Site-directed mutagenesis to improve the thermostability of tyrosine phenol-lyase

Journal Pre-proof Site-directed Mutagenesis to Improve the Thermostability of Tyrosine Phenol-Lyase Hongmei Han (Conceptualization) (Methodology) (Software) (Data curation) (Writing - original draft) (Visualization) (Investigation), Weizhu Zeng (Supervision) (Software) (Validation), Guocheng Du (Resources), Jian Chen (Funding acquisition), Jingwen Zhou (Writing - review and editing) (Project administration)

PII:

S0168-1656(20)30005-5

DOI:

https://doi.org/10.1016/j.jbiotec.2020.01.005

Reference:

BIOTEC 8582

To appear in:

Journal of Biotechnology

Received Date:

29 October 2019

Revised Date:

16 December 2019

Accepted Date:

9 January 2020

Please cite this article as: Han H, Zeng W, Du G, Chen J, Zhou J, Site-directed Mutagenesis to Improve the Thermostability of Tyrosine Phenol-Lyase, Journal of Biotechnology (2020), doi: https://doi.org/10.1016/j.jbiotec.2020.01.005

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier.

Site-directed Mutagenesis to Improve the Thermostability of Tyrosine Phenol-Lyase

Hongmei Han 1,2, Weizhu Zeng1,2,4, Guocheng Du1,3, Jian Chen1,2, Jingwen Zhou1,2,4*

1

Key Laboratory of Industrial Biotechnology, Ministry of Education, School of

Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China; National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan

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2

University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China; 3

The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of

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Education, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China;

Jiangsu Provisional Research Center for Bioactive Product Processing Technology,

* Corresponding author.

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Jingwen Zhou

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Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China.

Mailing address: School of Biotechnology, Jiangnan University, 1800 Lihu Road,

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Wuxi, Jiangsu 214122, China

Phone: +86-510-85914371, Fax: +86-510-85914371

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E-mail: [email protected].

Highlights:

  

The E313W and E313M TPL displayed improved activities and longer half-lives L-DOPA yield of E313W and E313M TPL increased by 107.3% and 174.8% Vmax values for E313W and E313M TPL improved by 102% and 88% 1



Melting temperatures for E313W and E313M TPL increased to 62.98°C and 65.86°C

Abstract 3,4-Dihydroxyphenyl-L-alanine (L-DOPA) is the most important antiparkinsonian drug, and tyrosine phenol-lyase (TPL)-based enzyme catalysis process is one of the

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most adopted methods on industrial scale production. TPL activity and stability represent the rate-limiting step in L-DOPA synthesis. Here, 25 TPL mutants were

predicted, and two were confirmed as exhibiting the highest L-DOPA production and

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named E313W and E313M. The L-DOPA production from E313W and E313M was

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47.5 g/L and 62.1 g/L, which was 110.2% and 174.8% higher, respectively, than that

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observed from wild-type (WT) TPL. The Km of E313W and E313M showed no apparent decrease, whereas the kcat of E313W and E313M improved by 45.5% and

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36.4%, respectively, relative to WT TPL. Additionally, E313W and E313M displayed improved thermostability, a higher melting temperature, and enhanced affinity

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between for pyridoxal-5′-phosphate. Structural analysis of the mutants suggested

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increased stability of the N-terminal region via enhanced interactions between the mutated residues and H317. Application of these mutants in a substrate fed-batch strategy as whole-cell biocatalysts allows realization of a cost-efficient short fermentation period resulting in high L-DOPA yield. Keywords: tyrosine phenol-lyase; Escherichia coli; L-DOPA; thermostability;

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site-directed mutagenesis. 1. Introduction 3,4-Dihydroxyphenyl-L-alanine (L-DOPA) is a top-selling drug for Parkinson’s disease, with a worldwide market requiring 250 tons of L-DOPA annually at $700/kg (Min et al., 2015). L-DOPA can be extracted from some leguminous plants, and since the Monsanto group developed asymmetric hydrogenation for L-DOPA production, most has been supplied by chemical asymmetric synthesis. However, chemical

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synthesis of L-DOPA has disadvantages, including poor conversion rates and the

requirement for diastereomer separation. This process usually requires a complicated reaction procedure and expensive metal catalysts, such as rubidium and palladium

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(Sayyed and Sudalai, 2004). Additionally, direct isolation and extraction is not an

environmentally friendly, sustainable, or economically viable solution. Therefore,

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biotechnological approaches are required to identify attractive alternatives for highly

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efficient heterologous biosynthesis of natural products (Valdes et al., 2004) to overcome the low conversion rates and low enantioselectivity while also increasing

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cost effectiveness (Li and Li, 2014).

Microorganisms exhibiting tyrosinase (Luo et al., 2015; Rao et al., 2011; Yamada and 1975),

tyrosine

phenol-lyase

(TPL)

(Foor

et

al.,

1993),

and

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Kumagai,

p-hydroxyphenylacetate 3-hydroxylase (PAHA) activity (Muñoz et al., 2011) have

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been studied as alternatives for chemically synthesizing L-DOPA. Brevundimonas sp. SGJ exhibiting 9201 U/mg of tyrosinase activity can synthesize 3.81 g/L L-DOPA (Surwase et al., 2012), which is much lower than microorganisms exhibiting TPL activity. Additionally, tyrosinase essentially requires a copper ion as a metal cofactor, and L-tyrosine is expensive. Compared with tyrosinase, PAHA uses the same

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substrate (L-tyrosine) to synthesize 1.51 g/L L-DOPA, although it requires NADH as a cofactor (Muñoz et al., 2011), the high cost of which represents a drawback for industrial application. Additionally, the substrates catechol, sodium pyruvate, and ammonium chloride are more soluble in an aqueous phase than L-tyrosine. Moreover, microorganisms exhibiting TPL activity usually display higher L-DOPA production than those exhibiting tyrosinase and PAHA activity, making them more advantageous for L-DOPA synthesis.

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TPL (E.C. 4.1.99.2) (Demidkina et al., 2006; Faleev et al., 2003; Milic et al., 2006;

Phillips et al., 2003) is a pyridoxal-5′-phosphate (PLP) dependent enzyme (Liu et al., 2017) and a homotetramer with four active sites. TPL was first identified in Erwinia

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herbicola, and its status as an L-tyrosine-inducible biocatalyst severely hindered

L-DOPA purification. To overcome the problem, the Kumagai group constructed a

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recombinant E. herbicola strain harboring a mutant transcriptional regulator (TyrR)

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that expressed TPL in the absence of tyrosine, resulting in yields as high as those produced by the wild-type (WT) strain and up to 11.1 g/L/h L-DOPA relative to 0.375

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g/L/h in the WT strain. Foor et al. (Foor et al., 1993)cloned the gene encoding TPL from E. herbicola into Escherichia coli, resulting in 105 mM L-DOPA production

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after 6 h. Lee et al (Lee et al., 1996; Lee et al., 1997) cloned and overexpressed thermostable TPL from thermophilic Symbiobacterium sp. in E. coli to obtain 29.8

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g/L of L-DOPA, which suggested that thermostability of TPL are important for L-DOPA synthesis. To improve TPL activity and thermostability, we generated 25 TPL mutants by site-directed mutagenesis and identified two (E313W and E313M) exhibiting 47.5 g/L and 62.1 g/L L-DOPA production representing 107.3% and 174.8% increases in yield

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as compared with WT TPL, respectively. Structural analysis showed that the mutations promoted higher-affinity interactions between H317 and residues in the N-terminus via W313 and M313, thereby decreasing the flexibility of the N-terminal region. Additionally, these mutants displayed increased kcat/Km values, half-lives, and melting temperatures, suggesting improvements in their respective catalytic rate and thermostability and their potential cost-effective efficacy for industrial-scale

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production of L-DOPA.

2. Materials and methods 2.1 Plasmids and strains

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The pET-28(a)+ plasmid was purchased from Novagen (Darmstadt, Germany). E. coli

JM109 cells and BL21 (DE3) cells were stored in our laboratory. Strains were grown

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in lysogeny broth (LB) and terrific broth (TB) (Oxoid, Basingstoke, UK), with 50

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g/mL of kanamycin added, as necessary. The TPL gene from Citrobacter freundii was codon-optimized and synthesized by GenScript (Nanjing, China). The nucleotide

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sequence of synthetic TPL gene has been accessed in National Center for Biotechnology Information (NCBI) and GenBank accession number was MN205565.

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There were no changes in protein level. The TPL gene (1371 bp) was digested and inserted

into

the

BamHI/HindIII

site

of

pET-28a(+),

and

the

resulting

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pET-28a(+)-TPL vector was used to transform E. coli JM109 and BL21 (DE3), respectively.

2.2 Site-directed mutagenesis

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Based on a 99.34% sequence identity between the TPL used in this study and that from C. freundii, the crystal structure of TPL from C. freundii (PDB: 2YHK; 1.85-Å resolution) was used as a homologous model (Fig. 1). Based on the three-dimension (3D) model of the holoenzyme TPL-PLP, define the residues that were surrounding the active site within a 5-Å sphere but excluding the active site as mutation group. Residues in mutation group were all virtually mutated to alanine by using the Calculate Mutation Energy (Stability) module of Discovery Studio (DS; Accelrys Inc.,

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San Diego, CA, USA). Mutations with greater mutation energy meant greater effects

and were key amino acids. Table S1 showed that Gly32, Gly73, Lys155, Gly326,

Gly342, Gly189 and Glu313 mutations might affect TPL thermostability and define

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them as key residues. Therefore, mutations located in Gly189 and Glu313 might

affect TPL thermostability. Saturation mutagenesis of key amino acids was then

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performed following screening according to the energy assessment in order to create

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thermostable TPL variants. Table S2 shows the 25 mutations that exhibited a stabilizing effect on TPL according to site-directed mutagenesis. Site-directed

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mutagenesis (Qin et al., 2017) were performed by using polymerase chain reaction (PCR) and primers designed according to the TPL nucleotide sequence (Table S3). The PCR included 50 ng of the template plasmid [pET28 (a)-TPL], 25 L of 2× Super

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pfu DNA polymerase (Biosci Biotech, Hangzhou, China), and 10 μM of the primer

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pairs in a final volume of 50 μL with the following cycle protocol: one cycle at 95°C for 3 min, followed by 35 cycles at 95°C for 30 s, 55°C for 30 s, 72°C for 3 min, and one cycle at 72°C for 4 min. PCR products were digested with DpnI (Thermo Fisher Scientific, Waltham, MA, USA) at 37°C for 1 h to remove the original template. Plasmid phosphorylation was performed using a Blunting Kination Ligation kit

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(Thermo Fisher Scientific), followed by self-linkage for 8 h. The constructed plasmids were used to transform into E. coli JM109 competent cells, and Sanger sequencing was performed by Sangon Biotech (Shanghai, China) for construction. The verified plasmids were used to transform competent E. coli BL21 (DE3) competent cells for exogenous protein expression.

2.3 Culture conditions and primary screening of mutations

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After site-directed mutagenesis, WT TPL and mutant TPL were grown at 37°C in TB

medium containing 50 μg/mL kanamycin. Upon reaching an optical density at 600 nm of between 0.6 and 0.8, the temperature was lowered to 20°C, and 0.4 mM

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isopropyl-β-thiogalactopyranoside (IPTG; final concentration) was added to induce

TPL expression. After 10 to 12 h, the culture was centrifuged at 5000 rpm for 10 min

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to collect cells, and strains capable of expressing thermostable TPL were screened

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according to the production of L-DOPA at 20°C and 40°C.

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2.4 Preparation of whole-cell biotransformation Catechol content >0.1 M inhibits both E. coli BL21 (DE3) growth (Park et al., 1998)

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and TPL activity; therefore, it was necessary to maintain low concentrations of sodium pyruvate and catechol according to the feeding strategy (Koyanagi et al.,

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2005). Cell pellets were resuspended in reaction liquid including 15 g/L sodium pyruvate, 10 g/L catechol, 30 g/L ammonium salt, 4 g/L sodium sulfite, 2 g/L EDTA, and 30 µM PLP. The pH value of reaction liquid was adjusted to 8.5 with ammonium hydroxide. During the initial 2 h reaction, sodium pyruvate was added at 9 g/L/h and catechol at 6 g/L/h, and from 2 to 4 h, sodium pyruvate was added at 6 g/L/h and

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catechol at 4 g/L/h. Reactions were incubated at 20°C and 40°C and shaken at 200 rpm with 9 g/L dried cell concentration. Optical density at 600 nm of cells in reaction liquid was between 30 to 31. The reaction was performed in the dark to protect L-DOPA from decomposition (Fordjour et al., 2019; Min et al., 2015), and samples were withdrawn and extracted with 20% (v/v) 0.1 M HCL (Ho et al., 2003). L-DOPA content was detected using an Agilent 1200-series high-performance liquid chromatography (HPLC; Agilent Technologies, Santa Clara, CA, USA) system

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equipped with a reversed-phase Gemini NX-C18 column (4.6 × 250 mm) according to

a previous described protocol (Ho et al., 2003). The maximal ultraviolet absorbance

was 270 nm. HPLC was performed with a 25 mM pH 2.5 phosphate buffer as the

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mobile phase at a constant flow rate of 0.8 mL/min. For each HPLC experiment,

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was represented with error bars.

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samples in three independent cultures were measured, and their standard deviation

2.5 Purification of 6×His-tagged TPL

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TPL harboring a 6×His tag was purified using a His-Trap FF 5-mL column on an AKTA Pure system (GE Healthcare, Piscataway, NJ, USA). An Ni-NTA Super flow

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column (5 mL) was washed with 50 mL 0.1 M NiSO4 followed by 10 mL of water and equilibrated with 20 mL of equilibration buffer [50 mM KH2PO4-K2HPO4 and

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150 mM NaCl (pH 8.5)]. Prior to purification, cell pellets were washed three times and resuspended in 50 mM PB buffer [50 mM KH2PO4-K2HPO4 (pH 8.5)]. Cells were sonicated for 20 min with a 10-s delay for each 20 s on ice, and the supernatant was collected by centrifugation at 9,000 rpm for 10 min. Samples were loaded with flow of 1 ml/min. And then, the column was washed until there was no peak at 280 nm

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with washing buffer [50 mM, KH2PO4-K2HPO4, 150 mM NaCl, and 20 mM imidazole (pH 8.5)], and 6×His-tagged TPL was eluted and collected with elution buffer [50 mM KH2PO4-K2HPO4, 150 mM NaCl, and 120 mM imidazole (pH 8.5)]. The column was washed clear with buffer [50 mM KH2PO4-K2HPO4, 150 mM NaCl, and 200 mM imidazole (pH 8.5)]. The collected enzyme fraction was desalted with a Sephadex-G column (2 mL). Sample was loaded with flow of 0.5 mL/min. Then desalted enzyme fraction was eluted and collected with PB buffer [50 mM

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KH2PO4-K2HPO4 (pH 8.5) at 2 mL/min. The purified and desalted enzyme fraction was stored in PB buffer at 4°C, and analyzed by sodium dodecyl sulfate

polyacrylamide gel electrophoresis (SDS-PAGE). Protein concentration was

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normalized using an enhanced BCA protein assay kit (P0009; Beyotime

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Biotechnology, Jiangsu, China).

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2.6 Enzyme assays and binding kinetics

The α- and β-elimination reactions for TPL were used as assays to determine enzyme activity (Chandel and Azmi, 2013). L-tyrosine was decomposed into phenol, pyruvate,

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and ammonia, and the resulting pyruvate concentration was measured by HPLC (Luo et al., 2018). The substrate was dissolved in 50 mM KH2PO4-K2HPO4 (pH 8.5),

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including 20 g/L L-tyrosine and 30 μΜ PLP. The substrate (900 μL) was introduced

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by adding 100 μL of enzyme solution diluted to 1 mg/mL, and the reaction was stopped by adding 100 μL HCL of 0.1 M. To determine the optimal pH value, L-tyrosine was dissolved in 50 mM KH2PO4-K2HPO4 (pH 6-8) and 50 mM glycine-NaOH (pH 8-10) buffer, respectively. One unit of TPL activity was defined as the amount of enzyme producing 1 µM of pyruvate/min

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Enzyme half-life was determined by maintenance at 20°C, 40°C, or 60°C for different time periods. A reference standard of 100% TPL represented was established and measured at the corresponding reaction temperature. Kinetic parameters were determination at 20°C using with 900 μL substrate and 100 μL of enzyme solution. The reaction substrate was L-DOPA at a gradient concentration range of 1 mM to 20 mM, and the reaction was initiated by the addition of 100 μL WT TPL and mutant TPL diluted to the appropriate concentration.

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Lineweaver–Burk plots were generated for analysis of Km, Vmax, and kcat/Km (Hoylaerts et al., 1990).

Absorbance spectra were obtained using a UV-2450 UV-VISIBLE spectrophotometer

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(Shimadzu, Kyoto Japan), and binding kinetics between TPL and the PLP coenzyme were determined by rapid scanning, stopped-flow spectroscopic analysis based on the

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maximal absorption spectra of the holoenzyme TPL–PLP at 420 nm (Lee et al., 2006).

2.7 Circular dichroism (CD) analyses

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CD spectra were obtained using a MOS-450 CD spectrometer (Bio-Logic, Claix, France) to identify the secondary structure of WT TPL, TPL E313W, and TPL

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E313M.

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2.8 Differential scanning calorimetry (DSC) analyses The melting temperature of WT TPL, TPL E313W, and TPL E313M was measured using a NANO DSC (TA Instruments–Waters Corp., Milford, MA, USA). The heating rate was kept at 2°C/min during all scans from 50°C to 70°C. Samples were degassed and heat capacity (Cp) were determined.

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3. Results 3.1 Primary screening of mutations After WT and mutated strains were induced at 20°C with 0.4 mM IPTG for 10 h, their growth suggested that the mutations did not affect strain growth. SDS-PAGE analysis (Fig. 2) suggested no difference in protein levels between WT TPL and the E313W and E313M mutants. Whole cells biotransformation for 4 h at 20°C and 40°C resulted

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in maximal L-DOPA production (Fig. 3). At 20°C, L-DOPA production from E313W

and E313M was 47.5 g/L and 62.1 g/L, exhibiting improvements of 107.3% and

174.8% relative to that of WT TPL (22.6 g/L), respectively. Due to the lower

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thermostability of WT TPL at 40°C, L-DOPA yield produced by whole cells

biotransformation at 40°C showed great fall, only 8 g/L. However, at 40°C, L-DOPA

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production by E313F, E313R, E313H, E313Y, E313Q, E313V, E313C, E313W, and

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E313M was higher than that of WT TPL. L-DOPA production of E313W and E313M with improved thermostability was 13.3 g/L and 16.7 g/L, representing improvements

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of 66.3% and 108.8% relative to that of WT, respectively.

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3.2 Effect of mutations on TPL activity and thermostability The optimal pH value for catalysis of TPL WT, E313W, and E313M all was 8.5 (Fig.

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4a). Temperature-specific activity measurements showed that catalytic activities of E313W and E313M displayed a little higher than that of TPL WT from 20°C to 40°C (Fig. 4b); however, E313W and E313M catalytic activities were 12- and 10-fold higher than that of TPL WT at 50°C. Additionally, TPL E313W and E313M exhibited catalytic activity at 60°C and 70°C close to their maximal activity, suggesting E313 as

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an important amino acid affecting TPL catalytic activity at temperatures >40°C. Moreover, WT TPL activity decreased more rapidly than that of TPL E313W or E313M with increasing incubation time at temperatures of 20°C, 40°C, and 60°C (Fig. 5). Furthermore, the half-life of E313W was 21.7 min at 20°C, 13.3 min at 40°C, and 7.7 min at 60°C, respectively, and that of TPL E313M was 37.9 min, 17.1 min, and 14.6 min, respectively. The higher half-life suggested an increase in the thermostability of the two mutants compared to TPL WT, thereby suggesting their

3.3 Analyses of TPL kinetics and binding to PLP

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great increase in L-DOPA yield and efficacy for industrial application.

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Kinetic analyses (Table S4) showed that the Km of both TPL E313W and E313M

showed no significant changes, whereas the kcat values increased by 45.5% and 36.4%,

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respectively, relative to that of WT TPL. Additionally, the Vmax values for TPL

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E313W and E313M improved by 102% and 88%, respectively, relative to that of WT TPL. Therefore, these results suggested that E313 substitutions might contribute to

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improved catalytic efficiency. Additionally, we found that TPL E313W bound to PLP with higher affinity relative to WT and E313M TPL (Fig. 6), as the later variants

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displayed substrate saturation after ~30 s relative to ~200 s for the E313W variant.

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3.4 Comparison of changes in secondary structure and melting temperature CD analysis indicated that helix secondary structures of E313W, and E313M were less than that of TPL WT (Fig. 7); however, the strand and turns secondary structures of E313W, and E313M were more than that of TPL WT. The increased strand and turns secondary structures played important roles in conformational stability of

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protein (Li et al., 2019). The melting temperatures of TPL WT, E313W, and E313M were 59.2°C, 63.0°C, and 65.9°C, respectively (Fig. 8). The increased melting temperatures for the E313W and E313M variants agreed with the increases in their respective half-lives and likely reflected their more compact structures.

4. Discussion Generally, enzyme with increased catalytic activity, improved thermostability is

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important to successful biosynthesis (Bankar et al., 2009; Choi et al., 2014; Takahashi

et al., 2019). Accordingly, TPL with higher catalytic activity was the key factor for

the application in L-DOPA production. A previous study indicated that TPL exhibits

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relative high activity in E. coli and is widely used for the bacterial biosynthesis of L-DOPA, as well as other amino acid derivatives (Satoh et al., 2012). In the present

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study we showed that the E313W and E313M TPL variants displayed improved

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activities relative to WT TPL, as well as longer half-lives, which are critical for improved L-DOPA production. These variants represent engineered TPL variants for

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optimal L-DOPA production.

To improve TPL thermostability, Lee et al. cloned a gene encoding TPL from the

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Symbiobacterium sp. SC-1 into plasmid pTrc99A for overexpression of thermostable TPL in E. coli, resulting in an enzyme stable at up to 70°C, although at lost ~70% of

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its initial activity at 80°C (Lee et al., 1997). Subsequently, a T15A mutation generated by random mutagenesis of C. freundii TPL exhibited a 2-fold increase in L-DOPA production along with a 16.8-fold increase in half-life at 45°C relative to the WT enzyme (Lee et al., 2006). Another study introduced increase stability via A13V, E83K, and T407A mutations along with improved activity via T129I or T451A

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mutations in TPL from Symbiobacterium toebii (Rha et al., 2009). The present study used computational methods to avoid random mutagenesis and screening, revealing E313 mutagenesis as capable of improving TPL thermostability (specifically E313W and E313M). Structural analysis provided critical information for modifications of industrial enzymes (Li et al., 2014; Seo et al., 2013). Previous studies show that most TPL mutations effecting stability are located around the N-terminus, whereas those

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affecting activity are located farther away from these areas. Therefore, minimizing any interference that would be detrimental to the co-improvements in stability and catalysis (Rha et al., 2009). Structural analysis showed the W313 was located

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proximal to H317 in the α-helix structure, and that the hydrogen-bond distance between W313 and H317 in the E313W was shorter than that between E313 and

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H317 in WT TPL, which also affected interactions between H317 and N-terminal

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residues (Fig. 1). Specifically, P4 and H317, as well as amide-pi-stacking interactions between A5, E6, and H317 and the Pi-alkyl interaction between P7 and H317 all

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increased in affinity due to the decreased bond lengths in TPL E313W relative to those in WT TPL. Notably, these same changes were observed in TPL E313M. P4,

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A5, E6, and P7 were all located on the intertwined N-terminal arm (residues 1–19) of the TPL structure (Lee et al., 2006). It is possible that the stronger interactions

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between H317 and N-terminal residues increased the stability of the N-terminal region to improve the overall thermostability and increase the melting temperatures of the TPL E313W and E313M variants (all residue numbers were derived from the structure of the TPL dimer; PDB: 2YHK).

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L-DOPA is the primary therapeutic intervention for Parkinson’s disease that addresses deficiencies in the neurotransmitter dopamine. There are multiple problems associated with increased L-DOPA production. Catechol is a substrate that inhibits not only cell growth but also TPL catalytic activity (Barbolina et al., 2018). An inactivation of TPL was observed when catechol was more 0.1 M, and that there is formation of byproducts by a non-enzymatic reaction between L-DOPA and pyruvate (Lee et al., 1996). Because fed-batch fermentation involving catechol and sodium pyruvate can

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control their respective concentration, this can address these issues. The culture period for E. coli BL21 (DE3) cells expressing TPL was 12 h, and whole-cell

biotransformation for maximal L-DOPA production required 4 h. These represented

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improvements in productivity and time; however, L-DOPA remained easily oxidized and reaction liquid become dark. Therefore, further research into the methods

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necessary to avoid L-DOPA oxidization is needed. Nevertheless, our results suggested

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protein engineering as an attractive approach for realizing highly efficient heterologous biosynthesis of L-DOPA.

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Compliance with ethical standards

This article does not contain any studies with human participants or animals

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performed by any of the authors.

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Sample CRediT author statement

Hongmei Han: Conceptualization, Methodology, Software. Hongmei Han:

Data curation, Writing-Original draft preparation. Hongmei Han: Visualization, Investigation. Guocheng Du: Resources. Weizhu Zeng: Supervision. Weizhu Zeng: Software, Validation. Jingwen Zhou: Writing-Reviewing and Editing. Jingwen Zhou: Project administration. Jian Chen: Funding acquisition.

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Declaration of Interest Statement There are no conflicts of interest to declare.

Acknowledgements This work was supported by the National Key Research and Development Program of China (2017YFC1600403), the National Science Fund for Excellent Young Scholars

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(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

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lP

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Engineering (LITE2018-08).

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and transfer in the reaction with substrate. Biochimie 147, 63-69. Chandel, M., Azmi, W., (2013) Purification and Characterization of Tyrosine Phenol Lyase from Citrobacter freundii. Appl Biochem Biotech 171, 2040-2052.

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Choi, S.-L., Rha, E., Lee, S.J., Kim, H., Kwon, K., Jeong, Y.-S., Rhee, Y.H., Song, J.J., Kim, H.-S., Lee, S.-G., (2014) Toward a generalized and high-throughput enzyme

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Figure legends Figure 1. 3D model of TPL and interactions between E313, H317, and N-terminal residues. (a) 3D model of TPL. (b) Magnified view of the boxed structure. Interactions between E313, H317, and N-terminal residues. P4, yellow stick model; A5, green stick model; pink stick model: E6; P7, purple stick model; E313, red stick model; and H317, blue stick model. The light-green broken line represents a carbon-hydrogen bond, the dark-green broken line represents a conventional hydrogen

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bond, a deep-red broken line represents an amide-pi-stacking interaction, and a pink

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broken line represents a pi-alkyl interaction.

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Figure 2. SDS-PAGE analysis of the heterologous expression of TPL in E. coli. Intracellular WT (lane 1), E313W (lane 3), and E313M (lane 5) levels. Purified TPL WT (lane 2), E313W (lane 4), and E313M (lane 6). Levels were normalized according

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to a 4-mg standard. Targets are denoted with a black arrow. M, standards (kDa).

Figure 3. L-DOPA yields produced by TPL WT and mutants. Whole-cell catalytic

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reaction of TPL WT and mutant variants at 20°C and 40°C. Experiments were performed in triplicate, and error bars represent the standard deviation.

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WT-TPL: codon-optimized WT TPL; G32L, G32Q, G73Q, K155R, G189C, G189I, G189N, G189V, G189W, E313C, E313F, E313H, E313I, E313L, E313M, E313Q,

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E313R, E313T, E313V, E313W, E313Y, G326F, G326H, G326Q, and G342T:

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mutants generated by site-directed mutagenesis.

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Figure 4. Effect of temperature on TPL WT, E313W, and E313M activities. (a) Enzyme activities at different pH value (pH 6-10). (b) Enzyme activities at 20°C, 30°C, 40°C, 50°C, 60°C, and 70°C. Experiments were performed in triplicate, and

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error bars represent the standard deviation.

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Figure 5. Relative activity of TPL WT, E313W, and E313M. Relative activities of TPL (a) WT, (b) E313W, and (c) E313M to evaluate thermostability. Enzymes were pre-incubated in 100 mM potassium phosphate buffer for different durations at various temperatures. Experiments were performed in triplicate, and error bars

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represent the standard deviation.

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Figure 6. Comparative analysis of binding kinetics between TPL and PLP. Maximal absorption spectra (420 nm) of the TPL–PLP holoenzyme. Purified and desalted TPL WT, E313W, and E313M diluted to same concentration in the presence

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of PLP (50 µM, final concentration).

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Figure 7. CD analysis of TPL WT, E313W, and E313M secondary structures. (a,b) CD analysis of TPL WT, E313W, and E313M secondary structures. Purified and desalted TPL WT, E313W, and E313M fractions were diluted to same concentration.

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PB buffer was used as the reference.

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Figure 8. Melting temperature of TPL WT, E313W, and E313M. Purified and desalted TPL WT, E313W, and E313M fractions were diluted to same concentration.

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PB buffer was used as the reference.

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