metabolic syndrome. Nutr. Res. Pr. 2014, 8, 3–10. 88. Drozak, J.; Veiga-da-Cunha, M.; Vertommen, D.; Stroobant, V.; Schaftingen, E.V. Molecular identification of carnosine synthase as ATP-grasp domain-containing protein 1 (ATPGD1). J. Biol. Chem. 2010, 285, 9346–9356. [CrossRef] Biomolecules 2019, 9, 733 22 of 23

89. Veiga-Da-Cunha, M.; Chevalier, N.; Stroobant, V.; Vertommen, D.; Van, S.E. Metabolite proofreading in carnosine and homocarnosine synthesis: Molecular identification of PM20D2 as β-alanyl-lysine dipeptidase. J. Biol. Chem. 2014, 289, 19726–19736. [CrossRef] 90. Yazawa, K.; Numata, K. Recent advances in chemoenzymatic peptide syntheses. Molecules 2014, 19, 13755–13774. [CrossRef] 91. Bialkowska, A.M.; Morawski, K.; Florczak, T. Extremophilic proteases as novel and efficient tools in short peptide synthesis. J. Ind. Microbiol. Biotechnol. 2017, 44, 1325–1342. [CrossRef] 92. Salam, S.M.A.; Kagawa, K.-i.; Matsubara, T.; Kawashiro, K. Protease-catalyzed dipeptide synthesis from N-protected amino acid carbamoylmethyl esters and free amino acids in frozen aqueous solutions. Enzym. Microb. Tech. 2008, 43, 537–543. [CrossRef] 93. Miyazawa, T.; Horimoto, T.; Tanaka, K. Kinetically controlled peptide synthesis mediated by papain using the carbamoylmethyl ester as an acyl donor. Int. J. Pept. Res. Ther. 2014, 20, 371–376. [CrossRef] 94. Harrison, M.J.; Burton, N.A.; Hillier, I.H. Catalytic mechanism of the enzyme papain: Predictions with a hybrid quantum mechanical/molecular mechanical potential. J. Am. Chem. Soc. 1997, 119, 12285–12291. [CrossRef] 95. Gudeangadi, P.G.; Tsuchiya, K.; Sakai, T.; Numata, K. Chemoenzymatic synthesis of polypeptides consisting of periodic di- and tri-peptide motifs similar to elastin. Polym. Chem. 2018, 9, 2336–2344. [CrossRef] 96. Wang, M.; Qi, W.; Yu, Q.; Su, R.; He, Z. Kinetically controlled enzymatic synthesis of dipeptide precursor of L-alanyl-L-glutamine. Biotechnol. Appl. Bioc. 2011, 58, 449–455. [CrossRef] 97. Cao, S.L.; Xu, H.; Li, X.H.; Lou, W.Y.; Zong, M.H. Papain@ magnetic nanocrystalline cellulose nanobiocatalyst: A highly efficient biocatalyst for dipeptide biosynthesis in deep eutectic solvents. ACS Sustain. Chem. Eng. 2015, 3, 1589–1599. [CrossRef] 98. Doekel, S.; Marahiel, M.A. Dipeptide formation on engineered hybrid peptide synthetases. Chem. Biol. 2000, 7, 373–384. [CrossRef] 99. Miyanaga, A.; Kudo, F.; Eguchi, T. Protein-protein interactions in polyketide synthase-nonribosomal peptide synthetase hybrid assembly lines. Nat. Prod. Rep. 2018, 35, 1185–1209. [CrossRef] 100. Duerfahrt, T.; Doekel, S.; Sonke, T.; Quaedflieg, P.J.; Marahiel, M.A. Construction of hybrid peptide synthetases for the production of alpha-l-aspartyl-l-phenylalanine, a precursor for the high-intensity sweetener aspartame. Eur. J. Biochem. 2003, 270, 4555–4563. [CrossRef] 101. Rui, Z.; Zhang, W. Engineering biosynthesis of non-ribosomal peptides and polyketides by directed evolution. Curr. Top. Med. Chem. 2016, 16, 1755–1762. [CrossRef] 102. Belshaw, P.J.; Walsh, C.T.; Stachelhaus, T. Aminoacyl-CoAs as probes of condensation domain selectivity in nonribosomal peptide synthesis. Science 1999, 284, 486–489. [CrossRef] 103. Niquille, D.L.; Hansen, D.A.; Mori, T.; Fercher, D.; Kries, H.; Hilvert, D. Nonribosomal biosynthesis of backbone-modified peptides. Nat. Chem. 2018, 10, 282–287. [CrossRef] 104. Ferrario, V.; Fischer, M.; Zhu, Y.; Pleiss, J. Modelling of substrate access and substrate binding to cephalosporin acylases. Sci. Rep. 2019, 9, 12402–12414. [CrossRef] 105. Sonawane, V.C. Enzymatic modifications of cephalosporins by cephalosporin acylase and other enzymes. Crit. Rev. Biotechnol. 2006, 26, 95–120. [CrossRef] 106. Sio, C.F.; Otten, L.G.; Cool, R.H.; Quax, W.J. Analysis of a substrate specificity switch residue of cephalosporin acylase. Biochem. Biophys. Res. Commun. 2003, 312, 755–760. [CrossRef] 107. Xiao, Y.; Huo, X.; Qian, Y.; Zhang, Y.; Chen, G.; Ouyang, P.; Lin, Z. Engineering of a CPC acylase using a facile pH indicator assay. J. Ind. Microbiol. Biotechnol. Y 2014, 41, 1617–1625. [CrossRef] 108. Ye, T.; Xu, Z.; Huang, X.; Zhu, Y. Computational design to improve catalytic activity of cephalosporin C acylase from Pseudomonas strain N176. RSC Adv. 2017, 7, 30370–30375. 109. Xu, Z.; Ye, T.; Zhu, Y. Computational design of thermostable mutants for cephalosporin C acylase from Pseudomonas strain SE83. Comput. Chem. Eng. 2018, 116, 112–121. [CrossRef] 110. Tian, Y.; Huang, X.; Li, Q.; Zhu, Y. Computational design of variants for cephalosporin C acylase fromPseudomonasstrain N176 with improved stability and activity. Appl. Microbiol. Biotechnol. 2017, 101, 621–632. [CrossRef] 111. Qing, L.; Huang, X.; Zhu, Y. Evaluation of active designs of cephalosporin C acylase by molecular dynamics simulation and molecular docking. J. Mol. R Model. 2014, 20, 2314–2325. Biomolecules 2019, 9, 733 23 of 23

112. He, J.; Huang, X.; Xue, J.; Zhu, Y. Computational redesign of penicillin acylase for cephradine synthesis with high kinetic selectivity. Green Chem. 2018, 20, 5484–5490. [CrossRef] 113. Huang, X.; Xue, J.; Lin, M.; Zhu, Y. Use of an improved matching algorithm to select scaffolds for enzyme design based on a complex active site model. PLoS ONE 2016, 11, e0156559. [CrossRef] 114. Zhang, H.; Li, J.; Huang, G.; Yang, Z.; Han, L. Understanding the synergistic effect and the main factors influencing the enzymatic hydrolyzability of corn stover at low enzyme loading by hydrothermal and/or ultrafine grinding pretreatment. Bioresour. Technol. 2018, 264, 327–334. [CrossRef] 115. Ovchinnikov, S.; Park, H.; Varghese, N.; Huang, P.S.; Pavlopoulos, G.A.; Kim, D.E.; Kamisetty, H.; Kyrpides, N.C.; Baker, D. Protein structure determination using metagenome sequence data. Science 2017, 355, 294–298. [CrossRef] 116. Hwang, H.; Dey, F.; Petrey, D.; Honig, B. Structure-based prediction of ligand-protein interactions on a genome-wide scale. Pro. Nati. Acad. Sci. USA 2017, 114, 13685–13690. [CrossRef] 117. Zheng, M.; Zhao, J.; Cui, C.; Fu, Z.; Li, X.; Liu, X.; Ding, X.; Tan, X.; Li, F.; Luo, X. Computational chemical biology and drug design: Facilitating protein structure, function, and modulation studies. Med. Res. Rev. 2018, 38, 914–950. [CrossRef] 118. Yamamoto, T.; Shimoyama, T.; Kuriyama, M. Dietary and enteral interventions for Crohn’s disease. Curr. Opini. Biotechnol. 2017, 44, 69–73. [CrossRef]

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