Genetically engineered bacterium, preparation method therefor and use thereof in de novo synthesis of salidroside

Abstract

Provided are a genetically engineered bacterium, a preparation method therefor and a use thereof in de novo synthesis of salidroside. The genetically engineered bacterium comprises an exogenously introduced phenylpyruvate decarboxylase gene ARO10 and a glycosyltransferase gene OfT8GT1, wherein the phenylpyruvate decarboxylase gene ARO10 has a nucleotide sequence as shown in SEQ ID NO: 3, and the glycosyltransferase gene OfT8GT1 has a nucleotide sequence as shown in SEQ ID NO: 4. The method for synthesizing salidroside from the genetically engineered bacterium by catalysis greatly reduces synthesis costs, simplifies subsequent separation and purification steps, and exhibits high efficiency of salidroside synthesis, thereby laying a foundation for industrial production of salidroside by using a microbial fermentation method.

Description

Genetically engineered bacteria, their preparation methods, and their application in the de novo synthesis of rhodioloside.

[0001] Cross-reference to related applications

[0002] This application claims priority to Chinese Patent Application No. 202410807229.X, filed on June 21, 2024, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0003] This invention relates to the field of bioengineering technology, and more specifically, to a genetically engineered bacterium, its preparation method, and its application in the de novo synthesis of rhodioloside. Background Technology

[0004] Rhodioloside (2-(4-Hydroxyphenyl)ethyl-betta-D-glucopyranoside, molecular formula C) 14 H 20 O7 is a plant-derived phenylethanol glycoside compound with functions such as anti-fatigue, anti-hypoxia, anti-aging, anti-cancer, anti-inflammatory, and protection of the cardiovascular and central nervous systems. It is now widely used in the pharmaceutical, food, and health product industries, and there are no reports of side effects or drug interactions of rhodioloside.

[0005] Currently, rhodioloside is mainly obtained through extraction from the plant Rhodiola rosea. Its yield is limited by the plant's unique high-altitude growing environment, its low rhodioloside content, and the scarcity of plant resources due to overexploitation. Chemical synthesis requires complex steps such as hydroxyl protection and deprotection, as well as expensive catalysts, and suffers from poor regioselectivity. Therefore, developing a mild and efficient biosynthetic method for rhodioloside is beneficial for reducing production costs and expanding market value.

[0006] With the development of synthetic biology and metabolic engineering, the use of microorganisms to ferment economical and renewable carbon sources (glucose, glycerol, sucrose) into high-value-added compounds has attracted increasing attention due to its low cost, high efficiency, and environmental friendliness. *Escherichia coli*, as a model organism, has advantages such as rapid growth, clear genetic background, mature genetic manipulation methods, and simple nutrient requirements for fermentation, making it a preferred chassis strain for synthesizing many natural products. Liu et al. constructed a plasmid-free engineered strain by integrating multiple copies of the genome into the rhodioloside synthesis pathway, achieving the synthesis of 2.42 g / L and 9.48 g / L rhodioloside at shake flask and fermenter levels, respectively (Liu S, Xia Y, Yang H, et al. Rational chromosome engineering of *Escherichia coli* for overproduction of salidroside[J]. *Biochemical Engineering Journal*, 2022, 184: 108474.). Shanghai Ruikang Biotechnology R&D Co., Ltd. constructed a method for the synergistic utilization of glycerol and glucose in *E. coli*. By uniformly feeding yeast extract and adding tyrosine as a precursor in batches, a 28.4 g / L rhodioloside was synthesized in a 10L fermenter over 72 hours, representing the highest reported yield for *E. coli* to date. However, the fermentation process requires strict dissolved oxygen control and a feeding strategy, increasing complexity and production costs (CN 115960811 A). *Saccharomyces cerevisiae*, as a model strain of eukaryotes, is also widely used in the synthesis of high-value compounds. Liu et al. divided the de novo synthesis pathway of rhodioloside in Saccharomyces cerevisiae into five modules. Through modular engineering optimization, 26.55 g / L of rhodioloside can be synthesized at the fermenter level. However, yeast extract needs to be added in batches and the fermentation cycle is relatively long, requiring 168 h (Liu H, Tian Y, Zhou Y, et al. Multi-modular engineering of Saccharomyces cerevisiae for high-titer production of tyrosol and salidroside[J]. Microbial Biotechnology, 2021, 14(6): 2605-2616.).

[0007] In summary, although rhodioloside has been synthesized de novo by microorganisms, it still suffers from low yield, high cost, complex process control, and long fermentation cycle. Therefore, constructing efficient, stable, and economical rhodioloside-synthesizing strains is crucial for realizing the industrial-scale microbial production of rhodioloside.

[0008] Summary of the Invention

[0009] The main objective of this invention is to provide a genetically engineered bacterium, its preparation method, and its application in the de novo synthesis of rhodioloside, so as to solve the problem of low synthesis efficiency of rhodioloside in the prior art.

[0010] To achieve the above objectives, according to a first aspect of the present invention, a genetically engineered bacterium is provided, the genetically engineered bacterium containing an exogenously introduced phenylpyruvate decarboxylase gene ARO10 and a glycosyltransferase gene OfT8GT1, wherein the phenylpyruvate decarboxylase gene ARO10 has a nucleotide sequence as shown in SEQ ID NO: 3; and the glycosyltransferase gene OfT8GT1 has a nucleotide sequence as shown in SEQ ID NO: 4.

[0011] Furthermore, the aforementioned genetically engineered bacteria do not contain any one or more of the following genes: poxB, pta, ackA, eutD, adhE, mhpF, eutE, ldhA, dld, lldD, aldA, fnr, arcA, ptsG, feaB, or puuC; and / or

[0012] The above-mentioned genetically engineered bacteria also contain any one or more of the following exogenous genes: glk, aroF, aroK, glf as shown in SEQ ID NO: 7, mutant aroG as shown in SEQ ID NO: 5, or mutant tyrA as shown in SEQ ID NO: 6; wherein the above-mentioned exogenous genes are all integrated into the genome of the above-mentioned genetically engineered bacteria.

[0013] Furthermore, the aforementioned genetically engineered bacteria are Escherichia coli, including MG1655, BW25113, W3110, DH5α, or BL21.

[0014] To achieve the above objectives, according to a second aspect of the present invention, a method for preparing genetically engineered bacteria is provided, the method comprising: transferring a plasmid carrying the phenylpyruvate decarboxylase gene ARO10 and the glycosyltransferase gene OfT8GT1 into a starting engineered bacterium to obtain the above-mentioned genetically engineered bacteria;

[0015] The phenylpyruvate decarboxylase gene ARO10 has the nucleotide sequence shown in SEQ ID NO: 3. The glycosyltransferase gene OfT8GT1 has the nucleotide sequence shown in SEQ ID NO: 4.

[0016] Furthermore, the aforementioned starting engineered bacteria are Escherichia coli, including MG1655, BW25113, W3110, DH5α, or BL21.

[0017] Furthermore, the above preparation method further includes: knocking out any one or more of the following endogenous genes in the starting engineered bacteria: poxB, pta, ackA, eutD, adhE, mhpF, eutE, ldhA, dld, lldD, aldA, fnr, arcA, ptsG, feaB, or puuC; and / or

[0018] Knock in any one or more of the following genes into the above-mentioned starting engineered bacteria: glk, aroF, aroK, glf as shown in SEQ ID NO: 7, mutant aroG as shown in SEQ ID NO: 5, or mutant tyrA as shown in SEQ ID NO: 6.

[0019] To achieve the above objective, according to a third aspect of the present invention, a method for de novo synthesis of rhodioloside is provided, the method comprising: using the above-described genetically engineered bacteria or the genetically engineered bacteria prepared by the above-described preparation method to ferment and synthesize the above-described rhodioloside in a fermentation medium.

[0020] Furthermore, the above fermentation is either shake-flask fermentation or fermenter fermentation.

[0021] Furthermore, the above-mentioned shake-flask fermentation includes: inoculating the above-mentioned genetically engineered bacteria that have been cultured overnight into the above-mentioned fermentation medium for expansion culture, culturing until the OD600 is 0.8-1, adding an inducer for induced fermentation for 22-94 hours, and obtaining the above-mentioned rhodioloside.

[0022] Further, the overnight culture conditions are 30-37℃, 200-220 rpm; the scale-up culture conditions are 30-37℃, 200-220 rpm; the induced fermentation conditions are 25-35℃, 200-220 rpm; the inducer is IPTG; and the final concentration of the inducer is 0.1-1 mM. Further, the fermentation medium for the shake-flask fermentation consists of 5-10 g / L NaCl, 2-5 g / L yeast extract, 5-10 g / L tryptone, and 10-20 g / L glucose.

[0023] Furthermore, the fermentation in the above-mentioned fermenter includes: inoculating the above-mentioned genetically engineered bacteria that have been cultured overnight into a seed tank for expansion culture until the OD600 is 10-20, stopping the fermentation, draining the liquid and transferring the culture for a second expansion culture until the OD600 is 10-20, adding an inducer for induced fermentation for 60-70 hours to obtain the above-mentioned rhodioloside.

[0024] Furthermore, the overnight culture conditions are 30-37℃; the scale-up culture and the secondary scale-up culture conditions are DO 30-40%, pH 6.8-7.0, 30-37℃; the induced fermentation conditions are DO 30-40%, pH 6.8-7.0, 25-35℃; the inducer is IPTG; and the final concentration of the inducer is 0.1-1mM.

[0025] Furthermore, the fermentation medium used in the above-mentioned fermenter consists of 10-30 g / L glucose monohydrate, 10-30 g / L glycerol, 2-4 g / L magnesium sulfate heptahydrate, 5-10 g / L yeast powder, 4-8 g / L corn steep liquor powder, 1-3 g / L sodium citrate, 4-8 g / L ammonium sulfate, 2-4 g / L dipotassium hydrogen phosphate, 2-4 g / L potassium dihydrogen phosphate, 1-2 mL / L trace element mixture, 0.1-0.3 g / L thiamine hydrochloride, and 0.2-0.4 mL / L foaming agent.

[0026] Applying the technical solution of this invention, the genetically engineered bacteria of this invention contain exogenously introduced phenylpyruvate decarboxylase gene ARO10 and glycosyltransferase gene OfT8GT1, wherein the phenylpyruvate decarboxylase gene ARO10 has the nucleotide sequence shown in SEQ ID NO: 3, and the glycosyltransferase gene OfT8GT1 has the nucleotide sequence shown in SEQ ID NO: 4. Such genetically engineered bacteria express phenylpyruvate decarboxylase and glycosyltransferase in vivo, thereby enabling the use of this genetically engineered bacteria to catalyze the synthesis of rhodioloside using fermented 4-hydroxyphenylpyruvate as a precursor. Using glucose as a carbon source, the method of synthesizing rhodioloside using this genetically engineered bacteria requires no antibiotics or intermediate substances, and yields high amounts of rhodioloside, significantly reducing synthesis costs, simplifying subsequent separation and purification steps, and increasing the efficiency of rhodioloside synthesis. It has good application prospects and lays the foundation for the industrial production of rhodioloside using microbial fermentation. Attached Figure Description

[0027] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0028] Figure 1 illustrates the relevant metabolic pathways for the synthesis of salidroside in this invention. In this diagram, glucose is referred to as glucose, salidroside as salidroside, glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), fructose-1,6-bisphosphate (F1,6P), glyceraldehyde-3-phosphate (GAP), phosphoenolpyruvate (PEP), erythrose-4-phosphate (E4P), 3-deoxy-D-arabinohepenulose-7-phosphate (DAHP), shikimate, shikimate-3-phosphate (S3P), cladonic acid (CHA), prephenate, 4-hydroxyphenylpyruvate (4-HPP), tyrosine, glycosyltransferase, 4-hydroxyphenylacetaldehyde (4-HPAA), alcohol dehydrogenase (ADH), tyrosol, 4-hydroxyphenylacetic acid (4-HPA), and the tricarboxylic acid cycle (TCA). Cycle, ethanol, Ac-CoA, CoA, FA, PYR, Acetate, Lactate, Acetyl-P, Glucose-1-phosphate, UDP-glucose, and PDH.

[0029] Figure 2 shows a comparative fermentation diagram of strain W3110 and byproduct knockout strain S75 according to Example 1 of the present invention. In the figure, A represents the fermentation result of strain W3110 and B represents the fermentation result of strain S75. Glucose is abbreviated as Glu, lactate is lactic acid, acetate is acetic acid, ethanol is ethanol, and byproduct is byproduct.

[0030] Figure 3 shows a schematic diagram of the efficient supply of chassis tyrosine production by the rhodioloside precursor according to Example 2 of the present invention.

[0031] Figure 4 shows the results of screening for the combination of phenylpyruvate decarboxylase and glycosyltransferase according to Example 3 of the present invention.

[0032] Figure 5 shows a schematic diagram of the fermentation curve of Sal085 according to Example 4 of the present invention, wherein (1) represents the yield of tyrosol, (2) represents the yield of salidroside, and (3) represents the yield of 4-hydroxyphenylacetic acid (4-HPA).

[0033] Figure 6 shows a schematic diagram of the Sal110 fermentation curve according to Example 5 of the present invention, wherein (1) represents the yield of 4-hydroxyphenylethanol, (2) represents the yield of rhodioloside, and (3) represents the yield of 4-hydroxyphenylacetic acid.

[0034] Figure 7 shows a schematic diagram of the Sal123 fermentation curve according to Example 6 of the present invention, wherein (1) represents the yield of 4-hydroxyphenylethanol, (2) represents the yield of rhodioloside, and (3) represents the yield of 4-hydroxyphenylacetic acid.

[0035] Figure 8 shows a schematic diagram of the Sal132 fermentation curve according to Example 6 of the present invention, wherein (1) represents the yield of 4-hydroxyphenylethanol, (2) represents the yield of rhodioloside, and (3) represents the yield of 4-hydroxyphenylacetic acid.

[0036] Figure 9 shows a schematic diagram of the effect of puuC knockout on 4-hydroxyphenylacetic acid according to Example 7 of the present invention. Detailed Implementation

[0037] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0038] As mentioned in the background section, rhodioloside is widely used in the pharmaceutical, food, and health product industries and has extremely high economic value. However, the yield of rhodioloside is very limited. Existing methods for synthesizing rhodioloside suffer from low efficiency, high cost, and complex operation. The technical solution of this invention utilizes Escherichia coli containing exogenously introduced phenylpyruvate decarboxylase gene ARO10 and glycosyltransferase gene OfT8GT1 to express phenylpyruvate decarboxylase and glycosyltransferase, using glucose as a carbon source to efficiently synthesize rhodioloside (related metabolic pathways are shown in Figure 1). This method requires no antibiotics or intermediate substances and yields high amounts of rhodioloside, significantly reducing production costs, simplifying subsequent separation and purification steps, and improving the efficiency of rhodioloside synthesis. It has good application prospects and lays the foundation for the industrial production of rhodioloside using microbial fermentation. Based on this, the inventors have proposed a series of protection schemes for this invention.

[0039] It is important to note that the writing format differs for proteins and genes from different sources. For gene names, if named as "Source + Gene Name," the first two letters represent the source, with the first letter capitalized and the second lowercase, both in italics (sources from *E. coli* are generally omitted by default). All subsequent letters in the gene name should be italicized; refer to the UniProt database for specific capitalization guidelines. If named as "Gene Number," those from plants should be capitalized and italicized (only some plant-derived glycosyltransferases in this patent have gene numbers). For protein names, if named as "Source + Protein Name," the first two letters represent the source, with the first letter capitalized and the second lowercase, both in italics (sources from *E. coli* are generally omitted by default). All subsequent letters in the protein name should not be italicized; refer to the UniProt database for specific capitalization guidelines. All protein and gene names involved in this invention follow this naming convention.

[0040] Only phenylpyruvate decarboxylases and glycosyltransferases from certain sources can efficiently synthesize rhodioloside. By combining phenylpyruvate decarboxylases from *Saccharomyces cerevisiae* and *Pichia pastoris* GS115 with glycosyltransferases from *Osmanthus fragrans*, *Arabidopsis thaliana*, *Camelina sativa*, *Rhodiola sachalinensis*, *Rhodiola rosea*, *Rehmannia glutinosa* Libosch, and *Bacillus licheniformis*, the optimal combination was selected: the phenylpyruvate decarboxylase gene ARO10 from *Saccharomyces cerevisiae* and the glycosyltransferase gene OfT8GT1 from *Osmanthus fragrans*. The combined synthesis yielded 29 g / L of rhodioloside, exceeding the levels achieved by other existing E. coli fermentation methods. It is important to note that the glycosyltransferase OfT8GT1 is a variant (V393L) derived from Osmanthus fragrans, with the wild-type glycosyltransferase having the NCBI ID: AYN74351.1.

[0041] In a first typical embodiment of the present invention, a genetically engineered bacterium is provided, which contains exogenously introduced phenylpyruvate decarboxylase gene ARO10 and glycosyltransferase gene OfT8GT1. By inducing the expression of phenylpyruvate decarboxylase and glycosyltransferase in this genetically engineered bacterium, rhodioloside can be efficiently synthesized from 4-hydroxyphenylpyruvate through in vivo fermentation. The amino acid sequences of the aforementioned phenylpyruvate decarboxylase and glycosyltransferase are SEQ ID NO: 1 and SEQ ID NO: 2, respectively, while the nucleotide sequences of the genes encoding these two proteins are SEQ ID NO: 3 and SEQ ID NO: 4, respectively, as shown below:

[0042] SEQ ID NO: 1 (Amino acid sequence of phenylpyruvate decarboxylase Aro10):

[0043] SEQ ID NO: 2 (Amino acid sequence of glycosyltransferase OfT8GT1):

[0044] SEQ ID NO: 3 (nucleotide sequence of ARO10, the gene encoding phenylpyruvate decarboxylase):

[0045] SEQ ID NO: 4 (nucleotide sequence of the gene OfT8GT1 encoding glycosyltransferase):

[0046] There are various ways to obtain the above-mentioned amino acid and nucleotide sequences, and they are not limited to obtaining them from the genomes of the above-mentioned species. In a preferred embodiment of the present invention, the phenylpyruvate decarboxylase gene ARO10, having the nucleotide sequence shown in SEQ ID NO: 3, is derived from Saccharomyces cerevisiae, and the glycosyltransferase gene OfT8GT1, having the nucleotide sequence shown in SEQ ID NO: 4, is derived from Osmanthus fragrans.

[0047] To further improve the efficiency of rhodioloside synthesis by the genetically engineered bacterium, in a preferred embodiment of the present invention, the genetically engineered bacterium does not contain any one or more of the following genes: poxB, pta, ackA, eutD, adhE, mhpF, eutE, ldhA, dld, lldD, aldA, fnr, arcA, ptsG, feaB, or puuC; and / or the genetically engineered bacterium contains any one or more exogenously introduced genes: glk, aroF, aroK, glf as shown in SEQ ID NO: 7, mutant aroG as shown in SEQ ID NO: 5, or mutant tyrA as shown in SEQ ID NO: 6; wherein, the exogenously introduced genes are all integrated into the genome of the genetically engineered bacterium. By exogenously introducing these genes, they are overexpressed in the bacteria. The nucleotide sequences of mutant aroG and mutant tyrA are as follows:

[0048] SEQ ID NO: 5 (nucleotide sequence of the mutant aroG gene):

[0049] SEQ ID NO: 6 (nucleotide sequence of mutant tyrA gene):

[0050] SEQ ID NO: 7 (nucleotide sequence of gene glf):

[0051] Of the genes mentioned above, poxB (Gene ID: 946132), pta (Gene ID: 946778), ackA (Gene ID: 946775), and eutD (Gene ID: 946940) are genes related to acetic acid synthesis; adhE (Gene ID: 945837), mhpF (Gene ID: 945008), and eutE (Gene ID: 946943) are genes related to ethanol synthesis; and ldhA (Gene ID: 946315), dld (Gene ID: 946653), lldD (Gene ID: 948121), and aldA (Gene ID: 945672) are genes related to lactic acid synthesis. The absence of these genes in the genetically engineered bacteria indicates that the synthesis pathways of acetic acid, ethanol, and lactic acid in the metabolic pathways have been interrupted, allowing more raw materials to be used for the synthesis of rhodioloside, removing fermentation byproducts, and simplifying the fermentation process.

[0052] The precursor 4-hydroxyphenylpyruvate is a precursor to both rhodioloside and tyrosine. Therefore, the yield of tyrosine is used to evaluate the efficient supply of rhodioloside precursors to the substrate. When the genetically engineered bacteria do not contain fnr (Gene ID: 945908), arcA (Gene ID: 948874), and ptsG (Gene ID: 945651), it indicates that the genetically engineered bacteria further reduce the metabolism of the phosphoenolpyruvate branch of the tyrosine synthesis precursor and promote oxygen utilization. Among them, fnr and arcA are global regulatory factors, and ptsG is a glucose transporter gene.

[0053] feaB (Gene ID: 945933) is the gene encoding the competitive pathway. When the genetically engineered bacteria do not contain feaB, they do not accumulate the byproduct 4-hydroxyphenylacetic acid, and the yield of rhodioloside is further increased. However, when the genetically engineered bacteria do not contain this gene, the intermediate product 4-hydroxyphenylethanol accumulates significantly. To eliminate the impact of 4-hydroxyphenylethanol accumulation on rhodioloside synthesis, the aforementioned genetically engineered bacteria must also not contain the gene puuC (Gene ID: 947003) encoding the competitive pathway; genetically engineered bacteria without this gene do not accumulate 4-hydroxyphenylacetic acid.

[0054] Of the overexpressed genes mentioned above, glf and glk (Gene ID: 946858) are glucose transport system genes, and overexpression of these genes in genetically engineered bacteria has a beneficial effect on promoting glucose utilization. aroF (Gene ID: 947084) and the mutant aroG shown in SEQ ID NO: 5 both encode 3-deoxy-D-arabinohepulose-7-phosphate synthase. This enzyme catalyzes the synthesis of 3-deoxy-D-arabinohepulose-7-phosphate from phosphoenolpyruvate and erythrose-4-phosphate. Introducing the gene encoding this enzyme into genetically engineered bacteria has a beneficial effect on promoting the synthesis of 4-hydroxyphenylpyruvate. aroK (Gene ID: 2847759) is the gene encoding shikimate kinase 1, which catalyzes the synthesis of shikimate from shikimate. Genetically engineered bacteria with this gene introduced have a beneficial effect on promoting the synthesis of 4-hydroxyphenylpyruvate. As shown in SEQ ID NO: 6, the mutant tyrA is a site-directed mutated cladoid mutase gene. This gene catalyzes the synthesis of 4-hydroxyphenylpyruvate from cladoids. Introducing this gene into genetically engineered bacteria has a beneficial effect of promoting 4-hydroxyphenylpyruvate synthesis. However, the two site-directed mutated genes mentioned above need to be validated before being introduced into the genome of the genetically engineered bacteria to ensure that the mutant 3-deoxy-D-arabinohepenoyl-7-phosphate synthase and cladoid mutase have higher enzyme activities than the wild-type enzymes.

[0055] Escherichia coli, as a common model organism, is a frequently used chassis bacterium in modern synthetic biology. The genetically engineered bacteria of this invention are Escherichia coli, including MG1655, BW25113, W3110, DH5α, or BL21. Using these Escherichia coli as a tool for synthesizing rhodioloside offers advantages such as a clear genetic background, simple operation, rapid growth, and economical and simple use of the required nutrients for fermentation.

[0056] Based on the above research, in a second typical embodiment of the present invention, a method for preparing genetically engineered bacteria is provided. This method includes: transferring a plasmid containing the phenylpyruvate decarboxylase gene ARO10 and the glycosyltransferase gene OfT8GT1 into a starting engineered bacterium to obtain the aforementioned genetically engineered bacteria. The aforementioned genetically engineered bacteria are *Escherichia coli*, including MG1655, BW25113, W3110, DH5α, or BL21. To further increase the yield of rhodioloside, the above preparation method further includes: knocking out any one or more of the following endogenous genes in the aforementioned starting engineered bacteria: poxB, pta, ackA, eutD, adhE, mhpF, eutE, ldhA, dld, lldD, aldA, fnr, arcA, ptsG, feaB, or puC; and / or

[0057] Knock in any one or more of the following genes into the starting engineered bacteria: glk, aroF, aroK, glf as shown in SEQ ID NO: 7, mutant aroG as shown in SEQ ID NO: 5, or mutant tyrA as shown in SEQ ID NO: 6.

[0058] Chassis bacteria modification is an important method for increasing the yield of target bacterial metabolites, involving gene knock-in or knock-out. Gene knock-out aims to reduce the accumulation of byproducts in metabolic pathways, while gene knock-in provides raw materials and impetus for the synthesis of the target product. Knock-in genes can be directly integrated into the bacterial genome or exist in the bacteria as plasmids. It is important to note that there is no specific limitation on the order of gene knock-in and knock-out; the timing can be chosen according to specific needs. Furthermore, for experimental convenience, multiple genes can be linked to the same expression vector during gene knock-in, followed by transformation. Finally, the integration of the plasmid carrying the target gene into the genome of the engineered bacteria can be considered based on the actual situation. The number of knock-ins of the same gene can also be selected according to actual needs. When multiple identical genes are knocked into a genetically engineered bacterium, the function of that gene in the bacterium will be enhanced.

[0059] The aforementioned genetically engineered bacteria and their preparation method provide an effective pathway for the efficient synthesis of rhodioloside. In a third typical embodiment of the present invention, a method for de novo synthesis of rhodioloside is provided. This method includes fermenting the aforementioned genetically engineered bacteria or the genetically engineered bacteria prepared using the above-mentioned method in a fermentation medium to synthesize the aforementioned rhodioloside. Depending on the fermentation system, shake-flask fermentation or fermenter fermentation can be selected according to actual needs. Specifically, the shake-flask fermentation includes: inoculating the aforementioned genetically engineered bacteria, cultured overnight, into the aforementioned fermentation medium for expansion culture until the OD600 reaches 0.8-1; adding an inducer for induced fermentation for 22-94 hours to obtain the aforementioned rhodioloside.

[0060] In some preferred embodiments, the overnight culture conditions are 30-37°C and 200-220 rpm; the scale-up culture conditions are 30-37°C and 200-220 rpm; the induced fermentation conditions are 25-35°C and 200-220 rpm; the inducer is IPTG; and the final concentration of the inducer is 0.1-1 mM.

[0061] In some other preferred embodiments, the fermentation medium for the shake flask fermentation is 5-10 g / L NaCl, 2-5 g / L yeast extract, 5-10 g / L tryptone, and 10-20 g / L glucose.

[0062] It should be noted that shake-flask fermentation is an experimental process before scale-up fermentation. The results of shake-flask fermentation allow for timely judgment and adjustment of fermentation conditions or materials. The system is small and easy to operate. In a preferred embodiment of the invention, the induction fermentation time is 22 hours. Under these conditions, screening for key enzymes in the synthesis of rhodioloside has the advantages of simplicity and speed. In another preferred embodiment of the invention, the induction fermentation time is 94 hours. Extending the induction fermentation time allows the genetically engineered bacteria sufficient time to synthesize and accumulate rhodioloside, maximizing the yield of rhodioloside.

[0063] To further expand the fermentation system and adapt it to the industrial production of rhodioloside, in some preferred embodiments, the fermentation can also be carried out in a fermenter. The fermenter fermentation includes: inoculating the genetically engineered bacteria cultured overnight into a seed tank for expansion culture until the OD600 reaches 10⁻²⁰; stopping the fermentation, draining the liquid, and transferring the culture for a second expansion culture to further increase bacterial density until the OD600 reaches 10⁻²⁰; adding an inducer for induced fermentation for 60-70 hours to obtain the rhodioloside. In a more preferred embodiment of the present invention, the induced fermentation time in the fermenter is 67 hours. Under these conditions, the genetically engineered bacteria fully synthesize rhodioloside, and the yield of rhodioloside is significantly improved compared to existing technologies, thus providing a preliminary exploration of the possibility of industrial production of rhodioloside.

[0064] The reaction conditions for each of the above steps can be obtained through reasonable optimization based on the above. In some preferred embodiments, the overnight culture conditions are 30-37°C; the scale-up culture and the secondary scale-up culture conditions are DO (dissolved oxygen) 30-40%, pH 6.8-7.0, 30-37°C; the induced fermentation conditions are DO 30-40%, pH 6.8-7.0, 25-35°C. Suitable temperature, pH, and dissolved oxygen are essential for normal bacterial growth. The inducer is IPTG; the final concentration of the inducer is 0.1-1 mM.

[0065] The fermentation medium required for fermentation in a fermenter can be reasonably adjusted based on existing conditions. In some preferred embodiments, the fermentation medium consists of 10-30 g / L glucose monohydrate, 10-30 g / L glycerol, 2-4 g / L magnesium sulfate heptahydrate, 5-10 g / L yeast powder, 4-8 g / L corn steep liquor powder, 1-3 g / L sodium citrate, 4-8 g / L ammonium sulfate, 2-4 g / L dipotassium hydrogen phosphate, 2-4 g / L potassium dihydrogen phosphate, 1-2 mL / L trace element mixture, 0.1-0.3 g / L thiamine hydrochloride, and 0.2-0.4 mL / L foaming agent.

[0066] The beneficial effects of the present invention will be explained in more detail below with reference to specific embodiments.

[0067] Example 1: Construction of strains for byproduct knockout chassis

[0068] To remove fermentation byproducts and simplify the fermentation process, *E. coli* W3110 was used as the starting strain. Genes related to acetic acid synthesis (poxB, pta, ackA, eutD), ethanol synthesis (adhE, mhpF, eutE), and lactic acid synthesis (ldhA, dld, lldD, aldA) were knocked out, resulting in strain S75. Both W3110 and S75 were fermented at 30℃. The fermentation results are shown in Figure 2. Under 20 g / L glucose + LB conditions, the wild-type strain W3110 consumed its carbon source and stopped growing after 30 h (Figure 2A), while strain S75 continued to grow until 52 h, with residual sugar still at 6.8 g / L (the OD600 of strain S75 was 4.3, greater than the wild-type strain's 3.8) (Figure 2B). This indicates that using the engineered strain S75 for fermentation can save significant carbon source costs. The amount of byproducts in the fermentation products was detected by liquid chromatography. The results showed that after 52 hours of fermentation, the acetic acid accumulation in the fermentation products of the wild-type strain reached 10.6 g / L, while that of the S75 strain was only 0.6 g / L. Ethanol and lactic acid were almost undetectable in the fermentation products of both strains, indicating that the engineered strain S75 has essentially interrupted the metabolic pathways of byproducts.

[0069] Example 2: Construction of a highly efficient supply system for rhodioloside precursors to chassis S209

[0070] To enhance the supply of 4-hydroxyphenylpyruvic acid, a precursor for rhodioloside synthesis, it is necessary to overexpress related genes. Using the *E. coli* W3110 genome as a template, the mutant tyrA (SEQ ID NO: 6) was amplified and assembled with the pACYC vector to construct pACYC-tyrA. Similarly, using the *E. coli* W3110 genome as a template, the mutant aroG (SEQ ID NO: 5) was amplified and assembled with the pACYC-tyrA vector to construct pACYC-tyrA-aroG. These were then introduced into the chassis strain S75 to construct strain S77.

[0071] To further reduce the metabolic pathway of phosphoenolpyruvate, a precursor for tyrosine synthesis, and promote oxygen utilization, the ptsG and fnr genes of strain S77 were knocked out to construct strain S114. Knocking out the arcA gene of strain S114 yielded strain S153. To eliminate the impact of ptsG knockout on carbon source utilization and to further enhance downstream driving forces, glf-glk, aroF, and aroK were integrated into the genome of strain S153, constructing strain S185.

[0072] To further reduce plasmid burden, the mutant aroG from the plasmid was integrated into the genome of strain S185, resulting in strain S203. Then, the mutant tyrA from the plasmid was integrated into the genome of strain S203, resulting in strain S208. Simultaneously, to enhance carbon source utilization, a second copy of glf-glk was integrated into strain S208, yielding the engineered strain S209.

[0073] Since tyrosine is also a product of 4-hydroxyphenylpyruvic acid, a precursor of rhodioloside, tyrosine was used to evaluate the efficient supply chassis for rhodioloside precursors. After 48 hours of shake-flask fermentation, the results are shown in Figure 3. With the superposition of modified target sites, the yield of tyrosine gradually increased. Among them, strains S208 and S209 could synthesize 2.62 g / L and 2.43 g / L of tyrosine, respectively. Although the tyrosine yield of S209 decreased, S209 was still selected as the synthesis chassis for rhodioloside to ensure the carbon source supply during subsequent scale-up production.

[0074] Example 3: Screening of key enzymes for rhodioloside synthesis

[0075] The synthesis of rhodioloside from 4-hydroxyphenylpyruvate requires two key exogenous enzymes: phenylpyruvate decarboxylase and glycosyltransferase. Screening enzymes from different sources for combination is crucial for the synthesis of the target product. Therefore, two phenylpyruvate decarboxylase genes were screened: the ARO10 gene from *Saccharomyces cerevisiae* and the KDC4 gene from *Pichia pastoris* GS115. Eight glycosyltransferase genes were also screened: the OfT8GT1 gene from *Osmanthus fragrans*, the UGT85A1 gene from *Arabidopsis thaliana*, the UGT71D1 gene from *Camelina sativa*, the UGT72B14 gene from *Rhodiola sachalinensis*, the UGT33 gene from *Rhodiola rosea*, and the gene from *Rehmannia glutinosa*. The RgUGT gene from Libosch, the UGT73B6 gene from Rhodiola sachalinensis, and the BlUGT gene from Bacillus licheniformis were combined to form a total of 16 combinations.

[0076] The phenylpyruvate decarboxylase gene and glycosyltransferase gene were constructed into the pACYC-Duet-1 vector, respectively, to obtain 16 combinatorial plasmids. These plasmids were then introduced into the chassis strain S209, resulting in 16 engineered strains as shown in Table 1. These 16 engineered strains were inoculated into LB liquid medium and cultured overnight at 37°C and 220 rpm to obtain a seed culture. This seed culture was then inoculated into fresh fermentation medium at a volume of 1%, and cultured at 37°C and 220 rpm for 2 hours. Afterward, IPTG was added to a final concentration of 0.5 mM for induction, and fermentation was induced at 30°C and 220 rpm for 22 hours. The yield of rhodioloside was detected using high-performance liquid chromatography (HPLC). The conditions were as follows: Atlantis T3 column 4.6*100mm 3μm, mobile phase H2O+0.1% TFA and ACN+0.1% TFA, flow rate 1.0mL / min, column temperature 40℃, UV detector, detection wavelength 220nm, detection time 12.0min.

[0077] Table 1. Screening strains for key enzymes in rhodioloside synthesis.

[0078] The fermentation results are shown in Figure 4. Among the 16 strains, the combination of ARO10 and OfT8GT1 was the best, and 163.5 mg / L of rhodioloside could be synthesized after 24 h of fermentation. Therefore, the combination of ARO10 and OfT8GT1 was selected for further modification to further improve the yield of rhodioloside.

[0079] Example 4: Integration of ARO10-OfT8GT1 and fermentation of the strain

[0080] The ARO10 fragment and OfT8GT1 from Example 1 were integrated into the *E. coli* S209 constructed in Example 2 to obtain strain Sal085. Sal085 was inoculated into LB liquid medium and cultured overnight at 37°C and 220 rpm to obtain a seed culture. This seed culture was then inoculated into fresh fermentation medium at a volume of 1% and cultured at 37°C and 220 rpm for 2 hours. After induction, 0.5 mM IPTG was added, and fermentation was induced at 30°C and 220 rpm for 94 hours. The yields of rhodioloside, 4-hydroxyphenylethanol, and 4-hydroxyphenylacetic acid were detected at different time points during fermentation using high-performance liquid chromatography (HPLC). The conditions were: Atlantis T3 column (4.6*100 mm, 3 μm); mobile phase: H2O + 0.1% TFA and ACN + 0.1% TFA; flow rate: 1.0 mL / min; column temperature: 40°C; UV detector; detection wavelength: 220 nm; detection time: 12.0 min.

[0081] The fermentation results are shown in Figure 5. Enzymes ARO10 and OfT8GT1 can effectively synthesize rhodioloside from glucose. After 96 hours of fermentation, 999.91 mg / L of rhodioloside can be synthesized (Figure 5(2)), while no intermediate 4-hydroxyphenylethanol accumulates (Figure 5(1)). However, a significant amount of the byproduct 4-hydroxyphenylacetic acid is synthesized during fermentation (Figure 5(3)), reaching 1.76 g / L after 96 hours. Therefore, further knocking out the gene encoding this pathway will further increase the yield of rhodioloside.

[0082] Example 5: Knockout of the feaB gene and fermentation

[0083] The feaB gene was knocked out in strain Sal085 to screen for the engineered strain Sal110. Sal110 was inoculated into LB liquid medium and cultured overnight at 37°C and 220 rpm to obtain a seed culture. This seed culture was then inoculated into fresh fermentation medium at a volume of 1% and cultured at 37°C and 220 rpm for 2 hours. After induction with 0.5 mM IPTG, fermentation was induced at 30°C and 220 rpm for 94 hours. The yields of rhodioloside, 4-hydroxyphenylethanol, and 4-hydroxyphenylacetic acid were detected at different time points during fermentation using high-performance liquid chromatography (HPLC). The conditions were: Atlantis T3 column (4.6*100 mm, 3 μm); mobile phase: H2O + 0.1% TFA and ACN + 0.1% TFA; flow rate: 1.0 mL / min; column temperature: 40°C; UV detector; detection wavelength: 220 nm; detection time: 12.0 min.

[0084] The fermentation results are shown in Figure 6. Compared with the engineered strain before knockout, after knocking out feaB, there was no accumulation of the corresponding byproduct 4-hydroxyphenylacetic acid (Figure 6(3)), while the yield of rhodioloside reached 1.67 g / L (Figure 6(2)). However, it was found that the intermediate product 4-hydroxyphenylethanol accumulated significantly after knocking out feaB (Figure 6(1)). Therefore, OfT8GT1 was further integrated to increase the downstream driving force, thereby further increasing the yield of rhodioloside.

[0085] Example 6: Integration and fermentation of OfT8GT1 strain

[0086] The OfT8GT1 fragment was reintegrated into Sal110 to obtain engineered strain Sal123. Further integration of OfT8GT1 into Sal123 yielded engineered strain Sal132. Sal123 and Sal132 were inoculated into LB broth and cultured overnight at 37°C and 220 rpm to obtain a seed culture. This seed culture was inoculated into fresh fermentation medium at a volume of 1% and cultured at 37°C and 220 rpm for 2 hours. Then, 0.5 mM IPTG was added for induction, and fermentation was induced at 30°C and 220 rpm for 94 hours. The yields of rhodioloside, 4-hydroxyphenylethanol, and 4-hydroxyphenylacetic acid were detected using high-performance liquid chromatography (HPLC) at different time points during fermentation. The conditions were as follows: Atlantis T3 column 4.6*100mm 3μm, mobile phase H2O+0.1% TFA and ACN+0.1% TFA, flow rate 1.0mL / min, column temperature 40℃, UV detector, detection wavelength 220nm, detection time 12.0min.

[0087] The fermentation results are shown in Figure 7. By further integrating the second copy of OfT8GT1, the yield of rhodioloside was further improved, reaching 2.36 g / L in 96 h (Figure 7(2)). At the same time, there was no significant accumulation of the toxic intermediate 4-hydroxyphenylethanol (Figure 7(1)) and almost no accumulation of the byproduct 4-hydroxyphenylacetic acid (Figure 7(3)). Although the yield of rhodioloside was not significantly improved after integrating the third copy of OfT8GT1 (Figure 8(2)) and there was almost no accumulation of 4-hydroxyphenylethanol (Figure 8(1)) and 4-hydroxyphenylacetic acid (Figure 8(3)), the Sal132 strain was used for subsequent studies to maximize downstream driving force.

[0088] Example 7: Knockout of the puuC gene and fermentation

[0089] To further remove the fermentation byproduct 4-hydroxyphenylacetic acid, puuC was knocked out in strain Sal132, resulting in the engineered strain Sal246. Sal246 was inoculated into LB liquid medium and cultured overnight at 37°C and 220 rpm to obtain a seed culture. This seed culture was then inoculated into fresh fermentation medium at a volume of 1%, and cultured at 37°C and 220 rpm for 2 hours. After induction, 0.5 mM IPTG was added, and fermentation was induced at 30°C and 220 rpm for 94 hours. The yield of 4-hydroxyphenylacetic acid was detected at different time points during fermentation using high-performance liquid chromatography (HPLC). The conditions were: Atlantis T3 column (4.6*100 mm, 3 μm); mobile phase: H2O + 0.1% TFA and ACN + 0.1% TFA; flow rate: 1.0 mL / min; column temperature: 40°C; UV detector; detection wavelength: 220 nm; detection time: 12.0 min.

[0090] The fermentation results are shown in Figure 9. Compared with the engineered strain before knockout, no corresponding byproduct 4-hydroxyphenylacetic acid was accumulated after knocking out puuC.

[0091] Example 8: Production of Rhodioloside in Fermentation Tanks

[0092] The engineered strain Sal246 was used to scale up the production of rhodioloside in a fermenter. Recombinant Escherichia coli Sal246 was inoculated into a seed tank and cultured at 37°C for 2-3 hours. The culture was then stopped, the liquid was drained, and the inoculum was transferred to the fermenter at a 5% inoculum level. During fermentation, the pH was controlled at 7.0, the temperature at 34-37°C, and the initial dissolved oxygen (DO) at 40%. The turbine speed and airflow were adjusted in conjunction with dissolved oxygen levels. After 3 hours of scale-up culture, IPTG was added to a final concentration of 0.5 mM, and fermentation was induced for another 67 hours, resulting in the synthesis of 29 g / L rhodioloside.

[0093] Compared with the results of this invention and those reported in the current literature (Table 2), it can be seen that the strain of this invention not only achieves the highest yield of Escherichia coli reported to date, but also has a short production cycle. The entire fermentation process does not require the addition of any antibiotics or precursors, and has great economic value and good industrialization prospects.

[0094] Table 2

[0095] Document 1: Liu S, Xia Y, Yang H, et al. Rational chromosome engineering of Escherichia coli for overproduction of salidroside[J]. Biochemical Engineering Journal, 2022,184:108474.

[0096] Document 2: Liu H, Tian Y, Zhou Y, et al. Multi-modular engineering of Saccharomyces cerevisiae for high-titer production of tyrosol and salidroside[J]. Microbial Biotechnology, 2021, 14(6): 2605-2616.

[0097] As can be seen from the above description, the embodiments of the present invention achieve the following technical effects: By optimizing the expression intensity of key genes in the rhodioloside synthesis pathway, enhancing precursor supply, knocking out competing pathways, and integrating related genes into the genome, the present invention constructs a plasmid-free, highly efficient de novo rhodioloside synthesis strain. This reduces the metabolic burden, increases rhodioloside yield and conversion rate, and eliminates the need for any antibiotics or intermediate substances during the entire fermentation process, significantly reducing production costs and simplifying subsequent separation and purification steps. Shake-flask fermentation can achieve a yield of 2.35 g / L, and a 1.5L fermenter can achieve 29 g / L, which is the highest reported yield in Escherichia coli to date. The engineered Escherichia coli provided by this invention has high production intensity, a simple fermentation process, and good application prospects, laying the foundation for the microbial fermentation production of rhodioloside.

[0098] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A genetically engineered bacterium, characterized in that, The genetically engineered bacteria contain exogenously introduced phenylpyruvate decarboxylase gene ARO10 and glycosyltransferase gene OfT8GT1, wherein the phenylpyruvate decarboxylase gene ARO10 has the nucleotide sequence shown in SEQ ID NO: 3; and the glycosyltransferase gene OfT8GT1 has the nucleotide sequence shown in SEQ ID NO:

4.

2. The genetically engineered bacterium according to claim 1, characterized in that, The genetically engineered bacteria do not contain any one or more of the following genes: poxB, pta, ackA, eutD, adhE, mhpF, eutE, ldhA, dld, lldD, aldA, fnr, arcA, ptsG, feaB, or puuC; and / or The genetically engineered bacteria also contain any one or more of the following exogenous genes: glk, aroF, aroK, glf as shown in SEQ ID NO: 7, mutant aroG as shown in SEQ ID NO: 5, or mutant tyrA as shown in SEQ ID NO: 6; wherein the exogenous genes are all integrated into the genome of the genetically engineered bacteria.

3. The genetically engineered bacteria according to claim 1 or 2, characterized in that, The genetically engineered bacteria is Escherichia coli.

4. A method for preparing genetically engineered bacteria, characterized in that, The preparation method includes: transferring a plasmid containing the phenylpyruvate decarboxylase gene ARO10 and the glycosyltransferase gene OfT8GT1 into the starting engineered bacteria to obtain the genetically engineered bacteria; The phenylpyruvate decarboxylase gene ARO10 has the nucleotide sequence shown in SEQ ID NO: 3, and the glycosyltransferase gene OfT8GT1 has the nucleotide sequence shown in SEQ ID NO:

4.

5. The preparation method according to claim 4, characterized in that, The starting engineered bacteria is Escherichia coli.

6. The preparation method according to claim 5, characterized in that, The preparation method further includes: knocking out any one or more of the following endogenous genes in the starting engineered bacteria: poxB, pta, ackA, eutD, adhE, mhpF, eutE, ldhA, dld, lldD, aldA, fnr, arcA, ptsG, feaB, or puuC; and / or Knock in any one or more of the following genes into the starting engineered bacteria: glk, aroF, aroK, glf as shown in SEQ ID NO: 7, mutant aroG as shown in SEQ ID NO: 5, or mutant tyrA as shown in SEQ ID NO:

6.

7. A method for de novo synthesis of rhodioloside, characterized in that, The method includes: fermenting the rhodioloside in a fermentation medium using the genetically engineered bacteria prepared by any one of claims 1 to 3 or by any one of claims 4 to 6.

8. The method according to claim 7, characterized in that, The fermentation is either shake flask fermentation or fermenter fermentation.

9. The method according to claim 8, characterized in that, The shake-flask fermentation includes: inoculating the genetically engineered bacteria cultured overnight into the fermentation medium for expansion culture, culturing until the OD600 is 0.8-1, adding an inducer for induced fermentation for 22-94 hours, and obtaining the rhodioloside.

10. The method according to claim 9, characterized in that, The overnight culture conditions are 30-37℃ and 200-220 rpm; the scale-up culture conditions are 30-37℃ and 200-220 rpm; the induced fermentation conditions are 25-35℃ and 200-220 rpm; the inducer is IPTG; and the final concentration of the inducer is 0.1-1 mM.

11. The method according to claim 9, characterized in that, The fermentation medium for the shake-flask fermentation consists of 5-10 g / L NaCl, 2-5 g / L yeast extract, 5-10 g / L tryptone, and 10-20 g / L glucose.

12. The method according to claim 8, characterized in that, The fermentation process in the fermenter includes: inoculating the genetically engineered bacteria cultured overnight into a seed tank for expansion culture until the OD600 reaches 10-20; stopping the fermentation, draining the liquid, transferring the culture, and performing a second expansion culture until the OD600 reaches 10-20; adding an inducer for induced fermentation for 60-70 hours to obtain the rhodioloside.

13. The method according to claim 12, characterized in that, The overnight culture conditions are 30-37℃; the expansion culture and the secondary expansion culture conditions are DO 30-40%, pH 6.8-7.0, 30-37℃; the induced fermentation conditions are DO 30-40%, pH 6.8-7.0, 25-35℃; the inducer is IPTG; the final concentration of the inducer is 0.1-1mM.

14. The method according to claim 12, characterized in that, The fermentation medium used in the fermenter consists of: glucose monohydrate 10-30 g / L, glycerol 10-30 g / L, magnesium sulfate heptahydrate 2-4 g / L, yeast powder 5-10 g / L, corn steep liquor powder 4-8 g / L, sodium citrate 1-3 g / L, ammonium sulfate 4-8 g / L, dipotassium hydrogen phosphate 2-4 g / L, potassium dihydrogen phosphate 2-4 g / L, a trace element mixture 1-2 mL / L, thiamine hydrochloride 0.1-0.3 g / L, and a foaming agent 0.2-0.4 mL / L.

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