Escherichia Coli from Glucose Wei Zhou1,2,3, Yibin Zhuang1,2, Yanfen Bai1,2,3, Huiping Bi1,2*, Tao Liu1,2* and Yanhe Ma1
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Zhou et al. Microb Cell Fact (2016) 15:149 DOI 10.1186/s12934-016-0549-9 Microbial Cell Factories RESEARCH Open Access Biosynthesis of phlorisovalerophenone and 4‑hydroxy‑6‑isobutyl‑2‑pyrone in Escherichia coli from glucose Wei Zhou1,2,3, Yibin Zhuang1,2, Yanfen Bai1,2,3, Huiping Bi1,2*, Tao Liu1,2* and Yanhe Ma1 Abstract Background: Type III polyketide synthases (PKSs) contribute to the synthesis of many economically important natural products, which are typically produced by direct extraction from plants or synthesized chemically. For exam- ple, humulone and lupulone (Fig. 1a) in hops (Humulus lupulus) account for the characteristic bitter taste of beer and display multiple pharmacological effects. 4-Hydroxy-6-methyl-2-pyrone is a precursor of parasorboside contributing to insect and disease resistance of plant Gerbera hybrida, and was recently demonstrated to be a potential platform chemical. Results: In this study, we achieved simultaneous biosynthesis of phlorisovalerophenone, a key intermediate of humulone biosynthesis and 4-hydroxy-6-isobutyl-2-pyrone in Escherichia coli from glucose. First, we constructed a biosynthetic pathway of isovaleryl-CoA via hydroxy-3-methylglutaryl CoA followed by dehydration, decarboxylation and reduction in E. coli. Subsequently, the type III PKSs valerophenone synthase or chalcone synthase from plants were introduced into the above E. coli strain, to produce phlorisovalerophenone and 4-hydroxy-6-isobutyl-2-pyrone at the highest titers of 6.4 or 66.5 mg/L, respectively. Conclusions: The report of biosynthesis of phlorisovalerophenone and 4-hydroxy-6-isobutyl-2-pyrone in E. coli adds a new example to the list of valuable compounds synthesized in E. coli from renewable carbon resources by type III PKSs. Keywords: Isovaleryl-CoA, Phlorisovalerophenone, 4-Hydroxy-6-isobutyl-2-pyrone, Valerophenone synthase, Escherichia coli Background pharmacological effects [4]. Hyperforin (Fig. 1a) is one A large number of natural products are synthesized by of the main active constituents in extracts of Hypericum type III polyketide synthases (PKSs). These compounds perforatum used for the treatment of depression [5]. The play an important role in human nutrition and health, 4-hydroxy-2-pyrone analogues display multiple biological and have recently expanded their roles as platform chem- activities and are valuable pharmaceutical precursors [6]. icals. Acylphloroglucinol derivatives have been isolated 4-Hydroxy-6-methyl-2-pyrone, also known as triacetic from a number of plants [1–3]. Their chemical struc- acid lactone (TAL, Fig. 1b) is a precursor of gerberin tures and intriguing biological activities have attracted and parasorboside, which could protect Gerbera hybrid increasing attention in recent years. Humulone and lupu- from fungal pathogens and attack by insects [7]. Fistupy- lone (Fig. 1a) in hops (Humulus lupulus) account for the rone (4-hydroxy-6-isovaleryl-2-pyrone) (Fig. 1b) was characteristic bitter taste of beer and display multiple isolated from Streptomyces sp. TP-A0569 and showed inhibition of the infection of Chinese cabbage by a fun- *Correspondence: [email protected]; [email protected] gus Alternaria brassicicola [8]. Germicidin homologs 1 Tianjin Institute of Industrial Biotechnology, Chinese Academy (Fig. 1b) arisen from streptomyces inhibit spore ger- of Sciences, Tianjin 300308, China mination and Na+/K+ ATPase [9, 10]. More recently, Full list of author information is available at the end of the article © 2016 The Author(s). This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Zhou et al. Microb Cell Fact (2016) 15:149 Page 2 of 11 Fig. 1 Examples of phloroglucinols (a) and 2-pyrones (b) synthesized by type III PKS. PIBP phlorisobutyrophenone; PIVP phlorisovalerophenone; TAL 4-hydroxy-6-methyl-2-pyrone (triacetic acid lactone); HIPP 4-hydroxy-6-isopropyl-2-pyrone; HIBP 4-hydroxy-6-isobutyl-2-pyrone 4-hydroxy-2-pyrones were reported to be potential plat- The acylphloroglucinol cores, phlorisovalerophenone form chemicals (Additional file 1: Figure S1a) [11–13]. (PIVP) and phlorisobutyrophenone (PIBP) are formed by Advances in synthetic biology and metabolic engineer- condensation of three malonyl-CoA-derived acetate units ing greatly promoted biosynthesis of a variety of valuable with isovaleryl-CoA or isobutyryl-CoA as the starter compounds, for instance, flavonoids produced by type III units [4, 20]. The enzyme valerophenone synthase (VPS) PKSs [14, 15] in microbial hosts. Biological production involved in the formation of PIVP in the biosynthesis of phloroglucinol (Fig. 1a) and TAL (Fig. 1b) from glu- of humulone has been characterized [21–23]. VPS is a cose has also been accomplished using genetically modi- homologue of chalcone synthase (CHS). They share the fied microorganisms [6, 16–19]. However, biosynthesis same reaction mechanism namely claisen condensation, of acylphloroglucinols and TAL analogues from renew- but differ in substrate specificity. CHS catalyzes conden- able feedstocks in Escherichia coli has not been well sation of one molecule of p-coumaroyl-CoA and three investigated. molecules of malonyl-CoA to form naringenin-chalcone. Zhou et al. Microb Cell Fact (2016) 15:149 Page 3 of 11 However, VPS preferentially uses isovaleryl-CoA or simultaneously subcloned into the multiple cloning isobutyryl-CoA instead of p-coumaroyl-CoA as the sites (MCS) 1 of pETDuet-1 by Golden Gate [25] clon- starter unit to condense with three molecules of malo- ing using AarI. Subsequently, the gene product of liuC nyl-CoA to form PIVP [21, 23]. Recently, dual functional was digested and ligated into the above resulting plas- CHS/VPS have also been reported. A CHS, FvCHS2-1 mid using HindIII/AflII to yield the final plasmid pET- from strawberry (Fragaria vesca) was identified to be A. Here, the sequences of T7 promoter, lac operator responsible for acylphloroglucinols synthesis in straw- and ribosome binding site (RBS) from pETDuet-1 were berry fruit [3]. Intriguingly, in vitro experiments demon- incorporated into the primer design, so each gene was strated that type III PKSs such as VPS and CHS usually driven by a single T7 promoter. ERG10 and ERG13 were also synthesize TAL analogues, 4-hydroxy-6-isobutyl- PCR-amplified from Saccharomyces cerevisiae BY4742 2-pyrone (HIBP) and 4-hydroxy-6-isopropyl-2-pyrone genome with primer pairs ERG10-F/ERG10-R or ERG13- (HIPP) (Fig. 1b), carrying isobutyl or isopropyl at posi- F/ERG13-R, respectively. First, the ERG13 fragment was tion 6 with isovaleryl-CoA or isobutyl-CoA as the starter digested and cloned into NcoI/BamHI sites of pCDF- unit and malonyl-CoA as the extender unit. Duet-1. The ERG10 fragment was digested and ligated In this work, we engineered the synthesis of isovaleryl- into the above plasmid via NdeI/XhoI sites, generating CoA in E. coli by recruiting a biosynthetic pathway via the recombinant plasmid pCDF-E. The HlVPS gene from hydroxy-3-methylglutaryl CoA (HMG-CoA), an inter- hops (GenBank: AB015430.1) was codon optimized for mediate of the mevalonate pathway (Fig. 2a) [24]. The expression in E. coli and synthesized by Shanghai Gen- VPS from hops (Humulus lupulus) HlVPS, the CHS eray Biotech Co., Ltd. (Shanghai, China), designated from strawberry (Fragaria vesca) FvCHS2-1 and a newly as HlVPSsyn (Additional file 1: Table S2). HlVPSsyn was cloned CHS from H. perforatum HpCHS were intro- PCR-amplified with primer pair VPS-F/VPS-R, and duced into the isovaleryl-CoA producing E. coli strain cloned into pET28a through NdeI/AflII sites generat- separately, led to the simultaneous production of PIVP ing the intermediate expression vector pET28a-HlVPS. and HIBP at different ratios. The highest titers of PIVP The T7-HlVPSsyn fragment with a T7 promoter and an and HIBP produced by the recombinant strains reached N-terminal His tag was PCR-amplified from pET28a- 6.4 and 66.5 mg/L, respectively. This work adds new HlVPS with primer pair T7VPS-F/T7VPS-R, digested products to the list of valuable compounds biosynthe- and ligated into pCDF-E via NotI/AflII to generate the sized in E. coli using type III PKSs and lays a foundation final recombinant plasmid pCDF-EV1. FvCHS2-1 from for microbial synthesis of not only acylphloroglucinol strawberry (GenBank: XM_004306495.1) was codon derivatives, but also potential platform chemicals. optimized for expression in E. coli and synthesized by Shanghai Generay Biotech Co., Ltd. (Shanghai, China), Methods designated as FvCHS2-1syn (Additional file 1: Table S2). Strains, plasmids and medium HpCHS was obtained from the mRNA of H. perforatum Strains and plasmids used in this study are listed in by RT-PCR. RNA of H. perforatum callus was isolated by Table 1. Luria–Bertani (LB) medium was used for the RNAprep pure Plant Kit from Tiangen Biotech Co., Ltd. propagation of E. coli cells for plasmid construction. Ter- (Beijing, China). Reverse transcription was performed rific Broth (TB) medium (12 g/L tryptone, 24 g/L yeast using the First Strand cDNA Synthesis Kit from Toyobo extract, 4 mL/L glycerol, 2.3 g/L KH2PO4, and 12.5 g/L Co., Ltd. (Osaka, Japan). PCR reactions were performed K2HPO4) and modified M9 medium (1 × M9 minimal using High Fidelity PCR system (Fermentas, Germany) salts, 20 g/L glucose, 10 g/L yeast extract, 2 mM MgSO4, with primer pair HpCHS-F/HpCHS-R. The HpCHS and 0.1 mM CaCl2) were used for protein expression and sequence was submitted into NCBI database (GenBank: PIVP production, respectively. Ampicillin (100 mg/L) KU180217).