Dynamic Regulation of Transporter Expression to Increase L-Threonine Production Using L-Threonine Biosensors

Abstract

The cytotoxicity of overexpressed transporters limits their application in biochemical production. To overcome this problem, we developed a feedback circuit for L-threonine production that uses a biosensor to regulate transporter expression. First, we used IPTG-induced rhtA regulation, L-threonine exporter, to simulate dynamic regulation for improving L-threonine production, and the results show that it had significant advantages compared with the constitutive overexpression of rhtA. To further construct a feedback circuit for rhtA auto-regulation, three L-threonine sensing promoters, P_cysJ, P_cysD, and P_cysJH, were characterized with gradually decreasing strength. The dynamic expression of rhtA with a threonine-activated promoter considerably increased L-threonine production (21.19 g/L) beyond that attainable by the constitutive expression of rhtA (8.55 g/L). Finally, the autoregulation method was used in regulating rhtB and rhtC to improve L-threonine production and achieve a high titer of 26.78 g/L (a 161.01% increase), a yield of 0.627 g/g glucose, and a productivity of 0.743 g/L/h in shake-flask fermentation. This study analyzed in detail the influence of dynamic regulation and the constitutive expression of transporters on L-threonine production. For the first time, we confirmed that dynamically regulating transporter levels can efficiently promote L-threonine production by using the end-product biosensor.

1. Introduction

L-threonine (Thr, CAS 72-19-5), an essential amino acid for mammals, is widely used in food, cosmetics, animal feed, and pharmaceuticals [1]. L-threonine is one of four major commercial amino acids, the others being L-glutamic acid, L-lysine, and DL-methionine. The global production of L-threonine totals 700,000 metric tons annually [2]. Currently, microbial fermentation with catalyzation by six enzymes from oxalacetate—including aspC-encoded aspartate aminotransferase, thrA (metL or lysC)-encoded aspartate kinase and homoserine dehydrogenase, asd-encoded aspartyl semialdehyde dehydrogenase, thrB-encoded homoserine kinase, and thrC-encoded threonine synthase—is the main method of L-threonine production (Figure 1) [3]. L-threonine belongs to the aspartate family of amino acids. First, L-aspartate is generated from oxaloacetate by L-aspartate aminotransferase. L-aspartate is then catalyzed by aspartate kinase to aspartate phosphate. This is followed by a two-step reduction reaction catalyzed by aspartyl semialdehyde dehydrogenase and homoserine dehydrogenase to generate L-homoserine. The homoserine kinase catalyzes the phosphorylation of L-homoserine to L-homoserine phosphate, which is finally catalyzed by threonine synthase to L-threonine.

Many strategies have been explored to synthesize L-threonine, including cofactor engineering, energy adjustment, the overexpression of rate-limiting steps, the removal of feedback inhibition, and the removal of competitive pathways [1,3,4,5]. Transporter engineering has also emerged as an attractive approach to improve L-threonine production [6,7,8,9,10]. The overexpression of transporters is a commonly used tactic to increase strain tolerance by pumping out products [3,7,8,11]. However, the overexpression of membrane proteins seriously hinders the homeostasis of proteins in the cytoplasm [12,13], which is not conducive to efficient catalytic anabolites. For example, overexpressing membrane proteins can reduce the levels of the respiratory chain complexes succinate dehydrogenase and cytochrome bd and bo₃ oxidases in Escherichia coli [13]. Therefore, the expression of transporters should be carefully modified for chemical production without disturbing the physiological metabolism. In addition, some transporter engineering strategies were also performed in microbial manufacturing, such as omics-guided transporter mining, transporter evolution to enhance transport efficiency and substrate specificity, and the heterologous expression of transporter [9]. However, transporters obtained from the above strategies still need to be overexpressed to improve product outflow. One promising method of performing this is to dynamically regulate transporter expression through a general biosensor. Dynamic regulation can automatically control the level of transporters according to the needs of cells, which can not only alleviate the adverse effects of overexpressed transporters on cells, but also timely discharge intracellular metabolites. To produce n-butanol and 1-alkene, previous studies have induced transporter expression by responding to membrane stress and exhausted glucose [14,15]. However, such a general biosensor needs to be coupled with the cell growth state to perform its regulatory functions. The cell growth state can be affected by many factors, such as hyperosmotic stress condition [16], which lead to the non-precise regulation of transporter expression by using the cell-growth-state-dependent biosensor. In contrast to the general biosensor, a product-specific biosensor finely regulates transporter expression without being affected by culture conditions and changes in cell physiology. Many end-product-specific biosensors have been designed and utilized to dynamically regulate metabolic pathways to improve chemical production [17,18]. Although Lange et al. found that the exporter BrnFE can be activated by the transcription factor lrp and that its expression increased with the addition of branched-chain amino acids [19], the effects of dynamically regulated transporter expression by using end-product biosensors have not been studied in detail. The comparison of the effects of dynamic regulation and the constitutive expression of transporters on product synthesis has not been reported.

In this study, we use the native L-threonine-sensing promoters P_cysJ, P_cysD, and P_cysJH to study in detail the effect of dynamically regulated transporter expression for L-threonine production (Figure 1). Compared with the constitutive expression of transporters, dynamic regulation significantly improved L-threonine production with an increase of approximately 147%. This regulation can also be extended to two other native transporters, rhtB and rhtC.

2. Materials and Methods

2.1. Chemicals and Reagents

The L-threonine standard was purchased from Sigma-Aldrich (St. Louis, MO, USA). Phanta HS Super-Fidelity DNA Polymerase was purchased from Vazyme Biotech (Nanjing, China). High-performance liquid chromatography grade acetonitrile (75-05-8) was obtained from Tedia (ACN, Tedia Company, Inc., Fairfield, OH, USA). Isopropyl-β-d-thiogalactoside (IPTG; CAS, 367-93-1) and spectinomycin dihydrochloride pentahydrate (CAS, 22189-32-8) were purchased from Sangon Biotech (Shanghai, China) Co., Ltd. Triethylamine (CAS, 121-44-8) and phenyl isothiocyanate (CAS, 103-72-0) were purchased from Aladdine. Other reagents were purchased from Sinopharm Chemical Reagent Beijing Co. Ltd. (Shanghai, China).

2.2. Construction of Strains and Plasmids

All strains and plasmids used in this study are listed in Tables S1 and S2. E. coli DH5α was used to reconstruct plasmids, and E. coli K-12 MG1655 was used for L-threonine sensor characterization. The L-threonine-producing Tm strain was provided by Fufeng Group. The strains Tm-100AR, Tm-101AR, and Tm-109AR containing plasmids cl-100AR, cl-101AR, and cl-109AR, respectively, were constructed to study the effect of rhtA levels on L-threonine production. We introduced pCL1920 and cl-rhtA into Tm to construct strains Tm-cl and Tm-AR to study the effects of rhtA expression times on cell growth and L-threonine production. MG-cl, MG-P_cysJ, MG-P_cysD, and MG-P_cysJH were constructed by transforming the plasmids pCL1920, cl-JAR, cl-DAR, and cl-JHAR into E. coli K-12 MG1655. Tm-P_cysJ, Tm-P_cysD, and Tm-P_cysJH were constructed by transforming the plasmids cl-JAR, cl-DAR, and cl-JHAR into Tm. Tm-JAR, Tm-DAR, Tm-JHAR, Tm-105AR, Tm-110AR, and Tm-116AR were engineered by introducing cl-JAR, cl-DAR, cl-JHAR, cl-105AR, cl-110AR, and cl-116AR into Tm to analyze the influence of the dynamic regulation of rhtA on L-threonine production. Tm-JB and Tm-JC were constructed by introducing cl-JB or cl-JC into Tm and used to further dynamically regulate the native transporters to improve L-threonine production. All engineered strains were constructed by transforming the corresponding plasmids into E. coli K-12 MG1655 or Tm with the calcium chloride (CaCl₂) or electroporation transformation methods.

The primers used for plasmid reconstruction are listed in Table S3. The low copy number plasmid pCL1920 with the pSC101 replicon was used as the cloning vector. All plasmids in this study were constructed using the Gibson assembly method [20]. To construct cl-rhtA, the backbone was amplified from pCL1920 with the primers cl-F1 and cl-R1, and rhtA was amplified from the E. coli K-12 MG1655 genome with the primers cl-rhtA-F and cl-rhtA-R by using Phanta HS Super-Fidelity DNA Polymerase (Vazyme Biotech, Nanjing, China). cl-rhtA was assembled by fusing the amplified backbone and rhtA with the 2X MultiF Seamless Assembly Mix (ABclonal Technology Co., Ltd. Wuhan, China). PcysJ-rfp, PcysD-rfp, and PcysJH-rfp were constructed through fusing the promoters P_cysJ, P_cysD, and P_cysJH amplified from the E. coli K-12 MG1655 genome with the reporter rfp and then was inserted into amplified pCL1920 backbone. The dynamically regulated rhtA plasmids containing cl-JAR, cl-DAR, and cl-JHAR were constructed with the frame of the P_cysJ/_cysD/_cysJH-B0034-rhtA-terminator BBa_B1006-P_cysJ B0034-rfp. To constitutively express rhtA, we selected three different transcription levels of promoters (J23105, J23110, and J23116) from iGEM (http://parts.igem.org/Main_Page /(accessed on December 2021)). cl-105AR, cl-110AR, and cl-116AR were constructed according to the form of the P_J23105/_J23110/_J23116-B0034-rhtA-terminator BBa_B1006-P_cysJ B0034-rfp. The native L-threonine exporters rhtB and rhtC were amplified from the E. coli K-12 MG1655 genome and cloned into pCL1920 to generate the recombinant plasmids cl-JB and cl-JC for dynamic regulation.

2.3. Characterization of the L-Threonine-Sensing Promoters

To characterize the promoters P_cysJ, P_cysD, and P_cysJH, the fluorescence intensity of the reporter gene rfp was monitored using a multidetection microplate reader (Synergy HT, BioTek, Winooski, VT, USA). Single colonies were grown in 12-well microassay plates with 2 mL of LB medium supplemented with spectinomycin dihydrochloride pentahydrate at 37 °C for approximately 12 h. The feeds were then transferred to a 96-well microassay plate containing 200 μL Luria–Bertani (LB) medium with 2% (v/v) inoculation at 37 °C for 48 h. Spectinomycin dihydrochloride pentahydrate was added during the cultivation process to maintain the stabilization of the plasmids. The fluorescence intensity of rfp was measured through excitation at 590 nm and emission at 645 nm. The characterization was calculated with the ratio of the fluorescence intensity of rfp to the optical density at 600 nm (OD₆₀₀). All measurements were performed in triplicate.

2.4. Culture Medium and L-Threonine Fermentation

A LB medium containing 10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl was used for plasmid construction and L-threonine sensor characterization. The L-threonine fermentation medium consisted of 15 g/L of (NH₄)₂SO₄, 2 g/L of KH₂PO₄, 1 g/L of MgSO₄·7H₂O, 2 g/L of yeast extract, and 0.02 g/L of FeSO₄ [8]. We also added 40 g/L of glucose as the initial carbon source and 20 g/L CaCO₃ to adjust the pH during fermentation. For shake-flask fermentation, single colonies were cultured in fresh LB medium at 37 °C for approximately 12 h. The precultured seeds were subsequently transformed into a 300 mL shake flask attached with flaps containing a 20 mL fermentation medium with 1% (v/v) inoculation. Fermentation was performed at 220 rpm, 37 °C for 36 h. When glucose was lower than 10 g/L, it was supplemented up to 40 g/L. rhtA expression was induced with 0.1/0.2/0.5 mM of IPTG when necessary.

Analytical Methods

A 1 mL culture was vigorously vortexed, and 0.1 mL of it was transferred to a 1.5 mL centrifuge tube. We added 0.9 mL of 1 mM HCl and mixed the solution to remove the residual CaCO₃. Subsequently, OD₆₀₀ was measured using a spectrophotometer (Shimazu, Kyoto, Japan). For glucose and L-threonine detection, the culture was centrifuged at 12,000 rpm for 2 min to collect the supernatant. The collected supernatant was filtered with a 0.22 μm aqueous membrane for glucose analysis. Glucose was quantified with a high-performance liquid chromatography (HPLC) system (Shimadzu, Kyoto, Japan) equipped with a refractive index detector (RID-10A; Shimadzu, Kyoto, Japan) and an Aminex HPX-87H ion exclusion column (Bio-Rad Laboratories, Hercules, CA, USA); 5 mM H₂SO₄ was used as the mobile phase at a flow rate of 0.6 mL/min [21].

For the detection of L-threonine, the collected supernatant was initially deproteinized with 5% trichloroacetic acid. Subsequently, the pretreated supernatant was derived with triethylamine and phenyl isothiocyanate and then extracted with n-hexane. Briefly, 0.2 mL of samples and standard L-threonine were pretreated with the mixture of triethylamine–acetonitrile (1.4 mL of triethylamine mixed with 8.6 mL of acetonitrile). Next, we added phenyl isothiocyanate–acetonitrile (25 μL of phenyl isothiocyanate mixed with 2 mL of acetonitrile) to pretreat the samples and standard L-threonine for 1 h at room temperature. We added 0.4 mL of n-hexane and vigorously shocked. The bottomed liquid (0.2 mL) was collected and diluted with 0.8 mL of deionized water. The solution was filtered with a 0.22 μm organic membrane, and samples were detected using an HPLC equipped with a diode array detector (SPD-M20A; Shimadzu, Kyoto, Japan), with the VenusilAA (4.6 × 250 mm, 5 μm, Agela Techanologies) column applied at 40 °C. The analysis was performed at a flow rate of 1 mL/min with mobile phases consisting of (A) 15.2 g of sodium acetate dissolved in 1850 mL of ultrapure water and mixed with 140 mL of acetonitrile and (B) 80% (v/v) acetonitrile and 20% (v/v) ultrapure water. The concentration of L-threonine was quantified with the peak area according to the corresponding standard curve.

2.5. Statistical Analysis

Results are presented as the mean ± SEM. Differences between the means were evaluated using a one-way ANOVA. p < 0.05 was considered statistically significant.

3. Results and Discussion

3.1. Simulating Dynamic rhtA Regulation with IPTG Induction

The membrane protein RhtA consists of 10 predicted transmembrane segments, which were identified and characterized as L-threonine and L-homoserine transporters [22]. Constitutive overexpressing rhtA is the commonly method used to improve L-threonine production and removing the toxic intermediate metabolite L-homoserine [22,23,24,25,26]. In this study, we used the industrial L-threonine production host Tm, engineered from E. coli K-12 MG1655, from the Fufeng Group (http://www.fufeng-group.com/ (accessed on January 2021)) as the parent strain.

To verify whether the dynamic regulation of transporter expression can improve L-threonine production, we firstly explored the effect of constitutive overexpressed rhtA on L-threonine production. Three promoters at strong (J23100, 1348), medium (J23101, 399), and weak (J23109, 18) from the Registry of Standard Biological Parts (http://parts.igem.org/Main_Page(accessed on January 2021)) were characterized in strains Tm-100AR, Tm-101AR, Tm-109AR, respectively (Figure 2A). As illustrated in Figure 2A, compared with the control (strain Tm-cl without rhtA overexpression), overexpressing rhtA enhanced L-threonine production due to relief from the toxicity of the accumulated products through the timely export of L-threonine [9,27]. Tm-100AR and Tm-109AR significantly increased the titer of L-threonine (p < 0.0001) than Tm-101AR. However, there was an irregular correlation between the increase in production and the intensity of rhtA levels. Then, we used IPTG-induced rhtA regulation to simulate the availability of dynamic regulation. rhtA was induced with 0.5 mM of IPTG in different growth phases (OD 0, 3, 6, or 9) (Figure 2B). When enhancing rhtA expression at different growth stages, host cells with delayed rhtA induction achieved a higher L-threonine titer than constitutive expression (Figure 2B). rhtA expressed at OD 3 achieved higher titers at 15.4 g/L than early at OD 0 (p < 0.05) with 13.4 g/L or delayed induction at OD 6 (p < 0.01) or 9 (p < 0.001) with 13.4 g/L and 10.7 g/L, respectively. The premature expression of high-level rhtA causes membrane burden. rhtA expresses too late to excrete the accumulated L-threonine with low-level rhtA in the cell timely, resulting in metabolic burden. Compared to the strength of the constitutive promoter shown in Figure 2A, the promoter strength of Plac-lacO (RFP/OD₆₀₀ values from 6.7 to 55.4) can be evaluated to be roughly between J23101 (RFP/OD₆₀₀ value, 399) and J23109 (RFP/OD₆₀₀ value, 18), suggesting that increased L-threonine productivity at OD 0–9 may be not due to higher intensity of Plac-lacO. Therefore, the dynamic regulation of rhtA in different fermentation phases can improve L-threonine production, and it had significant advantages compared with the constitutive overexpression of rhtA. Although the transporter rhtA was successfully regulated to increase L-threonine production, the addition of the exogenous inducer IPTG is impracticable in industrial production due to the high production cost [28].

3.2. Characterizing the L-Threonine-Sensing Promoters

Biosensors have emerged as an outstanding tool for dynamically adjusting gene expression in metabolic engineering [18,29]. Several E. coli-derived L-threonine-sensing promoters have been excavated through omics analysis [30]. In this paper, the L-threonine sensor was characterized by placing the report gene rfp under the promoter P_cysJ, P_cysD, or P_cysJH. We initially tested the response in E. coli K-12 MG1655 with the strains MG-P_cysJ, MG-P_cysD, and MG-P_cysJH. After removing the background value of RFP/OD₆₀₀ from strain MG-cl, MG1655 with empty plasmid pCL1920, all three promoters can be activated by adding L-threonine (Figure 3A). The intensity of expression continuously decreased for P_cysJ, P_cysD, and P_cysJH. To demonstrate that the three promoters were practicable in the L-threonine-producing strain and determine the lowest activation threshold, the strains Tm-P_cysJ, Tm-P_cysD, and Tm-P_cysJH were constructed by introducing the plasmid P_cysJ-rfp, P_cysD-rfp, or P_cysJH-rfp into the industrial strain Tm. The results demonstrate that the three promoters can be used to activate gene expression during the L-threonine production process (Figure 3B). Furthermore, expression levels increased with increased L-threonine concentration (Figure 3B,C). As indicated in Table S4, the three promoters had different minimum sensing thresholds during fermentation: P_cysJ and P_cysJH at 3 h with 0.05 g/L and P_cysD at 5 h with 0.25 g/L of L-threonine. The highest concentrations of L-threonine sensing were not measured because the fluorescence intensity of rfp continued to increase at the final concentration of L-threonine (Figure 3C). Nonetheless, we quantified the highest sensing threshold (~60 g/L) with MG-P_cysJ, MG-P_cysD, and MG-P_cysJH by exogenously adding different concentrations of L-threonine (Figure 3A).

3.3. Improving L-Threonine Production through the Dynamic Regulation of rhtA Expression

The characterized sensing promoters P_cysJ, P_cysD, and P_cysJH were used for gradually increasing rhtA expression in the L-threonine-producing strain Tm, corresponding to the strains Tm-JAR, Tm-DAR, and Tm-JHAR, respectively. To estimate the expression of rhtA, the linked rfp gene was activated by L-threonine-responsive promoters. Varying strengths of promoters J23105, J23110, and J23116 were selected to constitutively express rhtA in the strains Tm-105AR, Tm-110AR, and Tm-116AR, respectively. The strain Tm-cl contained the empty vector as the control. The expression levels of rhtA in strains with L-threonine-responsive promoters were gradually increased during the fermentation process with the analysis of RFP/OD₆₀₀ (Figure 4A). As illustrated in Figure 4A, the promoter J23110 had the same effect as P_cysJ on rhtA expression at 36 h. Comparing the constitutive expression of rhtA with the promoter J23100 in the strain Tm-110AR, dynamically regulating the strain Tm-JAR with P_cysJ resulted in a significantly improved L-threonine production of up to 21.19 g/L of titers, an increase of approximately 147% than the unexpressed strain (Figure 4B). The other L-threonine-sensing promoters P_cysD and P_cysJH had lower L-threonine titers than P_cysJ, 19.11 and 9.49 g/L, respectively, which are a result of the weaker expression of rhtA. These results suggest that stronger dynamic expression of rhtA is more conducive to L-threonine production. The difference in expression levels of rhtA may be due to the different transcription and translation strengths of P_cysJ, P_cysD and P_cysJH. It may also be caused by the different affinity for L-threonine of the currently unknown transcription regulators. Although a stronger constitutive expression of rhtA with the promoter J23116 can increase the L-threonine titer, it is still lower than the dynamic expression with the promoter P_cysJ and with a slower cell growth (Figure 4C). These results indicate that the dynamic regulation of rhtA expression is more conducive to promoting L-threonine production. Dynamic regulation is a successful strategy to improve biochemical production [4,31,32,33,34]. Recently, a study has developed a thermal switch system to increase L-threonine yield through regulating carbon distribution and cofactor supplementation by sensing temperature [4]. Apart from the generally utilized metabolic flux engineering, in this study, we confirmed in the first time that dynamically engineering the transporter rhtA is more effective than constitutive expression for improving L-threonine production.

3.4. Regulating rhtB and rhtC to Further Improve L-Threonine Production

In E. coli, three native permeases—RhtA, RhtB, and RhtC encoded with rhtA, rhtB, and rhtC, respectively—were confirmed as the membrane proteins to pump out L-threonine [3]. RhtA belong to the category of nonspecific transporters for exporting L-threonine and L-homoserine. RhtB also transports homoserine lactone (HSL) [7,35]. By contrast, RhtC is the specific transporter for L-threonine [7]. Studies have reported that the expression of all these exporters results in the efficient pumping of L-threonine and increased production [1,3,36]. In this study, we demonstrated that L-threonine titers were considerably increased through the dynamic regulation of rhtA expression (Figure 4B). The sensing promoter P_cysJ had the greatest impact on increasing L-threonine production. To explore the influence of the autoregulated expression of rhtB and rhtC on L-threonine production, rhtB and rhtC were controlled by P_cysJ by responding to L-threonine (Figure 5). The plasmids P_cysJ-rhtB and P_cysJ-rhtC were transformed into the parent strain Tm and formed Tm-JB or Tm-JC. As illustrated in Figure 5A and

Table 1. Titers, titer increase, yield, and productivity of L-threonine under dynamically regulated RhtB and RhtC.

	Titer (g/L)	Titer Increase (%)	Yield (g/g)	Productivity (g/L/h)
Tm-cl	10.26 (±0.68)	——	0.402 (±0.006)	0.285 (±0.019)
Tm-JB	26.78 (±0.67)	161.01 (±6.61)	0.627 (±0.023)	0.743 (±0.018)
Tm-JC	25.44 (±0.31)	147.95 (±3.02)	0.665 (±0.010)	0.706 (±0.008)

, compared with the unexpressed transporter and overexpressed rhtC, the dynamically expressed rhtB resulted in the highest L-threonine titers of 26.78 g/L, an approximately 161% increase, and a productivity up to 0.734 g/L/h. Because Tm-JB consumed more glucose during the 36 h fermentation (Figure 5C), the yield (0.627 g/g) was slightly lower than that of Tm-JC (0.665 g/g). Although Tm-JB grew less than Tm-JC during the 0 to 18 h period, the final biomass at 36 h was higher (Figure 5B). These results demonstrate that dynamically regulating rhtB expression is relatively beneficial for L-threonine production.

4. Conclusions

The toxicity of transporter expression to cells limits the application of transporter engineering in improving metabolite production. For the first time, we compared the effects of dynamic and constitutive expression transporters on product synthesis. We analyzed the effect of the dynamic regulation of transporters on L-threonine production based on end-product biosensors. These results demonstrate that the dynamic regulation of transporter expression was more conducive than constituent expression for L-threonine production, which increased by 147%. Therefore, the dynamic regulation of transporters is a promising strategy for improving biochemical production.