Metabolic Engineering Strategy for Synthetizing Trans-4-Hydroxy-L

Zhang et al. Microb Cell Fact (2021) 20:87 https://doi.org/10.1186/s12934-021-01579-2 Microbial Cell Factories REVIEW Open Access Metabolic engineering strategy for synthetizing trans-4-hydroxy-L-proline in microorganisms Zhenyu Zhang1†, Pengfu Liu1†, Weike Su1,2, Huawei Zhang2, Wenqian Xu1 and Xiaohe Chu1* Abstract Trans-4-hydroxy-L-proline is an important amino acid that is widely used in medicinal and industrial applications, par- ticularly as a valuable chiral building block for the organic synthesis of pharmaceuticals. Traditionally, trans-4-hydroxy- L-proline is produced by the acidic hydrolysis of collagen, but this process has serious drawbacks, such as low produc- tivity, a complex process and heavy environmental pollution. Presently, trans-4-hydroxy-L-proline is mainly produced via fermentative production by microorganisms. Some recently published advances in metabolic engineering have been used to efectively construct microbial cell factories that have improved the trans-4-hydroxy-L-proline biosynthetic pathway. To probe the potential of microorganisms for trans-4-hydroxy-L-proline production, new strategies and tools must be proposed. In this review, we provide a comprehensive understanding of trans-4-hydroxy-L-proline, including its biosynthetic pathway, proline hydroxylases and production by metabolic engineering, with a focus on improving its production. Keywords: Trans-4-hydroxy-L-proline, Biosynthetic pathway, Proline hydroxylases, Metabolic engineering Background t4Hyp being present only in the Y position [1]. T4Hyp Hydroxyprolines are a series of hydroxylated derivatives was previously considered to have little nutritional sig- of proline comprising six isomers—trans-4-hydroxy- nifcance but is now thought to be a major precursor for l-proline (t4Hyp), trans-3-hydroxy-l-proline (t3Hyp), the synthesis of glycine, pyruvate, and glucose. Some evi- cis-4-hydroxy-l-proline (c4Hyp), cis-3-hydroxy-l-pro- dence supports the ability of t4Hyp to scavenge oxidants, line (c3Hyp), trans-5-hydroxy-l-proline (t5Hyp), and regulate the state of cellular reduction and stimulate the cis-5-hydroxy-l-proline (c5Hyp) (Fig. 1). Furthermore, expression of anti-oxidative enzymes in the cell [2, 3]. t4Hyp is a major amino acid in collagen proteins, which T4Hyp is required for normal secretion after protein contain two α1 and one α2 polypeptide chain, to allow synthesis and contributes to the integrity of the triple- the sharp twisting of the collagen helix and are the major helical conformation [4]. In animals, t4Hyp is produced extracellular components of connective tissues, such as by the posttranslational hydroxylation of proline resi- skin, tendon, cartilage, blood vessels, and bone. Te heli- dues in proteins [5, 6]. T4Hyp is a nonessential α-amino cal region of collagen comprises Gly-X-Y repeats, with acid and is present in several secondary metabolites (e.g., echinocandins and etamycin) [7]. Additionally, t4Hyp has been widely used in the medicine, biochemistry, food, *Correspondence: [email protected] †Zhenyu Zhang and Pengfu Liu contributed equally to this work and cosmetic industries and is usually used as a valuable 1 Collaborative Innovation Center of Yangtze River Delta Region Green building block for the synthesis of many pharmaceu- Pharmaceuticals, Zhejiang University of Technology, Hangzhou 310014, ticals [8]. T4Hyp can be used to synthesize many com- Zhejiang, People’s Republic of China Full list of author information is available at the end of the article pounds, such as glutamic acid analogs, kainoid analogs © The Author(s) 2021. 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:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Zhang et al. Microb Cell Fact (2021) 20:87 Page 2 of 15 (a class of nonproteinogenic amino acids with a broad Artifcial biosynthetic pathway spectrum of biological activities), arginine analogs, and of trans‑4‑hydroxy‑L‑proline in Escherichia coli pipecolic acid and its derivatives (which play important and Corynebacterium glutamicum biological roles as components of peptides, proteins, and Te main types of microorganisms currently used to intermediates for the synthesis of conformationally con- produce t4Hyp are Escherichia coli and Corynebacte- strained molecular scafolds) through diastereoselective rium glutamicum (Fig. 3) [19]. In particular, because synthesis, N,Nʹ-dioxides, carbapenem antibiotics (imipe- of a lack of proline hydroxylases, proline hydroxylase nem, meropenem, panipenem), angiotensin-converting is introduced into E. coli and C. glutamicum and exog- enzyme inhibitors, N-aryl pyrroles (important organic enously expressed to form a complete t4Hyp synthe- compounds for medicinal and material sciences), oxace- sis pathway. Glucose as a substrate undergoes a series prol (an atypical inhibitor of infammation used for the of biochemical reactions to produce α-ketoglutarate treatment of conditions afecting the connective tissues, (α-KG). Subsequently, α-KG is catalyzed to form glu- such as osteoarthritis), oligomers, and macrocycles (usu- tamate via glutamate synthase. Te synthesis pathway ally used as valuable sources of bioactive molecules that from glutamate to proline requires three enzymatic have properties such as good solubility and metabolite reactions (Fig. 4) [20]. First, glutamate is catalyzed stability for drug discovery) (Fig. 2) [9–18]. to γ-glutamyl phosphate by γ-glutamyl kinase. Tis enzyme is the rate-limiting enzyme of proline synthesis and can be inhibited by proline feedback [21]. Typi- cally, the enzyme is a homotetramer of a 367-amino acid polypeptide comprising an N-terminal 257-residue amino acid kinase (AAK) domain along with a 110-residue PUA (named for pseudo uridine synthases Fig. 1 Structures of the hydroxyproline isomers. a trans-4-hydroxy-L-proline. b trans-3-hydroxy-L-proline. c cis-4-hydroxy-L-proline. d cis-3-hydroxy-L-proline, and e 5-hydroxy-L-proline Zhang et al. Microb Cell Fact (2021) 20:87 Page 3 of 15 Fig. 2 Compounds for which the parent structure is hydroxyproline and archaeosine-specifc transglycosylases) domain dehydrogenase occur in a complex, and γ-glutamyl [22]. Glutamate-γ-semialdehyde dehydrogenase cat- kinase activity is undetectable in the absence of alyzes the second reaction of proline biosynthesis glutamate-γ-semialdehyde dehydrogenase [24, 25]. and activates γ-glutamyl phosphate to glutamate-γ- Glutamate-γ-semialdehyde then cyclizes spontaneously semialdehyde. Te enzyme is not susceptible to either to form pyrroline-5-carboxylate (an internal Schif end-product prohibition or inhibition by proline [23]. base). In the third reaction of the proline biosynthetic γ-Glutamyl kinase and glutamate-γ-semialdehyde Zhang et al. Microb Cell Fact (2021) 20:87 Page 4 of 15 Fig. 3 Biosynthetic pathway of hydroxyproline pathway, pyrroline-5-carboxylate is reduced to proline of pyrroline-5-carboxylate reductase, and NADPH by pyrroline-5-carboxylate reductase. Pyrroline-5-car- demonstrates higher activity than NADH [26]. boxylate reductase mediates the pyridine nucleotide- linked reduction of pyrroline-5-carboxylate to proline Catabolism of proline but not the reverse reaction, even at high substrate Proline can be catabolized to glutamate through two concentrations. NADPH and NADH are both cofactors enzymatic steps (Fig. 5). Te E. coli PutA protein comprises 1320 amino acid residues, and its dimer has a Zhang et al. Microb Cell Fact (2021) 20:87 Page 5 of 15 Fig. 4 Synthesis pathway from glutamate to proline Fig. 5 Proline is catabolized to glutamate via two enzymatic steps. PRODH proline dehydrogenase, P5CDH pyrroline-5-carboxylate dehydrogenase molecular mass of 293 kDa [27]. PutA is a multifunc- to an acceptor in the electron transport chain to com- tional favor-protein that is both a membrane-associated plete the catalytic cycle. In the second step of reaction, proline catabolic enzyme and a transcriptional repressor non-enzymatic hydrolysis of pyrroline-5-carboxylate of a pair of genes encoding proline utilization proteins generates glutamic semi-aldehyde; subsequently, glu- [28]. Te frst enzymatic step is that proline is catalyzed tamic semi-aldehyde is oxidized to glutamate through to generate pyrroline-5-carboxylate by PutA. Te elec- the transfer of two electrons to reduce NAD + to NADH trons from reduced FAD are subsequently transferred [29]. Tere is some evidence that PutA has DNA-binding Zhang et al. Microb Cell Fact (2021) 20:87 Page 6 of 15 activity and serves as a transcriptional repressor of the Dactylosporangium sp.RH1) [42], l-proline cis-3-hydrox- putP and putA genes (proline utilization regulon) in E. ylase (cis-P3H, from the Streptomyces sp. strain TH1) coli [27]. In E. coli, the regulation of put genes relies on [43], and l-proline cis-4-hydroxylase (cis-P4H, from Mes- the concentration of proline and intracellular location orhizobium loti and Sinorhizobium meliloti) [44], and of the PutA protein [30]. In the presence of proline, the convert l-proline to generate trans-3-hydroxy-l-proline, localization of PutA changes from the cytoplasm to the trans-4-hydroxy-l-proline, cis-3-hydroxy-l-proline, membrane. Te transcription of put genes is then acti- and cis-3-hydroxy-l-proline, respectively.

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