Journal of Fungi

Review Reconstitution of Polyketide-Derived Meroterpenoid Biosynthetic Pathway in oryzae

Takayoshi Awakawa 1,2,* and Ikuro Abe 1,2,*

1 Laboratory of Natural Products Chemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan 2 Collaborative Research Institute for Innovative Microbiology, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan * Correspondence: [email protected] (T.A.); [email protected] (I.A.)

Abstract: The heterologous gene expression system with Aspergillus oryzae as the host is an effective method to investigate fungal secondary metabolite biosynthetic pathways for reconstruction to produce un-natural molecules due to its high productivity and genetic tractability. In this review, we focus on biosynthetic studies of fungal polyketide-derived meroterpenoids, a group of bioactive natural products, by means of the A. oryzae heterologous expression system. The heterologous expression methods and the biosynthetic reactions are described in detail for future prospects to create un-natural molecules via biosynthetic re-design.

Keywords: Aspergillus oryzae; heterologous expression; secondary metabolites; meroterpenoid

 

Citation: Awakawa, T.; Abe, I. Reconstitution of Polyketide-Derived 1. Introduction Meroterpenoid Biosynthetic Pathway Aspergillus oryzae is a that has been utilized for over 2000 years in the Japanese in Aspergillus oryzae. J. Fungi 2021, 7, fermentation industry to yield , , and , and for industrial enzyme pro- 486. https://doi.org/10.3390/jof duction. A. oryzae is an important heterologous expression host for biosynthetic genes from 7060486 Aspergillus species that produce numerous secondary metabolites, such as , aflatoxin, medicinal compounds, lovastatin, and pigments such as emodin [1–3], as well Academic Editors: as other secondary metabolite-producing fungi. For investigations on their biosynthesis, Katsuhiko Kitamoto and the heterologous expression system is a valuable tool, as it provides information about Yujiro Higuchi their reactions based on the products accumulated in the transformants. The A. oryzae includes some genes encoding secondary metabolite biosynthetic enzymes, such Received: 31 May 2021 as the non-ribosomal peptide synthetase, which is responsible for the production of toxic Accepted: 14 June 2021 compounds, such as cyclopiazonic acid. However, most of these genes, including cyclopi- Published: 16 June 2021 azonic acid synthetases, are dysfunctional or not expressed in the A. oryzae strains used in fermented food, and many have been recognized as safe strains that do not produce Publisher’s Note: MDPI stays neutral any toxic compounds [4]. In addition, A. oryzae possesses a strong metabolic flux to sup- with regard to jurisdictional claims in published maps and institutional affil- ply precursors of polyketides, terpenoids, and peptides, as judged by the high yields of iations. heterologous expression products. Furthermore, genetic manipulation methods, such as gene deletion or heterologous expression, strong promoters (e.g., PamyB and PenoA) and multicopy vectors [5,6], and the genetically tractable mutated strains A. oryzae M-2-3 and NSAR1, have been established [7,8]. The long-standing efforts of academic and industrial investigators have made this strain an excellent chassis for the heterologous expression of Copyright: © 2021 by the authors. secondary metabolite genes. By using this strain, we can easily access new biosynthetic Licensee MDPI, Basel, Switzerland. pathways and construct systems for the production of beneficial compounds. This article is an open access article distributed under the terms and The fungal meroterpenoids are one of the groups of natural products that include conditions of the Creative Commons various medicinally important compounds, such as the acyl-CoA:cholesterol acyltrans- Attribution (CC BY) license (https:// ferase inhibitor pyripyropene, the immunosuppressant mycophenolic acid, and the anti- creativecommons.org/licenses/by/ trypanosomiasis ascofuranone (Figure1)[ 9]. The biosynthesis of fungal meroterpenoids 4.0/).

J. Fungi 2021, 7, 486. https://doi.org/10.3390/jof7060486 https://www.mdpi.com/journal/jof J. Fungi 2021, 7, 486 2 of 10 J.J. Fungi Fungi 2021 2021, ,7 7, ,x x FOR FOR PEER PEER REVIEW REVIEW 22 ofof 1111

cancancan be bebe separated separatedseparated into intointo five fivefive parts: parts:parts: polyketide polyketidepolyketide sy synthesis,synthesis,nthesis, prenyl prenyl transfer, transfer, epoxidation, epoxidation,epoxidation, cy- cy- clization,clization,clization, and and post-cyclization post-cyclization post-cyclization modification modification modification [10–12], [10–12], [10–12 as], as asexemplified exemplified exemplified by by bypyripyropene pyripyropene pyripyropene A A bi- bi- A osynthesisbiosynthesisosynthesis (Figure (Figure (Figure 2). 2).2). By By By using using using the the the versatile versatile A. A.A. oryzae oryzae oryzae host, host,host, which which synthesizes synthesizes polyketide polyketidepolyketide andandand terpenoid terpenoidterpenoid compounds compounds with with high high yields, yields, we we can can create create a a fungal fungal meroterpenoid meroterpenoid pro- pro- duction system. This method includes important benefits, in that we can exploit the novel ductionduction system. system. This This method method includes includes important important benefits, benefits, in in that that we we can can exploit exploit the the novel novel biocatalysts by identifying the compounds accumulated in the heterologous expression biocatalystsbiocatalysts byby identifyingidentifying thethe compoundscompounds acaccumulatedcumulated inin thethe heterologousheterologous expressionexpression system, and also re-engineer the biosynthetic pathway easily, by simply swapping one part system,system, andand alsoalso re-engineerre-engineer thethe biosyntheticbiosynthetic pathwaypathway easily,easily, byby simplysimply swappingswapping oneone of the biosynthetic machinery or feeding an un-natural substrate. partpart of of the the biosynthetic biosynthetic machinery machinery or or feeding feeding an an un-natural un-natural substrate. substrate.

FigureFigureFigure 1. 1.1. Representative Representative bioactive bioactivebioactive fungal fungalfungal meroterpenoids. meroterpenoids.meroterpenoids.

FigureFigureFigure 2. 2.2. Biosynthetic Biosynthetic pathway pathway of of pyripyropene pyripyropenepyripyropene A. A.A. 2. Pyripyropene A Biosynthetic Pathway 2.2. Pyripyropene Pyripyropene A A Biosynthetic Biosynthetic Pathway Pathway The earliest study of fungal meroterpenoid biosynthesis was the reconstitution of TheThe earliest earliest study study of of fungal fungal meroterpenoid meroterpenoid biosynthesis biosynthesis was was the the reconstitution reconstitution of of the the the early pyripyropene A biosynthetic pathway in the Aspergillus oryzae M-2-3Δ strain earlyearly pyripyropene pyripyropene A A biosynthetic biosynthetic pathway pathway in in the the Aspergillus Aspergillus oryzae oryzae M-2-3 M-2-3 strain strain ( (ΔargBargB)) ([13].∆argB The)[13 pyr2]. The (polyketidepyr2 (polyketide synthase, synthase, PKS) gene PKS) was gene cloned was into cloned the into pTAex3 the pTAex3 vector [14], vec- [13]. The pyr2 (polyketidepyr1 synthase, PKS) gene was cloned into the pTAex3amyB vector [14], torand [14 the], andpyr1 the (CoA-ligase)(CoA-ligase) gene was gene cloned was cloned into pPTRI into pPTRI [15], with [15], withthe amyB the promoterpromoter and andand the terminator pyr1 (CoA-ligase) from pTAex3, gene to was yield cloned pTAex3- intopyr2 pPTRIand pPTRI-[15], withpyr1 the, respectively. amyB promoter Similarly, and terminator from pTAex3, to yield pTAex3-pyr2 and pPTRI-pyr1, respectively. Similarly, terminatorpyr5 (flavin-dependent from pTAex3, monooxygenase, to yield pTAex3- FMO)pyr2 wasand clonedpPTRI- intopyr1, pTAex3, respectively. and pyr4 Similarly,(trans- pyr5 (flavin-dependent monooxygenase, FMO) was cloned into pTAex3, and pyr4 (trans- pyr5membrane (flavin-dependent meroterpenoid monooxygenase, cyclase, CYC) FMO) and pyr6 was(UbiA-type cloned into prenyltransferase, pTAex3, and pyr4 PT) (trans- were membrane meroterpenoid cyclase, CYC) and pyr6 (UbiA-type prenyltransferase, PT) were membranecloned into meroterpenoid pPTRI with the cyclase,amyB promoter CYC) and and pyr6 terminator, (UbiA-type to yield prenyltransferase, pTAex3-pyr5 and PT) pPTRI- were cloned into pPTRI with the amyB promoter and terminator, to yield pTAex3-pyr5 and clonedpyr4+6 ,into respectively. pPTRI with Two the sets amyB of vectors, promoter pTAex3- and terminator,pyr2 and pPTRI- to yieldpyr1 pTAex3-and pTAex3-pyr5 pyr5and pPTRI-pyr4+6, respectively. Two sets of vectors, pTAex3-pyr2 and pPTRI-pyr1 and pPTRI-and pPTRI-pyr4+pyr46, respectively.+6, were introduced Two sets into ofAspergillus vectors, oryzaepTAex3-M-2-3pyr2 [7 ].and The pPTRI-A. oryzaepyr1/pyr1+2 and pTAex3-pyr5 and pPTRI-pyr4+6, were introduced into Aspergillus oryzae M-2-3 [7]. The A. pTAex3-was fedpyr5 nicotinic and pPTRI- acid andpyr4 produced+6, were 4-hydroxy-6-(3-pyridinyl)-2H-pyran-2-oneintroduced into Aspergillus oryzae M-2-3 [7]. (HPPO), The A. oryzae/pyr1+2 was fed nicotinic acid and produced 4-hydroxy-6-(3-pyridinyl)-2H-pyran- oryzaewhile/A.pyr1+2 oryzae was/pyr4+5+6 fed nicotinicwas fed acid HPPO and andproduced produced 4-hydroxy-6-(3-pyridinyl)-2H-pyran- deacetylpyripyropene E. The struc- 2-one (HPPO), while A. oryzae/pyr4+5+6 was fed HPPO and produced deacetylpyripyro- 2-onetures of(HPPO), these products while A. suggested oryzae/pyr4+5+6 that the was following fed HPPO biosynthetic and produced reactions, deacetylpyripyro- Pyr1 and Pyr2, pene E. The structures of these products suggested that the following biosynthetic reac- peneproduce E. The HPPO structures from nicotinic of these acid,products CoA, su andggested malonyl-CoA; that the following Pyr6 transfers biosynthetic the farnesyl reac- tions, Pyr1 and Pyr2, produce HPPO from nicotinic acid, CoA, and malonyl-CoA; Pyr6 tions,group Pyr1 at C-3 and to yieldPyr2, 3-farnesyl-HPPO;produce HPPO from Pyr5 nicotinic epoxidizes acid, the CoA, terminal and olefinmalonyl-CoA; of the farnesyl Pyr6 transfersgroup;transfers and the the Pyr4 farnesyl farnesyl facilitates group group theat at C-3 C-3 cyclization to to yield yield 3-farnesyl-HPPO; of3-farnesyl-HPPO; the epoxidated Pyr5 prenylPyr5 epoxidizes epoxidizes chain via the protonationthe terminal terminal olefintoolefin produce of of the the deacetylpyripyropene farnesyl farnesyl group; group; and and Pyr4 Pyr4 E (Figure facilit facilit2ates).ates This the the wascyclization cyclization the first of studyof the the epoxidated thatepoxidated identified prenyl prenyl the chainfunctionchain viavia of protonationprotonation the transmembrane toto produceproduce meroterpenoid deacetylpyripyropenedeacetylpyripyropene cyclase [16 E],E (Figure and(Figure it paved 2).2). ThisThis the waswas way thethe toward firstfirst studyinvestigationsstudy that that identified identified of the the fungalthe function function meroterpenoid of of the the tr transmembraneansmembrane biosynthetic meroterpenoid meroterpenoid pathway. The A.cyclase cyclase oryzae [16], [16],/pyr1+2 and and itstrainit pavedpaved was thethe fed wayway benzoic towardtoward acid investigationsinvestigations and generated ofof 4-hydroxy-6-phenyl-2H-pyran-2-one thethe fungalfungal meroterpenoidmeroterpenoid biosyntheticbiosynthetic (HpHpO), path-path- way.way. The The A. A. oryzae oryzae//pyr1+2pyr1+2 strain strain was was fed fed benzoic benzoic acid acid and and generated generated 4-hydroxy-6-phenyl- 4-hydroxy-6-phenyl-

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2H-pyran-2-one (HpHpO), which was then fed to A. oryzae/pyr4+5+6 as a substrate to yield which was then fed to A. oryzae/pyr4+5+6 as a substrate to yield the un-natural meroter- the un-natural meroterpenoid deacetyl-S14-95 (Figure 3) [13], illustrating the ease of mu- penoid deacetyl-S14-95 (Figure3)[ 13], illustrating the ease of mutasynthesis with this tasynthesisheterologous with expression this heterologous system. expression system.

Figure 3. Un-natural product synthesis from benzoic acid with Pyr12456. Figure 3. Un-natural product synthesis from benzoic acid with Pyr12456. 3. 3,5-Dimethylorselinic Acid (DMOA)-Derived Meroterpenoid Biosynthetic Pathways 3. 3,5-DimethylorselinicThe fungal meroterpenoids Acid derived (DMOA)-Derived from 3,5-dimethylorselinic Meroterpenoid acid (DMOA) Biosynthetic exhibit Path- waysstructural diversity derived from various kinds of biosynthetic enzymes, including terpene cyclases and oxygenases [10–12]. In most of our expression studies, we employed A. oryzae NSAR1The [8 fungal] as the heterologous meroterpenoids expression derived host. Since from this 3,5-dimethylorselinic strain possesses four auxotrophic acid (DMOA) exhibit structuralmutations (∆diversityargB, adeA -derived, sC-, and niaDfrom-), various we can introduce kinds of four biosynthetic vectors harboring enzymes, different including ter- peneauxotrophic cyclases markers, and oxygenases including argB [10–12].(pTAex3) In most [14], adeA of our(pAdeA) expression [17], sC studies,(pUSA) [ 18we], employed A. oryzaeand niaD NSAR1(pUNA) [8] [19 as]. the By employing heterologous two vectors expression with the host. pyrithiamine Since this resistance strain markerpossesses four aux- ptrA (pPTRI) [15] and the glufosinate resistance marker bar (pBAR) [20], we can introduce Δ - - - otrophicup to six mutations vectors into ( a singleargB, expressionadeA , sC , host. and Together,niaD ), we the can strong introduceamyB promoter four vectors on harboring differentthese vectors auxotrophic and the efficient markers, metabolite including efflux inargB the host(pTAex3) facilitate [14], the constructionadeA (pAdeA) [17], sC (pUSA)of a high-yield [18], and production niaD (pUNA) system. [19]. By expressing By employing the genes two encoding vectors Trt4 with (PKS), the Trt2 pyrithiamine re- sistance(PT), Trt8 marker (FMO), andptrA Trt5 (pPTRI) (methyltransferase) [15] and the in A.glufosinate oryzae NSAR1, resistance we prepared marker a strain bar (pBAR) [20], that produces 10’R-epoxyfarnesyl-3,5-DMOA methyl ester (Figure4)[ 21,22]. With the weadditionally can introduce expressed uptrt1 to six(CYC) vectors in the A.into oryzae a sisystem,ngle expression the strain produced host. Together, preterretonin the strong amyB promoterA (Figure4 on). When these we vectors swapped andtrt1 thewith efficientausL, a meta homologbolite of trt1 effluxclassified in the in host the same facilitate the con- structionclade of the of phylogenetic a high-yield tree, production the trt4285+ system.ausL transformant By expressing produced the protoaustinoid genes encoding A, Trt4 (PKS), Trt2a compound (PT), Trt8 with (FMO), a differently and cyclizedTrt5 (methyltransferase) terpene ring (Figure4 ).in Furthermore, A. oryzae NSAR1, replacing we prepared a straintrt1 with thatadrI producesin the transformant 10'R-epoxyfarnesyl-3,5-DMOA also led to the production methyl of another ester meroterpenoid, (Figure 4) [21,22]. With andrastin E [23]. These studies illustrate the usefulness of the heterologous gene expression thesystem additionally for metabolic expressed engineering, trt1 in (CYC) that we in can the create A. oryzae analogs system, by simply the swapping strain produced one preter- retoninenzyme A at a(Figure step in the4). biosyntheticWhen we reactions.swapped A trt1 similar with concept ausL was, a homolog also employed of trt1 in the classified in the samefungal clade indole of diterpene the phylogenetic combinatorial tree, biosynthesis the trt4285 [24].+ausL transformant produced protoausti- noid Recently,A, a compound we produced with novela differently meroterpenoids cyclized by terpene introducing ring meroterpenoid (Figure 4). Furthermore, cy- re- clases from four different biosynthetic pathways into the A. oryzae host producing 10’R- placingepoxyfarnesyl-3,5-DMOA trt1 with adrI in methylthe transformant ester, and obtained also led two to novel the mono-cyclizedproduction of meroter- another meroterpe- noid,penoids andrastin [25]. It is intriguingE [23]. These that the studies four different illustrat meroterpenoide the usefulness cyclases of produced the heterologous the gene expressionsame products system from the for non-native metabolic substrate. engineering, in that we can create analogs by simply swappingAnditomin one enzyme is also a DMOA-derivedat a step in the meroterpenoid biosynthetic withreactions. the characteristic A similar bicy-concept was also clo[2.2.2]octane structure (Figure5). In the anditomin biosynthetic study, we succeeded in employed in the fungal indole diterpene combinatorial biosynthesis [24]. the heterologous expression of 11 genes, andM(PKS)+K(P450 monooxygenase+hydrolase) +D(PT)+E(FMO)+B(CYC)+C(alcohol dehydrogenase)+A(α-ketoglutarate-dependent oxy- genase)+J(Baeyer-Villiger monooxygenase)+I(ketoreductase)+G(acetyltransferase)+H(ene- reductase). To manage the number of vectors, two expression cassettes of PamyB-ORF- TamyB were connected with a linear vector by three-fragment in-fusion recombination [20]. First, we introduced pTAex3-andM, pUSA-andK+D, and pAdeA-andE+B into A. oryzae NSAR1, and then transformed the resultant strain with pUNA-andC+A and pPTRI-andJ+I to construct the nine-gene expression strain. Finally, pBARI-andG+H was transformed into the nine-gene transformant to yield the 11-gene expression strain, which produced

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andilesin C (Figure5). pBARI was constructed by cloning the bar gene expression cassette with the gpd promoter from pBARGPE1 (FGSC) into pUC19. The transformants were selected with glufosinate, purified from a commercial herbicide [26]. To the best of our knowledge, the introduction of 11 genes into a single host is the largest number for A. oryzae heterologous expression. As we failed to introduce a 12th gene into the transformants, 11 genes might be the upper limit. The reaction of AndF was characterized through in vitro reaction by using andilesin C as a substrate. The characteristics of this biosynthesis are the generation of a 10S-epoxide, while the terretonin biosynthetic pathway generates a 10R- epoxide as a substrate for terpene cyclase, and the extensive terpene skeleton reconstruction by the α-ketoglutarate-dependent oxygenases AndA and AndF. The details of the cataly- sis by AndA have been extensively studied by a combination of X-ray crystal structural analysis and DFT calculations [27,28]. The generation of the 10S-epoxide in the Aspergillus J. Fungi 2021, 7, x FOR PEER REVIEW 4 of 11 expression system was also reported in the synthesis of the unique orthoester-bearing DMOA meroterpenoid novofumigatonin [29].

J. Fungi 2021, 7, x FOR PEER REVIEW 5 of 11 FigureFigure 4. 4. DiversificationDiversification of of DMOA-derived DMOA-derived meroterp meroterpenoidsenoids with with Trt1, Trt1, AusL, AusL, and and AdrI. AdrI. C-10’ C-10’R isR is highlightedhighlighted with with a a red red circle. circle.

Recently, we produced novel meroterpenoids by introducing meroterpenoid cyclases from four different biosynthetic pathways into the A. oryzae host producing 10'R- epoxyfarnesyl-3,5-DMOA methyl ester, and obtained two novel mono-cyclized mero- terpenoids [25]. It is intriguing that the four different meroterpenoid cyclases produced the same products from the non-native substrate. Anditomin is also a DMOA-derived meroterpenoid with the characteristic bicy- clo[2.2.2]octane structure (Figure 5). In the anditomin biosynthetic study, we succeeded in the heterologous expression of 11 genes, andM(PKS)+K(P450 monooxygenase+hydro- lase)+D(PT)+E(FMO)+B(CYC)+C(alcohol dehydrogenase)+A(α-ketoglutarate-dependent oxygenase)+J(Baeyer-Villiger monooxygenase)+I(ketoreductase)+G(acetyltransfer- ase)+H(ene-reductase). To manage the number of vectors, two expression cassettes of PamyB-ORF-TamyB were connected with a linear vector by three-fragment in-fusion re- combination [20]. First, we introduced pTAex3-andM, pUSA-andK+D, and pAdeA- andE+B into A. oryzae NSAR1, and then transformed the resultant strain with pUNA- andC+A and pPTRI-andJ+I to construct the nine-gene expression strain. Finally, pBARI- Figure 5. The 11-step anditomin biosynthetic pathway reconstructed in the A. oryzae heterologous expression system and andG+HFigure 5. Thewas 11-step transformed anditomin into biosynthetic the nine-gene pathway transformant reconstructed to yield in the the A. 11-gene oryzae heterologous expression the AndF reaction. C-10’S is highlighted with a red circle. strain,expression which system produced and the andilesin AndF reaction. C (Figure C-10’ S5). is pBARIhighlighted was with constructed a red circle. by cloning the bar gene expression cassette with the gpd promoter from pBARGPE1 (FGSC) into pUC19. The The D-ring of terretonin is highly oxygenated, and its biosynthesis is expected to transformants were selected with glufosinate, purified from a commercial herbicide [26]. include new biosynthetic enzymes. The expression of trt42851+trt9 (alcohol dehydrogenase) To the best of our knowledge, the introduction of 11 genes into a single host is the largest numberThe for D-ring A. oryzae of terretonin heterologous is highly expression. oxygenat Ased, we and failed its biosynthesis to introduce is a expected 12th gene to into in- theclude transformants, new biosynthetic 11 genes enzymes. might The be expressionthe upper limit.of trt42851+trt9 The reaction (alcohol of AndF dehydrogenase) was charac- terized+trt3 (flavin through monooxygenase) in vitro reaction resulted by using in andithe productionlesin C as a ofsubstrate. terrenoid, The which characteristics was further of thismodified biosynthesis to terretonin are the H, generation with the additional of a 10S-epoxide, expression while of trt6the (P450terretonin monooxygenase) biosynthetic pathway(Figure 6) generates [30]. The a membrane10R-epoxide fraction as a substrate expressing for terpene Trt6 was cyclase, also prepared and the extensive from A. ter-ory- penezae/trt6 skeleton and used reconstruction for the in vitro by assay, the α -ketoglutarate-dependentindicating that Aspergillus oxygenasesgene expression AndA system and AndF.can also The contribute details of tothe the catalysis preparation by AndA of membrane-boundhave been extensively enzymes studied that by are a combina- only ex- tionpressed of X-ray in the crystal eukaryotic structural host. analysis Trt6 cataly andzed DFT C-7 calculations hydroxylation, [27,28]. and The the generation generated of OH- the 107αS formed-epoxide a inhydrogen the Aspergillus bond with expression the carbonyl system oxygen was also of thereported methyl in ester. the synthesis This hydrogen of the uniquebond induces orthoester-bearing lactonization DMOA via the me nucleophilicroterpenoid attack novofumigatonin by OH-16 [30]. [29]. The resultant inter- mediate underwent retro-Claisen cleavage of the β-ketoester to the ring-expanded β-keto acid, and the following decarboxylation generated terretonin H. The additional expres- sion of Trt14 (isomerase) in A. oryzae/trt42851936 yielded terretonin D, which bears a me- thyl ester group, and facilitated the D-ring forming reaction. The molecular basis of Trt14 catalysis was reported in 2017, according to its crystal structure complexed with interme- diates [31].

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+trt3 (flavin monooxygenase) resulted in the production of terrenoid, which was further modified to terretonin H, with the additional expression of trt6 (P450 monooxygenase) (Figure6)[ 30]. The membrane fraction expressing Trt6 was also prepared from A. oryzae/trt6 and used for the in vitro assay, indicating that Aspergillus gene expression system can also contribute to the preparation of membrane-bound enzymes that are only expressed in the eukaryotic host. Trt6 catalyzed C-7 hydroxylation, and the generated OH-7α formed a hydrogen bond with the carbonyl oxygen of the methyl ester. This hydrogen bond induces lactonization via the nucleophilic attack by OH-16 [30]. The resultant intermediate underwent retro-Claisen cleavage of the β-ketoester to the ring-expanded β-keto acid, and the following decarboxylation generated terretonin H. The additional expression of Trt14 (isomerase) in A. oryzae/trt42851936 yielded terretonin D, which bears a methyl ester J. Fungi 2021, 7, x FOR PEER REVIEW 6 of 11 group, and facilitated the D-ring forming reaction. The molecular basis of Trt14 catalysis was reported in 2017, according to its crystal structure complexed with intermediates [31].

Figure 6. Biosynthesis of terretonin H and terretonin D from preterretonin A. Figure 6. Biosynthesis of terretonin H and terretonin D from preterretonin A. 4. Ascochlorin/Ascofuranone Biosynthetic Pathways 4. Ascochlorin/AscofuranoneAcremonium egyptiacum produces Biosynthetic two meroteropenoids, Pathways ascofuranone and ascochlorin, whichAcremonium are derived egyptiacum from the same produces intermediate, two meroteropenoids, illicicolin A epoxide ascofuranone [32] (Figure and7). ascochlo- To investigaterin, which are their derived biosynthetic from the pathways, same intermediat we expressede, illicicolinascC (polyketide A epoxide synthase), [32] (FigureascA 7). To (prenyltransferase),investigate their biosynthet and ascB (nonribosomalic pathways, peptidewe expressed synthetase-like ascC (polyketide reductase) synthase), in A. oryzae ascA NSAR1,(prenyltransferase), and the transformants and ascB produced(nonribosomal illicicolin peptide B with synthetase-like a yield of 0.71 mg/L.reductase) When in we A. ory- additionallyzae NSAR1, and expressed the transformantsascD (flavin-dependent produced illici halogenase),colin B with the transformanta yield of 0.71 yielded mg/L. When a smallwe additionally amount of ilicicolin expressed A (0.04 ascD mg/L). (flavin-dependent The lower yield halogenase might be caused), the transformant by the toxicity yielded of thea small product amount against of Aspergillusilicicolin A. Aspergillus(0.04 mg/L). soja Thewas lower also employed yield might as a be heterologous caused by expres-the toxicity sion host, and the proteins purified from this expression system were utilized for in vitro of the product against Aspergillus. Aspergillus soja was also employed as a heterologous assays. The information from these experiments clarified the biosynthetic pathways of expression host, and the proteins purified from this expression system were utilized for ascochlorin and ascofuranone, as shown in Figure7. In ascochlorin biosynthesis, illicicolin Ain epoxidevitro assays. was protonated The information and cyclized from these into a experiments (14S, 15R, 19 clarifiedR)-trimethylcyclohexanone the biosynthetic path- ringways structure of ascochlorin by AscF and to yieldascofuranone, ilicicolin C,as whichshown was in Figure then oxidized 7. In ascochlorin by AscG (P450 biosynthesis, oxi- dase)illicicolin to produce A epoxide ascochlorin. was protonated In ascofuranone and cyclized biosynthesis, into a C-16(14S, of15 illicicolinR, 19R)-trimethylcyclo- A epoxide washexanone hydroxylated ring structure by AscH by (P-450 AscF oxygenase),to yield ilicicolin and the C, productwhich was was then cyclized oxidized into asco-by AscG furanol(P450 oxidase) through to 6-endo-tet produce ascochlorin. cyclization by In AscI. ascofuranone The ascofuranol biosynthesis, was then C-16 oxidized of illicicolin by A AscJepoxide (NAD(P)-dependent was hydroxylated dehydratase) by AscH (P-450 to produce oxyge ascofuranone.nase), and the AscI product is a novel was cyclized meroter- into penoidascofuranol cyclase through sharing 6-endo-tet little similarity cyclization with knownby AscI. meroterpenoid The ascofuranol cyclases, was then while oxidized AscF by AscJ (NAD(P)-dependent dehydratase) to produce ascofuranone. AscI is a novel mero- terpenoid cyclase sharing little similarity with known meroterpenoid cyclases, while AscF has similarity with Trt1 and known meroterpenoid cyclases [16,32,33]. The gene deletion of ascF resulted in the higher production of ascofuranone (501 mg/L) as compared with the wild-type strain (388 mg/L), illustrating the effectiveness of bioengineering to create a high-yield metabolite production system based on biosynthetic knowledge.

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has similarity with Trt1 and known meroterpenoid cyclases [16,32,33]. The gene deletion of ascF resulted in the higher production of ascofuranone (501 mg/L) as compared with J. Fungi 2021, 7, x FOR PEER REVIEW 7 of 11 the wild-type strain (388 mg/L), illustrating the effectiveness of bioengineering to create a high-yield metabolite production system based on biosynthetic knowledge.

FigureFigure 7. BiosyntheticBiosynthetic pathways pathways of of ascochlorin ascochlorin and and ascofuranone. ascofuranone. Th Thee red red fonts fonts indicate indicate the the structural structural change change after after each each biosynthetic reaction. biosynthetic reaction.

5.5. Diterpene Diterpene Pyrone Pyrone Biosynthetic Biosynthetic Pathway Pathway TheThe most most successful successful and and thorough thorough study study on on the the combinatorial combinatorial biosynthesis biosynthesis of fungal of fun- meroterpenoidsgal meroterpenoids was performed was performed by Tsukada by Tsukada et al. et[34]. al. They [34]. Theyestablished established ten biosynthetic ten biosyn- pathwaysthetic pathways to yield to 22 yield diterpene 22 diterpene pyrones, pyrones, including including the immunosuppressive the immunosuppressive fungal mero- fungal terpenoidmeroterpenoid subglutinol subglutinol A from A Fusarium from Fusarium graminearum graminearum [35], and[35], 15 and novel 15 novel analogs. analogs. They Theyem- ployedemployed different different combinations combinations of the of thebiosynth biosyntheticetic genes genes from from five five fungi fungi isolated isolated from from a spider:a spider: F. graminearumF. graminearum, Macrophomina, Macrophomina phaseolina phaseolina, Colletotrichum, Colletotrichum higginsianum higginsianum, Metarhizium, Metarhiz- anisopliaeium anisopliae, and Arthrinium, and Arthrinium sacchari sacchari, in the, A. in oryzae the A. NSAR1 oryzae NSAR1heterologous heterologous expression expression system. Thesystem. genes The encoding genes encodingSubA/DpasA SubA/DpasA (PKS), SubD/DpasD (PKS), SubD/DpasD (geranylgeranylphosphate (geranylgeranylphos- syn- phate synthase), SubC/DpasC (PT), SubE/DpasE (FMO), and SubB/DpasB (CYC) from thase), SubC/DpasC (PT), SubE/DpasE (FMO), and SubB/DpasB (CYC) from these five these five clusters produced the common diterpene pyrone intermediate with a high yield clusters produced the common diterpene pyrone intermediate with a high yield (87 (87 mg/L). This intermediate structure was modified by the additional expression of mg/L). This intermediate structure was modified by the additional expression of DpmaF/DpasF/DpchF (flavin-dependent berberine bridge-like enzyme), to produce subg- DpmaF/DpasF/DpchF (flavin-dependent berberine bridge-like enzyme), to produce sub- lutinols A (89 mg/L) and B (6 mg/L) with their new analogs (Figure8). Furthermore, they glutinols A (89 mg/L) and B (6 mg/L) with their new analogs (Figure 8). Furthermore, they expressed DpfgG/DpmpG/DpchG and DpfgH/DpmpH/DpchH (NAD(P)-dependent expressed DpfgG/DpmpG/DpchG and DpfgH/DpmpH/DpchH (NAD(P)-dependent re- reductases/dehydratases) to produce the 8-OH epimerized higginsianin B. DpfgI/DpmpI ductases/dehydratases) to produce the 8-OH epimerized higginsianin B. DpfgI/DpmpI and DpfgK (methyltransferases), and DpmpJ and DpfgJ (P450 oxygenases) were employed and DpfgK (methyltransferases), and DpmpJ and DpfgJ (P450 oxygenases) were em- to further diverge the pathway, which yielded a total of 22 diterpene pyrones (Figure8) . ployed to further diverge the pathway, which yielded a total of 22 diterpene pyrones (Fig- The well-designed cloning strategies enabled the authors to clone three or four gene ex- ure 8). The well-designed cloning strategies enabled the authors to clone three or four pression cassettes into a single vector by two-step in-fusion cloning, and thus quickly geneconstruct expression diverse cassettes pathways. into Theya single reported vector aby broad two-step range in-fusion of bioactivity cloning, tests, and includ- thus quicklying anti-tumor, construct inhibition diverse pathways. of insect innate They repo immunerted system,a broad anti-HIV,range of bioactivity and anti-Alzheimer tests, in- cludingactivities. anti-tumor, Interestingly, inhibition the novel of insect compounds innate immune produced system, via the anti-HIV, un-natural and pathwaysanti-Alz- heimerpossess activities. different Interestingly, bioactivities fromthe novel the natural compounds compounds. produced This via studythe un-natural established path- the waysbasis possess of synthetic different biology bioactivities to supply from pharmacologically the natural compounds. active compounds This study by heterologous established theexpression basis of synthetic systems. biology to supply pharmacologically active compounds by heterol- ogous expression systems.

J.J. Fungi Fungi 20212021,, 77,, x 486 FOR PEER REVIEW 8 7of of 11 10 J. Fungi 2021, 7, x FOR PEER REVIEW 8 of 11

Figure 8. Combinatorial biosynthesis of diterpenoid pyrones in A. oryzae. FigureFigure 8.8. CombinatorialCombinatorial biosynthesisbiosynthesis ofof diterpenoidditerpenoid pyronespyrones ininA. A. oryzaeoryzae..

6.6.6. Biosynthetic BiosyntheticBiosynthetic Pathways PathwaysPathways of ofof Plant PlantPlant Meroterpenoids MeroterpenoidsMeroterpenoids in inin A. A.A. oryzae oryzaeoryzae InInIn addition additionaddition to to the the fungalfungal fungal meroterpenoids,meroterpenoids, meroterpenoids, plant plant plant meroterpenoids meroterpenoids meroterpenoids can can can be be be produced produced produced in in A.in A.oryzaeA. oryzae oryzaeby expressingby by expressing expressing the plantthe the plant enzymeplant enzyme enzyme with fungalwith with fungal meroterpenoidfungal meroterpenoid meroterpenoid biosynthetic biosynthetic biosynthetic enzymes. en- en- In zymes.2017,zymes. we In In successfully2017, 2017, we we successfully successfully constructed construct construct the planteded anti-HIVthe the plant plant meroterpenoidanti-HIV anti-HIV meroterpenoid meroterpenoid daurichromenic daurichro- daurichro- acid Rhododendron menic(1.23menic mg/L) acid acid (1.23 (1.23 by expressing mg/L) mg/L) by by DCASexpressing expressing (flavin-dependent DCAS DCAS (flavin-dependent (flavin-dependent oxidase) from oxidase) oxidase) the plant, from from the the plant, plant, dauricum, with StbA (PKS) and StbC (PT) from Stachybotrys bisbyi (Figure9)[ 36,37]. Further- RhododendronRhododendron dauricum dauricum, ,with with StbA StbA (PKS) (PKS) and and StbC StbC (PT) (PT) from from Stachybotrys Stachybotrys bisbyi bisbyi (Figure (Figure 9) 9) more, the additional expression of AscD from the Fusarium sp. [33] yielded its un-natural [36,37].[36,37]. Furthermore, Furthermore, the the addition additionalal expression expression of of AscD AscD from from the the Fusarium Fusarium sp. sp. [33] [33] yielded yielded halogenated analogs (2.06 mg/L). These results illustrate the potential of the Aspergillus itsits un-natural un-natural halogenated halogenated analogs analogs (2.06 (2.06 mg/L). mg/L). These These results results illustrate illustrate the the potential potential of of the the expression system to produce secondary metabolites. The recent successful syntheses of ba- AspergillusAspergillus expression expression system system to to produce produce second secondaryary metabolites. metabolites. The The recent recent successful successful sidiomycete and plant terpenes in A. oryzae further encourage the production of secondary synthesessyntheses of of basidiomycete basidiomycete and and plant plant terpenes terpenes in in A. A. oryzae oryzae further further encourage encourage the the produc- produc- metabolites from other organisms besides filamentous fungi [38,39]. tiontion of of secondary secondary metabolites metabolites from from other other organisms organisms besides besides filamentous filamentous fungi fungi [38,39]. [38,39].

FigureFigure 9. 9. ProductionProduction of of plant plant meroterpenoids meroterpenoids in A. oryzae. oryzae. Figure 9. Production of plant meroterpenoids in A. oryzae.

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7. Concluding Remarks Aspergillus oryzae is a powerful heterologous expression host for the production of polyketides and terpenoids. The high flux of its primary metabolism and the ease of genetic manipulation provide opportunities to produce various meroterpenoids. This powerful microbial platform will contribute to the generation of pharmaceutically important, un- natural molecules with relatively easy genetic and metabolic engineering. The recent development of a genetic manipulation system based on CRISPR-Cas9 in A. oryzae will further expand this versatile expression system, to produce high yields of diverse bioactive molecules, through engineering its primary metabolism and constructing multi-gene expression systems [40,41].

Author Contributions: Conceptualization, writing—review, editing, critical revising, and funding acquisition, T.A. and I.A. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (JSPS KAKENHI grant number JP16H06443, JP17H04763, JP19H04641, JP20H00490, JP20KK0173, and JP21H02636), the New Energy and Industrial Technology Development Organization (NEDO, Grant Number JPNP20011), UTEC- UTokyo FSI Research Grant Program, Kato Memorial Bioscience Foundation, and The Asahi Glass Foundation. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Conflicts of Interest: The authors declare no conflict of interest.

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