Biocatalytic asymmetric synthesis of l -phosphinothricin using a one-pot three enzyme system and a continuous substrate fed-batch strategy

Journal Pre-proof Biocatalytic asymmetric synthesis of L-phosphinothricin using a one-pot three enzyme system and a continuous substrate fed-batch strategy Haisheng Zhou, Lijun Meng, Xinjian Yin, Yayun Liu, Jianping Wu, Gang Xu, Mianbin Wu, Lirong Yang

PII:

S0926-860X(19)30394-1

DOI:

https://doi.org/10.1016/j.apcata.2019.117239

Reference:

APCATA 117239

To appear in:

Applied Catalysis A, General

Received Date:

9 June 2019

Revised Date:

6 August 2019

Accepted Date:

4 September 2019

Please cite this article as: Zhou H, Meng L, Yin X, Liu Y, Wu J, Xu G, Wu M, Yang L, Biocatalytic asymmetric synthesis of L-phosphinothricin using a one-pot three enzyme system and a continuous substrate fed-batch strategy, Applied Catalysis A, General (2019), doi: https://doi.org/10.1016/j.apcata.2019.117239

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Title page

Article title Biocatalytic asymmetric synthesis of L-phosphinothricin using a one-pot three enzyme

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system and a continuous substrate fed-batch strategy

Author names and affiliations

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Haisheng Zhou, Lijun Meng, Xinjian Yin, Yayun Liu, Jianping Wu, Gang Xu, Mianbin

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Wu, Lirong Yang*

Institute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang

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University, 310027 Hangzhou, PR China

Corresponding author Tel: +86-571-8795-2363; Fax: +86-571-8795-2363; E-mail:

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[email protected] (L. R. Yang)

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Graphical abstract

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Highlights 

A three-enzyme cascade reaction was developed for synthesis of Lphosphinothricin

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A continuous substrate fed-batch process was used to scale-up the reaction to 90 L 615.4 mM L-Phosphinothricin was produced in 99.7% yield and >99.9% ee



The method could be used for asymmetric synthesis of non-proteinogenic amino

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

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acids

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Abstract

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Transamination catalyzed by an aminotransferase is becoming a key tool for the production of chiral amine pharmaceuticals and agrochemicals owing to its excellent

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enantioselectivity and green credentials. To overcome the unfavorable thermodynamic equilibrium and the inhibition by the byproduct α-ketoglutaric acid in the transamination

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of 2-oxo-4-[(hydroxy)(methyl)phosphinoyl]butyric acid (PPO) to form the promising

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herbicide L-phosphinothricin (L-PPT), a tri-enzymatic cascade reaction system was developed by combining a robust glutamate dehydrogenase to recycle the byproduct to the amino donor L-glutamate in situ, together with a cofactor recycling process catalyzed by an alcohol dehydrogenase. Moreover, a continuous substrate fed-batch strategy was employed to alleviate the decomposition of PPO and applied to scale-up the cascade

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reaction to 90 L, yielding 111.4 g/L (615.4 mM) L-PPT in 99.7% yield and >99.9% ee with an productivity of 15.9 g/L•h. This combination of improved biocatalyst system and process engineering should prove to be economically competitive for industrial applications. Keywords: asymmetric synthesis; aminotransferase; continuous substrate fed-batch;

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enzymatic catalyze; herbicide; L-phosphinothricin.

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1. Introduction L-Phosphinothricin (L-PPT; L-2-amino-4-(hydroxy(methyl)-phosphonoyl)butanoic acid) is the active ingredient of many commercial herbicides used worldwide such as Basta, Buster, Challenge, Finale, Harvest, and Ignite [1]. As L-PPT resembles L-glutamic acid (L-Glu), it functions as a false substrate and irreversibly blocks glutamine synthetase, a

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vital enzyme on the nitrogen metabolic pathway of plants, and thus it possesses a broadspectrum, non-selective herbicidal activity [2-4]. Because of its low mammalian toxicity and ecological compatibility [1], it has the potential for huge market share growth with

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the development and promotion of genetically modified PPT-tolerant crops and the

shrinking market of its main competitors, glyphosate [5, 6] and paraquat [7]. Because

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almost all commercially available formulations consist of racemic D, L-PPT as the

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component and only the L-enantiomer has herbicidal efficacy [8], there is a persistent need for efficient methods to obtain enantiopure L-PPT.

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However, as L-PPT is a type of non-proteinogenic amino acid, which is rarely extracted from natural sources or produced through direct microbial fermentation [9], it is difficult

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to prepare at a low cost. Nevertheless, numerous attempts have been made to synthesize

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L-PPT including via chemical asymmetric synthesis using chiral auxiliaries [10, 11] or Lnatural amino acids obtained from a chiral pool [12, 13], enzymatic optical resolution of D, L-PPT derivatives [14-17] and enzymatic amination of the precursor PPO [18-23]. Because of the harsh operating conditions, complicated process, low yield, and insufficient enantiopurity, few chemical routes for industrial applications have been

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reported. Resolution of racemic phosphinothricin or its derivatives with the employment of enzymes such as deacetylase [14, 15], amidase [16], and nitrilase [17] is a feasible way to produce L-PPT with a high ee (enantiomeric excess) value, but the reaction is undermined by the theoretical maximum yield not exceeding 50% in a one reaction batch and the subsequent difficulties of recirculation of the racemate. Enzymatic amination of

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the corresponding keto acid PPO, including transamination catalyzed by an aminotransferase (transaminase, TA, EC 2.6.1.X) and reductive amination catalyzed by

an amino acid dehydrogenase (AADH, EC 1.4.1X), are promising asymmetric synthetic

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routes for manufacture of L-PPT based on the easy process and green credentials.

Because transamination to PPO is the major degradation product of naturally occurring

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L-PPT [24, 25], it was concluded that there are naturally occurring evolutionary TAs with

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robust activity and enantioselectivity for the synthesis of L-PPT from PPO. Indeed, most researchers have focused on transamination for the asymmetric synthesis of L-PPT [18-

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21]. However, one of the frequently cited drawbacks of transamination is its unfavorable thermodynamic equilibrium, that is, reactions catalyzed by TAs are reversible; although

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using a stoichiometric excess of the amino donor can easily shift the equilibrium. For

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example, Schulz et al. [18] used a four-fold molar excess of the amino donor L-Glu in the presence of PPO to produce L-PPT to achieve more than 90% yield, whereas the remaining PPO, excess L-Glu, and the byproduct α-KG made downstream purification extremely difficult and greatly elevated the process cost. Others have attempted to address this issue by coupling two transaminases, one utilized L-Glu as the amino donor, while

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the second recycled the byproduct α-KG back to L-Glu using aspartic acid as the amino donor, although only a slight excess of amino donors were used and a lower conversion of 85% was obtained [20]. Another ingenious approach was established by decomposing α-KG into ethylene and carbon dioxide in situ using an ethylene-forming enzyme with only an equivalent amount of the L-Glu amino donor to increase the conversion of PPO

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from <60% to >99% [21]. All these strategies effectively shifted the equilibrium to some extent, but all still required at least a stoichiometric amount of amino donor along with

the inevitable creation of some secondary byproducts, compromising the process facility

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and atom economy.

Reductive amination of PPO catalyzed by an AADH is considered another potential

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asymmetric synthetic route for production of L-PPT with advantages, such as complete

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conversion and no need for an organic amino donor because ammonium is used instead. Owing to the structural similarity between L-PPT and L-Glu, AADHs for reductive

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amination of PPO have been identified to be a glutamate dehydrogenase (GluDH) [22, 23]. However, this application is limited by the strict substrate specificity of natural

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GluDHs and plagued by their low activities and stereoselectivities toward PPO [22].

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Although a newly engineered NADPH-specific GluDH with an activity of 0.63 U/mgprotein toward PPO was recently used for a reductive amination process with a conversion of 99% (0.1 M PPO) and an L-PPT productivity of 1.35 g/h·L [23], extensive efforts are needed to enhance its activity and substitute the cofactor with the cheaper NADH. Therefore, a tri-enzymatic cascade reaction (Fig. 1) was constructed for asymmetric

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synthesis of L-PPT, which combined three naturally available enzymes, a TA that transfers the amino group from L-Glu to PPO to form L-PPT, a GluDH that recycles LGlu from α-KG by consuming ammonium and NADH, and an alcohol dehydrogenase (ADH, EC 1.1.1.X) that recycles NADH using the cheap reducing agent isopropanol. In contrast to previously reported transaminations that used at least a stoichiometric amount

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of amino donor, this cascade reaction system only requires a catalytic or sub-catalytic amount of amino donor, while shifting the reaction equilibrium to obtain a complete

conversion. Additionally, because it uses robust enzymes, this tri-enzymatic cascade

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reaction should be efficient and versatile for the synthesis of other non-proteinogenic

amino acids. Finally, by applying strategies of process engineering such as continuous

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substrate feeding, the cascade reaction was scaled up and the asymmetric synthesis of L-

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PPT was achieved with high yield and productivity.

2. Materials and Methods

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2.1. Strains, plasmids, enzymes, and chemicals E. coli W3110 was maintained in our laboratory and used to amplify the aminotransferase

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genes gamma-aminobutyrate aminotransferase (GABAT, GenBank: WP_001087611.1),

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branched-chain amino acid aminotransferase (BCAT, GenBank: CAQ34114.1), valinepyruvate

aminotransferase

(ValAT,

GenBank:

AM946981.2),

and

aspartate

aminotransferase (AspAT, GenBank: 945553). E. coli BL21 (DE3) was used for gene expression (Novagen, California, USA). The vector pET-28a (+) (Novagen, California, USA) was used to construct recombinant

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vectors for gene expression. PrimeSTAR Max DNA polymerase and restriction endonucleases were purchased from Takara (Dalian, China). Yeast extract, tryptone, kanamycin, and isopropyl β-D-1-thiogalactopyranoside (IPTG) were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). 2-Oxopentanoic acid and trimethylpyruvic acid were purchased from TCI (Shanghai) Development Co., Ltd. (Shanghai, China). 2-

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Oxo-4-[(hydroxy)(methyl)phosphinoyl]-butyric acid (PPO) was synthesized and purified in our laboratory in accordance with previously reported methods [26] and characterized

by our colleague X. J. Yin [23]. Nicotinamide adenine dinucleotide derivatives

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(NAD(P)H, NAD(P)+) were purchased from Bontac Bio-engineering Co., Ltd. (Shenzhen, China). All other chemicals and reagents were chemically pure and obtained

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commercially.

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2.2. Construction of recombinant vectors

The recombinant vectors GABAT and BCAT were previously constructed by our

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laboratory [21]. The genes ValAT and AspAT were amplified by polymerase chain reaction (PCR) using the genomic DNA of E. coli W3110 as the template. The primers restriction

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and

endonucleases

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CCGGAATTCATGACATTCTCCCTTTTTGGTGAC-3′

were (EcoR

5′-

I)

and

5′-

CCGCTCGAGTTAGTGACTTTCAGCCCAGGC-3′ (Xhol I) for ValAT, and 5′CCGGAATTCATGTTTGAGAACATTACCGCCGCTCC-3′

(EcoR

I)

and

5′-

CCCAAGCTTTTACAGCACTGCCACAATCGCTTCGC-3′ (Hind III) for AspAT. The PCR products were analyzed using 1% (w/v) agarose gel electrophoresis, and then were

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digested using their respective restriction endonucleases. The digested DNA fragments were inserted into the same restriction sites of the pET-28a (+) expression vector under the control of the T7 promoter. All of the other enzyme genes employed in this work were synthesized and inserted between the EcoR I and Hind III restriction sites of the vector pET-28a (+), including

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aromatic aminotransferase from Klebsiella michiganensis E718 (AroAT, GenBank: NC_018106.1) [27], glutamate dehydrogenase from Amphibacillus xylanus DSM 6626 (GluDH, GenBank: NP_391659.2) [28], and alcohol dehydrogenase from Geobacillus

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stearothermophilus (BsADH, GenBank: KR611715) [29]. 2.3 Expression of recombinant enzymes

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Recombinant E. coli BL21 (DE3) was cultivated at 37 °C in Luria–Bertani medium with

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kanamycin (50 μg/mL). The cells were induced for 16 h using IPTG (0.5 mM) at 25 °C, after which time OD600 had reached approximately 0.6. The cells were then harvested

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by centrifugation (12,000 × g, 5 min, 4 °C), washed with phosphate buffer (50 mM, pH 8.0), and resuspended in half the volume of buffer for ultrasonic cell disruption to obtain

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the crude enzyme extract.

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2.4 Purification of recombinant enzymes The collected cells were washed twice and resuspended in 50 mM phosphate buffer (pH = 8.0) at room temperature and subsequently disrupted through sonication (Sonicator 400, Misonix, USA) in an ice bath. The crude cell extract was centrifuged at 12,000 × g for 30 min to remove the insoluble debris. The obtained supernatant was loaded onto a nickel

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chelate affinity column (Ni-NTA Resin, Bio Basic) to purify the recombinant enzymes. Fractions containing the eluted product were identified by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), and then were desalted and replaced with phosphate buffer (50 mM, pH 8.0) by ultrafiltration. Finally, 20% (w/w) glycerol was added to the purified enzyme solution and it was stored at -20 °C.

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2.5. SDS-PAGE and protein concentration assays The expression and purification of the recombinant enzymes were analyzed by SDSPAGE. The gels were stained with Coomassie Brilliant Blue G-250. Molecular weight

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protein markers (PageRuler Prestained Protein Ladder, 10 to 170 kDa, Thermo Scientific, USA) were used as references. Protein concentrations were determined using a Bradford

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protein assay kit (Quick Start™, Bio-Rad, California, USA).

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2.6. Enzyme assays

The assay solution for TA activity measurements contained Tris-HCl buffer (100 mM, pH

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9.0), pyridoxal 5′-phosphate (PLP) (0.1 mM), amino donor (100 mM, adjusted to pH 9.0 with NaOH), and PPO (20 mM, adjusted to pH 9.0 with NaOH; Scheme 1). The reaction

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was initiated by adding TA enzyme solution and it was allowed to react for 10 min at

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35 °C. The enzymatic reaction was then quenched by adding 1 M HCl and the amount of L-PPT was detected by HPLC. The TA activity was defined as the amount of enzyme that catalyzed the formation of 1 μmol of L-PPT per min GluDH and BsADH activity were determined at 35 °C by monitoring at 340 nm with a TU-1810PC recording spectrophotometer equipped with a thermostated cell

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compartment (Beijing Puhua General Instrument Co., Ltd., China). The reaction mixtures for the GluDH assay contained Tris-HCl buffer (100 mM, pH 8.0), NADH (0.25 mM), αKG (20 mM, adjusted to pH 8.0 with ammonia), and 25 μL of enzyme solution in a total volume of 1 mL. The reaction mixtures for the BsADH assay contained Tris-HCl buffer (100 mM, pH 8.0), NAD+ (0.25 mM), isopropanol (100 mM), and 25 μL of enzyme

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solution in a total volume of 1 mL. Nonenzymatic reaction rates served as controls and were subtracted from the reaction rate in the presence of enzyme. One unit of enzyme

activity was defined as the amount of enzyme that catalyzed generation or degradation of

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1 μmol of NADH per min with a molar absorption coefficient of 6.22 mM-1•cm-1 under

2.7. Initial velocity measurements

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the above experimental conditions.

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The initial velocity (v0) of the reaction catalyzed by GABAT was measured in 100 mM Tris-HCl buffer (pH 8.0) in a 10 mL reactor placed in a thermostatic water bath under

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magnetic stirring at 35 °C. For measurement of the inhibition of GABAT by PPO, the 5 mL reaction mixture comprised 1000 mM L-Glu, 0.1 mM PLP, and a various

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concentration of PPO. To measure the inhibition of GABAT by α-KG, the reaction

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mixture was comprised of 1000 mM L-Glu, 10 mM PPO, 0.1 mM PLP, and a various concentration of α-KG. All the reactions were initiated by adding 25 μL of pure GABAT enzyme solution (0.1 U/mL). 2.8. Asymmetric synthesis of L-phosphinothricin using transamination catalyzed by gamma-aminobutyrate aminotransferase

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The reactions were performed in 10 mL EP tubes in a thermostatic water bath under magnetic stirring at 35 °C. For reactions with Glu/PPO >1, the reaction mixture was comprised of 1 U/mL GABAT crude extract, 1500 mM L-Glu (adjusted to pH 7.5 with NaOH beforehand), 0.1 mM PLP, and a various concentration of PPO (the mixture was neutralized by NaOH to pH 7.5). For reactions with Glu/PPO ≤1, the reaction mixture

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comprised 1 U/mL GABAT, 500 mM PPO (adjusted to pH 7.5 with NaOH beforehand), 0.1 mM PLP, and a various concentration of L-Glu (the mixture was neutralized by NaOH to pH 7.5).

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2.9. Asymmetric synthesis of L-phosphinothricin using the tri-enzymatic reaction

The reaction was performed in a 250 mL or 1000 mL round bottom flask reactor with a

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DG-150 industrial pH detector and controller (Hangzhou Lohand Biological Co., Ltd.,

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China) in a thermostatic water bath under magnetic stirring. For optimization of the trienzymatic reaction, the reaction volume was restricted to 100 mL and a 210 mM PPO

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aqueous solution adjusted to the desired pH with ammonia was added, and then 0.1 mM PLP, 0.1 mM NAD+, and 250 mM isopropanol were added into the reactor followed by

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the addition of 15 mM L-Glu, which was neutralized by ammonia to the desired pH value

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beforehand (during optimization of the amino donor/acceptor ratio, the concentration of L-Glu was varied from 60 mM to 0 mM). Finally, 1 U/mL crude enzyme extract was added to initiate the reaction (during optimization of the amino donor/acceptor ratio, 2 U/mL was used). The pH of the reaction mixture was monitored using a pH detector and controller using ammonia (about 15%, v/v). In the batch reaction mode, the reaction

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mixture was composed of about 543 mM PPO, 0.1 mM PLP, 0.1 mM NAD+, 660 mM isopropanol, 25 mM L-Glu, and 10 U/mL crude enzyme extract using the optimized reaction pH and temperature, respectively. In the intermittent fed-batch reaction mode, the reaction mixture was composed of 0.1 mM PLP, 0.1 mM NAD+, 600 mM isopropanol, and 25 mM L-Glu. PPO was added into the reactor as a solid, the other conditions were

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as same as for the batch reaction mode, and the pH was controlled as above using 15% ammonia. In the continuous fed-batch reaction mode, the reaction mixture was composed of 0.1 mM PLP, 0.1 mM NAD+, and 25 mM L-Glu. The 500 mL feeding solution of 0.60

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mol isopropanol, about 0.51 mol PPO, and a certain amount of pure water was

intermittent fed-batch reaction mode.

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continuously added into the reactor, while the other conditions were the same as for the

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2.10. Scale-up of the continuous fed-batch reaction

The reaction was performed in a 100 L stainless steel bio-reactor equipped with a pH

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detector and controller and a temperature detector and controller (Shanghai Baoxing biological equipment Engineering Co., Ltd., China). The crude enzyme extract and co-

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enzyme usage were 10 U/mL GABAT, 10 U/mL GluDH, 10 U/mL ADH, 0.1 mM PLP,

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and 0.1 mM NAD+. A total of 255 g L-Glu was neutralized with ammonia to pH 8.0 and then was added into the reactor together with the enzyme solution. The 50 L substrate feed solution was composed of 5 L isopropanol, 10.204 kg PPO (98% purity), and pure water and the feeding rate was manually monitored. The reaction pH was automatically controlled using ammonia (15%, v/v) at 8.0±0.1. The reaction temperature was

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automatically controlled using circulating cooling water at 35±0.5 °C. 2.11. Analysis The reactants and products were monitored using analytical HPLC (HP-1100 HPLC system; Hewlett-Packard Development Company, L.P., Houston, USA) equipped with a reverse-phase C18 column (Agilent Pursuit 5 C18, 5 μm, 4.6 × 250 mm). For glutamate

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acid, phosphinothricin, α-KG, and PPO detection, the flow rate was maintained at 1.0 mL/min with isocratic elution of 90% buffer A (50 mM (NH4)2HPO4 and 0.1% (w/v)

tetrabutylammonium hydroxide adjusted to pH 3.6 with 50% (w/v) phosphoric acid) and

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10% acetonitrile at a column temperature of 40 °C and detection at 205 nm.

The enantiomers D, L-PPT were identified by pre-column derivatization with o-

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phthalaldehyde and N-acetyl-L-cysteine [30]. The flow rate was maintained at 0.8

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mL/min with detection at 334 nm, and the column temperature was constant at 30 °C. D, L-PPT was eluted with buffer B (50 mM sodium acetate)/methanol (90:10, v/v).

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3. Results and Discussion

3.1. Screening, over-expression, and characterization of aminotransferases for

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asymmetric synthesis of L-phosphinothricin

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First, the feasibility of the tri-enzymatic cascade reaction for the asymmetric synthesis of L-PPT in one-pot was assessed based on the robust activity and biochemical characteristics of TAs toward PPO. Therefore, five representatives from all four subgroups of TAs [31] were included in the investigation. Among them, GABAT, BCAT, ValAT, and AspAT were cloned from E coli W3110 and expressed in E. coli BL21 (DE3).

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Additionally, the whole gene of an aromatic aminotransferase (AroAT) from Klebsiella michiganensis E718 was synthesized and also expressed in E. coli BL21 (DE3). All of these five TAs were successfully cloned and overexpressed (Fig. S1). Then the transamination activity of these TAs toward PPO with different amino donors were determined (Table 1). The results showed that GABAT possessed the best activity

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compared with the other TAs using L-Glu as the amino donor. Interestingly, BCAT also exhibited excellent catalytic activity toward PPO using L-Glu as the amino donor, indicating this enzyme also has the potency for catalyzing the asymmetric synthesis of L-

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PPT.

Nevertheless, GABAT was demonstrated to be the best catalyst and purified

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recombinant GABAT was biochemically characterized in more detail before using it in

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the cascade reaction. The effect of pH and temperature on the transamination of PPO and L-Glu was examined by using a standard enzyme assay as described herein. The

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recombinant GABAT was most active in the alkalescent region, with a maximum activity at pH 9.0, and almost no transaminase activity was detected below pH 6 or above pH 12

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(Fig. 2A). The reaction velocity dramatically increased with temperature and exhibited a

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maximum at 60 to 65 °C (Fig. 2B). The pH stability of the recombinant protein was examined at 35 °C. A pH of 8.0 was found to be the best for maintaining the stabilization of the recombinant GABAT (Fig. 2C). The enzyme was found to inactive at 45 °C but more stable below 35 °C (Fig. 2D) and possessed a half-life of 2.81 h at 35 °C (Table 2), indicating its characteristics were sufficient for industrial applications.

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3.2 Substrate and product inhibition of the gamma-aminobutyrate aminotransferase (GABAT) To examine the PPO substrate inhibition of GABAT, the initial velocity of the transamination was measured at various concentrations of PPO with saturated concentration of L-glutamate in the presence of recombinant enzyme. As depicted in Fig.

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3A, no obvious substrate inhibition was observed up to 35 mM, and then the substrate inhibition increased as the concentration of PPO increased from 35 mM to 65 mM, finally reaching a plateau with about a 31.5% decrease compared with the maximum initial

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velocity. Because common TAs are subject to product inhibition from the ketone

byproduct [32], the product inhibition by α-KG for the GABAT was also examined at

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invariant concentration of L-Glu and PPO but various concentrations of α-KG. The initial

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velocity continuously decreased as the concentration of α-KG increased, and lost up to 68.2% at 10 mM α-KG (Fig. 3B), suggesting that product inhibition by α-KG was severe

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and must be overcome for a reaction using a high concentration of substrate. 3.3 Asymmetric synthesis of L-phosphinothricin (L-PPT) using the gamma-

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aminobutyrate aminotransferase (GABAT)

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With stable and active biocatalysts identified, the asymmetric synthesis of L-PPT catalyzed only by GABAT at 35 °C was investigated. Accordingly for the transamination process, a 10-mL EP tube reactor was packed with the same amount of the recombinant enzyme crude extract, and various ratios of the substrate PPO and the amino donor L-Glu. When the molar ratio of L-Glu to PPO was varied from 15/1 to 3/1, the L-Glu

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concentration was held constant at 1500 mM and the PPO concentration was varied from 100 mM to 500 mM. Conversely, when the molar ratio of L-Glu to PPO was varied from 1/1 to 1/15, the L-Glu concentration was varied from 500 mM to 33 mM and the PPO concentration was held constant at 500 mM. The results (Fig. 4A) showed that only a 15fold molar excess of L-Glu with respect to PPO could drive the reaction to absolute

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completion. The conversion rate dropped as the ratio of L-Glu/PPO decreased and was nearly zero when the molar ratio of L-Glu/PPO was 1/15. That is, the asymmetric

synthesis of L-PPT using GABAT as the only catalyst alone did not occur when the

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proportion of the amino donor L-Glu to all substrates was lower than 6.3% (mol/mol).

After the yields of L-PPT under the above reaction conditions were determined, the

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results as shown in Fig. 4B indicated that almost all the yields of the product L-PPT did

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not match the conversion rates of the substrate PPO, and that deviations of the yield from the conversion rate increased as the concentration of PPO increased (when the molar ratio

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of L-Glu to PPO ranged from 15/1 to 3/1). This may be attributed to the employment of crude cell lysates as the catalysts, which always contains unknown background activities

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resulting in undesired side reactions (e.g., the reduction of a keto acid to a hydroxy acid

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by hydrogenases). However, the deviation still increased when the concentration of PPO was fixed at 500 mM and the molar ratio of L-Glu/PPO was varied from 1/1 to 1/15. Considering that the same dosage of crude cell lysates was used in all reactions, and similar results were obtained by using pure recombinant GABAT, it is likely that PPO probably underwent a chemical degradation to other chemicals under the experimental

17

condition. 3.4. Stability of 2-oxo-4-[(hydroxy)(methyl)phosphinoyl]butyric acid (PPO) in the biocatalytic environment The results obtained from the examination of the asymmetric synthesis of L-PPT using GABAT indicated that the stability of PPO in a biocatalytic environment (mild

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temperature and neutral or alkalescent pH) was essential to obtain a high yield of L-PPT whenever one or more enzymes were used in the reaction mixture, especially when a high

concentration of product was desired. Therefore, at a constant temperature of 35 °C, two

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influencing factors, namely the solution pH and the initial concentration of PPO, were

investigated. Fig. 5 shows that PPO became more unstable as the pH increased to the

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extent that only 17.4% remained after 12 h at pH 9.5 starting from an initial concentration

concentration of 300 mM.

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of 900 mM. PPO was definitely stable for 12 h from pH 7.0 to 7.5 at the lowest initial

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Moreover, addition of any other common alkaline species to neutralize PPO for the bio-reaction would also result in a considerable loss of PPO (Fig. S2-S5). All of these

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discoveries indicated that the bio-synthesis of L-PPT would be difficult, especially to

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obtain a high concentration of L-PPT with an acceptable yield for industrial manufacturing. Nevertheless, the experimental results also indicated that PPO had sufficient stability over an appropriate reaction time at an initial concentration lower than 300 mM and near a neutral pH value. 3.5. Construction of a tri-enzymatic cascade system for asymmetric synthesis of L-

18

phosphinothricin As suggested by the results from the synthesis of L-PPT using GABAT alone, it was necessary to construct the cascade reaction as shown in Fig. 1 to improve the manufacture of L-PPT by overcoming the disadvantages of the transamination. Thus, a GluDH and an ADH (BsADH) were both needed in addition to GABAT to fulfill the reaction scheme

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depicted in Fig. 1. Before optimization of the conditions for the cascade reaction, the enzymatic properties of the three recombinant enzymes were characterized (Table 3). The

results showed that both the selected GluDH and ADH were more active and stable than

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GABAT. Additionally, their similar optimal temperatures and pH stabilities supported the compatibility of these three enzymes in a single-pot reaction, indicating the great potential

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of this three-enzyme one-pot system for industrial application.

phosphinothricin

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3.6. Optimization of the tri-enzymatic reaction for asymmetric synthesis of L-

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Although the three enzymes were carefully selected and should have great compatibility, the cascade reaction conditions still needed to be optimized to demonstrate its feasibility

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and confirm the high conversion of the substrate PPO. Thus, the pH and temperature of

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the reaction mixture as well as the ratio of amino donor/amino acceptor (Glu/PPO) were investigated. As shown in Fig. 6A, the tri-enzymatic reaction accelerated as the reaction broth pH increased from 7.0 to 9.0. However, when the pH was 9.5, the reaction velocity dropped a little during the first hour compared with the maximum at pH 9.0 and then continually declined until the reaction stopped. Conversely, the yield of L-PPT dwindled

19

as the reaction pH increased, except for at pH 7.0 where the reaction was not yet complete within the experimental time. Based on these results, the variance in the reaction velocity with the reaction pH may be attributed to the pH profile of GABAT (Fig. 2A), while the variations in the yield probably resulted from the instability of PPO (described in Section 3.4). Considering the reaction velocity and the yield of the product, the optimal pH of the

ro of

cascade reaction was 8.0, at which a yield of 98.5% was achieved within 5 h, although a slightly higher yield of 99.1% was obtained at pH 7.5 but with a reaction time of 12 h.

Next, the temperatures of the cascade reaction at pH 8.0 were examined to find the

-p

optimal one for synthesis of L-PPT. As shown in Fig. 6B, the reaction velocity before 1.0

h increased as the reaction temperature increased and was attributed to the temperature

re

profile of GABAT (Fig. 2B). After 1 h, the variation of the reaction velocity with the

lP

temperature became more complicated because of the combined effect of the temperature profile and thermal stability of GABAT. Therefore, the conversion rates of PPO at 40 °C

na

and 45 °C were 74.4% and 90.9%, respectively, while the other reactions at 25 °C, 30 °C, and 35 °C reached complete conversion. Consequently, 35 °C was chosen as the best

ur

temperature for the cascade reaction.

Jo

Interestingly, the properties of GluDH and ADH exhibited nearly no influence on the optimal conditions of the tri-enzymatic cascade reaction, perhaps because of their better thermal stabilities (Table 3) compared with GABAT. That is, the stability GABAT was a limiting factor in this tri-enzymatic reaction system. Additionally, 210 mM PPO was transformed using only 15 mM of the amino donor L-Glu to yield complete conversion

20

at the optimal pH and temperature demonstrating the feasibility of the cascade reaction for the synthesis of L-PPT. To reduce the dosage of L-Glu to a catalytic amount, different reactions with various ratios of L-Glu/PPO were carried out at the same pH and temperature. The results indicated that the reactions with a ratio of Glu/PPO greater than or equal to 1/15 could achieve complete conversion and the reaction velocity accelerated

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rapidly as the ratio increased (Fig. 6C). Moreover, no α-KG was detected because of the robust catalytic capacity of GluDH and ADH, which immediately recycled α-KG to L-

Glu after it was produced. This system was far more productive than the situation with

-p

GABAT as the solo catalyst, in which the reaction was almost inert at a ratio of Glu/PPO

equal to 1/15. Although the reaction conversion at a Glu/PPO ratio of 1/35 was only

re

88.1%, a conversion near 100% using less amino donor might be expected by increasing

lP

the dosage of GABAT.

3.7. Mode of the tri-enzymatic cascade reaction

na

Use of the tri-enzymatic cascade reaction eliminated the issues of the unfavorable thermodynamic equilibrium, the obligatory excessive amino donor, and the inhibition by

ur

α-KG. However, the usefulness of the system for industrial applications was still limited

Jo

by inhibition from the substrate PPO and its instability. As shown in Fig. 7A, in a batch reaction of this tri-enzymatic system for transformation of 543.6 mM PPO, 491.7 mM LPPT was formed and 26.1 mM PPO remained with a conversion rate of 95%, but the yield of L-PPT was only 90%. Therefore, the traditional batch reaction mode is not suitable for the biotransformation of PPO to L-PPT, and thus, the supply or delivery of substrates for

21

the biocatalysis must be investigated. First, an intermittent fed-batch reaction was carried out (Fig. 7B). Based on the result from the stability research of PPO in Section 3.4, the initial concentration of PPO was set as 100 mM. When the residual PPO in the medium was nearly 0, the feed PPO (solid) was added until the PPO concentration reached 100 mM. The conversion rate decreased

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when the next feeding was added. After 5 feedings, 500 mM PPO was added to the reaction mixture and a complete conversion was reached, forming 485 mM L-PPT with a 97% yield and a productivity of 8.8 g/L/h. Compared with the batch mode, the

-p

intermittent fed-batch mode remarkably improved the substrate conversion and product yield. However, the reaction time decreased only a little from 12 h to 10 h. Additionally,

re

this intermittent feeding of a strongly acidic solid substrate to the biocatalysis system can

lP

easily result in inactivation of the biocatalyst and bring about operating difficulties, impeding its application for large-scale industrial manufacturing.

na

Therefore, a continuous fed-batch reaction mode was proposed in which PPO was continuously fed into the reaction mixture in an aqueous solution and the feeding rate was

ur

monitored by controlling the concentration of PPO to be lower than 80 mM. As shown in

Jo

Fig. 7C, 508.8 mM PPO was completely converted to L-PPT in 6 h using the continuous fed-batch mode, and the product yield was 99.8% with a productivity of 15.3 g/L/h, nearly two-fold more than the intermittent fed-batch mode. Compared with a previously described two coupled transaminase process [20] where 500 mM PPO was consumed in 24 h when a final reaction broth was composed of 413.5 mM L-PPT, 86 mM glutamic

22

acid, 93.5 mM aspartic acid, 62.5 mM alanine, and 55 mM PPO, the system described herein was more advanced. 3.8. Scale-up of the tri-enzymatic cascade reaction using continuous fed-batch mode Finally, a continuous fed-batch reaction system as depicted in Fig. 8A was developed to scale-up the cascade reaction to verify its potency for large-scale industrial manufacturing.

ro of

Based on the results obtained above, a pilot-scale of 90 L was performed in a 100 L stainless steel tank with continuously feeding of 10.204 kg PPO in aqueous solution. This allowed a low concentration of PPO to be maintained in the reaction broth to avoid

-p

substrate inhibition and minimize the degradation of PPO. Fig. 8B shows the result of this pilot-scale reaction in detail. A product concentration of 615.4 mM (111.4 g/L) was

re

achieved with a yield of 99.7% and a complete conversion in 7 h. Thus, a productivity of

lP

15.9 g/L•h was obtained, which was enhanced by more than an order of magnitude compared with the productivity (1.35 g/L•h) in the study reported by X. J. Yin and

na

colleagues [23] using a new engineered NADPH-specific GluDH and a glucose dehydrogenase. Additionally, only 21.2 mM L-Glu remained in the reaction mixture at

ur

the end of the reaction, accounting for a proportion of 2.7% (wt/wt) in the mixture of L-

Jo

Glu and L-PPT. Although a small amount of the excessive co-substrate isopropanol and byproduct acetone might have remained in the mixture, which can be conveniently removed by evaporation in vacuo, no other compounds requiring complicated separation procedures existed in the reaction broth. Taken together, the asymmetric synthesis of LPPT proposed herein was demonstrated to be practical for industrial applications, and its

23

process simplicity and economic value were tested and verified by the pilot-scale experiment. 3.9. Application of this method to other non-proteinogenic amino acids With the successful synthesis of L-PPT and considering that the aminotransferase BCAT was already demonstrated to have a broad substrate spectrum [33] and was used to

ro of

synthesize a series of optical pure amino acids using L-Glu as amino donor [21], it was feasible to test the versatility of the developed method by employing BCAT instead of

GABAT to asymmetrically synthesize other compounds. Among them, L-norvaline,

-p

which is a vital intermediate of Perindopril and is mainly produced by chemical synthesis

with low purity [34], and L-tert-leucine, which is a component of several pharmaceutical

re

development projects focused on tumor-fighting agents and HIV protease inhibitors [35],

lP

are valuable targets and therefore were selected for this study. Except for PPO, for which the activity of BCAT is shown in Table 1, two α-keto acids, namely 2-oxopentanoic acid

na

and trimethylpyruvic acid, were identified as substrates of BCAT. Therefore, the threeenzymatic reaction was used to convert 2-oxopentanoic acid and trimethylpyruvic acid to

ur

produce L-norvaline and L-tert-leucine, respectively, using the continuous fed-batch

Jo

reaction mode. A complete conversion of the 500 mM α-keto acid substrate (limited by resources, these reactions were performed only at a 1000 mL level) with a product yield and ee value over 99% was obtained.

4. Conclusion In summary, we have developed an irreversible transamination procedure that can be

24

applied as an efficient and economic process for the industrial manufacture of optically pure non-proteinogenic amino acids. This approach is based on using a tri-enzymatic cascade reaction system to solve the unfavorable thermodynamic equilibrium and to eliminate the inhibition of the byproduct (α-KG) that commonly plagues transamination catalyzed by an aminotransferase. Compared with the conventional procedure, which uses

ro of

at least a stoichiometric amount of amino donor for the transamination, only a subcatalytic amount of amino donor is required and no byproducts requiring complicated separation procedures are formed. Furthermore, despite the fact that it remains unknown

-p

what mechanism is involved in the decomposition of PPO under biocatalytic conditions,

a continuous substrate fed-batch strategy was validated to alleviate the decomposition of

re

PPO and was successfully applied to the scale-up of the tri-enzymatic cascade reaction

lP

with complete conversion of PPO to L-PPT in 99.7% yield and >99.9% ee. Further work is needed to investigate a wider range of products by selecting appropriate

na

enzymes for the cascade. In fact, the direct asymmetric synthesis of optically pure amines from the corresponding keto compounds by transamination has emerged as a particularly

ur

promising process [36]. Considering this and the unstable properties of keto substrates

Jo

[37], this cascade reaction using a continuous substrate fed-batch strategy may provide the engineering tools necessary to move this technology toward a rational approach for developing large-scale applications of aminotransferases.

25

Acknowledgments

This work was financially supported by the National Natural Science Foundation of China

Jo

ur

na

lP

re

-p

ro of

(No. 21476199, 21676240).

26

References [1] G. Hoerlein, in: George W. Ware (Eds.), Reviews of Environmental Contamination and Toxicology, volume 138, Springer, 1994, pp. 73-145. [2] E. Bayer, K.H. Gugel, K. Haegele, H. Hagenmaier, S. Jessipow, W.A. Koenig, H Zaehner, Helv. Chim. Acta. 55 (1972) 224-239.

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[3] W.R. Ullrich, C.I. Ullrich-Eberius, H. Koecher, Pestic. Biochem. Physiol. 37 (1990) 1-11.

[4] C. Wendler, M. Barniske, A. Wild, Photosynth. Res. 24 (1990) 55-61.

-p

[5] C. Gillezeau, M. van Gerwen, R.M. Shaffer, I. Rana, L. Zhang, L. Sheppard, and E. Taioli, Environ. Health. (2019) 18:2.

re

[6] C.M. Benbrook, Environ. Sci. Eur. (2016) 28:3.

lP

[7] T. Baltazar, R.J. Dinis-Oliveira, J.A. Duarte, M. de Lourdes Bastos, F. Carvalho, Br. J. Pharmacol. 168 (2013) 44-45.

na

[8] L. Maier, P.J. Lea, Phosphorus. Sulfur. 17 (1983) 1-19. [9] R.P. Hicks, A.L. Russell in: L. Pollegioni, S. Servi (Eds.), Unnatural Amino Acids,

ur

Springer, 2012, pp. 135-167.

Jo

[10] N. Minowa, M. Hirayama, S. Fukatsu, Bull. Chem. Soc. Jpn. 60 (1987) 1761-1766. [11] H.J. Zeiss, Tetrahedron Lett. 28 (1987) 1255-1258. [12] H.J. Zeiss, Tetrahedron 48 (1992) 8263-8270. [13] M.G. Hoffmann, H.J. Zeiss, Tetrahedron Lett. 33 (1992) 2669-2672. [14] S. Grabley, K. Sauber, US Patent 4389488 (1982), to Hoechst AG.

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[15] K. Bartsch, US Patent 6686181 (2004), to Aventis Corporation Gmbh. [16] L. Willms, G. Fuelling, R. Keller, Europe Patent 382113 (1989), to Hoechst AG. [17] K.F. Albizati, S. Kambourakis, A. Grubbs, B.C. Borer, US Patent 8981142 (2015), to Strategic Enzyme Application, Inc. [18] A. Schulz, P. Taggeselle, D. Tripier and K. Bartsch, Appl. Environ. Microbiol. 56

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(1990) 1-6. [19] K. Bartsch, R. Dichmann, P. Schmitt, E. Uhlmann and A. Schulz, Appl. Environ. Microbiol. 56 (1990) 7-12.

-p

[20] K. Bartsch, A. Schulz, Appl. Environ. Microbiol. 62 (1996) 3794-3799.

Catal. Lett. 148 (2018) 3309-3314.

re

[21] L.J. Meng, Y.Y. Liu, H.S. Zhou, X.J. Yin, J.P. Wu, M.B. Wu, G. Xu, L.R. Yang,

lP

[22] J.M. Fang, C.H. Lin, C.W. Bradshaw, J. Chem. Soc. Perkin Trans. 1 (1995) 967978.

na

[23] X.J. Yin, J.P. Wu, L.R. Yang, Appl. Microbiol. Biotechnol. 102 (2018) 4425–4433. [24] K. Bartsch, C.C. Tebbe, Appl. Environ. Microbiol. 55 (1989) 711-716.

ur

[25] C.C. Tebbe, H.H. Reber, Appl. Microbiol. Biotechnol. 29 (1988) 103-105.

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[26] N. Minowa, N. Nakanishi, M. Mitomi, US Patent 8017797 (2011), to Meiji Seika Kaisha Ltd.

[27] T.L. Liao, A.C. Lin, E. Chen, T.W. Huang, Y.M. Liu, Y.H. Chang, J.F. Lai, T.L. Lauderdale, J.T. Wang, S.C. Chang, S.F. Tsai, and Y.T. Chen, J. Bacteriol. 194 (2012) 5454.

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[28] T. Jahns, Antonie van Leeuwenhoek. 70 (1996) 89-95. [29] L. Kirmair, D.L. Seiler, A. Skerra, Appl. Microbiol. Biotechnol. 99 (2015) 1050110513. [30] H.J. Zeiss, Pest Manag. Sci. 41 (1994) 269-277. [31] P.K. Mehta, T.I. Hale, and P. Christen, Eur. J. Biochem. 214(1993) 549-561.

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[32] I. Slabu, J.L. Galman, R.C. Lloyd, and N.J. Turner, ACS Catal. 7 (2017) 8263-8284. [33] X.J. Yu, X.G. Wang, P.C. Engel, FEBS J. 281 (2014) 391-400.

[34] Y. Qi, T. Yang, J. Zhou, J. Zheng, M. Xu, X. Zhang, Z. Rao, S.T. Yang, Process

-p

Biochem. 55 (2017) 104-109.

[35] P. Lehr, A. Billich, B. Charpiot, P. Ettmayer, D. Scholz, B. Rosenwirth, H. Gstach,

re

J. Med. Chem. 39 (1996) 2060–2067.

lP

[36] P. Tufvesson, J. Lima-Ramos, J.S. Jensen, N. Al-Haque, W. Neto, J.M. Woodley, Biotechnol. Bioeng. 108 (2011) 1479-1493.

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ur

na

[37] A.J.L. Cooper, J.Z. Ginos, and A. Meister, Chem. Rev. 83 (1983) 321-358.

29

Figure Captions Fig. 1. Outline of the cascade reaction for the asymmetric synthesis of L-phosphinothricin (L-PPT). PLP: pyridoxal 5’-phosphate; GluDH: glutamate dehydrogenase; NAD+: nicotinamide adenine dinucleotide in the oxidized form; NADH: nicotinamide adenine

na

lP

re

-p

ro of

dinucleotide in the reductive form; ADH: alcohol dehydrogenase.

Fig. 2. Enzymology studies of the recombinant gamma-aminobutyrate aminotransferase

ur

(GABAT). The pH profile (A), the temperature profiles (B), the pH stability (C) at 35°C

Jo

and thermal stability (D) at pH 8.0 of the recombinant GABAT.

30

ro of -p

Jo

ur

na

lP

aminobutyrate aminotransferase.

re

Fig. 3. Substrate inhibition (A) and byproduct inhibition (B) of recombinant gamma-

31

ro of -p re lP na

Fig. 4. Asymmetric synthesis of L-phosphinothricin (L-PPT) by recombinant gammaaminotransferase.

ur

aminobutyrate

A:

the

conversion

process

of

2-oxo-4-

Jo

[(hydroxy)(methyl)phosphinoyl]butyric acid (PPO); B: the reaction conversion rate of PPO and yield of L-PPT after 24 h. For experimental details see Section 2.8.

32

ro of -p re lP na ur Jo Fig. 5. Stability of 2-oxo-4-[(hydroxy)(methyl)phosphinoyl]butyric acid (PPO) in neutral or alkalescent solution. Various concentration of PPO was neutralized using ammonia to

33

the corresponding pH value, and the residual concentration was detected by HPLC after

-p

ro of

12 h at a constant temperature of 35°C. All measurements were done in at least duplicate.

re

Fig. 6. Asymmetric synthesis of L-phosphinothricin by the tri-enzymatic cascade reaction.

lP

A: optimization of the reaction pH at a reaction temperature of 35°C. B: optimization of the reaction temperature at pH 8.0. C: optimization of the amino donor/acceptor ratio at

Jo

ur

na

pH 8.0 and a reaction temperature of 35°C. Experimental details see section 2.9.

34

ro of -p re lP na ur Jo

Fig. 7. Reaction mode of the tri-enzymatic cascade. A: batch reaction. B: intermittent fedbatch reaction. C: continuous fed-batch reaction.

35

ro of -p re lP na ur Jo

Fig. 8. Asymmetric synthesis of L-phosphinothricin by the tri-enzymatic cascade reaction with a continuous substrate fed-batch process. A: the sketch map of the substrate fed-

36

batch reactor. B: the process data of the substrate fed-batch reaction. For experimental

Jo

ur

na

lP

re

-p

ro of

details see Section 2.10.

37

Tables Table 1 Specific activity of aminotransferases toward 2-oxo-4[(hydroxy)(methyl)phosphinoyl]butyric acid (PPO) with different amino donors. Amino donor TA L-Val

L-Glu

L-Phe

GABAT

0.17±0.01

<0.001

41.8±1.3

<0.001

BCAT

0.03±0.01

<0.001

13.7±0.3

<0.001

ValAT

<0.001

<0.001

<0.001

AspAT

<0.001

<0.001

<0.001

AroAT

<0.001

<0.001

<0.001

ro of

L-Ala

<0.001

<0.001 <0.001

-p

All measurements were done at least in duplicate using a pure enzyme solution. The

na

lP

re

specific activity is expressed as units per milligram protein (U/mg).

Half-life of activity (h)

25

11.6

30

5.4

35

2.8

40

1.5

45

0.6

Table 2 Thermal stability of the recombinant gamma-aminobutyrate aminotransferase.

Jo

ur

Temperature (°C)

38

Table 3. Enzymatic properties of the three enzymes involved in this study. GABAT

GluDHa

BsADHb

Optimal reaction temperature

60-65 C

60 C

60-65 C

pH 9.0

pH 8.0

pH 9.5

41.8±1.3 U/mg

903.1±24.6 U/mg

206.4±4.5 U/mg

t1/2=2.8 h (35 C)

t1/2=167 h (60 C)

t1/2=11.3 h (40 C)

Stable at pH 7.0–9.0

Stable at pH7.0–8.0

Stable at pH 7.0–9.0

Optimal reaction pH Activity in optimal condition Thermal stability pH stability

-p

t1/2 is the half-life of activity at the specific temperature.

ro of

Enzymes

for more detail see Figs. S6 and Table S1.

b

for more detail see Figs. S7 and Table S2.

Jo

ur

na

lP

re

a

39