Wang et al. Microb Cell Fact (2020) 19:110 https://doi.org/10.1186/s12934-020-01367-4 Microbial Cell Factories REVIEW Open Access Biosynthesis of aromatic polyketides in microorganisms using type II polyketide synthases Jia Wang1, Ruihua Zhang2, Xin Chen1, Xinxiao Sun1, Yajun Yan2, Xiaolin Shen1* and Qipeng Yuan1* Abstract Aromatic polyketides have attractive biological activities and pharmacological properties. Diferent from other polyke- tides, aromatic polyketides are characterized by their polycyclic aromatic structure. The biosynthesis of aromatic pol- yketides is usually accomplished by the type II polyketide synthases (PKSs), which produce highly diverse polyketide chains by sequential condensation of the starter units with extender units, followed by reduction, cyclization, aroma- tization and tailoring reactions. Recently, signifcant progress has been made in characterization and engineering of type II PKSs to produce novel products and improve product titers. In this review, we briefy summarize the architec- tural organizations and genetic contributions of PKS genes to provide insight into the biosynthetic process. We then review the most recent progress in engineered biosynthesis of aromatic polyketides, with emphasis on generating novel molecular structures. We also discuss the current challenges and future perspectives in the rational engineering of type II PKSs for large scale production of aromatic polyketides. Keywords: Aromatic polyketides, Type II polyketide synthases, Starter units, Chain length, Tailoring reactions Background Te biosynthesis of polyketides is initiated by loading the Polyketides are structurally diverse and biologically active starter unit acyl-Coenzyme A (CoA) on the acyl carrier secondary metabolites derived from natural sources such protein (ACP) catalyzed by the AT domain [2]. Te KS as animals, plants, fungi and bacteria [1]. Tese com- domain subsequentially elongates the carbon chain by pounds are widely used as clinical medicines for the treat- decarboxylative Claisen condensation. Te β-keto group ment of various acute and chronic diseases [2]. Examples can be further modifed to generate diferent polyketide include antibacterial (erythromycin and tetracycline) [3], structures by additional domains, including ketoreduc- antitumor (anthracycline and doxorubicin) [4], antifungal tase (KR), dehydratase (DH) and enoylreductase (ER) (amphotericin and griseofulvin) [5], antiparasitic (aver- domains. Finally, the TE domain hydrolyzes or cyclizes mectin) [6] and anti-cholesterol (lovastatin) [7] drugs. the completed polyketide chain from the ACP-domain Polyketides are synthesized by polyketide synthases to terminate the elongation [9]. Although sharing a simi- (PKSs) which are multi-domain enzymes or enzyme lar synthetic process, PKSs can be classifed into three complexes [8], consisting of acyltransferase (AT), keto- types, type I, II, and III PKSs (Fig. 1) [10, 11]. Type I synthase (KS), thioesterase (TE) and optional domains. PKSs are multifunctional peptides containing linearly arranged and covalently fused domains. Te type I PKSs can be further classifed into iterative type I PKSs (iPKSs) *Correspondence: [email protected]; [email protected] (Fig. 1a) and modular type I PKSs (mPKSs) (Fig. 1b) [12]. 1 State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, 15 Beisanhuan East Road, Chaoyang iPKSs are mainly found in fungi and the domains are used District, Beijing 100029, China repeatedly to catalyze multiple rounds of elongation [13, Full list of author information is available at the end of the article 14]. While, mPKSs are primarily found in bacteria and © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://crea- tivecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdo- main/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Wang et al. Microb Cell Fact (2020) 19:110 Page 2 of 11 Fig. 1 Catalytic reaction of PKSs. a Iterative type I PKSs; b Modular type I PKSs; c Type II PKSs; d Typical type III PKSs use acyl-CoA as the starter unit and malonyl-CoA as the extender unit. KS ketoacyl synthase, AT acyltransferase, DH dehydratase, ER enoylreductase, KR ketoreductase, ACP acyl carrier protein, TE thioesterase, CYC cyclase, ARO aromatase, CHS chalcone synthase, STS stilbene synthase, AS acridone synthase function in an assembly line similar to the nonribosomal and functional group regeneration [16]. Type II PKSs are peptide synthases [15]. mPKSs consist of several domains multi-enzyme complexes composed of monofunctional with defned functions that are separated by short spacer proteins (Fig. 1c). Tey are found predominantly in bac- regions. Te distinct domains work cooperatively and teria and produce diverse aromatic polyketides [17]. Dif- non-iteratively to catalyze the carbon chain elongation ferent from both type I and type II PKSs, type III PKSs Wang et al. Microb Cell Fact (2020) 19:110 Page 3 of 11 are mainly found in plants as simple homodimers that production and broadening the spectrum of aromatic use CoA rather than ACP as an anchor for chain exten- polyketides. sion (Fig. 1d) [18], while both type II and type III PKSs are iterative. Production of aromatic polyketides by employing diferent Type II PKSs are mainly responsible for producing aro- starter units matic polyketides by catalyzing iterative Claisen conden- Te frst step of aromatic polyketide biosynthesis is load- sation reaction usually using acetate as the starter unit ing of a starter unit, mostly being acetate, onto ACP. A [19]. Aromatic polyketides are polycyclic compounds typical case is the biosynthesis of kinamycins, a group of harboring at least one aromatic ring [17]. Tey are an bacterial polyketide secondary metabolites containing important type of natural products with antibacterial, a diazo group [30], which begins with the condensation anticancer and antiviral bioactivities, with representative of 1 acetyl-CoA and 9 malonyl-CoA molecules to form examples being the above-mentioned drugs tetracyclines the intermediate dehydrorabelomycin [31]. While only a and anthracyclines [3, 4]. Te clinical and environmental few type II PKSs have been identifed to use other starter potential of aromatic polyketides has attracted increasing units, the employment of diferent starter units could attention from researchers to conduct studies on the bio- provide additional structurally diverse products. Propio- synthesis of these polyketides. Te production process of nyl-CoA has been reported to be the starter unit for sev- aromatic polyketides includes the following reactions. (1) eral aromatic polyketides. For instance, lomaiviticins, an α-Carboxylated precursor loading: acetate is loaded onto important class of diazo-containing aromatic polyketides, ACP, forming acyl-ACP. (2) Iterative chain extension have gained great interest because of their antibiotic and and ketone reduction: acyl-ACP is transferred to the KS antitumor activities [32]. Waldman and his colleagues subunit and iteratively elongated with the extender unit identifed a gene cluster responsible for the biosynthe- malonyl-CoA to form the poly-β-keto chain. (3) Cycliza- sis of lomaiviticin in Salinispora pacifca DPJ-0019. tion and/or aromatization: the poly-β-keto chain is cata- Tis cluster contains a bifunctional enzyme (encoded lyzed by aromatases (ARO) and oxygenases to generate by Lom62) that has both acyltransferation and decar- the aromatic polyketide core. (4) Post-modifcation: oxy- boxylation activities and can catalyze the conversion of genases, methyltransferases and glycosyltransferases cat- methylmalonyl-CoA to propionyl-CoA, which serves as a alyze this aromatic polyketide core to generate the fnal starter unit for lomaiviticin production (Fig. 2a) [19]. In product [17, 20]. Te characteristic of aromatic polyke- addition, Otten et al. identifed a gene cluster dnr encod- tides biosynthesis is the employment of a set of iteratively ing type II PKS which can also employ propionyl-CoA as used enzymes. Tis set of enzymes is called minimal type the starter unit for the production of daunorubicin and II PKS, which contains KSα, chain length factors (CLF or doxorubicin, two famous anthracycline topoisomerase KSβ) and ACP subunits. Te sequences of KS α and CLF inhibitors, in Streptomyces spp. [33]. However, expres- components are highly similar, except for a cysteine-con- sion of this cluster was found restrained by the lack of taining active site in KS α that is essential for assembling bldA-tRNA in S. peucetius to read a rare TTA codon in aromatic polyketides [17, 21]. A minimal PKS can solely dnrO, which is a transcriptional