An Efficient Prephenate Dehydrogenase Gene for the Biosynthesis of L-tyrosine: Gene Mining, Sequence Analysis, and Expression Optimization

L-tyrosine is a key precursor for synthesis of various functional substances, but the microbial production of L-tyrosine faces huge challenges. The development of new microbial chassis cell and gene resource is especially important for the biosynthesis of L-tyrosine. In this study, the optimal host strain Bacillus amyloliquefaciens HZ-12 was firstly selected by detecting the production capacity of L-tyrosine. Subsequently, the recombinant expression of 15 prephenate dehydrogenase genes led to the discovery of the best gene, Bao-tyrA from B. amyloliquefaciens HZ-12. After the overexpression of Bao-tyrA, the L-tyrosine yield of the recombinant strain HZ/P43-Bao-tyrA reach 411 mg/L, increased by 42% compared with the control strain (HZ/pHY300PLK). Moreover, the nucleic acid sequence and deduced amino acid sequence of the gene Bao-tyrA were analyzed, and their conservative sites and catalytic mechanisms were proposed. Finally, the expression of Bao-tyrA was regulated through a promoter and 5′-UTR sequence to obtain the optimal expression elements. Thereby, the maximum L-tyrosine yield of 475 mg/L was obtained from HZ/P43-UTR3-Bao-tyrA. B. amyloliquefaciens was applied for the first time to produce L-tyrosine, and the optimal prephenate dehydrogenase gene Bao-tyrA and corresponding expression elements were obtained. This study provides new microbial host and gene resource for the construction of efficient L-tyrosine chassis cells, and also lays a solid foundation for the production of various functional tyrosine derivatives.


Introduction
The aromatic amino acid L-tyrosine is a nutritionally essential amino acid for humans, and it has been widely used in food additives, dietary supplements, pharmaceuticals, and chemicals [1][2][3]. Moreover, L-tyrosine is a common precursor for the synthesis of various high-value-added natural active substances such as salvianic acid A, resveratrol, caffeic acid, hydroxytyrosol, salidroside, curcumins, benzylisoquinoline alkaloids (BIAs), and so on [4][5][6][7][8][9]. These derivatives are endowed with various functions, such as antioxidant, anticoagulation and anti-inflammatory activities [10,11]. Therefore, L-tyrosine has prospects for broad application as a platform compound, and the demand for nutritional chemicals derived from L-tyrosine is constantly increasing [12][13][14][15]. For example, the market-sharing of the common L-tyrosine derivivates of flavonoids will reach 1.26 billion USD by 2026 according to market research reports [16].

Recombinant Expression of the Prephenate Dehydrogenase Gene tyrA
The cloned prephenate dehydrogenase gene was identified using heterologous expression in B. amyloliquefaciens HZ-12 following the procedure reported in our previous study. Thereby, taking the gene Bao-tyrA expression strain as an example, the gene fragment of Bao-tyrA was amplified with a pair of primers, Bao-tyrA-F and Bao-tyrA-R, using the B. amyloliquefaciens HZ-12 DNA template, and then the gene Bao-tyrA was fused with the P43 promoter amplified from B. subtilis 168 and the TamyL terminator obtained from B. licheniformis WX-02 using overlap extension PCR (SOE-PCR). After digestion of fusion fragment and pHY300PLK plasmid by restriction enzymes of BamHI and XbaI, they were ligated to obtain the expression plasmid pHY-P43-Bao-tyrA. Finally, this expression plasmid was electro-transformed into the competent cells of B. amyloliquefaciens HZ-12, generating the recombinant strain HZ/P43-Bao-tyrA. Other recombinant strains were obtained using the same procedure in this study. Moreover, synthetic 5 -UTRs were designed using UTR Library Designer (http://sbi.postech.ac.kr/utr_library, accessed on 6 July 2022) to fine-tune gene expression levels [43].

Chemicals
In this study, the TransStartFastPfu DNA polymerase and TransStartR easyTaq DNA polymerase were purchased from TransGen Biotech Co., Ltd. (Beijing, China). DNA restriction enzymes, T4 ligase, dNTPs, RNase, and DL5000 Marker were provided by

Determination of L-tyrosine
To measure L-tyrosine, 1 mL of fermentation broth was vortexed with 0.6 mL of 1 M HCl for 1 h, and centrifuged at 10,000× g for 3 min. The supernatant was then filtered using a 0.22 µm membrane. The HPLC analysis conditions are listed as follows: Agilent 1100 HPLC chromatograph, ZORBAX Eclipse XDB-C18 (4.6 mm × 250 mm, 5 µm) column, mobile phase of 10% methanol, 90% sodium acetate (100 mM, pH 4.0), flow rate 0.6 mL/min, injection volume 10 µL, column temperature 30 • C, and UV detector with a detection wavelength of 280 nm. The L-tyrosine standard was used to calculate the concentration.

Statistical Analysis
Each fermentation experiment was carried out in three independent replicates. SPSS 20.0 (IBM, Armonk, NY, USA) was used to calculate the means and standard deviations, and observe the significance. GraphPad Prism 8 (GraphPad Software Inc., San Diego, CA, USA) was applied to deal with the data and plot the graphs.

Screening the Optimal Host Strain for the Synthesis of the Platform Compound L-tyrosine
At present, the host strains for the production of platform compound L-tyrosine by metabolic engineering are concentrated in E. coli [26,44]. Juminaga et al. constructed a modular biosynthesis pathway in E. coli MG1655, and each module was optimized to achieve the optimal combination. Finally, the L-tyrosine yield reached 2.6 g/L, 79% of the maximum theoretical yield under corresponding fermentation conditions [26]. By using global transcription machinery engineering and high-throughput screening strategies, the rpoA mutant E. coli strains encoding RNA polymerase subunits were obtained, which could produce 13.8 g/L L-tyrosine in a 2 L fermenter [44]. However, the existence of endotoxins in E. coli hinders its applicability in the food industry [35]. In contrast, Bacillus species have become a promising alternative due to their advantages of their Generally Recognized As Safe (GRAS) status, good growth on cheap carbon sources, distinct endogenous metabolism, and robustness in industrial fermentations [34]. Hence, a series of Bacillus strains (shown in Table 1) stored in this laboratory were verified with L-tyrosine as the monitoring target for obtaining the optimal host strain. As shown in Figure 1, the L-tyrosine yield of the strain B. amyloliquefaciens HZ-12 reached 297 mg/L after fermentation for 36 h, much higher than other strains. B. amyloliquefaciens HZ-12 has a high initial L-tyrosine yield, and it has also been broadly applied in the biosynthesis of functional substances due to its genetic transformation potential, being able to transform into organic compounds such as spermidine and S-adenosylmethionine [39,40]. Therefore, the B. amyloliquefaciens HZ-12 was selected for engineering modification in the subsequent experimental operations.
transform into organic compounds such as spe [39,40]. Therefore, the B. amyloliquefaciens HZ-12 cation in the subsequent experimental operations.

Effects of Different Prephenate Dehydrogenase Gene
At present, the L-tyrosine biosynthesis pathwa organisms have been analyzed, which has laid a subsequent optimization of L-tyrosine production [ is a key pathway enzyme for the synthesis of L-tyr thesis of metabolic end products [33,34]. Kim
The L-tyrosine yields of all the 15 engineered strains were measured after shake-flask cultivation for 36 h under the same experimental conditions. As shown in Figure 2, the highest yield (411 mg/L) was obtained after the enhanced expression of the gene tyrA from B. amyloliquefaciens HZ-12, which was 42% higher than that of the control strain HZ/pHY300PLK. In addition, the expression of the gene from E. coli, B. subtilis, B. licheni-formis, B. pumilus, Bacillus megaterium, B. thuringiensis, and Bacillus cereus also has a significant improvement effect on the synthesis of L-tyrosine. The data indicate that the efficient expression of the prephenate dehydrogenase gene is crucial for the synthesis of L-tyrosine, and various prephenate dehydrogenase gene resources suitable for L-tyrosine synthesis were obtained, especially the gene Bao-tyrA from B. amyloliquefaciens HZ-12.
from B. amyloliquefaciens HZ-12, which was 42% higher tha HZ/pHY300PLK. In addition, the expression of the gene from formis, B. pumilus, Bacillus megaterium, B. thuringiensis, and B nificant improvement effect on the synthesis of L-tyrosine. T ficient expression of the prephenate dehydrogenase gene is c tyrosine, and various prephenate dehydrogenase gene resou synthesis were obtained, especially the gene Bao-tyrA from B Figure 2. Effects of enhanced expression of the key enzyme genes ty tyrosine production. Note: * means significant difference (p < 0.05) ference (p < 0.01), and ns means no significant difference.

Amino Acid Sequence Analysis and Possible Catalytic Mechan
Prephenate dehydrogenase catalyzes the synthesis of ping prephenate and NAD + as substrates [45]. To explain the dehydrogenase gene Bao-tyrA from B. amyloliquefaciens HZbp) and corresponding amino acids were further analyzed. tyrA was translated into a sequence containing 368 amino ac software ( Figure 3). Then, the sequence was aligned with the acid sequences (Figure 4), and the similarities with E. col (CAF18797), and S. cerevisiae (NC_001134) were 20.89%, 25 spectively. In addition, the TyrA protein from E. coli is a bidisplay the activities of chorismate mutase/prephenate deh protein from B. amyloliquefaciens HZ-12 only shows the activ genase [34,46]. Sequence alignment revealed that these amin the same conserved sites. Therefore, His131 might assist in t H Z / p H Y 3 0 0 P L K Effects of enhanced expression of the key enzyme genes tyrA from different species on L-tyrosine production. Note: * means significant difference (p < 0.05), ** means very significant difference (p < 0.01), and ns means no significant difference.

Amino Acid Sequence Analysis and Possible Catalytic Mechanism
Prephenate dehydrogenase catalyzes the synthesis of p-hydroxyphenylpyruvate using prephenate and NAD + as substrates [45]. To explain the function of the prephenate dehydrogenase gene Bao-tyrA from B. amyloliquefaciens HZ-12, the gene sequence (1107 bp) and corresponding amino acids were further analyzed. The gene sequence of Bao-tyrA was translated into a sequence containing 368 amino acids by using BioEdit v7.0.9.0 software ( Figure 3). Then, the sequence was aligned with the previously reported amino acid sequences (Figure 4), and the similarities with E. coli (NC_000913), C. glutamate (CAF18797), and S. cerevisiae (NC_001134) were 20.89%, 25.86%, and no similarity, respectively. In addition, the TyrA protein from E. coli is a bi-functional enzyme that can display the activities of chorismate mutase/prephenate dehydrogenase, while the TyrA protein from B. amyloliquefaciens HZ-12 only shows the activity of prephenate dehydrogenase [34,46]. Sequence alignment revealed that these amino acid sequences contained the same conserved sites. Therefore, His131 might assist in the transfer of hydride from prephenate to NAD + in the dehydrogenase reaction [45]. Arg294 might interact specifically with the cyclic carboxylate at C-1 of prephenate [47]. This further illustrates the catalytic mechanism of the prephenate dehydrogenase coded by the gene Bao-tyrA in B. amyloliquefaciens HZ-12.

Effects of Different Promoters of Bao-tyrA on Biosynthesis of L-tyrosine
The promoter is one of the key factors that can affect the gene expres it constitutes an important genetic regulatory element in the complex fram scriptional control [48,49]. An efficient promoter, SPL-21, was screened from to control the expression of toyF, which significantly increased the produ camycin [50]. A high-strength promoter, PtnrQ, was mined from B. subtilis b scriptome data, and it could double the amylase expression [51]. Based on microarray analyses, a toolbox of novel promoters was obtained from B. offer versatile promoter strength, and the final progesterone yields of found to be increased compared with the control promoter [52]. This indi cient promoters can further improve the production of metabolites. There to further optimize the expression level of the gene Bao-tyrA, promoters

Effects of Different Promoters of Bao-tyrA on Biosynthesis of L-tyrosine
The promoter is one of the key factors that can affect the gene expression level, an it constitutes an important genetic regulatory element in the complex framework of tra scriptional control [48,49]. An efficient promoter, SPL-21, was screened from Streptomyc to control the expression of toyF, which significantly increased the production of toy camycin [50]. A high-strength promoter, PtnrQ, was mined from B. subtilis based on tra scriptome data, and it could double the amylase expression [51]. Based on genome-wi microarray analyses, a toolbox of novel promoters was obtained from B. megaterium offer versatile promoter strength, and the final progesterone yields of 3.6 mM we found to be increased compared with the control promoter [52]. This indicates that ef cient promoters can further improve the production of metabolites. Therefore, in ord to further optimize the expression level of the gene Bao-tyrA, promoters with differe strengths were used, and corresponding recombinant strains were obtained, includin HZ/PsrfA-Bao-tyrA, HZ/PytzE-Bao-tyrA, HZ/Pylb-Bao-tyrA, HZ/Pbay-Bao-tyr HZ/PykzA-Bao-tyrA, HZ/PykzA-PRBS6-Bao-tyrA, HZ/PmmgA-Bao-tyrA, HZ/PabrB-Ba tyrA, HZ/PbacA-Bao-tyrA, HZ/PUTR12-Bao-tyrA, HZ/P43-Pylb-Bao-tyrA, HZ/PR5-Ba tyrA, and HZ/PRBS6-Bao-tyrA. The L-tyrosine yields of all the 13 engineered strains we

Effects of Different Promoters of Bao-tyrA on Biosynthesis of L-tyrosine
The promoter is one of the key factors that can affect the gene expression level, and it constitutes an important genetic regulatory element in the complex framework of transcriptional control [48,49]. An efficient promoter, SPL-21, was screened from Streptomyces to control the expression of toyF, which significantly increased the production of toyocamycin [50]. A high-strength promoter, P tnrQ , was mined from B. subtilis based on transcriptome data, and it could double the amylase expression [51]. Based on genomewide microarray analyses, a toolbox of novel promoters was obtained from B. megaterium to offer versatile promoter strength, and the final progesterone yields of 3.6 mM were found to be increased compared with the control promoter [52]. This indicates that efficient promoters can further improve the production of metabolites. Therefore, in order to further optimize the expression level of the gene Bao-tyrA, promoters with different strengths were used, and corresponding recombinant strains were obtained, including HZ/PsrfA-Bao-tyrA, HZ/PytzE-Bao-tyrA, HZ/Pylb-Bao-tyrA, HZ/Pbay-Bao-tyrA, HZ/PykzA-Bao-tyrA, HZ/PykzA-PRBS6-Bao-tyrA, HZ/PmmgA-Bao-tyrA, HZ/PabrB-Bao-tyrA, HZ/PbacA-Bao-tyrA, HZ/PUTR12-Bao-tyrA, HZ/P43-Pylb-Bao-tyrA, HZ/PR5-Bao-tyrA, and HZ/PRBS6-Bao-tyrA. The L-tyrosine yields of all the 13 engineered strains were measured ( Figure 5); it was found that different promoters had different effects on the expression of prephenate dehydrogenase. In comparison, the P43 promoter could drive the maximum L-tyrosine production in B. amyloliquefaciens HZ-12, while other promoters did not further increase L-tyrosine production. The data indicate that the constitutive promoter P43 has great advantages in the sustained and efficient expression of its regulated genes.

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promoter P43 has great advantages in the sustained and lated genes.

Redesign of the 5′-UTR of the Gene Bao-tyrA
In addition to the promoter, 5′-UTR is also a factor can play a role in post transcriptional regulation of genes constitutive promoter and a synthetic 5′-UTR into each g pathway, and then used UTR designer to control the exp After optimization, the L-tyrosine yield of E. coli was inc introduced a strong inducible tac promoter with synth expression to the genes hemA and hemL, and the yield o creased to 0.74 g/L after optimization [55]. This demonstr an effective strategy for optimizing the biosynthetic path mize the expression of gene Bao-tyrA at the translation . Effect of replacement of promoter of the gene Bao-tyrA on L-tyrosine production. Note: * means significant difference (p < 0.05), ** means very significant difference (p < 0.01), and ns means no significant difference.

Redesign of the 5 -UTR of the Gene Bao-tyrA
In addition to the promoter, 5 -UTR is also a factor affecting gene expression, and can play a role in post transcriptional regulation of genes [53,54]. Kim et al. introduced a constitutive promoter and a synthetic 5 -UTR into each gene of the L-tyrosine synthesis pathway, and then used UTR designer to control the expression level of PEP synthetase. After optimization, the L-tyrosine yield of E. coli was increased to 3.0 g/L [2]. Noh et al. introduced a strong inducible tac promoter with synthetic 5 -UTRs designed for high expression to the genes hemA and hemL, and the yield of 5-aminolevulinic acid was increased to 0.74 g/L after optimization [55]. This demonstrates that the 5 -UTR redesign is an effective strategy for optimizing the biosynthetic pathway. Therefore, to further optimize the expression of gene Bao-tyrA at the translation level, the fiveterminal UTR sequence of gene Bao-tyrA was redesigned to precisely regulate gene expression. The corresponding gene-expression plasmids pHY-P43-UTR1-Bao-tyrA, pHY-P43-UTR2-Bao-tyrA, pHY-P43-UTR3-Bao-tyrA, pHY-P43-UTR4-Bao-tyrA, pHY-P43-UTR5-Bao-tyrA, and pHY-P43-UTR6-Bao-tyrA were constructed. These expression plasmids were then converted into B. amyloliquefaciens HZ-12, and corresponding recombinant strains were constructed, including HZ/P43-UTR1-Bao-tyrA, HZ/P43-UTR2-Bao-tyrA, HZ/P43-UTR3-Bao-tyrA, HZ/P43-UTR4-Bao-tyrA, HZ/P43-UTR5-Bao-tyrA, and HZ/P43-UTR6-Bao-tyrA. The L-tyrosine yields of all these six engineered strains after shake-flask cultivation for 36 h are shown in Figure 6. The redesigned UTR3 could significantly increase the L-tyrosine production to 475 mg/L, which was 16% higher than the control strain HZ/P43-Bao-tyrA. The data indicated that the redesign of the five-terminal UTR was a feasible strategy in the metabolic engineering of Bacillus. The 5 -UTR redesign can be used to fine-tune the level of target gene expression within cells, and has been widely used in E. coli and C. glutamate. Lee et al. constructed a plug-in inhibitor expression library based on 5 -UTR redesign, and verified the expression range of the library. Subsequently, the library was applied to accurately control the throughput of key metabolic nodes in the synthesis pathway of target substances. Finally, the yield of 3-HP and lycopene reached 2.59 g/L and 11.66 mg/L, which, respectively, increased 16.5 times and 2.82 times compared with the parental strains [56]. Jiang et al. developed two plug-in repressor expression libraries by diversifying the translation levels of phlF and mcbR based on the 5 -UTR variants to accurately control the metabolic flux, which can reduce the production of L-lysine as a by-product and balance extracellular protein synthesis and cell growth. The two libraries resulted in a 28% and 12% increase in the production of Ectoine compared with the control strain, respectively [57]. This indicates that the redesign of 5 -UTR is important for the precise control of intracellular carbon flux, and can provide some guidance for the subsequent optimization of biosynthesis. However, few studies have been conducted to fine-tune the expression level of target genes in B. amyloliquefaciens by redesigning 5 -UTR sequences. This study fills in this gap and verifies that this strategy of redesigning 5 -UTR sequences can fine-tune the intracellular gene expression level in B. amyloliquefaciens, which can be further applied to the production of more target compounds.

Conclusions
This study obtained a food-safe strain, B. amyloliquefaciens HZ-12, with a high initial yield of L-tyrosine. A total of 15 prephenate dehydrogenase genes were successfully expressed to screen the optimal gene suitable for the host strain, and the gene Bao-tyrA from B. amyloliquefaciens HZ-12 was confirmed to be an efficient gene resource. In addition, promoter replacement and five-terminal UTR redesign were also carried out, and it was found that the use of a 5 -UTR-3 sequence driven by a P43 promoter could enhance the expression of prephenate dehydrogenase in B. amyloliquefaciens. This study provides new microbial and genetic resources for construction of a L-tyrosine chassis cell, which will be beneficial for metabolic engineering.