In situ insertion of one or two hydroxy-rich unnatural amino acid into sfGFP to alter its performance

In situ insertion of one or two hydroxy-rich unnatural amino acid into sfGFP to alter its performance | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article In situ insertion of one or two hydroxy-rich unnatural amino acid into sfGFP to alter its performance Xuanhe Fan, Yumei Liu, Zhenya Chen This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4824485/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Unnatural amino acids (unAAs) possess unique properties owing to their distinct functional groups, and their insertion into proteins can significantly alter protein function and properties. Currently, the predominant method for inserting unAAs into proteins is through genetic code expansion (GCE), which mimics the natural translation process within cells and necessitates the exogenous supplementation of unAAs. However, in many instances, microbial cells do not recognize unAAs as essential nutrients and lack specific transporters for their uptake across the cell membrane, thereby greatly reducing their insertion efficiency. To address this issue, our study developed an in situ insertion method for enhancing the efficiency of unAAs insertion into proteins and further explored the feasibility of simultaneously inserting two different unAAs into one protein. Firstly, the orthogonal translation system for hydroxy-rich unAAs 5-hydroxytryptophan (5-HTP) or 4-hydroxyisoleucine (4-HiL) were constructed and then transformed into microbial cells to achieve the insertion of 5-HTP or 4-HiL into sfGFP by feeding 5-HTP or 4-HiL. Subsequently, the biosynthetic pathways of 5-HTP or 4-HiL were constructed in E. coli which contained the corresponding orthogonal translation system, resulting in the in situ insertion of 5-HTP or 4-HiL into sfGFP.Further, we developed a co-insertion method based on codons UGA and UAG. Introduction of the biosynthetic pathways and the orthogonal translation systems of 5-HTP and 4-HiL in the same cells achieved the in situ co-insertion of 5-HTP and 4-HiL in one sfGFP. This work provided a representative example for in situ insertion of unAAs into protein to increase the insertion efficiency, and explored the possibility of co-inserting two types of unAAs into one protein. unnatural amino acid protein engineering in situ insertion signal molecule Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Proteins are macromolecules that are indispensable for maintaining structure and function of organisms. Native proteins are composed of 20 natural amino acids (nAAs), which could ensure the basic growth and metabolism of organisms[ 1 – 3 ]. To complete some specific physiological or biochemical reactions in organisms, some nAAs such as L-Ser, L-Thr or L-Arg etc. in specific proteins need to be added with phosphate, methyl, acetyl, glycosyl or hydroxyl group via a process of post-translational modification. These groups can endow the proteins with unique functions, which in turn activate the corresponding specific reactions[ 4 – 6 ]. Hydroxylation is an important post-translational modification process for proteins. Hydroxyl groups can form hydrogen bonds with nearby amino groups, which helps to maintain the structural stability of proteins[ 7 ]. Moreover, hydroxylation reactions can alter the activity of proteins, affecting their function[ 8 , 9 ]. These post-translational modifications only target a few nAAs in some specific endogenous proteins and require the participation of multiple enzymes and the complex regulation of protein-dependent regulatory network[ 10 – 13 ]. Unnatural amino acids (unAAs), as derivatives of nAAs, have specific functions derived from the redundant side-chain groups, such as hydroxyl, carboxyl, acetyl, phosphate, luminescence, etc [ 14 – 16 ]. Currently, insertion of unAAs into proteins is an attractive alternative to post-translational modification in order to confer new properties or alter existing properties of proteins, including biocatalytic activity, structure, thermal stability, and substrate specificity[ 17 , 18 ]. Compared to the complex process of post-translational modification, the method of inserting unAAs into proteins, such as genetic code expansion (GCE), is much simpler. The GCE works similarly to the natural translation process within the cells, requiring efficient and rigorous translation mechanism, with a ‘codon’ for each unAA and an orthogonal pair aaRS/tRNA[ 19 – 23 ]. The aminoacyl-tRNA synthetase aaRS loads unAAs on tRNA via aminoacylation reactions. The aminoacylated tRNA carries unAA to complementally pair with the reassigned codon, which in turn inserts the unAA into the extended peptide chain in an orderly manner[ 24 – 27 ]. Currently, the commonly used reassigned codons for unAAs are the stop codons UAG, UGA or UAA[ 28 – 30 ]. Utilization of microbial cells to express unAA-contained proteins usually requires the feeding of the corresponding unAAs into the culture[ 31 , 32 ]. In many cases, microbial cells do not consider the unAAs as essential substances for cells, and do not have unique transporters for transporting unAAs in cells[ 33 – 35 ]. The cell membrane acts as a natural barrier to unAAs, significantly decreasing the efficiency of unAAs entry cells. Specially, some unAAs with complex functional groups are still unable to enter cells, and currently their insertions are only achieved outside the cells[ 36 ]. Therefore, the in situ biosynthesis and insertion of unAAs in the same cells is necessary to be developed in order to enhance the utilization efficiency of unAAs. The function of protein is often affected by the interaction between multiple amino acid residues, the two or more site mutations of protein can optimize the overall performance of the protein through the synergistic effect between different sites[ 37 , 38 ]. In general, the site-directed mutagenesis method commonly used in protein modification can realize the simultaneous mutagenesis of multiple sites, and the original amino acids can be replaced with other 19 nAAs. However, there is currently a lack of method for the co-insertion of two or more unAAs. In this study, the unAA 5-hydroxytryptophan (5-HTP) and 4-hydroxyisoleucine (4-HiL) were inserted into sfGFP, in an attempt to use the functional groups carried by 5-HTP and 4-HiL to endow sfGFP with special properties. The unAA 5-HTP, a precursor for the human neurotransmitter serotonin, has one more hydroxyl group at the 5th carbon atom than L-Trp[ 39 , 40 ]. 4-HiL is a nonproteinogenic amino acid possessing insulinotropic biological activity, which be able to increase glucose-induced release of Insulin, has one more hydroxyl group at the 3rd carbon atom than L-Ile[ 41 ]. Base on the similar hydroxy-rich structural characteristics, these unAAs insertion into sfGFP have high probability of influencing the protein properties. Base on this, specific sites were performed on sfgfp to replace the original codons with stop codon UAG and UGA, in order to insert 5-HTP and 4-HiL at specific sites of sfGFP. After verification, 5-HTP-contained sfGFP mutant, 4-HiL-contained sfGFP mutant and 5-HTP/4-HiL-contained sfGFP mutant were isolated. Further, to avoid the barrier effect of the cell membrane on in-out of 5-HTP and 4-HiL, the pathways for both of unAAs biosynthesis were constructed separately within cells, achieving the in situ insertion of 5-HTP or 4-HiL into sfGFP. Further, we explored the potential of the in situ insertion of two types of unAAs into proteins. The biosynthesis pathways for 5-HTP and 4-HiL, and the corresponding orthogonal insertion system were co-expression in the same cells, enabling the co-insertion of 5-HTP and 4-HiL into sfGFP. These provided guidance for the insertion of two types of hydroxy-rich unAAs into protein in order to change the characteristics of protein. This work provided an example of co-insertion of unAAs into protein and achieved the in situ insertion of unAAs into sfGFP, significantly expanding the application scope of unAAs and investigating the possibility of co-inserting two types of unAAs into one protein. Materials and methods Strains and growth conditions Escherichia coli JCL16, Escherichia coli JM109, was used for plasmid construction as well as fluorescence screening. The details of the strains and plasmids used in this study are listed in Table 1 . E. coli strains were grown aerobically at 37 ℃ in LB broth. Ampicillin (100 g/mL), kanamycin (50 g/mL) and spectinomycin (50 g/mL) were added when required. Table 1 Strains and plasmids used in this study Strain or plasmid Genotype or description Reference or Source E. coli strains JM109 recA1, endA1, gyrA96, thi-1, hsdR17(rk-mk+), e14 − (mcrA − ), supE44, relA1, Δ(lac-proAB)/F´ [traD36, proAB + , lacI q , lacZΔM15] lab source JCL16 ΔrhaBADLD 78 [F’ traD36 proAB lacI q ZΔM15 Tn10 (Tet R )] lab source Plasmid pET-28a ColE1 origin; Kan r ; P lacI : lacI ; 5369bp lab source pYH1 ColE1 origin; Amp r ; P bmoR : bmoR ; P bmo : gfp lab source pFL-1 ColE1 origin; Amp r ; P L lacO 1 : XcP4H ; P lacI : lacI This study pFL-2 ColA origin; Cm r ; P lpp : PCD This study pFL-3 ColA origin; Amp r ; P L lacO 1 : XcP4H - PCD ; P lacI : lacI This study pFL-4 ColA origin; Amp r ; P L lacO 1 : XcP4H (W179F)- PCD ; P lacI : lacI This study pFL-5 ColE1 origin; Amp r ; P L lacO 1 : XcP4H (W179F)- PCD ; P lacI : lacI ; P tac : HRE342(ScW aaRS); P LeuV : ScW tRNA-40A This study pFL-9 P15A origin; Cm r ; P lpp : 4-Hil tRNA; ivbL; P lacI : lacI ; P L lacO 1 : GFP This study pLF-12 P15A origin; Spec r ; P bmoR : sfGFP (AAT153TAG) This study pLF-13 P15A origin; Spec r ; P bmoR : sfGFP (AAT153TAG/ATT150TGA) This study pLF-23 P15A origin; Spec r ; P bmoR : sfGFP This study pFL-24 P15A origin; Spec r ; P bmoR : sfGFP (AAT150TAG) This study pLF-25 P15A origin; Spec r ; P bmoR : sfGFP (AAT150TGG) This study pLF-26 ColE1 origin; Amp r ; P tac : HRE342(ScW aaRS); P LeuV : ScW tRNA-40A; P lacI : lacI This study Note: Tet, Tetracycline; Amp, Ampicilli; Cm, Chloramphenicol; Kan, Kanamycin; Spec, spectacularinomycin; r, resistance. Media, and materials LB medium (10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl) with a pH of 7.0 was used for strain incubation. M9 medium (6 g/L Na 2 HPO 4 , 3 g/L KH 2 PO 4 , 1 g/L NH 4 Cl, and 0.5 g/L NaCl, 1 mM MgSO 4 , 0.1 mM CaCl 2 , 10 mg/L VB 1 , 4 g/L yeast extract, and 40 g/L glucose) with a pH of 7.0 was used for the fermentation experiment to produce 5-HTP and 4-HiL. GMML medium (6.78 g/L NaH 2 PO 4 , 3.00g/L KH 2 PO 4 , 0.5g/L NaCl, 1.0g/L NH 4 Cl, 1mmol/L MgSO 4 , 0.1 mmol/L CaCl 2 , 0.3 mmol/L L-Leucine solution, 1% glycerin) with a pH of 7.0 was used for Validation of 5-HTP and 4-HiL orthogonal pair. Plasmid construction The lacI fragment was amplified from plasmid pET-28a and inserted into the backbone of pYH1 (ColE1-amp) by Gibson assembly, generating the high-copy plasmid pLF-26 which could express the exogenous 5-HTP orthogonal system. A pair of upstream and downstream primers containing the anticodon of 4-HiL tRNA were designed, with Circular plasmid PCR directed-site mutation technique, the plasmid template containing L-Ile tRNA was amplified to generate the plasmid pLF-9 which could express the exogenous 4-HiL orthogonal system. To achieve the expression of XcP4H W179F in E. coli , its wild type gene phhA was amplified from the genomic DNA of X. Campestris , and then L-Trp at position 179th of the gene was replaced with L-Phe. Thus, the plasmid pLF-4 was cloned under the control of IPTG inducible promoter P L lacO 1 . We synthesized PCD, encoded by phhB by OE-PCR and inserted it into the backbone of pLF-4 ( LacI - HRE342 - Amp - phha ) by Gibson assembly to generate a high-copy plasmid pLF-5. Gene ido was synthesized by OE-PCR and inserted into the backbone of pYH-1 (LacI-Amp) by Gibson assembly to generate high-copy production plasmid pLF-6 which could express 4-HiL. To detect the effect of amino acid insertion on the expression of the fluorescent protein, directed-cite circular PCR of the wild type sfGFP plasmid pLF-23 was performed to obtain the sfGFP N150HTP plasmid pLF-8, the sfGFP I153HiL plasmid pLF-12, and the sfGFP N150HTP/I153HiL plasmid pLF-13. UHPLC analysis In the determination of 5-HTP or yield, L-Trp and 5-HTP were used as standards. Both the standards and samples were quantified by UHPLC (Agilent Technologies 1290 Infinity II) equipped with a reverse phase column (Agilent ZORBAX SB-C18, 5 µm, 4.6×250 mm). methanol and water (adding 0.2% TFA) were used as mobile phase, the flow rate was 1.0 mL/min, the column temperature was 30 ℃, and the detection wavelength was 276 nm. By gradient elution, the methanol increased from 5–30% in 0-15min. Methanol increased from 30–100% in 15-16min. The methanol remained 100% for 16–18 min. 18–19 min, methanol decreased from 100–5%. The methanol remained at 5% for 19–21 min. In the determination of 4-HiL yield, L-Ile and 4-HiL were used as standards. The detection wavelengths were 210 nm, 250 nm, and 395 nm. The UHPLC detection methods were the same as those for 5-HTP. SfGFP protein expression Single colonies of strain JCL16 harboring pLF-24, pLF-12, or pLF-13 were cultivated in 4 mL LB medium containing the appropriate antibiotic at 37 ℃ at 220 rpm for 8 hours. Then, 1 mL culture was inoculated into 100 mL M9 Y and cultivated at 37 ℃ and 220 rpm. When the OD 600 value reached 0.8, IPTG was added to the culture at a final concentration of 0.5 mM, and the sfGFP expression was induced at 30 ℃ and 220 rpm for 6 hours. After expression, cells were collected and resuspended in Tris-HCl buffer containing 20 mM imidazole, followed by ultrasonic treatment to obtain the crude enzyme extract. Fluorescence assay Fluorescence detection experiments. Single colonies were pre-inoculated into 5 mL LB medium containing antibiotics and incubated overnight at 37℃. Then, 50 µL of the seed culture was added to 5 mL of fresh LB medium containing antibiotics and 1 mM IPTG. Afterward, that culture was placed in the incubator at 37 ℃ for 24 h. Samples were taken at 24 h, 48 h, and 72 h, and sfGFP fluorescence and OD600 values were detected by microplate reader (BioTek Cytation 3). The sfGFP fluorescence intensity was measured using an excitation wavelength of 488 nm and an emission wavelength of 510 nm. The sfGFP fluorescence values were normalized to sfGFP/OD 600 and the background fluorescence of the medium was subtracted. Purification of different sfGFP proteins After induction (IPTG concentration was 0.5 mmol/L), 1L strain was collected by centrifugation at 5000 r/min, and 20 mL storage buffer (20 mmol/L Tris-HCl, pH = 8.0). The thalli were broken by ultrasonic wall-breaking instrument with 40% power until the bacterial solution became relatively clear, centrifuged at 12000 r/min for 30 mins, and the supernatant of the bacterial solution was retained for purification by Ni 2+ -NTA affinity chromatography. After the sample was loaded, 50 mmol/L and 75 mmol/L imidazole were used to wash away the impurity protein, and then 200 mmol/L imidazole was used to elute, and then SDS-PAGE was used to detect the bacterial supernatant, breakthrough liquid, 40 and 200 mmol/L imidazole eluent. Samples were finally concentrated using a 10 kDa molecular weight cut-off Microcon centrifugal filter (EMD Millipore). Mass spectrometry Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS) was performed using the model Autoflex Speed. The detection mode was set to positive ion detection, in linear mode. The saturated solution of 3,5-dimethoxy-4-hydroxycinnamic acid (SA) was prepared: trifluoroacetic acid (0.1%), ultra-pure water (70%), and acetonitrile (30%). For the sample application, the SA was mixed with the sample in a 1:1 ratio, and 1 µL was applied onto the target plate. The laser power was set to 100%, and the voltage was set to 3100 V for detection. Results Construction of 5-HTP or 4-HiL orthogonal pair To insert the unAA 5-HTP into the sfGFP, we utilized the orthogonal translation system-genetic code expansion approach (Fig. 2 a). The orthogonal translation system could recognize the aminoacyl-tRNA synthetase (aaRS)/tRNA orthogonal pair of 5-HTP, while don’t cross-react with the orthogonal system of 20 endogenous natural amino acids. The orthogonality of 5-HTP orthogonal pair WRS-R3-13/ \(\:{\text{t}\text{R}\text{N}\text{A}}_{\text{C}\text{U}\text{A}}^{\text{T}\text{r}\text{p}}\) -40A, which came from native Saccharomyces cerevisiae L-Trp orthogonal pair, was further modified to improve its orthogonality because the source of the orthogonal pair was far away from the host phylogeny[ 42 ]. In this 5-HTP orthogonal pair, the aaRS WRS-R3-13 had the mutations of T107C, P254T and C255A as compared to the native L-Trp aaRS, while the \(\:{\text{t}\text{R}\text{N}\text{A}}_{\text{C}\text{U}\text{A}}^{\text{T}\text{r}\text{p}}\) -40A had an anticodon of CUA to complementary pair with the stop codon UAG, and nine base mutations as compared to the native L-Trp tRNA. The lacI fragment was amplified from plasmid pET-28a and inserted into the backbone of pYH1 (ColE1-amp) by Gibson assembly, generating the high-copy plasmid pLF-26 which could express the exogenous 5-HTP orthogonal system. The gene sequence information of sfgfp was obtained from the plasmid pBad-sfGFP (Addgene plasmid # 85482)[ 43 ], and the sfgfp fragment was obtained by primers synthesis method. Finally, the plasmid pLF-23 and the plasmid pLF-24 that contained the gene of sfGFP N150HTP by replacing the L-Asn codon AAU at 150th of sfGFP in plasmid pLF-23 with UAG were both generated (Fig. 2 b). Like the principle of 5-HTP orthogonal pair WRS-R3-13/ \(\:{\text{t}\text{R}\text{N}\text{A}}_{\text{C}\text{U}\text{A}}^{\text{T}\text{r}\text{p}}\) -40A, the orthogonal pair of 4-HiL in E. coli was constructed in order to accurately insert 4-HiL into the specific site of sfGFP in order to change its protein characteristics (Fig. 2 a). The 4-HiL orthogonal pair used in this study was derived from the native IleRS/L-ile tRNA orthogonal pair of E. coli . Because the source of the L-Ile orthogonal pair was the same as the host system, only the anticodon of the tRNA was modified. The 4-HiL tRNA had a CUA anticodon complementary to the stop codon UGA at position 153th of sfGFP. A pair of upstream and downstream primers containing the anticodon of 4-HiL tRNA were designed, with circular plasmid PCR directed-site mutation technique, the plasmid template containing L-Ile tRNA was amplified to generate the plasmid pLF-9 which could express the exogenous 4-HiL orthogonal system(Fig. 2 b). In Addition, plasmid pLF-12 that contained the gene of sfGFP I153HiL by replacing the L-Ile codon AUU at 153th of sfGFP in plasmid pLF-23 with UGA as generated by PCR circular amplification (Fig. 2 b). Validation of 5-HTP orthogonal pair Plasmid pLF-26 was individually co-transformed with pLF-23 and pLF-24 into E. coli XL10-Gold to form the control strain and the experimental strain, respectively. As shown in Fig. 2 c, for the control strain, sfGFP could be normally expressed, showing a sfGFP/OD 600 value of 491 ± 17.9 within 24 hours. For the experimental strain, the 5-HTP orthogonal pair could not be expressed in the absence of inducer isopropyl β-D-1-thiogalactopyranoside (IPTG) feeding, and correspondingly, the significant fluorescence could not be detected regardless of whether 5-HTP was added. This result illustrated that the tRNA lacking the anticodon to pair with the stop codon UAG, could not enable the insertion of Trp or 5-HTP into sfGFP N150HTP . The cells could express 5-HTP orthogonal pair in the presence of IPTG feeding and could not express sfGFP N150HTP in the absence of 5-HTP feeding, suggesting aaRS WRS-R3-13 could not load the intercellular L-Trp on \(\:{\text{t}\text{R}\text{N}\text{A}}_{\text{C}\text{U}\text{A}}^{\text{T}\text{r}\text{p}}\) -40A. The cells could only express sfGFP N150HTP in the presence of IPTG and 5-HTP feeding, showing a sfGFP/OD 600 value of 303 ± 3.50, 60% lower than that of the control strain. To confirm the absolute loading of 5-HTP orthogonal pair on 5-HTP, we mutated the L-Asn codon AAU at 150th of sfGFP in pLF-23 to L-Trp codon TGG, forming plasmid pLF-25. E. coli XL10-Gold harboring the pLF-25 could normally express sfGFP N150W in the presence or absence of L-Trp feeding, showing comparable sfGFP/OD 600 values to the control strain E. coli XL10-Gold harboring the pLF-23. These results indicated that substitution of L-Asn to L-Trp at 150th did not significantly change the fluorescence intensity of sfGFP, while substitution of L-Asn to 5-HTP at 150th significantly decreased the fluorescence intensity, demonstrating the 5-HTP orthogonal pair rigorously recognized 5-HTP and 5-HTP was then precisely inserted into the 150th of sfGFP to produce sfGFP N150HTP . Subsequently, sfGFP and sfGFP N150HTP were purified and identified using the MALDI-MS mass spectrometer. The generated results showed that the molecular weight of sfGFP N150HTP was 27934, 88 more than that of sfGFP (27847), consistent with the theoretical molecular weight. The purity of sfGFP N150HTP was almost 100%, indicating the experimental strain could precisely insert 5-HTP into 150th of sfGFP to produce sfGFP N150HTP . To confirm the absolute loading of 5-HTP orthogonal pair on 5-HTP, the L-Asn codon AAU at 150th of sfGFP in pLF-23 was mutated to L-Trp codon TGG, forming plasmid pLF-25. E. coli XL10-Gold harboring the pLF-25 could normally express sfGFP N150HTP . As shown in Fig. 2 c, in the presence or absence of 1 mM L-Trp feeding, showing comparable sfGFP/OD 600 values to the control strain E. coli XL10-Gold harboring the pLF-23. These results indicated that substitution of L-Asn to L-Trp at 150th did not significantly change the fluorescence intensity of sfGFP, while substitution of L-Asn to 5-HTP at 150th significantly decreased the fluorescence intensity, demonstrating the 5-HTP orthogonal pair rigorously recognized 5-HTP and 5-HTP was then precisely inserted into the 150th of sfGFP to produce sfGFP N150HTP . Subsequently, sfGFP and sfGFP N150HTP were purified and identified using the Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS) (Fig. 2 d). The generated results showed that the molecular weight of sfGFP N150HTP was 27934, 88 more than that of sfGFP (27847), consistent with the theoretical molecular weight. The purity of sfGFP N150HTP was almost 100%, indicating the experimental strain could precisely insert 5-HTP into 150th of sfGFP to produce sfGFP N150HTP . Validation of 4-HiL orthogonal pair In order to validate the 4-HiL orthogonal translation system, the plasmid pLF-9 was co-transformed with pLF-23 and pLF-12 into E. coli JCL16 to form the control strain and the experimental strain, respectively. As shown in the Fig. 2 c, for the control strain, sFGFP WT was normally expressed without the inducer IPTG or 4-HiL feeding, showing a sfGFP/OD 600 value of 863 ± 62 within 24 hours. The IPTG feeding could induce the expression of 4-HiL tRNA, when the IPTG was added and 4-HiL was not added, there was no significant change in the fluorescence value of sfGFP. The results showed that the value of sfGFP/OD 600 was 884 ± 62. These results indicated that sfGFP WT could not insert 4-HiL in the absence of 4-HiL. After the feeding of 1 mM 4-HiL, whether the IPTG was added to induce the expression of 4-HiL, the sfGFP/OD 600 (288 ± 6.00) was significantly decreased compared with the value of the previous two control groups. These results might be due to the fact that the structure of 4-HiL has only one more hydroxyl functional group than L-Ile, and the endogenous L-Ile tRNA in this E. coli JCL16 strain could recognize 4-HiL and insert it into the 153rd of sfGFP, thus changing the characteristics of sfGFP. For the experimental strains, the sfGFP/OD 600 values of the sfGFP mutants in the absence of 4-HiL, with or without the addition of the inducer IPTG (352 ± 1.83 or 340 ± 7.83), were much lower than those of normal expression, but still higher than those of sfGFP I153HiL that could be inserted into 4-HiL. These results indicated that the stop coden UGA and the absence of the amino acid L-Ile at position 153rd of sfGFP resulted in premature termination of sfGFP expression. With the addition of IPTG, the cells could normally express the 4-HiL orthogonal pair, and the experimental group with the addition of 1 mM 4-HiL showed lower fluorescence than the group without the addition of 4-HiL, showing a sfGFP/OD 600 value of 217 ± 2.83, which was 75% lower than that of the control strain. These results indicated that 4-HiL could be successfully inserted into sfGFP I153Hil in the presence of both the 4-HiL tRNA and 4-HiL. Subsequently, sfGFP I153HIL was purified and identified using the MALDI-MS (Fig. 2 d). As a result of the generation, the molecular weight of sFGFP I153Hil was 27863, which was 16 greater than the molecular weight of sfGFP WT (27847) and was consistent with the theoretical molecular weight. The lower peak in the MALDI-MS results might be because the 4-HiL orthogonal pair was derived from the L-Ile orthogonal pair, and the competitive binding of the two tRNAs to the IleRs resulted in a decrease in the insertion efficiency of the 4-HiL. Moreover, the purity of sfGFP I153HiL was almost 100%, indicating that the experimental strain was able to precisely insert 4-HiL into the 153rd of sfGFP to produce sfGFP I153HiL . Biosynthesis and in situ insertion of 5-HTP into sfGFP After verifying that exogenously added 5-HTP could be inserted into sfGFP N150HTP , we constructed the pathway for the biosynthesis of 5-HTP in E. coli , with the aim of avoiding the barrier effect of the cell membrane on the entry and exit of 5-HTP (Fig. 3 a). In this pathway, the enzyme XcP4H W179F was used to catalyze the production of 5-HTP from L-Trp, with Pterin 4A-methanolamine dehydratase (PCD) as the cofactor[ 40 ]. To achieve the expression of XcP4H W179F in E. coli , its wild type gene phhA was amplified from the genomic DNA of X. Campestris , and then L-Trp at position 179th of the gene was replaced with L-Phe. Thus, the plasmid pLF-4 was cloned under the control of IPTG inducible promoter P L lacO 1 . In addition, previous experimental evidence suggested that bacterial P4Hs might utilize tetrahydromonopterin (MH4) as a natural pterin coenzyme, while endogenous MH4 in E. coli might be used as the coenzyme[ 40 ]. PCD, encoded by phhB , was responsible for the regeneration of dihydromonopterin (MH2), which could be further reduced to MH4. Therefore, we synthesized phhB by OE-PCR and inserted it into the backbone of pLF-4 ( LacI - HRE342 - Amp - phha ) by Gibson assembly to generate a high-copy plasmid pLF-5. Subsequently, we transformed pLF-5 into E. coli JCL16 to enable endogenous production of 5-HTP. As shown in the Fig. 3 b, in the absence of L-Trp feeding, the engineered bacteria produced only 1.00 ± 0.714 mg/L 5-HTP in 48h by using the endogenously accumulated L-Trp as the substrate, while in the presence of 10 mM L-Trp, the engineered bacteria produced 25.9 ± 0.245 mg/L 5-HTP in 48h. During 24 h, 48 h and 72 h of fermentation, the OD 600 values of exogenous L-Trp and endogenous 5-HTP were the lowest among all the experimental groups, which might be due to the fact that the growth of the bacteria was slowed down because L-Trp in the culture was catalyzed by XcP4H W179F to form 5-HTP in time. At the same time, we observed that the color of the culture gradually darkened after 6 hours at 37 ℃, which probably because of the oxidation of 5-HTP and tryptophan under aerobic conditions. Subsequently, the plasmid pLF-26 that contained the genes of 5-HTP orthogonal pair and the plasmid pLF-8 containing the gene of sfGFP N150HTP were co-transformed into E. coli JCL16 harboring plasmid pLF-4, in order to insert the endogenously biosynthesized 5-HTP into 150th of sfGFP to produce sfGFP N150HTP . As shown in Fig. 3 c, in the absence of 5-HTP feeding, the sfGFP/OD 600 value of the engineered strain reached 96.6 ± 18.3 within 48 hours, 8.1-fold higher than that of the strain without 5-HTP biosynthetic pathway, indicating that the endogenously produced 5-HTP was successfully inserted into 150th of sfGFP. In addition, this value was 0.52-fold lower than that in the presence of 10 mM 5-HTP, which might be due to the low efficiency of 5-HTP biosynthetic pathway, ultimately limiting the expression amount of sfGFP N150HTP . We then compared the insertion efficiency of 5-HTP obtained via exogenously addition or endogenously biosynthesis, and the results showed that the insertion efficiency of 5-HTP obtained via endogenously biosynthesis was 54.4-fold higher than that of 5-HTP obtained via exogenously addition. To further investigate the specificity of the incorporation of 5-HTP feeding and bio- synthesized 5-HTP, sfGFP WT and sfGFP N150HTP containing exogenous or biosynthetic 5-HTP were purified by Ni 2+ -NTA affinity chromatography and characterized by Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Fig. 3 c). Subsequently, we used the MALDI-MS to verify that the molecular weight of the SFGFP N150HTP protein of the biosynthetic 5-HTP was 27935, which was consistent with the theoretical value (Fig. 3 d). Meanwhile, the purity was almost 100%. These results suggested that the endogenous biosynthesis and in-situ insertion of 5-HTP in the same cell could significantly improve the utilization efficiency of 5-HTP, but the low biosynthesis efficiency of 5-HTP limited the amount of corresponding proteins. In the future, it is necessary to focus on improving the biosynthesis efficiency of unAAs, including the design of unAAs biosynthetic pathways, the modification of crucial enzymes in pathways, and the selection of optimal chassis hosts. Biosynthesis and in situ insertion of 4-HiL into sfGFP Like the effect of the 5-HTP production pathway, after verifying that exogenously added 4-HiL could be inserted into sfGFP I153HiL , we constructed the pathway for the biosynthesis of 4-HiL in E. coli , with the aim of avoiding the barrier effect of the cell membrane on the entry and exit of 4-HiL (Fig. 4 a). Gene ido was synthesized by OE-PCR and inserted into the backbone of pYH-1 (LacI-Amp) by Gibson assembly to generate high-copy plasmid pLF-6. Subsequently, we transformed pLF-6 into E. coli JCL16 to enable endogenous production of 4-HiL. As shown in Figure A, in the absence of L-Trp feeding, the engineered strains only produced 47.2 ± 0.219 mg/L 4-HiL within 48 hours by using the endogenous accumulation of L-Trp as substrate, while in the presence of 10 mM L-Trp feeding, the engineered strains produced 1.12 \(\:\times\:\) 10 3 ± 0.312 mg/L 4-HiL within 48 hours. Subsequently, the plasmid pLF-9 that contained the gene of 4-HiL orthogonal pair and the plasmid pLF-12 containing the gene of sfGFP N153HTP were co-transformed into E. coli JCL16 harboring pLF-6, in order to insert the endogenous 4-HiL into 153rd of sfGFP to produce sfGFP I153HiL . As shown in Fig. 4 b, in the absence of 4-HiL feeding, the sfGFP/OD 600 value of the engineered strain reached 540 ± 16.3 within 48 hours, 1.41-fold higher than that of the strain without 4-HiL biosynthetic pathway, indicating that the endogenously produced 4-HiL was successfully inserted into 153th of sfGFP. We then compared the insertion efficiency of 4-HiL obtained via exogenously addition or endogenously biosynthesis, and the results showed that the insertion efficiency of 4-HiL obtained via endogenously biosynthesis was 1.89-fold higher than that of 4-HiL obtained via exogenously addition. Subsequently, we purified sfGFP I153HiL containing exogenous or biosynthetic 4-HiL by Ni 2+ -NTA affinity chromatography and characterized by SDS-PAGE (Fig. 4 c). Moreover, we verified that the molecular weight of the sfGFP I153HiL protein of the biosynthetic 4-HiL was 27863 by using MALDI-MS, which was consistent with the theoretical value (Fig. 4 d). Co-insertion and in situ co-insertion of two unAAs into sfGFP Finally, we explored the possibility of in situ insertion of two types of unAAs into the same proteins (Fig. 5 a). The experiments of co-feeding 5-HTP and 4-HiL and co-synthesizing 5-HTP and 4-HiL to detect the fluorescence of sfGFP were carried out. As shown in Fig. 5 b, when 10 mM 5-HTP and 10 mM 4-HiL were added simultaneously, the value of sfGFP/OD 600 was up to 1.13 \(\:\times\:\) 10 3 ± 131. When 5-HTP or 4-HiL was added alone, the value of sfGFP/OD 600 was higher than that without any of the unAAs, indicating that the exogenous supplementation of 5-HTP and 4-HiL simultaneously inserted sfGFP protein and changed the characteristics of protein. As shown in Fig. 5 c, in the case of endogenous co-biosynthesis of 5-HTP and 4-HiL, the value of sfGFP/OD 600 was the highest at 2.26 \(\:\times\:\) 10 3 ± 254. In addition, the engineered bacteria containing only the 5-HTP biosynthetic pathway or the 4-HiL biosynthetic pathway had a lower sfGFP/OD 600 , but they were 2.1-fold and 1.2-fold separately higher than that of the engineered bacteria not containing any unAA biosynthetic pathway. This result indicated that the biosynthesis of 5-HTP and 4-HiL were successfully co-inserted into sfGFP. Subsequently, we purified SFGFP N150HTP/I153HiL containing exogenous (Fig. 5 b) or biosynthetic 5HTP and 4-HiL (Fig. 5 c) by Ni 2+ -NTA affinity chromatography and characterized by SDS-PAG. Moreover, we used the MALDI-MS to verify that the molecular weights of the proteins from the two sources were 27950 (Fig. 5 b) and 27950 (Fig. 5 c), respectively, which were consistent with the theoretical values. The purity was close to 100%. Discussion In conclusion, we have achieved the insertion of 5-HTP and 4-HiL into sfGFP, using the hydroxyl functional groups carried by these unAAs to endow sfGFP with special properties. In addition, in order to avoid the barrier effect of cell membrane on the entry and exit of unAAs, we have constructed two pathways of unAAs biosynthesis in cells and have realized the in situ insertion of 5-HTP or 4-HiL into sFGFP. More importantly, we have also explored the possibility of in situ co-insertion of two types of unAAs into proteins, which provides guidance for inserting two hydroxyl-rich unAAs into proteins to modify the properties of proteins. In subsequent research, efforts will focus on enhancing the binding efficiency between 4-HiL tRNA and IleRs by optimizing the 4-HiL orthogonal pair. This refinement is expected to markedly increase the expression of sfGFP I153HiL and enhance the peak intensity detected by MALDI-MS. This study has provided an example of co-insertion of unAAs into proteins, and realized in situ insertion of unAAs into sfGFP, significantly expanded the application scope of unAAs, and explored the possibility of co-insertion of two types of unAAs into one protein. At present, most unAAs are obtained by chemical synthesis. However, the steps of chemical synthesis are complex and the generated unAAs are usually racemic mixtures of L- and D-type, bringing challenges to the subsequent separation and purification[ 44 , 45 ]. Specially, unAAs with large molecular weight produced by chemical synthesis have obstacles to enter cells, and are difficult to be taken up by cells, ultimately affecting the insertion efficiency of unAAs in target proteins. In addition, due to the limitations of key technologies such as the screening and preparation of catalysts, the construction of synthetic routes and the regulation of catalytic processes, the chemical synthesis of unAAs has high technical barriers and production costs[ 46 ]. Therefore, the development of green and efficient method for unAAs synthesis to replace the chemical synthesis is important. So far, the microbial-based metabolic engineering emerges as an effective method for the biosynthesis of unAAs. Metabolic engineering for unAAs biosynthesis usually uses cheap glucose as the starting carbon source, and the corresponding biosynthesis process is inexpensive and environmentally friendly[ 47 ]. The efficient biosynthesis of unAAs in microbial cells can be achieved by constructing the artificial biosynthesis pathway of unAAs, designing and modifying key enzymes, regulating precursor biosynthesis, knocking out the competing pathway, constructing the cofactor regeneration system, and intelligently regulating fermentation process[ 48 – 50 ]. For example, Mora-Villalobos et al. [ 51 , 52 ]used sequence analysis, phylogenetic analysis and functional difference analysis tools to predict, screen and design the site-directed mutations for substrate specific sites of aromatic amino acid hydroxylase ( Ct AAAH) from Cupriavidus taiwanensis . The substrate preference of Ct AAAH was transferred from L-Phe to L-Trp, achieving the formation of 5-HTP. In addition, the synthesis efficiency and yield of unAAs, such as enzymes involved in unAAs biosynthetic pathway, and enhancement the metabolic flux of unAAs biosynthetic pathways. However, only a few of unAAs biosynthetic pathways have been validated in microbial cells, and the majority of unAAs biosynthetic pathways remain unclear[ 40 , 51 , 52 ]. Therefore, how to design and create artificial unAAs biosynthetic pathways is still a challenge to be solved. At present, advanced bioinformation tools or biosynthesis simulation tools can be used to mine unknown biosynthetic pathways and enzymes in nature, providing more components for the design of artificial unAAs biosynthetic pathways. Some special unAAs are biosynthetically dependent on cofactors, which significantly increases the cost of biosynthesis. Building an effective cofactor recycling pathway can significantly improve the supply of cofactors. In addition, some biotoxic unAAs can inhibit the growth of chassis hosts, limiting the high-level biosynthesis of unAAs. Therefore, screening or engineering chassis hosts with stronger tolerance to unAAs can significantly increase the biosynthesis efficiency of unAAs. At present, advances in synthetic and computational biology techniques provide effective tools for the design of high efficiency unAAs biosynthesis strategies. In the future, the precise design of the unAAs biosynthetic pathway can be accomplished by advanced bioinformatics or biosynthetic simulation tools. In addition, directed design and screening of chassis with high tolerance to specific unAAs to increase compatibility between microbial cells and unAAs biosynthetic pathways will significantly improve the efficiency of metabolic engineering production of unAAs. At present, the structure and function of proteins are probed in the base of that the approximately 300 unAAs has been added to proteins[ 13 , 53 – 57 ]. Although this technique is widely used, incorporation of these unAAs into proteins requires exogenous feeding of unAAs as well as their successful uptake into cells, a process that significantly limits the application of this method to protein modification studies and the preparation of new proteins. Therefore, the creation of cells with the endogenous capacity to biosynthesize unAAs and their use for new protein synthesis is of great significance for improving the efficiency of producing proteins containing unAAs and expanding the application of genetic code expansion at the level of more comprehensive cell-organisms. By optimizing bio-orthogonal translational components and unAA biosynthetic machinery, we hope to provide powerful tools for the modification of novel proteins containing unAAs. Declarations Acknowledgements The authors thank the Hebei Natural Science Foundation (grant no. 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Xiao H, Schultz PG: At the Interface of Chemical and Biological Synthesis: An Expanded Genetic Code. Cold Spring Harbor Perspectives in Biology 2016, 8 . Young DD, Schultz PG: Playing with the Molecules of Life. ACS Chemical Biology 2018, 13: 854-870. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4824485","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":345606187,"identity":"06a66653-0f86-4a84-ad61-fa178e03d9a2","order_by":0,"name":"Xuanhe Fan","email":"","orcid":"","institution":"Beijing Institute of Technology","correspondingAuthor":false,"prefix":"","firstName":"Xuanhe","middleName":"","lastName":"Fan","suffix":""},{"id":345606188,"identity":"6c5444bd-d366-496c-bb9a-b25733a8160c","order_by":1,"name":"Yumei Liu","email":"","orcid":"","institution":"Beijing Institute of Technology","correspondingAuthor":false,"prefix":"","firstName":"Yumei","middleName":"","lastName":"Liu","suffix":""},{"id":345606189,"identity":"75b982fd-767d-4b6c-8741-7156de3fca65","order_by":2,"name":"Zhenya Chen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6UlEQVRIiWNgGAWjYDACCSBmbACxmI8xJIDoA8RrYUtjSEggTQuPGQMDMVr4Zzcfe/hzh02ewfEz3x48/MEgx3cjgfFzAT5L7hxLN5A8k1ZscCZ3uwHQYcaSNxKYpWfg0WIgkWMmYdh2OHHDgdxtEkAtiRtuJLAx8+DVkv9NIrHtf+KG82+egbTUE6Elh03iYNsBoOFABlBLggEhLRI30swkG9uSE2feeGZukJAmYTjzzMNmaXxa+GckP5P82WaX2Hc++dnDHzY28nzHkw9+xqcFDhQOQGxlgEUTYSBPpLpRMApGwSgYgQAADq5Rf6H59jgAAAAASUVORK5CYII=","orcid":"","institution":"Beijing Institute of Technology","correspondingAuthor":true,"prefix":"","firstName":"Zhenya","middleName":"","lastName":"Chen","suffix":""}],"badges":[],"createdAt":"2024-07-29 21:39:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4824485/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4824485/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":63463461,"identity":"01f16f8e-be3b-4c50-bbff-b8d1f2563001","added_by":"auto","created_at":"2024-08-28 11:55:34","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1767273,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eIn situ\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e insertion of one or two unAAs into sfGFP.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFirstly, the orthogonal translation system for 5-HTP and 4-HiL were constructed and transformed into microbial cells to achieve the insertion of 5-HTP or 4-HiL into sfGFP by feeding 5-HTP or 4-HiL. Then, the biosynthetic pathways of 5-HTP or 4-HiL were constructed in \u003cem\u003eE. coli\u003c/em\u003e to achieve the \u003cem\u003ein situ\u003c/em\u003e insertion of 5-HTP or 4-HiL into sfGFP. Finally, the co-insertion based on the biosynthetic pathways and the orthogonal translation systems of 5-HTP and 4-HiL in the same cells achieved the \u003cem\u003ein situ\u003c/em\u003e co-insertion of 5-HTP and 4-HiL in one sfGFP. GTP, guanosine 5′-triphosphate; XcP4H\u003csup\u003eW179F\u003c/sup\u003e, phenylalanine 4-hydroxylase mutant from \u003cem\u003eX. campestris\u003c/em\u003e; DHMR, dihydromonapterin reductase; PCD, pterin-4\u003cimg width=\"10\" height=\"19\" src=\"file:///C:/Users/nawa05/AppData/Local/Temp/msohtmlclip1/01/clip_image002.png\"/\u003e-carbinolamine dehydratase; MH4, tetrahydromonapterin; MH2, dihydromonapterin; IDO, L-Ile dioxygenase;\u003cem\u003e \u003c/em\u003e\u003cimg width=\"10\" height=\"19\" src=\"file:///C:/Users/nawa05/AppData/Local/Temp/msohtmlclip1/01/clip_image002.png\"/\u003e-KG, \u003cimg width=\"10\" height=\"19\" src=\"file:///C:/Users/nawa05/AppData/Local/Temp/msohtmlclip1/01/clip_image002.png\"/\u003e-Ketoglutaric acid.\u003c/p\u003e","description":"","filename":"OnlineFigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4824485/v1/6cba9da7af42b7527a6063df.png"},{"id":63464359,"identity":"812bd872-7fd5-4e1b-9d94-03a15cbd1416","added_by":"auto","created_at":"2024-08-28 12:03:34","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":946674,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eConstruction and validation of the orthogonal translation system for 5-HTP and 4-HiL.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e the principle of the orthogonal translation system for 5-HTP and 4-HiL and the structure of 5-HTP tRNA WRS-R3-13/-40A and 4-HiL tRNA. In the tRNA clover structure, pink is the anti-codon, yellow indicates the tRNA optimization position. SC:\u003cem\u003e Saccharomyces cerevisiae\u003c/em\u003e. \u003cstrong\u003eb\u003c/strong\u003e The plasmids used in the experiment. Plasmid pLF-26 contained the orthogonal pair for 5-HTP. Plasmid pLF-24 contained the gene for sfGFP \u003csup\u003eN150HTP\u003c/sup\u003e. Plasmid pLF-9 contained the orthogonal pair for 4-HiL. Plasmid pLF-12 contained the gene for sfGFP\u003csup\u003eI153HiL\u003c/sup\u003e. \u003cstrong\u003ec\u003c/strong\u003e The fluorescence change results for the insertion of 5-HTP into sfGFP \u003csup\u003eN150HTP\u003c/sup\u003e and the insertion of 4-HiL into sfGFP\u003csup\u003eI153HiL\u003c/sup\u003e. With (+) or without (-) was used to indicate whether the cells were supplemented with the aaRS/tRNA pair, 0.1 mM IPTG, 1 mM 5-HTP or 4-HiL. \u003cstrong\u003ed\u003c/strong\u003e Mass spectra of sfGFP\u003csup\u003eWT\u003c/sup\u003e, sfGFP\u003csup\u003eN150HTP\u003c/sup\u003e, and sfGFP\u003csup\u003eI153HiL\u003c/sup\u003e expressed from E. coli with exogenously fed 5-HTP and 4-HiL.\u003c/p\u003e","description":"","filename":"OnlineFigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4824485/v1/b26dd2b491b40a30c8dbcd77.png"},{"id":63464360,"identity":"3f1927f5-3ec7-4eac-9d32-57183d680fab","added_by":"auto","created_at":"2024-08-28 12:03:34","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":798811,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBiosynthesis and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein situ\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e insertion of 5-HTP into sfGFP.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e The Reconstitution of XcP4H\u003csup\u003eW179F\u003c/sup\u003e activity in \u003cem\u003eE. coli \u003c/em\u003eand the principle \u003cem\u003ein situ\u003c/em\u003e insertion of 5-HTP into sfGFP\u003csup\u003eN150HTP\u003c/sup\u003e. Subsequently, the 5-HTP tRNA inserted 5-HTP that produced endogenously into sfGFP\u003csup\u003eN150HTP\u003c/sup\u003e. \u003cstrong\u003eb\u003c/strong\u003e bioconversion of L-Trp into 5-HTP using XcP4H\u003csup\u003eW179F\u003c/sup\u003e in \u003cem\u003eE. coli\u003c/em\u003e. The change in Trp concentration, in 5-HTP yield and in cell growth (OD\u003csub\u003e600\u003c/sub\u003e) from glucose over time during the production process. All data were reported as means ± the standard deviation from three independent experiments. Error bars were defined as the standard deviation. \u003cstrong\u003ec\u003c/strong\u003e The role of XcP4H\u003csup\u003eW179F\u003c/sup\u003e, ScTrpRS/tRNA and 10 mM 5-HTP in producing 5-HTP-containing sfGFP. The purification steps of sfGFP and its mutants, with each substance being analyzed by Coomassie Brilliant Blue-stained SDS-PAGE. Marker: prestained SDS–PAGE standards, 14-120kDa, Lane 1: sfGFP\u003csup\u003eWT\u003c/sup\u003e. lanes 2: Feeding and insertion 5-HTP into sfGFP\u003csup\u003eN150HTP\u003c/sup\u003e. Lanes 3: Biosynthesis of 5-HTP and insertion it into sfGFP\u003csup\u003eN150HTP\u003c/sup\u003e. \u003cstrong\u003ed\u003c/strong\u003e Mass spectra of sfGFP\u003csup\u003eN150HTP\u003c/sup\u003e expressed from\u003cem\u003e E. coli\u003c/em\u003e with endogenously produced 5-HTP．\u003c/p\u003e","description":"","filename":"OnlineFigure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4824485/v1/1a272292c27b975fb66fe2fe.png"},{"id":63465070,"identity":"4e9f20df-5d02-462a-9189-b60aabeee5dc","added_by":"auto","created_at":"2024-08-28 12:11:34","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":703887,"visible":true,"origin":"","legend":"\u003cp\u003eBiosynthesis and in situ insertion of 4-HiL into sfGFP.\u003c/p\u003e\n\u003cp\u003ea The Reconstitution of IDO activity in E. coli and the principle in situ insertion of 4-HiL into sfGFPI153HiL. Glucose-6-P, D-Glucose 6-phosphate;PEP, Phosphoenolpyruvate; Pyr, Pyruvic acid ; OAA, Oxalacetic acid ;Asp, L-Aspartic acid ;Asp-P, Aspartic acid phosphate; ASA, L-Aspartate-4-semialdehyde; Hom, L-Homoserine;Hom-P, L-Homoserine phosphate;Thr, Threonine;-KB, Α-ketobutyric acid; AHB, -Acetyl- -hydroxybutyric acid; DMV, - -dihydroxy- - methylpentanoic acid; KMV, -keto- -methylpentanoic acid. b bioconversion of L-Ile into 4-HiL using IDO in E. coli. The change in L-Ile concentration, in 4-HiL yield and in cell growth (OD600) from glucose over time during the production process. All data were reported as means ± the standard deviation from three independent experiments. Error bars were defined as the standard deviation. c The role of IDO, 4-HiL tRNA and 10 mM 4-HiL in producing 4-HiL-containing sfGFP. The purification steps of sfGFP and its mutants, with each substance being analyzed by SDS-PAGE. Marker: prestained SDS–PAGE standards, 14-120kDa, Lane 1: Feeding and insertion 4-HiL into sfGFPI153HiL. Lanes 2: Biosynthesis of 4-HiL and insertion it into sfGFPI153HiL. d Mass spectra of sfGFPI153HiL expressed from E. coli with endogenously produced 4-HiL．\u003c/p\u003e","description":"","filename":"OnlineFigure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4824485/v1/9873a6e491324ce3bad2e1ce.png"},{"id":63463464,"identity":"b3f2ee16-d8bf-496f-90bb-ca0e50ba7035","added_by":"auto","created_at":"2024-08-28 11:55:34","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3481752,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBiosynthesis and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein situ\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e co-insertion of 5-HTP and 4-HiL into sfGFP.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e The principle \u003cem\u003ein situ\u003c/em\u003e co-insertion of 5-HTP and 4-HiL into sfGFP\u003csup\u003eN150HTP/I153HiL\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe role of XcP4H\u003csup\u003eW179F\u003c/sup\u003e , IDO, 5-HTP and 4-HiL in producing 5-HTP-4-HiL-containing sfGFP. \u003cstrong\u003eb\u003c/strong\u003e The purification steps of biosynthesis of 5-HTP and 4-HiL: sfGFP\u003csup\u003eN150HTP/I153HiL\u003c/sup\u003e, with the substance being analyzed by SDS-PAGE. Marker: prestained SDS–PAGE standards, 14-120kDa, Lane 1: biosynthesis of 5-HTP and 4-HiL: sfGFP\u003csup\u003eN150HTP/I153HiL\u003c/sup\u003e. Mass spectra of this sfGFP\u003csup\u003eN150HTP/I153HiL\u003c/sup\u003e. \u003cstrong\u003ec\u003c/strong\u003e The purification steps of feeding 5-HTP and 4-HiL: sfGFP\u003csup\u003eN150HTP/I153HiL\u003c/sup\u003e, with the substance being analyzed by SDS-PAGE. Marker: prestained SDS–PAGE standards, 14-120kDa, Lane 1: feeding 5-HTP and 4-HiL: sfGFP\u003csup\u003eN150HTP/I153HiL\u003c/sup\u003e. Mass spectra of this sfGFP\u003csup\u003eN150HTP/I153HiL\u003c/sup\u003e.\u003c/p\u003e","description":"","filename":"OnlineFigure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4824485/v1/37a0b2f44ffcde2e45a9cd2f.png"},{"id":64240766,"identity":"cfdb49bd-cbfb-4b73-86e5-5ea17b6db984","added_by":"auto","created_at":"2024-09-10 17:53:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7000279,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4824485/v1/ccfb4b2c-4548-427a-a767-7b179ce43de1.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"In situ insertion of one or two hydroxy-rich unnatural amino acid into sfGFP to alter its performance","fulltext":[{"header":"Introduction","content":"\u003cp\u003eProteins are macromolecules that are indispensable for maintaining structure and function of organisms. Native proteins are composed of 20 natural amino acids (nAAs), which could ensure the basic growth and metabolism of organisms[\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. To complete some specific physiological or biochemical reactions in organisms, some nAAs such as L-Ser, L-Thr or L-Arg \u003cem\u003eetc.\u003c/em\u003e in specific proteins need to be added with phosphate, methyl, acetyl, glycosyl or hydroxyl group via a process of post-translational modification. These groups can endow the proteins with unique functions, which in turn activate the corresponding specific reactions[\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Hydroxylation is an important post-translational modification process for proteins. Hydroxyl groups can form hydrogen bonds with nearby amino groups, which helps to maintain the structural stability of proteins[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Moreover, hydroxylation reactions can alter the activity of proteins, affecting their function[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. These post-translational modifications only target a few nAAs in some specific endogenous proteins and require the participation of multiple enzymes and the complex regulation of protein-dependent regulatory network[\u003cspan additionalcitationids=\"CR11 CR12\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eUnnatural amino acids (unAAs), as derivatives of nAAs, have specific functions derived from the redundant side-chain groups, such as hydroxyl, carboxyl, acetyl, phosphate, luminescence, \u003cem\u003eetc\u003c/em\u003e[\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Currently, insertion of unAAs into proteins is an attractive alternative to post-translational modification in order to confer new properties or alter existing properties of proteins, including biocatalytic activity, structure, thermal stability, and substrate specificity[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Compared to the complex process of post-translational modification, the method of inserting unAAs into proteins, such as genetic code expansion (GCE), is much simpler. The GCE works similarly to the natural translation process within the cells, requiring efficient and rigorous translation mechanism, with a \u0026lsquo;codon\u0026rsquo; for each unAA and an orthogonal pair aaRS/tRNA[\u003cspan additionalcitationids=\"CR20 CR21 CR22\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The aminoacyl-tRNA synthetase aaRS loads unAAs on tRNA via aminoacylation reactions. The aminoacylated tRNA carries unAA to complementally pair with the reassigned codon, which in turn inserts the unAA into the extended peptide chain in an orderly manner[\u003cspan additionalcitationids=\"CR25 CR26\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Currently, the commonly used reassigned codons for unAAs are the stop codons UAG, UGA or UAA[\u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eUtilization of microbial cells to express unAA-contained proteins usually requires the feeding of the corresponding unAAs into the culture[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In many cases, microbial cells do not consider the unAAs as essential substances for cells, and do not have unique transporters for transporting unAAs in cells[\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. The cell membrane acts as a natural barrier to unAAs, significantly decreasing the efficiency of unAAs entry cells. Specially, some unAAs with complex functional groups are still unable to enter cells, and currently their insertions are only achieved outside the cells[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Therefore, the \u003cem\u003ein situ\u003c/em\u003e biosynthesis and insertion of unAAs in the same cells is necessary to be developed in order to enhance the utilization efficiency of unAAs.\u003c/p\u003e \u003cp\u003eThe function of protein is often affected by the interaction between multiple amino acid residues, the two or more site mutations of protein can optimize the overall performance of the protein through the synergistic effect between different sites[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. In general, the site-directed mutagenesis method commonly used in protein modification can realize the simultaneous mutagenesis of multiple sites, and the original amino acids can be replaced with other 19 nAAs. However, there is currently a lack of method for the co-insertion of two or more unAAs.\u003c/p\u003e \u003cp\u003eIn this study, the unAA 5-hydroxytryptophan (5-HTP) and 4-hydroxyisoleucine (4-HiL) were inserted into sfGFP, in an attempt to use the functional groups carried by 5-HTP and 4-HiL to endow sfGFP with special properties. The unAA 5-HTP, a precursor for the human neurotransmitter serotonin, has one more hydroxyl group at the 5th carbon atom than L-Trp[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. 4-HiL is a nonproteinogenic amino acid possessing insulinotropic biological activity, which be able to increase glucose-induced release of Insulin, has one more hydroxyl group at the 3rd carbon atom than L-Ile[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Base on the similar hydroxy-rich structural characteristics, these unAAs insertion into sfGFP have high probability of influencing the protein properties. Base on this, specific sites were performed on \u003cem\u003esfgfp\u003c/em\u003e to replace the original codons with stop codon UAG and UGA, in order to insert 5-HTP and 4-HiL at specific sites of sfGFP. After verification, 5-HTP-contained sfGFP mutant, 4-HiL-contained sfGFP mutant and 5-HTP/4-HiL-contained sfGFP mutant were isolated.\u003c/p\u003e \u003cp\u003eFurther, to avoid the barrier effect of the cell membrane on in-out of 5-HTP and 4-HiL, the pathways for both of unAAs biosynthesis were constructed separately within cells, achieving the \u003cem\u003ein situ\u003c/em\u003e insertion of 5-HTP or 4-HiL into sfGFP. Further, we explored the potential of the \u003cem\u003ein situ\u003c/em\u003e insertion of two types of unAAs into proteins. The biosynthesis pathways for 5-HTP and 4-HiL, and the corresponding orthogonal insertion system were co-expression in the same cells, enabling the co-insertion of 5-HTP and 4-HiL into sfGFP. These provided guidance for the insertion of two types of hydroxy-rich unAAs into protein in order to change the characteristics of protein. This work provided an example of co-insertion of unAAs into protein and achieved the \u003cem\u003ein situ\u003c/em\u003e insertion of unAAs into sfGFP, significantly expanding the application scope of unAAs and investigating the possibility of co-inserting two types of unAAs into one protein.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStrains and growth conditions\u003c/h2\u003e \u003cp\u003e \u003cem\u003eEscherichia coli\u003c/em\u003e JCL16, \u003cem\u003eEscherichia coli\u003c/em\u003e JM109, was used for plasmid construction as well as fluorescence screening. The details of the strains and plasmids used in this study are listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. \u003cem\u003eE. coli\u003c/em\u003e strains were grown aerobically at 37 ℃ in LB broth. Ampicillin (100 g/mL), kanamycin (50 g/mL) and spectinomycin (50 g/mL) were added when required.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eStrains and plasmids used in this study\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStrain or plasmid\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGenotype or description\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eReference or Source\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e strains\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eJM109\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003erecA1, endA1, gyrA96, thi-1, hsdR17(rk-mk+), e14\u003csup\u003e\u0026minus;\u003c/sup\u003e(mcrA\u003csup\u003e\u0026minus;\u003c/sup\u003e), supE44, relA1, Δ(lac-proAB)/F\u0026acute; [traD36, proAB\u003csup\u003e+\u003c/sup\u003e, lacI\u003csup\u003eq\u003c/sup\u003e, lacZΔM15]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003elab source\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eJCL16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eΔrhaBADLD\u003csub\u003e78\u003c/sub\u003e [F\u0026rsquo; traD36 proAB lacI\u003csup\u003eq\u003c/sup\u003eZΔM15 Tn10 (Tet\u003csup\u003eR\u003c/sup\u003e)]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003elab source\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePlasmid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epET-28a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eColE1 origin; Kan\u003csup\u003er\u003c/sup\u003e; P\u003csub\u003e\u003cem\u003elacI\u003c/em\u003e\u003c/sub\u003e: \u003cem\u003elacI\u003c/em\u003e; 5369bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003elab source\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epYH1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eColE1 origin; Amp\u003csup\u003er\u003c/sup\u003e; P\u003csub\u003e\u003cem\u003ebmoR\u003c/em\u003e\u003c/sub\u003e: \u003cem\u003ebmoR\u003c/em\u003e; P\u003csub\u003e\u003cem\u003ebmo\u003c/em\u003e\u003c/sub\u003e: \u003cem\u003egfp\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003elab source\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epFL-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eColE1 origin; Amp\u003csup\u003er\u003c/sup\u003e; \u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eL\u003c/em\u003e\u003c/sub\u003e\u003cem\u003elacO\u003c/em\u003e\u003csub\u003e\u003cem\u003e1\u003c/em\u003e\u003c/sub\u003e: \u003cem\u003eXcP4H\u003c/em\u003e; P\u003csub\u003e\u003cem\u003elacI\u003c/em\u003e\u003c/sub\u003e: \u003cem\u003elacI\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epFL-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eColA origin; Cm\u003csup\u003er\u003c/sup\u003e; P\u003csub\u003e\u003cem\u003elpp\u003c/em\u003e\u003c/sub\u003e: \u003cem\u003ePCD\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epFL-3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eColA origin; Amp\u003csup\u003er\u003c/sup\u003e; \u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eL\u003c/em\u003e\u003c/sub\u003e\u003cem\u003elacO\u003c/em\u003e\u003csub\u003e\u003cem\u003e1\u003c/em\u003e\u003c/sub\u003e: \u003cem\u003eXcP4H\u003c/em\u003e-\u003cem\u003ePCD\u003c/em\u003e; P\u003csub\u003e\u003cem\u003elacI\u003c/em\u003e\u003c/sub\u003e: \u003cem\u003elacI\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epFL-4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eColA origin; Amp\u003csup\u003er\u003c/sup\u003e; \u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eL\u003c/em\u003e\u003c/sub\u003e\u003cem\u003elacO\u003c/em\u003e\u003csub\u003e\u003cem\u003e1\u003c/em\u003e\u003c/sub\u003e: \u003cem\u003eXcP4H\u003c/em\u003e(W179F)-\u003cem\u003ePCD\u003c/em\u003e; P\u003csub\u003e\u003cem\u003elacI\u003c/em\u003e\u003c/sub\u003e: \u003cem\u003elacI\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epFL-5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eColE1 origin; Amp\u003csup\u003er\u003c/sup\u003e; \u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eL\u003c/em\u003e\u003c/sub\u003e\u003cem\u003elacO\u003c/em\u003e\u003csub\u003e\u003cem\u003e1\u003c/em\u003e\u003c/sub\u003e: \u003cem\u003eXcP4H\u003c/em\u003e(W179F)-\u003cem\u003ePCD\u003c/em\u003e; P\u003csub\u003e\u003cem\u003elacI\u003c/em\u003e\u003c/sub\u003e: \u003cem\u003elacI\u003c/em\u003e; P\u003csub\u003e\u003cem\u003etac\u003c/em\u003e\u003c/sub\u003e: HRE342(ScW aaRS); P\u003csub\u003e\u003cem\u003eLeuV\u003c/em\u003e\u003c/sub\u003e: ScW tRNA-40A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epFL-9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP15A origin; Cm\u003csup\u003er\u003c/sup\u003e; P\u003csub\u003e\u003cem\u003elpp\u003c/em\u003e\u003c/sub\u003e: 4-Hil tRNA; ivbL; P\u003csub\u003e\u003cem\u003elacI\u003c/em\u003e\u003c/sub\u003e: \u003cem\u003elacI\u003c/em\u003e; \u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eL\u003c/em\u003e\u003c/sub\u003e\u003cem\u003elacO\u003c/em\u003e\u003csub\u003e\u003cem\u003e1\u003c/em\u003e\u003c/sub\u003e: GFP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epLF-12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP15A origin; Spec\u003csup\u003er\u003c/sup\u003e; P\u003csub\u003e\u003cem\u003ebmoR\u003c/em\u003e\u003c/sub\u003e: \u003cem\u003esfGFP\u003c/em\u003e(AAT153TAG)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epLF-13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP15A origin; Spec\u003csup\u003er\u003c/sup\u003e; P\u003csub\u003e\u003cem\u003ebmoR\u003c/em\u003e\u003c/sub\u003e: \u003cem\u003esfGFP\u003c/em\u003e(AAT153TAG/ATT150TGA)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epLF-23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP15A origin; Spec\u003csup\u003er\u003c/sup\u003e; P\u003csub\u003e\u003cem\u003ebmoR\u003c/em\u003e\u003c/sub\u003e: \u003cem\u003esfGFP\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epFL-24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP15A origin; Spec\u003csup\u003er\u003c/sup\u003e; P\u003csub\u003e\u003cem\u003ebmoR\u003c/em\u003e\u003c/sub\u003e: \u003cem\u003esfGFP\u003c/em\u003e(AAT150TAG)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epLF-25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP15A origin; Spec\u003csup\u003er\u003c/sup\u003e; P\u003csub\u003e\u003cem\u003ebmoR\u003c/em\u003e\u003c/sub\u003e: \u003cem\u003esfGFP\u003c/em\u003e(AAT150TGG)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epLF-26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eColE1 origin; Amp\u003csup\u003er\u003c/sup\u003e; P\u003csub\u003e\u003cem\u003etac\u003c/em\u003e\u003c/sub\u003e: HRE342(ScW aaRS); P\u003csub\u003e\u003cem\u003eLeuV\u003c/em\u003e\u003c/sub\u003e: ScW tRNA-40A; P\u003csub\u003e\u003cem\u003elacI\u003c/em\u003e\u003c/sub\u003e: \u003cem\u003elacI\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003eNote: Tet, Tetracycline; Amp, Ampicilli; Cm, Chloramphenicol; Kan, Kanamycin; Spec, spectacularinomycin; r, resistance.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eMedia, and materials\u003c/h2\u003e \u003cp\u003eLB medium (10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl) with a pH of 7.0 was used for strain incubation. M9 medium (6 g/L Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, 3 g/L KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 1 g/L NH\u003csub\u003e4\u003c/sub\u003eCl, and 0.5 g/L NaCl, 1 mM MgSO\u003csub\u003e4\u003c/sub\u003e, 0.1 mM CaCl\u003csub\u003e2\u003c/sub\u003e, 10 mg/L VB\u003csub\u003e1\u003c/sub\u003e, 4 g/L yeast extract, and 40 g/L glucose) with a pH of 7.0 was used for the fermentation experiment to produce 5-HTP and 4-HiL. GMML medium (6.78 g/L NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 3.00g/L KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 0.5g/L NaCl, 1.0g/L NH\u003csub\u003e4\u003c/sub\u003eCl, 1mmol/L MgSO\u003csub\u003e4\u003c/sub\u003e, 0.1 mmol/L CaCl\u003csub\u003e2\u003c/sub\u003e, 0.3 mmol/L L-Leucine solution, 1% glycerin) with a pH of 7.0 was used for Validation of 5-HTP and 4-HiL orthogonal pair.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePlasmid construction\u003c/h3\u003e\n\u003cp\u003eThe \u003cem\u003elacI\u003c/em\u003e fragment was amplified from plasmid pET-28a and inserted into the backbone of pYH1 (ColE1-amp) by Gibson assembly, generating the high-copy plasmid pLF-26 which could express the exogenous 5-HTP orthogonal system. A pair of upstream and downstream primers containing the anticodon of 4-HiL tRNA were designed, with Circular plasmid PCR directed-site mutation technique, the plasmid template containing L-Ile tRNA was amplified to generate the plasmid pLF-9 which could express the exogenous 4-HiL orthogonal system. To achieve the expression of XcP4H\u003csup\u003eW179F\u003c/sup\u003e in \u003cem\u003eE. coli\u003c/em\u003e, its wild type gene \u003cem\u003ephhA\u003c/em\u003e was amplified from the genomic DNA of \u003cem\u003eX. Campestris\u003c/em\u003e, and then L-Trp at position 179th of the gene was replaced with L-Phe. Thus, the plasmid pLF-4 was cloned under the control of IPTG inducible promoter \u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eL\u003c/em\u003e\u003c/sub\u003e\u003cem\u003elacO\u003c/em\u003e\u003csub\u003e\u003cem\u003e1\u003c/em\u003e\u003c/sub\u003e. We synthesized PCD, encoded by \u003cem\u003ephhB\u003c/em\u003e by OE-PCR and inserted it into the backbone of pLF-4 (\u003cem\u003eLacI\u003c/em\u003e-\u003cem\u003eHRE342\u003c/em\u003e-\u003cem\u003eAmp\u003c/em\u003e-\u003cem\u003ephha\u003c/em\u003e) by Gibson assembly to generate a high-copy plasmid pLF-5. Gene \u003cem\u003eido\u003c/em\u003e was synthesized by OE-PCR and inserted into the backbone of pYH-1 (LacI-Amp) by Gibson assembly to generate high-copy production plasmid pLF-6 which could express 4-HiL. To detect the effect of amino acid insertion on the expression of the fluorescent protein, directed-cite circular PCR of the wild type sfGFP plasmid pLF-23 was performed to obtain the sfGFP\u003csup\u003eN150HTP\u003c/sup\u003e plasmid pLF-8, the sfGFP\u003csup\u003eI153HiL\u003c/sup\u003e plasmid pLF-12, and the sfGFP\u003csup\u003eN150HTP/I153HiL\u003c/sup\u003e plasmid pLF-13.\u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eUHPLC analysis\u003c/h2\u003e \u003cp\u003eIn the determination of 5-HTP or yield, L-Trp and 5-HTP were used as standards. Both the standards and samples were quantified by UHPLC (Agilent Technologies 1290 Infinity II) equipped with a reverse phase column (Agilent ZORBAX SB-C18, 5 \u0026micro;m, 4.6\u0026times;250 mm). methanol and water (adding 0.2% TFA) were used as mobile phase, the flow rate was 1.0 mL/min, the column temperature was 30 ℃, and the detection wavelength was 276 nm. By gradient elution, the methanol increased from 5\u0026ndash;30% in 0-15min. Methanol increased from 30\u0026ndash;100% in 15-16min. The methanol remained 100% for 16\u0026ndash;18 min. 18\u0026ndash;19 min, methanol decreased from 100\u0026ndash;5%. The methanol remained at 5% for 19\u0026ndash;21 min. In the determination of 4-HiL yield, L-Ile and 4-HiL were used as standards. The detection wavelengths were 210 nm, 250 nm, and 395 nm. The UHPLC detection methods were the same as those for 5-HTP.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eSfGFP protein expression\u003c/h2\u003e \u003cp\u003eSingle colonies of strain JCL16 harboring pLF-24, pLF-12, or pLF-13 were cultivated in 4 mL LB medium containing the appropriate antibiotic at 37 ℃ at 220 rpm for 8 hours. Then, 1 mL culture was inoculated into 100 mL M9 Y and cultivated at 37 ℃ and 220 rpm. When the OD\u003csub\u003e600\u003c/sub\u003e value reached 0.8, IPTG was added to the culture at a final concentration of 0.5 mM, and the sfGFP expression was induced at 30 ℃ and 220 rpm for 6 hours. After expression, cells were collected and resuspended in Tris-HCl buffer containing 20 mM imidazole, followed by ultrasonic treatment to obtain the crude enzyme extract.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eFluorescence assay\u003c/h2\u003e \u003cp\u003eFluorescence detection experiments. Single colonies were pre-inoculated into 5 mL LB medium containing antibiotics and incubated overnight at 37℃. Then, 50 \u0026micro;L of the seed culture was added to 5 mL of fresh LB medium containing antibiotics and 1 mM IPTG. Afterward, that culture was placed in the incubator at 37 ℃ for 24 h. Samples were taken at 24 h, 48 h, and 72 h, and sfGFP fluorescence and OD600 values were detected by microplate reader (BioTek Cytation 3). The sfGFP fluorescence intensity was measured using an excitation wavelength of 488 nm and an emission wavelength of 510 nm. The sfGFP fluorescence values were normalized to sfGFP/OD\u003csub\u003e600\u003c/sub\u003e and the background fluorescence of the medium was subtracted.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003ePurification of different sfGFP proteins\u003c/h2\u003e \u003cp\u003eAfter induction (IPTG concentration was 0.5 mmol/L), 1L strain was collected by centrifugation at 5000 r/min, and 20 mL storage buffer (20 mmol/L Tris-HCl, pH\u0026thinsp;=\u0026thinsp;8.0). The thalli were broken by ultrasonic wall-breaking instrument with 40% power until the bacterial solution became relatively clear, centrifuged at 12000 r/min for 30 mins, and the supernatant of the bacterial solution was retained for purification by Ni\u003csup\u003e2+\u003c/sup\u003e-NTA affinity chromatography. After the sample was loaded, 50 mmol/L and 75 mmol/L imidazole were used to wash away the impurity protein, and then 200 mmol/L imidazole was used to elute, and then SDS-PAGE was used to detect the bacterial supernatant, breakthrough liquid, 40 and 200 mmol/L imidazole eluent. Samples were finally concentrated using a 10 kDa molecular weight cut-off Microcon centrifugal filter (EMD Millipore).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eMass spectrometry\u003c/h2\u003e \u003cp\u003eMatrix-Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS) was performed using the model Autoflex Speed. The detection mode was set to positive ion detection, in linear mode. The saturated solution of 3,5-dimethoxy-4-hydroxycinnamic acid (SA) was prepared: trifluoroacetic acid (0.1%), ultra-pure water (70%), and acetonitrile (30%). For the sample application, the SA was mixed with the sample in a 1:1 ratio, and 1 \u0026micro;L was applied onto the target plate. The laser power was set to 100%, and the voltage was set to 3100 V for detection.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eConstruction of 5-HTP or 4-HiL orthogonal pair\u003c/h2\u003e \u003cp\u003eTo insert the unAA 5-HTP into the sfGFP, we utilized the orthogonal translation system-genetic code expansion approach (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). The orthogonal translation system could recognize the aminoacyl-tRNA synthetase (aaRS)/tRNA orthogonal pair of 5-HTP, while don\u0026rsquo;t cross-react with the orthogonal system of 20 endogenous natural amino acids. The orthogonality of 5-HTP orthogonal pair WRS-R3-13/\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\text{t}\\text{R}\\text{N}\\text{A}}_{\\text{C}\\text{U}\\text{A}}^{\\text{T}\\text{r}\\text{p}}\\)\u003c/span\u003e\u003c/span\u003e-40A, which came from native \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e L-Trp orthogonal pair, was further modified to improve its orthogonality because the source of the orthogonal pair was far away from the host phylogeny[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. In this 5-HTP orthogonal pair, the aaRS WRS-R3-13 had the mutations of T107C, P254T and C255A as compared to the native L-Trp aaRS, while the \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\text{t}\\text{R}\\text{N}\\text{A}}_{\\text{C}\\text{U}\\text{A}}^{\\text{T}\\text{r}\\text{p}}\\)\u003c/span\u003e\u003c/span\u003e-40A had an anticodon of CUA to complementary pair with the stop codon UAG, and nine base mutations as compared to the native L-Trp tRNA. The lacI fragment was amplified from plasmid pET-28a and inserted into the backbone of pYH1 (ColE1-amp) by Gibson assembly, generating the high-copy plasmid pLF-26 which could express the exogenous 5-HTP orthogonal system. The gene sequence information of \u003cem\u003esfgfp\u003c/em\u003e was obtained from the plasmid pBad-sfGFP (Addgene plasmid # 85482)[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], and the \u003cem\u003esfgfp\u003c/em\u003e fragment was obtained by primers synthesis method. Finally, the plasmid pLF-23 and the plasmid pLF-24 that contained the gene of sfGFP\u003csup\u003eN150HTP\u003c/sup\u003e by replacing the L-Asn codon AAU at 150th of sfGFP in plasmid pLF-23 with UAG were both generated (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eLike the principle of 5-HTP orthogonal pair WRS-R3-13/\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\text{t}\\text{R}\\text{N}\\text{A}}_{\\text{C}\\text{U}\\text{A}}^{\\text{T}\\text{r}\\text{p}}\\)\u003c/span\u003e\u003c/span\u003e-40A, the orthogonal pair of 4-HiL in \u003cem\u003eE. coli\u003c/em\u003e was constructed in order to accurately insert 4-HiL into the specific site of sfGFP in order to change its protein characteristics (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). The 4-HiL orthogonal pair used in this study was derived from the native IleRS/L-ile tRNA orthogonal pair of \u003cem\u003eE. coli\u003c/em\u003e. Because the source of the L-Ile orthogonal pair was the same as the host system, only the anticodon of the tRNA was modified. The 4-HiL tRNA had a CUA anticodon complementary to the stop codon UGA at position 153th of sfGFP. A pair of upstream and downstream primers containing the anticodon of 4-HiL tRNA were designed, with circular plasmid PCR directed-site mutation technique, the plasmid template containing L-Ile tRNA was amplified to generate the plasmid pLF-9 which could express the exogenous 4-HiL orthogonal system(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). In Addition, plasmid pLF-12 that contained the gene of sfGFP\u003csup\u003eI153HiL\u003c/sup\u003e by replacing the L-Ile codon AUU at 153th of sfGFP in plasmid pLF-23 with UGA as generated by PCR circular amplification (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eValidation of 5-HTP orthogonal pair\u003c/h2\u003e \u003cp\u003ePlasmid pLF-26 was individually co-transformed with pLF-23 and pLF-24 into \u003cem\u003eE. coli\u003c/em\u003e XL10-Gold to form the control strain and the experimental strain, respectively. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec, for the control strain, sfGFP could be normally expressed, showing a sfGFP/OD\u003csub\u003e600\u003c/sub\u003e value of 491\u0026thinsp;\u0026plusmn;\u0026thinsp;17.9 within 24 hours. For the experimental strain, the 5-HTP orthogonal pair could not be expressed in the absence of inducer isopropyl β-D-1-thiogalactopyranoside (IPTG) feeding, and correspondingly, the significant fluorescence could not be detected regardless of whether 5-HTP was added. This result illustrated that the tRNA lacking the anticodon to pair with the stop codon UAG, could not enable the insertion of Trp or 5-HTP into sfGFP\u003csup\u003eN150HTP\u003c/sup\u003e. The cells could express 5-HTP orthogonal pair in the presence of IPTG feeding and could not express sfGFP\u003csup\u003eN150HTP\u003c/sup\u003e in the absence of 5-HTP feeding, suggesting aaRS WRS-R3-13 could not load the intercellular L-Trp on \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\text{t}\\text{R}\\text{N}\\text{A}}_{\\text{C}\\text{U}\\text{A}}^{\\text{T}\\text{r}\\text{p}}\\)\u003c/span\u003e\u003c/span\u003e-40A. The cells could only express sfGFP\u003csup\u003eN150HTP\u003c/sup\u003e in the presence of IPTG and 5-HTP feeding, showing a sfGFP/OD\u003csub\u003e600\u003c/sub\u003e value of 303\u0026thinsp;\u0026plusmn;\u0026thinsp;3.50, 60% lower than that of the control strain. To confirm the absolute loading of 5-HTP orthogonal pair on 5-HTP, we mutated the L-Asn codon AAU at 150th of sfGFP in pLF-23 to L-Trp codon TGG, forming plasmid pLF-25. \u003cem\u003eE. coli\u003c/em\u003e XL10-Gold harboring the pLF-25 could normally express sfGFP\u003csup\u003eN150W\u003c/sup\u003e in the presence or absence of L-Trp feeding, showing comparable sfGFP/OD\u003csub\u003e600\u003c/sub\u003e values to the control strain \u003cem\u003eE. coli\u003c/em\u003e XL10-Gold harboring the pLF-23. These results indicated that substitution of L-Asn to L-Trp at 150th did not significantly change the fluorescence intensity of sfGFP, while substitution of L-Asn to 5-HTP at 150th significantly decreased the fluorescence intensity, demonstrating the 5-HTP orthogonal pair rigorously recognized 5-HTP and 5-HTP was then precisely inserted into the 150th of sfGFP to produce sfGFP\u003csup\u003eN150HTP\u003c/sup\u003e. Subsequently, sfGFP and sfGFP\u003csup\u003eN150HTP\u003c/sup\u003e were purified and identified using the MALDI-MS mass spectrometer. The generated results showed that the molecular weight of sfGFP\u003csup\u003eN150HTP\u003c/sup\u003e was 27934, 88 more than that of sfGFP (27847), consistent with the theoretical molecular weight. The purity of sfGFP\u003csup\u003eN150HTP\u003c/sup\u003e was almost 100%, indicating the experimental strain could precisely insert 5-HTP into 150th of sfGFP to produce sfGFP\u003csup\u003eN150HTP\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo confirm the absolute loading of 5-HTP orthogonal pair on 5-HTP, the L-Asn codon AAU at 150th of sfGFP in pLF-23 was mutated to L-Trp codon TGG, forming plasmid pLF-25. \u003cem\u003eE. coli\u003c/em\u003e XL10-Gold harboring the pLF-25 could normally express sfGFP\u003csup\u003eN150HTP\u003c/sup\u003e. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec, in the presence or absence of 1 mM L-Trp feeding, showing comparable sfGFP/OD\u003csub\u003e600\u003c/sub\u003e values to the control strain \u003cem\u003eE. coli\u003c/em\u003e XL10-Gold harboring the pLF-23. These results indicated that substitution of L-Asn to L-Trp at 150th did not significantly change the fluorescence intensity of sfGFP, while substitution of L-Asn to 5-HTP at 150th significantly decreased the fluorescence intensity, demonstrating the 5-HTP orthogonal pair rigorously recognized 5-HTP and 5-HTP was then precisely inserted into the 150th of sfGFP to produce sfGFP\u003csup\u003eN150HTP\u003c/sup\u003e. Subsequently, sfGFP and sfGFP\u003csup\u003eN150HTP\u003c/sup\u003e were purified and identified using the Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). The generated results showed that the molecular weight of sfGFP\u003csup\u003eN150HTP\u003c/sup\u003e was 27934, 88 more than that of sfGFP (27847), consistent with the theoretical molecular weight. The purity of sfGFP\u003csup\u003eN150HTP\u003c/sup\u003e was almost 100%, indicating the experimental strain could precisely insert 5-HTP into 150th of sfGFP to produce sfGFP\u003csup\u003eN150HTP\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eValidation of 4-HiL orthogonal pair\u003c/h2\u003e \u003cp\u003eIn order to validate the 4-HiL orthogonal translation system, the plasmid pLF-9 was co-transformed with pLF-23 and pLF-12 into \u003cem\u003eE. coli\u003c/em\u003e JCL16 to form the control strain and the experimental strain, respectively. As shown in the Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec, for the control strain, sFGFP\u003csup\u003eWT\u003c/sup\u003e was normally expressed without the inducer IPTG or 4-HiL feeding, showing a sfGFP/OD\u003csub\u003e600\u003c/sub\u003e value of 863\u0026thinsp;\u0026plusmn;\u0026thinsp;62 within 24 hours. The IPTG feeding could induce the expression of 4-HiL tRNA, when the IPTG was added and 4-HiL was not added, there was no significant change in the fluorescence value of sfGFP. The results showed that the value of sfGFP/OD\u003csub\u003e600\u003c/sub\u003e was 884\u0026thinsp;\u0026plusmn;\u0026thinsp;62. These results indicated that sfGFP\u003csup\u003eWT\u003c/sup\u003e could not insert 4-HiL in the absence of 4-HiL. After the feeding of 1 mM 4-HiL, whether the IPTG was added to induce the expression of 4-HiL, the sfGFP/OD\u003csub\u003e600\u003c/sub\u003e (288\u0026thinsp;\u0026plusmn;\u0026thinsp;6.00) was significantly decreased compared with the value of the previous two control groups. These results might be due to the fact that the structure of 4-HiL has only one more hydroxyl functional group than L-Ile, and the endogenous L-Ile tRNA in this \u003cem\u003eE. coli\u003c/em\u003e JCL16 strain could recognize 4-HiL and insert it into the 153rd of sfGFP, thus changing the characteristics of sfGFP. For the experimental strains, the sfGFP/OD\u003csub\u003e600\u003c/sub\u003e values of the sfGFP mutants in the absence of 4-HiL, with or without the addition of the inducer IPTG (352\u0026thinsp;\u0026plusmn;\u0026thinsp;1.83 or 340\u0026thinsp;\u0026plusmn;\u0026thinsp;7.83), were much lower than those of normal expression, but still higher than those of sfGFP\u003csup\u003eI153HiL\u003c/sup\u003e that could be inserted into 4-HiL. These results indicated that the stop coden UGA and the absence of the amino acid L-Ile at position 153rd of sfGFP resulted in premature termination of sfGFP expression. With the addition of IPTG, the cells could normally express the 4-HiL orthogonal pair, and the experimental group with the addition of 1 mM 4-HiL showed lower fluorescence than the group without the addition of 4-HiL, showing a sfGFP/OD\u003csub\u003e600\u003c/sub\u003e value of 217\u0026thinsp;\u0026plusmn;\u0026thinsp;2.83, which was 75% lower than that of the control strain. These results indicated that 4-HiL could be successfully inserted into sfGFP\u003csup\u003eI153Hil\u003c/sup\u003e in the presence of both the 4-HiL tRNA and 4-HiL.\u003c/p\u003e \u003cp\u003eSubsequently, sfGFP\u003csup\u003eI153HIL\u003c/sup\u003e was purified and identified using the MALDI-MS (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). As a result of the generation, the molecular weight of sFGFP\u003csup\u003eI153Hil\u003c/sup\u003e was 27863, which was 16 greater than the molecular weight of sfGFP\u003csup\u003eWT\u003c/sup\u003e (27847) and was consistent with the theoretical molecular weight. The lower peak in the MALDI-MS results might be because the 4-HiL orthogonal pair was derived from the L-Ile orthogonal pair, and the competitive binding of the two tRNAs to the IleRs resulted in a decrease in the insertion efficiency of the 4-HiL. Moreover, the purity of sfGFP\u003csup\u003eI153HiL\u003c/sup\u003e was almost 100%, indicating that the experimental strain was able to precisely insert 4-HiL into the 153rd of sfGFP to produce sfGFP\u003csup\u003eI153HiL\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eBiosynthesis and\u003c/b\u003e \u003cb\u003ein situ\u003c/b\u003e \u003cb\u003einsertion of 5-HTP into sfGFP\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAfter verifying that exogenously added 5-HTP could be inserted into sfGFP\u003csup\u003eN150HTP\u003c/sup\u003e, we constructed the pathway for the biosynthesis of 5-HTP in \u003cem\u003eE. coli\u003c/em\u003e, with the aim of avoiding the barrier effect of the cell membrane on the entry and exit of 5-HTP (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). In this pathway, the enzyme XcP4H\u003csup\u003eW179F\u003c/sup\u003e was used to catalyze the production of 5-HTP from L-Trp, with Pterin 4A-methanolamine dehydratase (PCD) as the cofactor[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. To achieve the expression of XcP4H\u003csup\u003eW179F\u003c/sup\u003e in \u003cem\u003eE. coli\u003c/em\u003e, its wild type gene \u003cem\u003ephhA\u003c/em\u003e was amplified from the genomic DNA of \u003cem\u003eX. Campestris\u003c/em\u003e, and then L-Trp at position 179th of the gene was replaced with L-Phe. Thus, the plasmid pLF-4 was cloned under the control of IPTG inducible promoter \u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eL\u003c/em\u003e\u003c/sub\u003e\u003cem\u003elacO\u003c/em\u003e\u003csub\u003e\u003cem\u003e1\u003c/em\u003e\u003c/sub\u003e. In addition, previous experimental evidence suggested that bacterial P4Hs might utilize tetrahydromonopterin (MH4) as a natural pterin coenzyme, while endogenous MH4 in \u003cem\u003eE. coli\u003c/em\u003e might be used as the coenzyme[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. PCD, encoded by \u003cem\u003ephhB\u003c/em\u003e, was responsible for the regeneration of dihydromonopterin (MH2), which could be further reduced to MH4. Therefore, we synthesized \u003cem\u003ephhB\u003c/em\u003e by OE-PCR and inserted it into the backbone of pLF-4 (\u003cem\u003eLacI\u003c/em\u003e-\u003cem\u003eHRE342\u003c/em\u003e-\u003cem\u003eAmp\u003c/em\u003e-\u003cem\u003ephha\u003c/em\u003e) by Gibson assembly to generate a high-copy plasmid pLF-5. Subsequently, we transformed pLF-5 into \u003cem\u003eE. coli\u003c/em\u003e JCL16 to enable endogenous production of 5-HTP.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs shown in the Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, in the absence of L-Trp feeding, the engineered bacteria produced only 1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.714 mg/L 5-HTP in 48h by using the endogenously accumulated L-Trp as the substrate, while in the presence of 10 mM L-Trp, the engineered bacteria produced 25.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.245 mg/L 5-HTP in 48h. During 24 h, 48 h and 72 h of fermentation, the OD\u003csub\u003e600\u003c/sub\u003e values of exogenous L-Trp and endogenous 5-HTP were the lowest among all the experimental groups, which might be due to the fact that the growth of the bacteria was slowed down because L-Trp in the culture was catalyzed by XcP4H\u003csup\u003eW179F\u003c/sup\u003e to form 5-HTP in time. At the same time, we observed that the color of the culture gradually darkened after 6 hours at 37 ℃, which probably because of the oxidation of 5-HTP and tryptophan under aerobic conditions.\u003c/p\u003e \u003cp\u003eSubsequently, the plasmid pLF-26 that contained the genes of 5-HTP orthogonal pair and the plasmid pLF-8 containing the gene of sfGFP\u003csup\u003eN150HTP\u003c/sup\u003e were co-transformed into \u003cem\u003eE. coli\u003c/em\u003e JCL16 harboring plasmid pLF-4, in order to insert the endogenously biosynthesized 5-HTP into 150th of sfGFP to produce sfGFP\u003csup\u003eN150HTP\u003c/sup\u003e. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec, in the absence of 5-HTP feeding, the sfGFP/OD\u003csub\u003e600\u003c/sub\u003e value of the engineered strain reached 96.6\u0026thinsp;\u0026plusmn;\u0026thinsp;18.3 within 48 hours, 8.1-fold higher than that of the strain without 5-HTP biosynthetic pathway, indicating that the endogenously produced 5-HTP was successfully inserted into 150th of sfGFP. In addition, this value was 0.52-fold lower than that in the presence of 10 mM 5-HTP, which might be due to the low efficiency of 5-HTP biosynthetic pathway, ultimately limiting the expression amount of sfGFP\u003csup\u003eN150HTP\u003c/sup\u003e. We then compared the insertion efficiency of 5-HTP obtained via exogenously addition or endogenously biosynthesis, and the results showed that the insertion efficiency of 5-HTP obtained via endogenously biosynthesis was 54.4-fold higher than that of 5-HTP obtained via exogenously addition.\u003c/p\u003e \u003cp\u003eTo further investigate the specificity of the incorporation of 5-HTP feeding and bio-\u003c/p\u003e \u003cp\u003esynthesized 5-HTP, sfGFP\u003csup\u003eWT\u003c/sup\u003e and sfGFP\u003csup\u003eN150HTP\u003c/sup\u003e containing exogenous or biosynthetic 5-HTP were purified by Ni\u003csup\u003e2+\u003c/sup\u003e-NTA affinity chromatography and characterized by Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). Subsequently, we used the MALDI-MS to verify that the molecular weight of the SFGFP\u003csup\u003eN150HTP\u003c/sup\u003e protein of the biosynthetic 5-HTP was 27935, which was consistent with the theoretical value (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). Meanwhile, the purity was almost 100%.\u003c/p\u003e \u003cp\u003eThese results suggested that the endogenous biosynthesis and \u003cem\u003ein-situ\u003c/em\u003e insertion of 5-HTP in the same cell could significantly improve the utilization efficiency of 5-HTP, but the low biosynthesis efficiency of 5-HTP limited the amount of corresponding proteins. In the future, it is necessary to focus on improving the biosynthesis efficiency of unAAs, including the design of unAAs biosynthetic pathways, the modification of crucial enzymes in pathways, and the selection of optimal chassis hosts.\u003c/p\u003e \u003cp\u003e \u003cb\u003eBiosynthesis and\u003c/b\u003e \u003cb\u003ein situ\u003c/b\u003e \u003cb\u003einsertion of 4-HiL into sfGFP\u003c/b\u003e\u003c/p\u003e \u003cp\u003eLike the effect of the 5-HTP production pathway, after verifying that exogenously added 4-HiL could be inserted into sfGFP\u003csup\u003eI153HiL\u003c/sup\u003e, we constructed the pathway for the biosynthesis of 4-HiL in \u003cem\u003eE. coli\u003c/em\u003e, with the aim of avoiding the barrier effect of the cell membrane on the entry and exit of 4-HiL (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Gene \u003cem\u003eido\u003c/em\u003e was synthesized by OE-PCR and inserted into the backbone of pYH-1 (LacI-Amp) by Gibson assembly to generate high-copy plasmid pLF-6. Subsequently, we transformed pLF-6 into \u003cem\u003eE. coli\u003c/em\u003e JCL16 to enable endogenous production of 4-HiL. As shown in Figure A, in the absence of L-Trp feeding, the engineered strains only produced 47.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.219 mg/L 4-HiL within 48 hours by using the endogenous accumulation of L-Trp as substrate, while in the presence of 10 mM L-Trp feeding, the engineered strains produced 1.12\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\times\\:\\)\u003c/span\u003e\u003c/span\u003e10\u003csup\u003e3\u003c/sup\u003e \u0026plusmn; 0.312 mg/L 4-HiL within 48 hours.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSubsequently, the plasmid pLF-9 that contained the gene of 4-HiL orthogonal pair and the plasmid pLF-12 containing the gene of sfGFP\u003csup\u003eN153HTP\u003c/sup\u003e were co-transformed into \u003cem\u003eE. coli\u003c/em\u003e JCL16 harboring pLF-6, in order to insert the endogenous 4-HiL into 153rd of sfGFP to produce sfGFP\u003csup\u003eI153HiL\u003c/sup\u003e. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, in the absence of 4-HiL feeding, the sfGFP/OD\u003csub\u003e600\u003c/sub\u003e value of the engineered strain reached 540\u0026thinsp;\u0026plusmn;\u0026thinsp;16.3 within 48 hours, 1.41-fold higher than that of the strain without 4-HiL biosynthetic pathway, indicating that the endogenously produced 4-HiL was successfully inserted into 153th of sfGFP. We then compared the insertion efficiency of 4-HiL obtained via exogenously addition or endogenously biosynthesis, and the results showed that the insertion efficiency of 4-HiL obtained via endogenously biosynthesis was 1.89-fold higher than that of 4-HiL obtained via exogenously addition. Subsequently, we purified sfGFP\u003csup\u003eI153HiL\u003c/sup\u003e containing exogenous or biosynthetic 4-HiL by Ni\u003csup\u003e2+\u003c/sup\u003e-NTA affinity chromatography and characterized by SDS-PAGE (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). Moreover, we verified that the molecular weight of the sfGFP\u003csup\u003eI153HiL\u003c/sup\u003e protein of the biosynthetic 4-HiL was 27863 by using MALDI-MS, which was consistent with the theoretical value (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003e \u003cb\u003eCo-insertion and\u003c/b\u003e \u003cb\u003ein situ\u003c/b\u003e \u003cb\u003eco-insertion of two unAAs into sfGFP\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFinally, we explored the possibility of \u003cem\u003ein situ\u003c/em\u003e insertion of two types of unAAs into the same proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). The experiments of co-feeding 5-HTP and 4-HiL and co-synthesizing 5-HTP and 4-HiL to detect the fluorescence of sfGFP were carried out. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb, when 10 mM 5-HTP and 10 mM 4-HiL were added simultaneously, the value of sfGFP/OD\u003csub\u003e600\u003c/sub\u003e was up to 1.13\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\times\\:\\)\u003c/span\u003e\u003c/span\u003e10\u003csup\u003e3\u003c/sup\u003e \u0026plusmn; 131. When 5-HTP or 4-HiL was added alone, the value of sfGFP/OD\u003csub\u003e600\u003c/sub\u003e was higher than that without any of the unAAs, indicating that the exogenous supplementation of 5-HTP and 4-HiL simultaneously inserted sfGFP protein and changed the characteristics of protein. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec, in the case of endogenous co-biosynthesis of 5-HTP and 4-HiL, the value of sfGFP/OD\u003csub\u003e600\u003c/sub\u003e was the highest at 2.26\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\times\\:\\)\u003c/span\u003e\u003c/span\u003e10\u003csup\u003e3\u003c/sup\u003e \u0026plusmn; 254. In addition, the engineered bacteria containing only the 5-HTP biosynthetic pathway or the 4-HiL biosynthetic pathway had a lower sfGFP/OD\u003csub\u003e600\u003c/sub\u003e, but they were 2.1-fold and 1.2-fold separately higher than that of the engineered bacteria not containing any unAA biosynthetic pathway. This result indicated that the biosynthesis of 5-HTP and 4-HiL were successfully co-inserted into sfGFP. Subsequently, we purified SFGFP\u003csup\u003eN150HTP/I153HiL\u003c/sup\u003e containing exogenous (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb) or biosynthetic 5HTP and 4-HiL (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec) by Ni\u003csup\u003e2+\u003c/sup\u003e-NTA affinity chromatography and characterized by SDS-PAG. Moreover, we used the MALDI-MS to verify that the molecular weights of the proteins from the two sources were 27950 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb) and 27950 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec), respectively, which were consistent with the theoretical values. The purity was close to 100%.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn conclusion, we have achieved the insertion of 5-HTP and 4-HiL into sfGFP, using the hydroxyl functional groups carried by these unAAs to endow sfGFP with special properties. In addition, in order to avoid the barrier effect of cell membrane on the entry and exit of unAAs, we have constructed two pathways of unAAs biosynthesis in cells and have realized the \u003cem\u003ein situ\u003c/em\u003e insertion of 5-HTP or 4-HiL into sFGFP. More importantly, we have also explored the possibility of \u003cem\u003ein situ\u003c/em\u003e co-insertion of two types of unAAs into proteins, which provides guidance for inserting two hydroxyl-rich unAAs into proteins to modify the properties of proteins. In subsequent research, efforts will focus on enhancing the binding efficiency between 4-HiL tRNA and IleRs by optimizing the 4-HiL orthogonal pair. This refinement is expected to markedly increase the expression of sfGFP\u003csup\u003eI153HiL\u003c/sup\u003e and enhance the peak intensity detected by MALDI-MS. This study has provided an example of co-insertion of unAAs into proteins, and realized \u003cem\u003ein situ\u003c/em\u003e insertion of unAAs into sfGFP, significantly expanded the application scope of unAAs, and explored the possibility of co-insertion of two types of unAAs into one protein.\u003c/p\u003e \u003cp\u003eAt present, most unAAs are obtained by chemical synthesis. However, the steps of chemical synthesis are complex and the generated unAAs are usually racemic mixtures of L- and D-type, bringing challenges to the subsequent separation and purification[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Specially, unAAs with large molecular weight produced by chemical synthesis have obstacles to enter cells, and are difficult to be taken up by cells, ultimately affecting the insertion efficiency of unAAs in target proteins. In addition, due to the limitations of key technologies such as the screening and preparation of catalysts, the construction of synthetic routes and the regulation of catalytic processes, the chemical synthesis of unAAs has high technical barriers and production costs[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Therefore, the development of green and efficient method for unAAs synthesis to replace the chemical synthesis is important. So far, the microbial-based metabolic engineering emerges as an effective method for the biosynthesis of unAAs.\u003c/p\u003e \u003cp\u003eMetabolic engineering for unAAs biosynthesis usually uses cheap glucose as the starting carbon source, and the corresponding biosynthesis process is inexpensive and environmentally friendly[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. The efficient biosynthesis of unAAs in microbial cells can be achieved by constructing the artificial biosynthesis pathway of unAAs, designing and modifying key enzymes, regulating precursor biosynthesis, knocking out the competing pathway, constructing the cofactor regeneration system, and intelligently regulating fermentation process[\u003cspan additionalcitationids=\"CR49\" citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. For example, Mora-Villalobos et al. [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]used sequence analysis, phylogenetic analysis and functional difference analysis tools to predict, screen and design the site-directed mutations for substrate specific sites of aromatic amino acid hydroxylase (\u003cem\u003eCt\u003c/em\u003eAAAH) from \u003cem\u003eCupriavidus taiwanensis\u003c/em\u003e. The substrate preference of \u003cem\u003eCt\u003c/em\u003eAAAH was transferred from L-Phe to L-Trp, achieving the formation of 5-HTP. In addition, the synthesis efficiency and yield of unAAs, such as enzymes involved in unAAs biosynthetic pathway, and enhancement the metabolic flux of unAAs biosynthetic pathways.\u003c/p\u003e \u003cp\u003eHowever, only a few of unAAs biosynthetic pathways have been validated in microbial cells, and the majority of unAAs biosynthetic pathways remain unclear[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Therefore, how to design and create artificial unAAs biosynthetic pathways is still a challenge to be solved. At present, advanced bioinformation tools or biosynthesis simulation tools can be used to mine unknown biosynthetic pathways and enzymes in nature, providing more components for the design of artificial unAAs biosynthetic pathways. Some special unAAs are biosynthetically dependent on cofactors, which significantly increases the cost of biosynthesis. Building an effective cofactor recycling pathway can significantly improve the supply of cofactors. In addition, some biotoxic unAAs can inhibit the growth of chassis hosts, limiting the high-level biosynthesis of unAAs. Therefore, screening or engineering chassis hosts with stronger tolerance to unAAs can significantly increase the biosynthesis efficiency of unAAs. At present, advances in synthetic and computational biology techniques provide effective tools for the design of high efficiency unAAs biosynthesis strategies. In the future, the precise design of the unAAs biosynthetic pathway can be accomplished by advanced bioinformatics or biosynthetic simulation tools. In addition, directed design and screening of chassis with high tolerance to specific unAAs to increase compatibility between microbial cells and unAAs biosynthetic pathways will significantly improve the efficiency of metabolic engineering production of unAAs.\u003c/p\u003e \u003cp\u003eAt present, the structure and function of proteins are probed in the base of that the approximately 300 unAAs has been added to proteins[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan additionalcitationids=\"CR54 CR55 CR56\" citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Although this technique is widely used, incorporation of these unAAs into proteins requires exogenous feeding of unAAs as well as their successful uptake into cells, a process that significantly limits the application of this method to protein modification studies and the preparation of new proteins. Therefore, the creation of cells with the endogenous capacity to biosynthesize unAAs and their use for new protein synthesis is of great significance for improving the efficiency of producing proteins containing unAAs and expanding the application of genetic code expansion at the level of more comprehensive cell-organisms. By optimizing bio-orthogonal translational components and unAA biosynthetic machinery, we hope to provide powerful tools for the modification of novel proteins containing unAAs.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank the Hebei Natural Science Foundation (grant no. B2023105008), and the Biological \u0026amp; Medical Engineering Core Facilities (Beijing Institute of Technology)\u0026nbsp;for providing advanced equipment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompliance with ethical standards\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis article does not contain any studies with human participants or animals performed by any of the authors.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAcevedo‐Rocha CG, Budisa N: \u003cstrong\u003eXenomicrobiology: a roadmap for genetic code engineering.\u003c/strong\u003e \u003cem\u003eMicrobial Biotechnology \u003c/em\u003e2016, 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protein engineering, in situ insertion, signal molecule","lastPublishedDoi":"10.21203/rs.3.rs-4824485/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4824485/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eUnnatural amino acids (unAAs) possess unique properties owing to their distinct functional groups, and their insertion into proteins can significantly alter protein function and properties. Currently, the predominant method for inserting unAAs into proteins is through genetic code expansion (GCE), which mimics the natural translation process within cells and necessitates the exogenous supplementation of unAAs. However, in many instances, microbial cells do not recognize unAAs as essential nutrients and lack specific transporters for their uptake across the cell membrane, thereby greatly reducing their insertion efficiency. To address this issue, our study developed an \u003cem\u003ein situ\u003c/em\u003einsertion method for enhancing the efficiency of unAAs insertion into proteins and further explored the feasibility of simultaneously inserting two different unAAs into one protein. Firstly, the orthogonal translation system for hydroxy-rich unAAs 5-hydroxytryptophan (5-HTP) or 4-hydroxyisoleucine (4-HiL) were constructed and then transformed into microbial cells to achieve the insertion of 5-HTP or 4-HiL into sfGFP by feeding 5-HTP or 4-HiL. Subsequently, the biosynthetic pathways of 5-HTP or 4-HiL were constructed in \u003cem\u003eE. coli\u003c/em\u003e which contained the corresponding orthogonal translation system, resulting in the \u003cem\u003ein situ\u003c/em\u003e insertion of 5-HTP or 4-HiL into sfGFP.Further, we developed a co-insertion method based on codons UGA and UAG. Introduction of the biosynthetic pathways and the orthogonal translation systems of 5-HTP and 4-HiL in the same cells achieved the \u003cem\u003ein situ\u003c/em\u003e co-insertion of 5-HTP and 4-HiL in one sfGFP. This work provided a representative example for\u003cem\u003e in situ\u003c/em\u003einsertion of unAAs into protein to increase the insertion efficiency, and explored the possibility of co-inserting two types of unAAs into one protein.\u003c/p\u003e","manuscriptTitle":"In situ insertion of one or two hydroxy-rich unnatural amino acid into sfGFP to alter its performance","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-28 11:55:29","doi":"10.21203/rs.3.rs-4824485/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2ef8fd04-c981-447d-a958-5d775d75ef58","owner":[],"postedDate":"August 28th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-09-10T17:45:10+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-28 11:55:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4824485","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4824485","identity":"rs-4824485","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}