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Systems Microbiology and Biomanufacturing (2021) 1:234–244https://doi.org/10.1007/s43393-020-00021-9

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ORIGINAL ARTICLE

Cascade biocatalysis for production of enantiopure (S)‑2‑hydroxybutyric acid using recombinant Escherichia coli with a tunable multi‑enzyme‑coordinate expression system

Lingzhi Tian1 · Junping Zhou1 · Taowei Yang1 · Xian Zhang1 · Meijuan Xu1 · Zhiming Rao1

Received: 3 November 2020 / Revised: 13 December 2020 / Accepted: 21 December 2020 / Published online: 25 January 2021 © Jiangnan University 2021

AbstractRacemize 2-hydroxybutyric acid is usually synthesized by organic methods and needs additional deracemization to obtain optically pure enantiomers for industrial application. Here we present a cascade biocatalysis system in Escherichia coli BL21 which employed L-threonine deaminase (TD), NAD-dependent L-lactate dehydrogenase (LDH) and alcohol dehydrogenase (ADH) for producing optically pure (S)-2-hydroxybutyric acid ((S)-2-HBA) from bulk chemical L-threonine. To solve the mismatch in the conversion rate and the consumption rate of intermediate 2-oxobutyric acid (2-OBA) formed in the multi-enzyme catalysis reaction, ribosome binding site regulation strategy was explored to control TD expression levels, achieving an eightfold alteration in the conversion rate of 2-OBA. With the optimized activity ratio of the three enzymes and using ADH for NADH regeneration, the recombinant strain ADH-r53 showed increased production of (S)-2-HBA with the highest titer of 129 g/L and molar yield of 93% within 24 h, which is approximately 1.65 times that of the highest yield reported so far. Moreover, (S)-2-HBA could easily be purified by distillation, making it have great potential for industrial application. Additionally, our results indicated that constructing a tunable multi-enzyme-coordinate expression system in single cell had great significance in biocatalysis of hydroxyl acids.

Keywords Cascade biocatalysis · (S)-2-hydroxybutyric acid · Multi-enzyme-coordinate expression system · NADH regeneration · Ribosome binding site strength

Introduction

2-hydroxybutyric acid (2-HBA) is an essential intermediate for synthesizing biodegradable materials and various types of medicines [1, 2]. Synthesis of enantiopure 2-HBA can be achieved by traditional chemical methods [3, 4], particularly

from petrochemical resources which results in formation of two optical isomers [5]. These racemic 2-HBA usually require additional deracemization to obtain optically pure enantiomers which are used for chiral intermediates, biode-gradable materials and in the chemical industry. (R)-2-HBA along with other (R)-hydroxyl acids are also applied for the synthesis of polyhydroxyalkanoates (PHAs) [6–8], which belong to one type of fundamental materials for biodegrad-able polymers [1, 6, 9–11]. Moreover, 2-HBA containing PHAs, which include P(2HB-co-3HB) and P(2HB), are mainly used for biomedical materials like medical implants and stent materials [12] due to its better biocompatibility, and thus have become a relatively active research hotspot in the understanding of biomaterials [13]. On the other hand, (S)-2-HBA can be used to synthesize levetiracetam and brivaracetam (two anti-epileptic drugs) [14], the per-oxisome proliferator-activated receptor a (PPARa) agonist (R)-K-13675 [15] and the PPARg agonist MK-0533 [16]. Therefore, the development of an efficient and economical

Lingzhi Tian and Junping Zhou contributed equally to this work.

Supplementary Information The online version contains supplementary material available at https ://doi.org/10.1007/s4339 3-020-00021 -9.

* Meijuan Xu xumeijuan@jiangnan.edu.cn

* Zhiming Rao raozhm@jiangnan.edu.cn

1 The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, Jiangsu Province, China

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method for the production of optically pure 2-HBA is in high demand.

Synthesis of optically pure 2-HBA has been achieved by enzymatic kinetic resolution of racemic mixtures [17], and asymmetric bioreduction of 2-OBA [18]. However, both of the substrates supplied in these methods are non-commercial chemicals. With the development of fermentation technol-ogy and metabolic engineering, L-Thr, the third bulky amino acid has mainly been produced through fermentation with a global annual output of approximately 300 thousand metric tons in 2014 [19]. Until recently, an efficient approach for (R)- or (S)-2-HBA production from L-threonine (L-Thr) was developed using recombinant Escherichia coli cells express-ing separately or co-expressing L-threonine deaminase (TD), formate dehydrogenase (FDH) and lactate dehydrogenase (LDH) [20]. However, the mismatch in conversion rates for different steps in the multi-enzyme catalysis reactions often present a challenge to efficient bioconversion, hence requir-ing further optimization/ regulation of the different catalytic steps to get a dynamic equilibrium [21]. Thus, the develop-ment of reliable and efficient alternative routes to produce enantiopure 2-HBA is attractive.

Nature has evolved ways in which cells balance meta-bolic processes, such as the feedback inhibition of enzymes by end products or intermediates [22], presence of oper-ons to facilitate coordinated expression of multiple genes [23], promoters to control gene transcription rates [24, 25],

terminators to terminate transcription [26], and ribosome binding sites (RBS) sequence to regulate enzyme expres-sion [27, 28]. Such precise control is used by organisms to balance their in vivo components and reactions, and also to prevent the accumulation of toxic intermediate metabolites along the pathway[26, 29, 30]. At present, RBS optimiza-tion is regularly applied to the coordinated optimization of protein expression levels [31, 32]. Using the predictive design method, RBS sequences with specific strengths for specific genes can be obtained, leading to regulated protein expression levels, hence achieving precise regulation and rational control of a protein’s production rate on a compa-rable scale [33]. Regulating enzyme expression levels by RBS optimization could better solve the existing problems in the last step from R&D to industrialization. By coor-dinating the expression level of enzymes, the rate mis-matching problem in multi-enzyme catalysis was solved, and the kinetic equilibrium of the reaction was achieved. Therefore, in this study (as shown in Fig. 1), we con-tstructed a tunable multi-enzyme-coordinate expression system for efficient whole-cell biocatalysis of the bulky chemical L-Thr to produce (S)-2-HBA, by exploring suit-able expression of TD, regulating enzymatic expression strengths of RBS, and coexpressed NAD-dependent LDH to convert intermediate 2-OBA to (S)-2-HBA. Addition-ally, ADH was introduced for NADH regeneration, hence, the byproduct acetone with a low boiling point could quickly be removed by distillation.

Fig. 1 Scheme of cascade biocatalysis for producing enantiopure (S)-2-hydroxybutyric acid using recombinant E. coli with a tunable multi-enzyme-coordinate expression system

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Materials and methods

Microorganisms and chemicals

All strains, plasmids, and primers are mentioned in Table S1. E. coli BL21 (DE3) served as ivlA-TD gene source [34] was preserved in our laboratory, and it was also used for cloning and the propagation of all plas-mids. Both Staphylococcus aureus CGMCC 1.282 (Gene ID: 3921373) and Enterococcus faecalis CGMCC 1.9 (Gene ID: 1199146) served as the genetic source of ldh-L- LDH and were provided by China General Micro-biological Culture Collection Center, (Beijing, China). Other genes encoding LDH from Plasmodium falciparum and Solanum tuberosum were synthesized and ordered from Sangon Biotech Co., Ltd (Shanghai, China). G. stearothermophilus (CICIM F1013), used as the source of adhT-ADH gene, was purchased from the Culture & Information Center of Industrial Microorganism of China Universities (CICIM, Wuxi, China). The genetic source of fdh-FDH enzyme was preserved in our lab [35]. The restriction enzymes, the PCR kit, and T4 DNA ligase, were ordered from TaKaRa Bio. Co. (Dalian, China). The ClonExpress MultiS One Step Cloning Kit for ligation of multiple gene fragments to plasmids was obtained from Vazyme Biotech. (Nanjing, China). Other high analyti-cal grade chemicals were purchased from commercial sources.

Plasmid construction

All constructed plasmids used in this study are mentioned in Table S1. Both pET-28a and pETDuet-1 plasmids used for gene expression were stored in our laboratory. Custom DNA oligonucleotides were provided by Sangon Biotech Co., Ltd (Shanghai, China). Genomic DNA (gDNA) was extracted from cell cultures using the Fungal/Bacterial DNA MiniPrep (Generay Biotech., Shanghai, China) according to manufacturer instructions. Using standard protocols of PCR, all genes were amplified with Extaq DNA Polymerase (TaKaRa Bio. Co. Dalian, China). The amplified and digested linear DNA fragments were purified using DNA Clean & Concentrator Kit (Generay Biotech.) following the manufacturer’s instructions. The RBS Calculator (https ://www.denov odna.com/softw are/desig n_rbs_calcu lator ) was used to calculate the different strengths of RBS, and gene ivlA with different RBSs was PCR amplified from pET28a-ivlA with designed primers in the Table S1 to form the vectors pET28a-r9ivlA to pET28a-r79ivlA. Prior to the transformation, the resulted plasmids

and genes were all digested by enzymes, and then the gene fragments were ligated using T4 DNA ligase. After that, the ligation product was transformed into E. coli BL21 (DE3). All positive transformants were selected by plating them on Luria–Bertani (LB) solid agar medium containing antibiotics (kanamycin or ampicillin). Colony PCR and as well as restriction digest mapping were performed to ensure the successful screening. The recombinant strains containing plasmids pETDuet-adhT + r40ivlA + Pfldh, p E T D u e t - a d h T + r 5 3 i v l A + P f l d h , p E T D u e t -adhT + r79ivlA + Pfldh, pET28a-fdh + Pfldh + r53ivlA, and pET28a-fdh + Pfldh + r79ivlA were named as ADH-r40, ADH-r53, ADH-r79, FDH-r53 and FDH-r79, respectively.

Recombinant proteins expression

The recombinant proteins with the genes inserted into the plasmid pET-28a were expressed in E. coli BL21 (DE3) as His6-tagged proteins. All the recombinant E. coli strains were inoculated at 37 °C in Luria–Bertani medium contain-ing 50 μg ml−1 antibiotics. To induce protein expression, we added isopropyl-β-D-thiogalactopyranoside (IPTG) with a final concentration of 1 mM when the OD600 value of the inoculum reached 0.6–0.8. The cultures were inoculated at 28 °C for another 8 h and harvested by centrifugation (8000 rpm, 10 min). Finally, the harvested cells were resus-pended in 50 mM Phosphate Buffered Saline (PBS) buffer (pH 7.5) followed by cell lysis using sonication with an ultra-sonic oscillator (Sonic Materials Co., USA). Subsequently, the cell debris was removed by centrifugation (12,000 rpm, 40 min) at 4 °C. The supernatant was then subjected to a HisTrap HP affinity column (GE Healthcare, Piscat-away, NJ, USA) in equilibrium with the buffer (20 mM Tris–HCl, 0.3 M NaCl; pH 7.5). The elution was carried out with the elution buffer (20 mM Tris–HCl, 0.3 M NaCl, 0.3 M imida-zole; pH 7.5) in the ÄKTA purifier system (GE Healthcare, Piscataway, USA). The protein content of the supernatant was measured using the Bradford assay. The homogeneity of purified enzymes was determined by Coomassie brilliant blue staining of SDS-PAGE gels.

Enzyme activity assay

The crude and purified enzymes from different recombinant strains were used for enzyme activity mearsurement. For the recombinant strains with multi-enzyme co-expressed, only the crude enzyme activities were measured.

The activity assay of TD was carried out by measuring 2-oxobutyric acid (2-OBA) formation at 230 nm using a UV–VIS Spectrophotometer (Aoesh, Shanghai, China) [36]. The reaction was held at 30 °C with a mixture of 100 mM

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L-Thr, 20 μM PLP, and a suitable amount of TD enzyme in 50 mM potassium phosphate buffer (pH 7.5). The amount of enzyme that produced 1 μmol 2-OBA per minute was used to define one unit of enzyme activity.

The enzyme activities of LDHs and ADH were measured by detecting the change of NAD(H) concentration at 340 nm using Aoesh UV–VIS Spectrophotometer [20]. The reaction was held at 30 °C with a mixture of 5 mM substrates, 1 mM NAD+ or 0.2 mM NADH, and a suitable amount of enzyme in 50 mM potassium phosphate buffer (pH 7.5). One unit of enzyme activity was defined as the amount of enzyme that produced 1 mmol of NADH or NAD+ per minute.

Optimization of the enzyme ratio for multi‑enzyme catalysis in (S)‑2‑HBA production

The purified enzymes EcTD, PfLDH and GsADH were added in different proportions into buffer for optimization of the enzyme ration in (S)-2-HBA production. The EcTD activity was maintained at 20 U/ml and excess GsADH with activity over 30 U/mL, the ratio of EcTD to PfLDH was changed from 0.5:1 to 4:1. Then we maintained the enzyme activity ratio of EcTD to PfLDH at the optimal ratio (EcTD activity at the same 20 U/ml) and changed the enzyme activ-ity ratio of EcTD to GsADH, which was ranged from 1:0.1 to 1:0.5. The bioconversion was carried out in 50 mL flask with 20 mL 50 mM PBS buffer (pH 7.5) containing 60 g/L L-Thr, and the reaction mixture was stirred at 160 rpm with pH maintained at 7.5 using 1 M NaOH in 30 °C water bath. The residual L-Thr, intermediate 2-OBA and productivity of 2-OBA, (S)-2-HBA in the reaction solutions were measured by HPLC.

Production of (S)‑2‑HBA from L‑Thr by recombinant E. coli whole‑cell biocatalyst

For measuring the consumption rate of L-Thr and the conversion rate of 2-OBA to (S)-2-HBA, the transfor-mation was accomplished using whole-cell biocatalyst. The recombinant E. coli with different RBS strengths for TD expression and the recombinant strain coexpressing PfLDH and ADH were cultured in LB medium, IPTG was added when the OD600 reached 0.6–0.8, then the cultures were subsequently inoculated at 28 °C for another 12 h and harvested using centrifugation (8000 rpm, 10 min). After that, the cell with different RBS strengths for TD expression was resuspended in 100 mL sodium phosphate buffer (0.1 M, pH 7.5) with 30 g/L substrate L-Thr, while the cell with coexpressing PfLDH and ADH was resus-pended in 100 mL sodium phosphate buffer (0.1 M, pH 7.5) with 10 g/L 2-OBA, respectively. The OD600 for both

were adjusted to 5. The transformation was performed for 1 h at 30 °C and 160 rpm with the pH-controlled at pH 7.5 using 1 M NaOH solution. The samples were taken every 15 min; the residual L-Thr, 2-OBA and productivity of 2-OBA, (S)-2-HBA in transformation solutions were measured by HPLC.

For whole-cell biocatalysis of (S)- 2-HBA from L-Thr, the recombinant E. coli BL21 with three enzymes co-expression was cultured in 250 ml flasks using 50 ml TY medium (Com-position, g/L: yeast extract, 8; tryptone, 12; K3PO4, 4.02; NaCl, 3; citric acid monohydrate, 2.1; ferric ammonium cit-rate, 0.3; glycerol, 10; (NH4)2SO4, 2.5; MgSO4·7H2O, 0.5) with same cultivation method reported by literature [37]. The cell was recovered and resuspended in 50 mL sodium phos-phate buffer (0.1 M, pH 7.5) with the cell concentration as 8 g dry cell weight per liter (g DCW/L). The substrate L-Thr were added into the reaction buffer with a final concentration at 60 g/L. The molar ratio of added cosubstrates isopropanol to L-Thr was 1:1. The transformation was carried out at 30 °C and 160 rpm for 12 h. Solution of 1 M NaOH was added to maintain the reaction medium pH at 7.5 during the transfor-mation process. The residual L-Thr, intermediate 2-OBA, and final product (S)-2-HBA were analyzed using HPLC.

The choosen tunable multi-enzyme-coordinate strains were cultured in 5 L fermentor using 2 L the same TY medium with the same cultivation method as above. The cell was recovered and resuspended in 2 L sodium phosphate buffer (0.1 M, pH 7.5) with the cell concentration as 25 g DCW/L. The substrates, including L-Thr, isopropanol were added into the buffer at 0, 2, 5, 8, 12 and 16 h. While the ratio of L-Thr to isopropanol was 1:1. The transformation was carried out at 30 °C and 160 rpm. The pH was also maintained at 7.5 using 1 M NaOH during the transformation process. The samples were taken at specific time intervals, the residual L-Thr, inter-mediate 2-OBA, and final product (S)-2-HBA were analyzed using HPLC.

Analytical methods

The accurate concentrations of L-Thr, isopropanol, 2-OBA, acetone, and (S)-2-HBA were analyzed by HPLC (Agilent 1260 series, Hewlett-Packard, USA). The L-Thr was derivat-ized with OPA/BOC-Cys and analyzed at 338 nm by HPLC using a LC-18DB column (5 μm, 4.6*250 mm, Agilent) [37]. The isopropanol, 2-OBA, acetone, and (S)-2-HBA were detected using refractive index detector (RID) with a Bio-Rad Aminex HPX-87 H column with the mobile phase consisted of 10 mM H2SO4 pumped at 0.5 ml min−1 (55 °C), while the stereoselective assay of (S)-2-HBA was performed using a chi-ral column (MCI GEL CRS10W, Japan) with a mobile phase mixture (water and acetonitrile (90:10), containing 2 mM cop-per sulfate) pumped at 0.5 ml min−1 (25 °C) [5].

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Results and discussion

Cascade reaction design and construction of the (S)‑2‑HBA synthesis system using L‑Thr as substrate in vitro

The cascade reaction from L-Thr to produce (S)-2-HBA proceeds in two steps. The first step is the formation of 2- OBA from L-Thr catalyzed by TD enzyme which removes its hydroxyl and amino groups. In the second step, LDH enzyme asymmetrically reduced the carbonyl group of 2-OBA to a hydroxyl group with the help of NADH to produce (S)-2-HBA, which required the coupling of NADH coenzyme regeneration system. We found that TD from E. coli BL21 (DE3) had high enzyme activity [34] hence EcTD was selected as the first enzyme. LDHs have been widely studied for producing 2-hydroxyacids by ste-reospecific conversion of 2-keto acids [18, 38]. Mainly, kinetic parameters and highly enantioselectivity of LDHs from five animal sources have been carefully studied for the reduction of approximately 20 α-keto acid substrates [18]. However, these LDHs were extracted with the high cost and were unknown to be successfully expressed in recombinant strains. Thus, here we studied the reduction of 2-OBA with LDH from different species. LDH from S. aureus [39], E. faecalis, P. falciparum [40], and S. tubero-sum [41] were compared for measuring their activities towards 2-OBA reduction. As shown in Table S2, PfLDH in the recombinant strain BL21/pET28a-Pfldh showed the highest specific activities with 262 ± 4.7 U/mg using 2-OBA as substrate. ADH from G. stearothermophilus was introduced for NADH regeneration, the byproduct acetone with a low boiling point could quickly be removed by distillation.

Enzyme EcTD, PfLDH, and GsADH were then puri-fied by HisTrap HP affinity column (as shown in Fig. S1). To obtain the optimal ratio of the three enzymes for realizing kinetic equilibrium of the reaction, the purified enzymes were added in different proportions into 20 mL transformation mixture with 60 g/L L-Thr. The enzyme reactions were optimized by altering the ratio of EcTD to PfLDH while maintaining the activity of EcTD at 20 U/ml as shown in Fig. 2a. When the activity ratio of EcTD to PfLDH was 0.5:1–1:1, the titer and molar conversion rate for (S)-2-HBA both increased with an increasein the enzyme ratio. However, changing the ratio of EcTD to PfLDH from 1.5:1–4:1, the titer and molar conversion rate for (S)-2-HBA both decreased with increase in enzyme ratio. When the ratio of EcTD to PfLDH was changed from 1:1 to 1.5:1, the titer of (S)-2-HBA was 51 g /L and the highest molar conversion rate of 98.0% ± 0.5% was reached. This indicated that balancing the conversion rate of L-Thr to 2-OBA and the consumption rate of 2-OBA

is very important, and the best ratio of EcTD to PfLDH was 1:1. As shown in Fig.  2b, the titer of (S)-2-HBA reached more than 51.5 g/L when the activity ratio of

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Fig. 2 Optimization of activity ratio of EcTD, PfLDH and GsADH for enzyme catalysis in  vitro. a Optimization of activity ration of EcTD and PfLDH for enzyme catalysis in  vitro. The purified enzymes EcTD and PfLDH were added in different proportions into buffer for optimization of the enzyme ration in (S)-2-HBA produc-tion. maintained the EcTD activity at 20 U/ml and excess GsADH with activity over 30 U/mL, the ratio of EcTD to PfLDH was changed from 0.5:1 to 4:1. b Optimization of activity ration of EcTD and GsADH for enzyme catalysis in  vitro. The purified enzymes EcTD and GsADH were added in different proportions into buffer for opti-mization of the enzyme ration in (S)-2-HBA production. We main-tained the enzyme activity ratio of EcTD to PfLDH at the optimal ratio (EcTD activity at the same 20 U/ml) and changed the enzyme activity ratio of EcTD to GsADH, which was ranged from 1:0.1 to 1:0.5. The bioconversion was carried out in 50 mL flask with 20 mL 50 mM PBS buffer (pH 7.5) containing 60 g/L L-Thr, and the reac-tion mixture was stirred at 160 rpm with pH maintained at 7.5 using 1  M NaOH in 30  °C water bath. The residual L-Thr, intermediate 2-OBA and productivity of 2-OBA, (S)-2-HBA in the reaction solu-tions were measured by HPLC

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EcTD to GsADH was above 1:0.25. When the activity ratio of EcTD to GsADH was below 1:0.25, the titer and molar conversion rate for (S)-2-HBA both decreased with a decreased enzyme ratio. This indicated that the GsADH enzyme activity in the system should be maintained at least 25% of EcTD, so that sufficient coenzyme NADH can be supplied. Therefore, it was determined that the best enzyme activity ratio of EcTD, PfLDH and GsADH was 1:1:0.25, which showed efficient conversion of L-Thr for (S)-2-HBA.

The construction of single cell multi‑enzyme biocatalysis system and study of the conversion productivity balance from L‑Thr to (S)‑2‑HBA

To reduce the cost of cell cultivation and improve conveni-ence of operation, a single cell multi-enzyme biocatalysis strain E.coli BL21/pETduet-adhT + ivlA + Pfldh was con-structed by co-expressing EcTD, PfLDH, and GsADH in E.coli BL21, and the conversion of L-Thr to (S)-2-HBA was determined. As shown in Fig. S2 (Lane 4), SDS-PAGE gels of recombinant E.coli BL21/pETduet-adhT + ivlA + Pfldh showed the EcTD protein band of 38 KDa, which belonged to the overlapping bands of PfLDH (34.1 kDa) and GsADH (38.9  kDa). To evaluate the catalytic effeciency of the recombinant strain, 10–60 g/L L-Thr was added to 50 ml transformation system with the cell concentration at 8 g DCW/L. As the L-Thr concentration increased, the molar conversion rate dropped from 98 to 34.8%, while the concen-tration of 2-OBA increased to 38 g/L (as shown in Fig. 3). This indicated the imbalance of conversion rate during this multi-enzyme catalysis in the single recombinant cell with-out any stragety for regulating the enzyme expression levels.

Thus the rate of conversion of L-Thr to 2-OBA and con-sumption rate of 2-OBA using the whole cell was investi-gated. As shown in Fig. 4, we found that the conversion rate of 2-OBA reached 25.73 ± 0.91 g/L in 1 h using a recom-binant whole-cell expressing EcTD, with the transforma-tion carried out in 0.1 M sodium phosphate buffer (pH 7.5) and the cell concentration of 2.5 g DCW/L. While using the recombinant whole-cell coexpressing PfLDH and ADH, the consumption rate of 2-OBA was only 4.32 ± 0.28 g/L in 1 h under the same conditions, which was approximately 17% of the 2-OBA conversion rate by recombinant whole-cell expressing EcTD. Analysis of the effect of 2-OBA on the activity of PfLDH indicated that 2-OBA exerted substrate inhibition, as the concentration of 2-OBA reached 16 g/L, resulting in a 60% decrease in the relative enzyme activity of PfLDH (as shown in Fig. S3), which was in accordance with the results reported [20]. As 2-OBA can inhibit the enzymatic activity of PfLDH, the unbalanced rate of trans-formation from substrate L-Thr to (S)-2-HBA resulted in accumulation of intermediate 2-OBA leading to inefficient

transformation. Thus, it was essential to balance the pro-duction and consumption rates of 2-OBA so as to solve the mismatch in multi-enzyme catalysis.

Optimizing the RBS strengths of TD expression to regulate the conversion rate of 2‑OBA

One efficient way of controlling the recombinant enzyme expression is regulating the RBS strengths [42]. As shown in Fig. 5a, we got eight RBS with different strengths rang-ing from 94,670 to 789,197 T.I.R by submitting the TD base sequence to the https ://salis lab.net/softw are/ website based on the tunable control of the translation initiation rate [33, 43]. The TD enzyme with different RBS was expressed under the control of T7 promoter in E. coli BL21. The cata-lytic efficiencies of recombinant strains were determined by transformation for 1 h in 0.1 M sodium phosphate buffer (pH 7.5) with cell concentration at 2.5 g DCW/L and results indicated that the catalytic efficiencies were proportional to the RBS strengths in the expression system except with r23ivlA. The multi-enzyme-coordinate expression system in this reaction produced the same or lower than ~ 4.3 g/L 2-OBA, meaning that the recombinant strain expressing EcTD produced approximately ~ 13 g/L 2-OBA. Referring to the results shown in Fig. 5b, the recombinant strains with r40ivlA and r53ivlA were suitable for use in a multi-enzyme-coordinate expression system, as they showed 9.48 ± 0.55

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Fig. 3 Effect of substrate L-Thr concentration on (S)-2-HBA pro-duction by recombinant E.coli BL21/ /pETDuet-adhT + ivlA + Pfldh. The recombinant E. coli BL21 with three enzymes co-expression was cultured and recovered then resuspended in 50 mL sodium phosphate buffer (0.1 M, pH 7.5) with the cell concentration as 8 gDCW/L. The substrate L-Thr were added into the reaction buffer with a final con-centration at 60  g/L. The molar ratio of added cosubstrates isopro-panol to L-Thr was 1:1. The transformation was carried out at 30 °C and 160 rpm for 12 h. Solution of 1 M NaOH was added to maintain the reaction medium pH at 7.5 during the transformation process. The residual L-Thr, intermediate 2-OBA, and final product (S)-2-HBA were analyzed using HPLC

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and 11.80 ± 0.68 g/L 2-OBA productivity, respectively. In addition, the recombinant strain with r79ivlA was also cho-sen for use in a multi-enzyme-coordinate expression system.

Construction of a tunable multi‑enzyme‑coordinate expression system for high efficiency (S)‑2‑HBA production

Construction of a tunable multi-enzyme-coordinate expres-sion system could help not only in balancing the rates of the individual catalytic steps, but also relieve the nega-tive effect of intermediate 2-OBA on enzyme activities. We cloned the gene of EcTD enzyme with the chosen RBS strength r40, r53, and r79 then ligated them onto the

plasmid pETDuet-adhT + Pfldh (the order of gene posi-tions shown in Fig. 6a). Three recombinant strains includ-ing ADH-r40, ADH-r53, and ADH-r79 were constructed. The protein expression levels of these recombinant strains were shown in SDS-PAGE (Fig. S2). The recombinant strain harboring pETDuet-adhT + ivlA + Pfldh plasmid showed a prominent band of EcTD and in contrast, other recombi-nant strains showed lower EcTD enzyme expression levels which confirmed that controlled protein expression could be achieved by altering the RBS sequence. The enzyme activities of EcTD, PfLDH, and GsADH were measured, as shown in Fig. 6b, with the weakening strengths of RBS, the EcTD enzyme activity decreased from 5689 U/g DCW (strain harboring pETDuet-adhT + ivlA + Pfldh) to 3372 U/g DCW (ADH-r79), 2827 U/g DCW (ADH-r53) and 1522 U/g DCW (ADH-r40), while both PfLDH and GsADH showed a slight upward trend. As shown in Table 1, the ratio of EcTD, PfLDH, and GsADH activity in strain ADH-r53 was 0.97:1:0.22, which were almost similar to the best ratio 1:1:0.25 in vitro. The transformation results of these strains in 50 mL bottles were shown in Table 2, ADH-r40 showed lower (S)-2-HBA yield, and a large amount of L-Thr remained, which was due to the insufficient conversion of L-Thr to 2-OBA by TD enzyme. ADH-r79 also showed lower (S)-2-HBA yield because of accumulated 2-OBA titer of about 27.46 ± 1.8 g/L, while there was barely any L-Thr left in ADH-r79 strain, indicating imbalanced con-sumption of 2-OBA. With good enzyme activity ratios of the three enzymes, ADH-r53 showed 83.5% molar yield and 43.8 ± 3.5 g/L (S)-2-HBA production within 8 h. Further applying this strategy with FDH for NADH regeneration, the ratio of EcTD, PfLDH, and GsADH activity in strain FDH-r79 was 1.12:1:0.27, and under these conditions, FDH-r79 showed 88.2% molar yield and 46.5 ± 2.3 g/L (S)-2-HBA production in 8 h. These results indicated the importance of fine-tuning expression of multi-enzyme cascades in multi-enzyme biocatalysis reactions.

To further improve the production of (S)-2-HBA, recom-bianant strains ADH-r53 (with a comparatively excellent enzyme ratio of the three enzymes) were chosen to be cul-tured and used for conversion of L-Thr to (S)-2-HBA in 5 L fermentor. As shown in Fig. 7, the strain ADH-r53 showed the ability for (S)-2-HBA bioconversion with 129 g/L titer and 93% molar yield within 24 h, and the (S)-2-HBA yield was 1.65-fold of the current reported highest yield of 78 g/L [20], signifying the high efficiency of a multi-enzyme-coor-dinate expression system. However, the molar yield of (S)-2-HBA could not reach 100%, which might be due to part of the intermediate 2-OBA would be converted into L- Iso-leucine [44] or propionyl-CoA [45]. For the self-sufficient cofactor system using ADH, byproduct acetone produced could be separated by distillation. due to its low melting point, making product purification easy and cheap, which

0 15 30 45 600

8

16

24

32

0

8

16

24

32

2-O

BA

(g/L

)

)L/g(

rhT-

L

Time (min)

L-Thr2-OBA

0 15 30 45 600

3

6

9

12

0

3

6

9

12

(S)-2

-HBA

(g/L

)

)L/g(AB

O-2

Time (min)

2-OBA(S)-2-HBA

A

B

Fig. 4 The productivity of 2-OBA from L-Thr by recombinant E. coli BL21/pET28a-ivlA. a The transformation was carried out in 0.1  M sodium phosphate buffer (pH 7.5) with the cell OD600 = 5 and 30 g/L substrate L-Thr. The transformation proceeded for 1 h at 30  °C and 160 rpm with the pH-controlled at pH 7.0 using 1 M NaOH solution, and the samples were taken every 15  min. b The consumption rate of 2-OBA for (S)-2-HBA production by recombinant E. coli BL21/pETDuet-adhT + Pfldh. The transformation was carried out in 0.1 M sodium phosphate buffer (pH 7.5) with the cell OD600 = 5 and 10 g/L substrate 2-OBA. The transformation proceeded for 1 h at 30 °C and 160 rpm with the pH-controlled at pH 7.5 using 1 M NaOH solution, and the samples were taken every 15 min

241Systems Microbiology and Biomanufacturing (2021) 1:234–244

1 3

0

8

16

24

r32ivlA

ABO-2fo

ytivitcudorP(g

/Lh)

r9ivlA

r16ivlA

r23ivlA

r40ivlA

r53ivlA

r79ivlA

ivlA

B

A

Fig. 5 a The synthetic RBS sequence with varying strength, includ-ing original and the synthetic RBS sequence with predicted T.I.R. from 94,670 to 789,197. b The productivity of 2-OBA by recombi-nant BL21/pET28a-ivlA with different strength RBSs. The transfor-

mation was carried out in 0.1  M sodium phosphate buffer (pH 7.5) with the cell OD600 = 5 and 30 g/L substrate L-Thr. The transforma-tion proceeded for 1 h at 30 °C and 160 rpm with the pH-controlled at pH 7.5 using 1 M NaOH solution

242 Systems Microbiology and Biomanufacturing (2021) 1:234–244

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further enhanced the potential for industrial application of this strategy.

Conclusions

In summary, a multi-enzyme coordinate expression system for chiral (S)-2-HBA production from the bulk chemical L-Thr was successfully achieved, by regulating the expres-sion level of the enzyme TD, using different strengths RBS to coordinate the expression of proteins. This multi-enzyme-coordinate expression system effectively solved the rate

ADH-rbs ADH-r79 ADH-r53 ADH-r400

1000

2000

3000

4000

5000

6000

TD,L

DH

and

AD

H(U

/gD

CW

) TD LDH ADH

A

B

Fig. 6 a The gene ligation orders in the recombinant strains with tun-able multi-enzyme-coordinate expression systems. b The activity of EcTD、PfLDH、GsADH for recombinant E.coli with TD enzyme expressed under different strength of RBS were measured. Strain har-boring pETDuet-adhT + ivlA + Pfldh, strains ADH-r79, ADH-r53 and ADH-r40 were cultured in 250  ml flasks using 50  ml TY medium with the same cultivation method mentioned in above methods. The cell was recovered and resuspended with those strains at the same cell concentration as 8 g DCW/L. Then lysised cell using sonication with an ultrasonic oscillator and removed the cell debris by centrifugation (12,000  rpm, 40 min) at 4  °C. The supernatant crude enzyme solu-tion were used to measure the activities of the enzymes, respectively. The methods for measuring the activity of EcTD、PfLDH、GsADH were the same as previous mensioned

Table 1 The activity of EcTD、PfLDH、GsADH for recombinant E.coli with TD enzyme expressed under different strength of RBS

Strains Enzyme activity (U/g DCW) EcTD:PfLDH:GsADH

EcTD PfLDH GsADH

ADH-RBS 5689 ± 70 2199 ± 34 453 ± 17 2.59: 1.0:0.21ADH-R79 3372 ± 86 2745 ± 76 572 ± 16.4 1.23:1.0:0.21ADH-R53 2827 ± 63 2920 ± 47 645 ± 13.8 0.97:1.0:0.22ADH-R40 1522 ± 32 3112 ± 53 692 ± 11.4 0.49:1.0:0.22

Table 2 The production of (S)-2-HBA for recombinant E.coli with TD enzyme expressed under different strength of RBS

Strains L-Thr (g/L) 2-OBA (g/L) L-2-HBA (g/L) Conversion rate (%)

ADH-RBS ND 34.72 ± 2.9 16.9 ± 1.4 32.4% ± 2.8ADH-R79 ND 27.46 ± 1.8 24.5 ± 2.3 46.5% ± 1.9ADH-R53 5.6 ± 0.7 4.7 ± 0.9 43.8 ± 3.5 83.5% ± 3.1ADH-R40 23.9 ± 2.1 4.3 ± 0.3 27.2 ± 1.7 51.7% ± 2.2

0 4 8 12 16 20 240

15

30

45

60 L-Thr2-OBA

)L/g(

AB

O-2dna

rhT-

L

Time (h)

ADH-R53

0

30

60

90

120

150

(S)-2-HBA

(S)-2

-HB

A(g

/L)

Fig. 7 The transformation result of ADH-r53. The transformations were carried out in 0.1 M sodium phosphate buffer (pH 7.5) with the cell concentration was 25 g DCW/L and proceeded for 24 h at 30 °C and 160 rpm with the pH-controlled at pH 7.5 using 1 M NaOH solu-tion. The substrates L-Thr and isopropanol (The molar ratio is 1:1) were added in batches and the samples were taken at different times

243Systems Microbiology and Biomanufacturing (2021) 1:234–244

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mismatch problem in multi-enzyme biocatalysis reactions. The whole-cell transformation using the strain ADH-r53 with the three enzymes TD, LDH and ADH coordinated expression showed 129 g/L (S)-2-HBA production with the molar yield reached 93%. The whole-cell transforma-tion with this multi-enzyme-coordinate expression system did not require the addition of expensive cofactor NADH. Moreover, compared with the system using FDH for NADH regeneration, using ADH to regenerate NADH was more suitable for hydroxyl acid biosynthesis as the product could easily be separated from byproduct acetone through distil-lation. Our work demonstrated the importance of a tunable multi-enzyme-coordinate expression system in cascade bio-catalysis reactions. With applying this system in cascade biocatalysis, it would be more economically feasible for all kinds of biochemical production on industrial scale.

Author contributions LT investigation, methodology, writing—origi-nal draft. JZ investigation, formal analysis, writing—review & editing. XZ writing—review & editing. TY writing—review & editing. MX project administration, writing—review & editing. ZR project admin-istration, validation.

Funding This work was funded by the National Key Research and Development Program of China (2018YFA0900300), the National Natural Science Foundation of China (31770058, 32070035), Natu-ral Science Foundation of Jiangsu Province (BK20181205), the Key Research and Development Program of Ningxia Hui Autonomous Region (No. 2019BCH01002), the national first-class discipline pro-gram of Light Industry Technology and Engineering (LITE2018-06) and the 111 Project (111-2-06).

Compliance with ethical standards

Conflict of interest The authors declare no conflict of interest.

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