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

doi:10.21203/rs.3.rs-4824485/v1

Download PDF

Research Article

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

https://doi.org/10.21203/rs.3.rs-4824485/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

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 situinsertion 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 situinsertion 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

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.

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	ΔrhaBADLD₇₈ [F’ traD36 proAB lacI^qZΔM15 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_LlacO₁: XcP4H; P_lacI: lacI	This study
pFL-2	ColA origin; Cm^r; P_lpp: PCD	This study
pFL-3	ColA origin; Amp^r; P_LlacO₁: XcP4H-PCD; P_lacI: lacI	This study
pFL-4	ColA origin; Amp^r; P_LlacO₁: XcP4H(W179F)-PCD; P_lacI: lacI	This study
pFL-5	ColE1 origin; Amp^r; P_LlacO₁: 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_LlacO₁: 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 Na₂HPO₄, 3 g/L KH₂PO₄, 1 g/L NH₄Cl, and 0.5 g/L NaCl, 1 mM MgSO₄, 0.1 mM CaCl₂, 10 mg/L VB₁, 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 NaH₂PO₄, 3.00g/L KH₂PO₄, 0.5g/L NaCl, 1.0g/L NH₄Cl, 1mmol/L MgSO₄, 0.1 mmol/L CaCl₂, 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_LlacO₁. 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 OD₆₀₀ 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/OD₆₀₀ 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 Ni²⁺-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.

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. 2a). 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. 2b).

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. 2a). 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. 2b). 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. 2b).

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. 2c, for the control strain, sfGFP could be normally expressed, showing a sfGFP/OD₆₀₀ 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/OD₆₀₀ 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/OD₆₀₀ 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. 2c, in the presence or absence of 1 mM L-Trp feeding, showing comparable sfGFP/OD₆₀₀ 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. 2d). 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. 2c, for the control strain, sFGFP^WT was normally expressed without the inducer IPTG or 4-HiL feeding, showing a sfGFP/OD₆₀₀ 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/OD₆₀₀ 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/OD₆₀₀ (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/OD₆₀₀ 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/OD₆₀₀ 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. 2d). 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. 3a). 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_LlacO₁. 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. 3b, 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 OD₆₀₀ 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. 3c, in the absence of 5-HTP feeding, the sfGFP/OD₆₀₀ 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 Ni²⁺-NTA affinity chromatography and characterized by Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Fig. 3c). 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. 3d). 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. 4a). 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\(\:\times\:\)10³ ± 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. 4b, in the absence of 4-HiL feeding, the sfGFP/OD₆₀₀ 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 Ni²⁺-NTA affinity chromatography and characterized by SDS-PAGE (Fig. 4c). 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. 4d).

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. 5a). 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. 5b, when 10 mM 5-HTP and 10 mM 4-HiL were added simultaneously, the value of sfGFP/OD₆₀₀ was up to 1.13\(\:\times\:\)10³ ± 131. When 5-HTP or 4-HiL was added alone, the value of sfGFP/OD₆₀₀ 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. 5c, in the case of endogenous co-biosynthesis of 5-HTP and 4-HiL, the value of sfGFP/OD₆₀₀ was the highest at 2.26\(\:\times\:\)10³ ± 254. In addition, the engineered bacteria containing only the 5-HTP biosynthetic pathway or the 4-HiL biosynthetic pathway had a lower sfGFP/OD₆₀₀, 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. 5b) or biosynthetic 5HTP and 4-HiL (Fig. 5c) by Ni²⁺-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. 5b) and 27950 (Fig. 5c), respectively, which were consistent with the theoretical values. The purity was close to 100%.

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 (CtAAAH) from Cupriavidus taiwanensis. The substrate preference of CtAAAH 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.

Acknowledgements

The authors thank the Hebei Natural Science Foundation (grant no. B2023105008), and the Biological & Medical Engineering Core Facilities (Beijing Institute of Technology) for providing advanced equipment.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Acevedo‐Rocha CG, Budisa N: Xenomicrobiology: a roadmap for genetic code engineering. Microbial Biotechnology 2016, 9:666-676.
Ros E, Torres AG, Ribas de Pouplana L: Learning from Nature to Expand the Genetic Code. Trends in Biotechnology 2021, 39:460-473.
Sun Y, Wu J, Xu J, Yang L: Metabolic Engineering of Escherichia coli for the Production of l-Homoserine. Chem & Bio Engineering 2024, 1:223-230.
Shi S, Wang L, Cao M, Chen G, Yu J: Proteomic analysis and prediction of amino acid variations that influence protein posttranslational modifications. Briefings in Bioinformatics 2019, 20:1597-1606.
Wang S, Osgood AO, Chatterjee A: Uncovering post-translational modification-associated protein–protein interactions. Current Opinion in Structural Biology 2022, 74.
Maas MN, Hintzen JCJ, Mecinović J: Probing lysine posttranslational modifications by unnatural amino acids. Chemical Communications 2022, 58:7216-7231.
Chen J, Dong S, Fang W, Jiang Y, Chen Z, Qin X, Wang C, Zhou H, Jin L, Feng Y, et al: Regiodivergent and Enantioselective Hydroxylation of C−H bonds by Synergistic Use of Protein Engineering and Exogenous Dual‐Functional Small Molecules. Angewandte Chemie International Edition 2022, 62.
Cramer J, Sager CP, Ernst B: Hydroxyl Groups in Synthetic and Natural-Product-Derived Therapeutics: A Perspective on a Common Functional Group. Journal of Medicinal Chemistry 2019, 62:8915-8930.
Palone A, Casadevall G, Ruiz-Barragan S, Call A, Osuna S, Bietti M, Costas M: C–H Bonds as Functional Groups: Simultaneous Generation of Multiple Stereocenters by Enantioselective Hydroxylation at Unactivated Tertiary C–H Bonds. Journal of the American Chemical Society 2023, 145:15742-15753.
Frost JR, Smith JM, Fasan R: Design, synthesis, and diversification of ribosomally derived peptide macrocycles. Current Opinion in Structural Biology 2013, 23:571-580.
Macek B, Forchhammer K, Hardouin J, Weber-Ban E, Grangeasse C, Mijakovic I: Protein post-translational modifications in bacteria. Nature Reviews Microbiology 2019, 17:651-664.
Peng T, Das T, Ding K, Hang HC: Functional analysis of protein post‐translational modifications using genetic codon expansion. Protein Science 2023, 32.
Chen Y, Tang J, Wang L, Tian Z, Cardenas A, Fang X, Chatterjee A, Xiao H: Creation of Bacterial Cells with 5-Hydroxytryptophan as a 21st Amino Acid Building Block. Chem 2020, 6:2717-2727.
Martínez-Rodríguez S, Torres JM, Sánchez P, Ortega E: Overview on Multienzymatic Cascades for the Production of Non-canonical α-Amino Acids. Frontiers in Bioengineering and Biotechnology 2020, 8.
Adhikari A, Bhattarai BR, Aryal A, Thapa N, Kc P, Adhikari A, Maharjan S, Chanda PB, Regmi BP, Parajuli N: Reprogramming natural proteins using unnatural amino acids. RSC Advances 2021, 11:38126-38145.
Guo Q-r, Cao YJ: Applications of genetic code expansion technology in eukaryotes. Protein & Cell 2024, 15:331-363.
Li Y, Dalby Paul A: Engineering of enzymes using non-natural amino acids. Bioscience Reports 2022, 42.
Goettig P, Koch NG, Budisa N: Non-Canonical Amino Acids in Analyses of Protease Structure and Function. International Journal of Molecular Sciences 2023, 24.
Chin JW: Expanding and reprogramming the genetic code. Nature 2017, 550:53-60.
Dien VT, Morris SE, Karadeema RJ, Romesberg FE: Expansion of the genetic code via expansion of the genetic alphabet. Current Opinion in Chemical Biology 2018, 46:196-202.
Guo J, Niu W: Genetic Code Expansion Through Quadruplet Codon Decoding. Journal of Molecular Biology 2022, 434.
Zhao S, Liu D: Applications of genetic code expansion and photosensitive UAAs in studying membrane proteins. Open Life Sciences 2023, 18.
Sisila V, Indhu M, Radhakrishnan J, Ayyadurai N: Building biomaterials through genetic code expansion. Trends in Biotechnology 2023, 41:165-183.
Ros E, Bellido M, Verdaguer X, Ribas de Pouplana L, Riera A: Synthesis and Application of 3-Bromo-1,2,4,5-Tetrazine for Protein Labeling to Trigger Click-to-Release Biorthogonal Reactions. Bioconjugate Chemistry 2020, 31:933-938.
Stieglitz JT, Kehoe HP, Lei M, Van Deventer JA: A Robust and Quantitative Reporter System To Evaluate Noncanonical Amino Acid Incorporation in Yeast. ACS Synthetic Biology 2018, 7:2256-2269.
Zheng Y, Mukherjee R, Chin MA, Igo P, Gilgenast MJ, Chatterjee A: Expanding the Scope of Single- and Double-Noncanonical Amino Acid Mutagenesis in Mammalian Cells Using Orthogonal Polyspecific Leucyl-tRNA Synthetases. Biochemistry 2017, 57:441-445.
Zamanian-Daryoush M, Gogonea V, DiDonato AJ, Buffa JA, Choucair I, Levison BS, Hughes RA, Ellington AD, Huang Y, Li XS, et al: Site-specific 5-hydroxytryptophan incorporation into apolipoprotein A-I impairs cholesterol efflux activity and high-density lipoprotein biogenesis. Journal of Biological Chemistry 2020, 295:4836-4848.
Lajoie MJ, Rovner AJ, Goodman DB, Aerni H-R, Haimovich AD, Kuznetsov G, Mercer JA, Wang HH, Carr PA, Mosberg JA, et al: Genomically Recoded Organisms Expand Biological Functions. Science 2013, 342:357-360.
Chen J, Tsai Y-H: Applications of Genetic Code Expansion in Studying Protein Post-translational Modification. Journal of Molecular Biology 2022, 434.
Belin D, Puigbò P: Why Is the UAG (Amber) Stop Codon Almost Absent in Highly Expressed Bacterial Genes? Life 2022, 12.
Wu KL, Moore JA, Miller MD, Chen Y, Lee C, Xu W, Peng Z, Duan Q, Phillips GN, Uribe RA, Xiao H: Expanding the eukaryotic genetic code with a biosynthesized 21st amino acid. Protein Science 2022, 31.
Chen Y, Jin S, Zhang M, Hu Y, Wu K-L, Chung A, Wang S, Tian Z, Wang Y, Wolynes PG, Xiao H: Unleashing the potential of noncanonical amino acid biosynthesis to create cells with precision tyrosine sulfation. Nature Communications 2022, 13.
Zhang MS, Brunner SF, Huguenin-Dezot N, Liang AD, Schmied WH, Rogerson DT, Chin JW: Biosynthesis and genetic encoding of phosphothreonine through parallel selection and deep sequencing. Nature Methods 2017, 14:729-736.
Kim S, Sung BH, Kim SC, Lee HS: Genetic incorporation ofl-dihydroxyphenylalanine (DOPA) biosynthesized by a tyrosine phenol-lyase. Chemical Communications 2018, 54:3002-3005.
Marchand JA, Neugebauer ME, Ing MC, Lin CI, Pelton JG, Chang MCY: Discovery of a pathway for terminal-alkyne amino acid biosynthesis. Nature 2019, 567:420-424.
Neugebauer ME, Sumida KH, Pelton JG, McMurry JL, Marchand JA, Chang MCY: A family of radical halogenases for the engineering of amino-acid-based products. Nature Chemical Biology 2019, 15:1009-1016.
Kimoto M, Hirao I: Genetic Code Engineering by Natural and Unnatural Base Pair Systems for the Site-Specific Incorporation of Non-Standard Amino Acids Into Proteins. Frontiers in Molecular Biosciences 2022, 9.
He X, Gao T, Chen Y, Liu K, Guo J, Niu W: Genetic Code Expansion in Pseudomonas putida KT2440. ACS Synthetic Biology 2022, 11:3724-3732.
Turner EH, Loftis JM, Blackwell AD: Serotonin a la carte: Supplementation with the serotonin precursor 5-hydroxytryptophan. Pharmacology & Therapeutics 2006, 109:325-338.
Lin Y, Sun X, Yuan Q, Yan Y: Engineering Bacterial Phenylalanine 4-Hydroxylase for Microbial Synthesis of Human Neurotransmitter Precursor 5-Hydroxytryptophan. ACS Synthetic Biology 2014, 3:497-505.
Hibi M, Kawashima T, Kasahara T, Sokolov PM, Smirnov SV, Kodera T, Sugiyama M, Shimizu S, Yokozeki K, Ogawa J: A novel Fe(II)/α-ketoglutarate-dependent dioxygenase fromBurkholderia ambifariahas β-hydroxylating activity ofN-succinyl l-leucine. Letters in Applied Microbiology 2012, 55:414-419.
Ellefson JW, Meyer AJ, Hughes RA, Cannon JR, Brodbelt JS, Ellington AD: Directed evolution of genetic parts and circuits by compartmentalized partnered replication. Nature Biotechnology 2013, 32:97-101.
Miyake-Stoner SJ, Refakis CA, Hammill JT, Lusic H, Hazen JL, Deiters A, Mehl RA: Generating Permissive Site-Specific Unnatural Aminoacyl-tRNA Synthetases. Biochemistry 2010, 49:1667-1677.
Nowak MG, Skwarecki AS, Milewska MJ: Amino Acid Based Antimicrobial Agents – Synthesis and Properties. ChemMedChem 2021, 16:3513-3544.
Skwarecki AS, Nowak MG, Milewska MJ: Amino Acid and Peptide‐Based Antiviral Agents. ChemMedChem 2021, 16:3106-3135.
Karbalaei-Heidari HR, Budisa N: Combating Antimicrobial Resistance With New-To-Nature Lanthipeptides Created by Genetic Code Expansion. Frontiers in Microbiology 2020, 11.
Chen L, Xin X, Zhang Y, Li S, Zhao X, Li S, Xu Z: Advances in Biosynthesis of Non-Canonical Amino Acids (ncAAs) and the Methods of ncAAs Incorporation into Proteins. Molecules 2023, 28.
Xu D, Fang M, Wang H, Huang L, Xu Q, Xu Z: Enhanced production of 5-hydroxytryptophan through the regulation of L-tryptophan biosynthetic pathway. Applied Microbiology and Biotechnology 2020, 104:2481-2488.
Liu Z, Cai M, Zhou S, You J, Zhao Z, Liu Z, Xu M, Rao Z: High-efficient production of L-homoserine in Escherichia coli through engineering synthetic pathway combined with regulating cell division. Bioresource Technology 2023, 389.
Li N, Zeng W, Zhou J, Xu S: O-Acetyl-L-homoserine production enhanced by pathway strengthening and acetate supplementation in Corynebacterium glutamicum. Biotechnology for Biofuels and Bioproducts 2022, 15.
Mora‐Villalobos JA, Zeng AP: Protein and pathway engineering for the biosynthesis of 5‐hydroxytryptophan in Escherichia coli. Engineering in Life Sciences 2017, 17:892-899.
Mora-Villalobos J-A, Zeng A-P: Synthetic pathways and processes for effective production of 5-hydroxytryptophan and serotonin from glucose in Escherichia coli. Journal of Biological Engineering 2018, 12.
Wang L, Xie J, Schultz PG: Expanding the Genetic Code. Annual Review of Biophysics and Biomolecular Structure 2006, 35:225-249.
Ambrogelly A, Palioura S, Söll D: Natural expansion of the genetic code. Nature Chemical Biology 2006, 3:29-35.
Liu CC, Schultz PG: Adding New Chemistries to the Genetic Code. Annual Review of Biochemistry 2010, 79:413-444.
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.

No competing interests reported.

Download PDF

Version 1

posted

You are reading this latest preprint version

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

Status:

Version 1

Abstract

Figures

Introduction

Materials and methods

Strains and growth conditions

Media, and materials

Plasmid construction

UHPLC analysis

SfGFP protein expression

Fluorescence assay

Purification of different sfGFP proteins

Mass spectrometry

Results

Construction of 5-HTP or 4-HiL orthogonal pair

Validation of 5-HTP orthogonal pair

Validation of 4-HiL orthogonal pair

Discussion

Declarations

References

Additional Declarations

Status:

Version 1