Elsevier

Journal of Biotechnology

Volume 321, 10 September 2020, Pages 68-77
Journal of Biotechnology

Regulating the biosynthesis of pyridoxal 5'-phosphate with riboswitch to enhance L-DOPA production by Escherichia coli whole-cell biotransformation

https://doi.org/10.1016/j.jbiotec.2020.05.009Get rights and content

Highlights

  • The co-expression of TPL and pdxST genes for biosynthesis of L-DOPA and PLP.

  • Regulating the biosynthesis of PLP with riboswitch at translation level.

  • The titer and productivity of L-DOPA was improved to 69.8 g/L, 13.96 g/L/h.

  • Maintaining lower catechol concentration by continuous-flow in 3–L fermentor.

Abstract

Pyridoxal 5′-phosphate (PLP) is an essential cofactor that participates in ∼4% enzymatic activities cataloged by the Enzyme Commission. The intracellular level of PLP is usually lower than that demanded in industrial catalysis. To realize the self-supply of PLP cofactor in whole-cell biotransformation, the de novo ribose 5-phosphate (R5P)-dependent PLP synthesis pathway was constructed. The pdxST genes from Bacillus subtilis 168 were introduced into the tyrosine phenol-lyase (TPL)-overexpressing Escherichia coli BL21(DE3) strain. TPL and PdxST were co-expressed with a double-promoter or a compatible double-plasmid system. The 3,4-dihydroxyphenylacetate-L-alanine (L-DOPA) titer did not increase with the increase in the intracellular PLP concentration in these strains with TPL and PdxST co-expression. Therefore, it is necessary to optimize the intracellular PLP metabolism level so as to achieve a higher L-DOPA titer and avoid the formation of L-DOPA–PLP cyclic adducts. The thi riboswitch binds to PLP and forms a complex such that the ribosome cannot have access to the Shine-Dalgarno (SD) sequence. Therefore, this metabolite-sensing regulation system was applied to regulate the translation of pdxST mRNA. Riboswitch was introduced into pET–TPL–pdxST-2 to downregulate the expression of PdxST and biosynthesis of PLP at the translation level by sequestering the ribosome-binding site. As a result, the titer and productivity of L-DOPA using the strain BL21–TPLST–Ribo1 improved to 69.8 g/L and 13.96 g/L/h, respectively, with a catechol conversion of 95.9% and intracellular PLP accumulation of 24.8 μM.

Introduction

Pyridoxal 5′-phosphate (PLP) is one of the most multifunctional cofactors with a wide range of applications in ∼4% enzymatic activities that are cataloged by the Enzyme Commission, including oxidoreductases, transferases, hydrolases, lyases, and isomerases (di Salvo et al., 2012; Percudani and Peracchi, 2003). PLP that is found in eukaryotes serves vital roles in organs such as liver and heart and is involved mainly in basic metabolism (Schneider et al., 2000). PLP is also distributed among mitochondrial and cytosolic isoforms of microorganisms (Christen and Mehta, 2001). PLP-dependent enzymes are involved in diverse reactions, such as decarboxylation (Lee et al., 1999), transamination (Leipold et al., 2019; Rudat et al., 2012), racemization (Ahn et al., 2018; Awad et al., 2017), Cα–Cβ bond cleavage (Frey and Reed, 2011; Giger et al., 2012), and α,β-elimination reactions (Dajnowicz et al., 2015; Wang et al., 2016). PLP-dependent reactions play wide-ranging roles in catalyzing diverse processes and biosynthesis of natural products, such as 1,4-diaminobutane (Konst et al., 2011), L-erythro (3S,4S) ketoses (Lorilliere et al., 2017), amines (Baud et al., 2017), and γ-aminobutyric acid (Lammens et al., 2009). Generally, the theme of PLP in these enzymes remains constant, that is, these enzymes critically rely on the formation of a quinonoid intermediate (Du and Ryan, 2019). On the whole, PLP plays important roles in stabilizing the transition state and forming a quinonoid intermediate for the enzymatic reactions.

3,4-Dihydroxyphenylacetate-L-alanine (L-DOPA) has been widely used as a drug for Parkinson’s disease caused by the deficiency of the neurotransmitter dopamine since the 1970s (Rekdal et al., 2019). Metabolic engineering has been applied to generate Escherichia coli for L-DOPA biosynthesis from carbon source (Fordjour et al., 2019). There are inevitable issues for L-DOPA biosynthesis, such as low titer/yield and by-product accumulation (L-tyrosine and melanin caused by oxidation). Elimination of L-tyrosine from L-DOPA is a big challenge until now. Therefore, it is important to further improve the biotransformation process for L-DOPA production. In the last decade, production of pyruvic acid, which is the substrate for enzymatic synthesis of L-DOPA, has been significantly improved (Luo et al., 2019). This significantly lowered the cost for the biotransformation process by tyrosine phenol-lyase (TPL, E.C. 4.1.99.2) (Min et al., 2015a). TPL was widely found from Citrobacter freudii and Erwinia herbicola. TPL is homo-tetrameric and composed of two catalytic dimers, which are held together by intertwined N-terminal arms and a hydrophobic cluster formed in the middle of the tetramer (Fig. 1a) (Milic et al., 2008, 2006; Phillips et al., 2016). PLP binds with Arg100, Phe123, Asp214, Arg217, and Lys257 from active pockets of TPL and forms a TPL–PLP holoenzyme (Fig. 1b). PLP is involved in the electron sink effect from the pyridine ring, the conformational changes when the substrate binds with TPL, and the stabilization of several catalytic intermediates during the TPL catalytic reaction (Milic et al., 2012). Therefore, it is necessary to enhance intracellular PLP accumulation in the whole-cell biotransformation.

Two distinct de novo PLP synthesis pathways exist (Raschle et al., 2005). One is the deoxyglucose 5-phosphate (DXP)-dependent pathway, which is found in E. coli and a few members of the γ subdivision of Proteobacteria (Fitzpatrick et al., 2007; Strohmeier et al., 2006). This pathway involves seven enzymes and uses DXP as a precursor (Mittenhuber, 2001; Moccand et al., 2011). The other is the ribose 5-phosphate (R5P)-dependent pathway, which is widely distributed among fungi, plants, eubacteria, and archaea (Mukherjee et al., 2011; Zhang et al., 2010). Differentially, it just involves two enzymes, PdxS (also referred to as Pdx1, SnzP, or YaaD) and PdxT (also referred to as Pdx2, SnoP, or YaaE) (El Qaidi et al., 2013). In this study, the R5P-dependent PLP synthesis pathway was constructed with a common protein expression system, such as pCDFDuet–1, pETDuet–1, and pRSFDuet–1. These vectors were effective for protein expression to ensure biological activity. The PLP synthesis pathway was introduced into E. coli BL21 harboring TPL to construct a PLP self-supply catalysis system. Moreover, based on the similar molecular structure between PLP and thiamine pyrophosphate (TPP), riboswitch was introduced into the Shine-Dalgarno (SD) region of pdxST gene to regulate intracellular level of PLP by self-feedback control. The introduction of the PLP synthesis pathway and regulation system can not only avoid the exogenous addition of cofactors but is also highly advantageous to the downstream extraction of L-DOPA.

In this study, the co-expression of TPL and pdxST genes (Fig. 2) was applied to biosynthesize L-DOPA by whole-cell biotransformation. However, a previous study showed the formation of condensation adducts between L-DOPA and PLP (Lee et al., 2006). Moreover, the L-DOPA titer did not increase with the increase in the PLP metabolism level. Therefore, regulating the intracellular PLP synthesis level is a key issue in whole-cell biotransformation. For example, TPP–specific riboswitch from eukaryotic Arabidopsis thaliana is involved in regulating the biosynthesis of thiamine (Sudarsan et al., 2003). Due to the similar structure of PLP and TPP, thiM riboswitch and thiC riboswitch were introduced into pET–TPL–pdxST–2, followed by the SD region of pdxST genes. The thi riboswitch could be specially recognized and bound by PLP, bringing about a structural change in the SD region. This could result in the formation of a complex and, consequently, the ribosome had no access to the SD sequence. When PLP bound to thi riboswitch, the formation of a complex prevented the ribosome from combining with the SD region, and the translation of PdxST was inhibited. Accordingly, the intracellular PLP metabolism level was downregulated by repressing the initiation of translation. By regulating the biosynthesis of PLP with riboswitch, the titer and productivity of L-DOPA by E. coli whole-cell biotransformation were significantly enhanced. The strategy described in this study might help achieve the dynamic rational regulation of PLP-dependent whole cell transformation processes.

Section snippets

Cloning and enzymatic cascade construction

The pdxST genes (GenBank, Accession No. KR821087) were amplified from the genomic DNA of Bacillus subtilis 168 with primer pair pdxST F/R (Table 1) and inserted into the NdeI and XhoI sites of pCDFDuet–1′, pCDFDuet–1, pETDuet–1, and pRSFDuet–1 to generate the pCDFDuet–pdxST′, pCDFDuet–pdxST, pETDuet–pdxST, and pRSFDuet–pdxST plasmids, respectively. The second promoter of the pCDFDuet–1 plasmid was substituted by the Trc promoter, generating the pCDFDuet–1′ plasmid. All plasmids were saved in

Effect of PLP supplementation on whole-cell biotransformation

As shown in Fig. 3, when PLP was absent, the yield of L-DOPA was 5.2 ± 0.9 g/L after 1 h, with no obvious increase as the reaction proceeded. In comparison, with the addition of 30 μM PLP, the L-DOPA titer reached 13.2 ± 1.0 g/L at 1 h, 23.6 ± 0.9 g/L at 3 h, and 31.1 ± 1.0 g/L at 5 h. When PLP was increased to 60 μM and 90 μM, the variation tendency of biosynthesized L-DOPA was similar during the biotransformation process. There was no obvious L-DOPA titer increase at higher concentrations of

Regulation of the intracellular PLP level with a riboswitch sensor

A TPP-dependent regulatory protein was proposed to control the expression of thiamine biosynthetic genes (Miranda-Rios et al., 2001). Because of the similarity in structure between PLP and TPP, PLP could also bind with the riboswitch to cause a structural change at the RNA level. In addition, this binding also suggested that a significant portion of genetic control observed with “thi box” occurred at the translation level. Fig. 5a and 5c showed that although the intracellular PLP level of the

Discussion

The aim of this study was to create a platform for an E. coli whole-cell biotransformation system for the biosynthesis ofL-DOPA from sodium pyruvate, catechol, and ammonium salt. A model of the metabolic pathway flux of PLP accumulation was constructed by the heterologous expression of R5P-dependent pathway genes (Burns et al., 2005), leading to the enhanced PLP biosynthesis level. The results showed that the L-DOPA titer did not increase as the intracellular PLP biosynthesis level increased in

CRediT authorship contribution statement

Hongmei Han: Conceptualization, Methodology, Software, Data curation, Writing - original draft, Visualization, Investigation, Software, Validation, Writing - review & editing. Bingbing Xu: Software, Validation. Weizhu Zeng: Resources, Supervision. Jingwen Zhou: Writing - review & editing, Project administration, Funding acquisition.

Declaration of Competing Interest

The authors declare no competing financial interests.

Acknowledgements

This work was supported by the National Key Research and Development Program of China (YFC1600403), the National Science Fund for Excellent Young Scholars (21822806), the National Natural Science Foundation of China (31670095, 31770097), the Fundamental Research Funds for the Central Universities (JUSRP51701A), the National First-class Discipline Program of Light Industry Technology and Engineering (LITE2018-08).

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