Optogenetics-assisted continuous evolution screening of engineered strains and screening methods and applications

Abstract

The present invention discloses an optogenetics-assisted continuous evolution screening engineered strain and a screening method and application, which belong to the field of bioengineering technology. The engineered strain includes two units: a mutation-reporter unit, which is used to introduce point mutations in the promoter target protein region and change the fluorescence properties of the target protein or change the fluorescence properties of the downstream reporter protein regulated by the target protein; a lethal-screening unit, which is used to protect the target bacteria by synthesizing antibiotics in response to light, and at the same time, generate reactive oxygen species in response to light to kill non-target bacteria. The present invention can well solve the problem of a large number of independent (high-throughput) optogenetic tests in biology. Its overall structure is simple, its applicability is wide, and its actual operation is convenient. It has great significance for improving the development efficiency of high-throughput optogenetic gene circuits and studying the basic principles of light-sensitive genes.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering technology, and specifically relates to an optogenetics-assisted continuous evolution screening engineered strain, a screening method, and an application. Background Art

[0002] Optogenetics is a discipline that combines light and genetic engineering to manipulate target genes. By expressing light-responsive target proteins in a target host, it is possible to modulate the host's physiological functions or behaviors through light stimulation. Technologies related to optogenetics are widely used in scientific research and manufacturing.

[0003] Directed evolution, a recently developed protein engineering method that mimics Darwinian evolution, involves artificially introducing large numbers of mutations and then screening for desired proteins based on specific needs and goals. It plays a significant role in metabolic engineering and synthetic biology. Common strategies for directed evolution include random evolution, shuffling techniques, semi-rational evolution, and rational evolution. The key to these strategies lies in mutation screening. Therefore, the key to direct evolution screening is how to introduce large numbers of mutations and select those that meet the desired requirements.

[0004] Existing directed evolution screening methods vary, including a library construction-screening cycle. For example, patent CN109943581A provides a plasmid- and phage-assisted directed evolution screening system and method; patent CN111269822A discloses a multifunctional high-throughput directed evolution system and method; and patent CN211227159U discloses a DNA polymerase continuous directed evolution strain screening chip, which unitizes repetitive steps onto the chip, increasing automation and reducing manual errors. It can be seen that existing technologies for evolutionary screening typically require multiple channels, multiple chambers, and multiple units to perform screening continuously. The entire system and method require numerous and complex devices, channels, and connectors.

[0005] Currently, there are few reports on technologies combining optogenetics with targeted screening. Summary of the Invention

[0006] In order to overcome the shortcomings of the above-mentioned prior art, the purpose of the present invention is to provide an optogenetics-assisted continuous evolution screening engineered strain and screening method and application, so as to broaden the strategy of the directed evolution screening method and provide a directed screening engineered strain and screening method that is easy to modify and simple to operate.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] The present invention discloses an optogenetics-assisted continuous evolution screening engineered strain, which includes two units:

[0009] A mutation-reporter unit is used to introduce a point mutation in the promoter target protein region and to change the fluorescence properties of the target protein or the downstream reporter protein regulated by the target protein;

[0010] The lethal-selection unit is used to protect target bacteria by synthesizing antibiotics in response to light, while simultaneously generating reactive oxygen species in response to light to kill non-target bacteria.

[0011] If the target protein in the mutant-reporter unit is non-fluorescent, a downstream fluorescent reporter protein induced by the target protein can be used as a reporter system.

[0012] Preferably, the mutation-reporter unit is expressed by one plasmid, and the lethal screening unit is expressed by one plasmid, forming a dual-plasmid expression system capable of simultaneous expression.

[0013] Further preferably, the mutation-reporter unit is expressed by plasmid pJN105.

[0014] More preferably, the plasmid pJN105 carries an arabinose promoter for expressing the mutant protein AID732 fused with T7 polymerase RNAP.

[0015] Preferably, in the mutation-reporter unit, the mutant protein AID732 fused to the expressed T7 polymerase RNAP can be replaced by any single-base mutant protein, or by multiple single-base mutant proteins expressed simultaneously.

[0016] Preferably, the lethal selection unit selects for expression of plasmid pSUPAR.

[0017] Further preferably, the plasmid pSUPAR expresses a blue light responsive protein that can screen target bacteria that survive antibiotics through blue light response, and also expresses a red light responsive protein that can kill non-target bacteria by inducing the production of reactive oxygen species through red light.

[0018] More preferably, the blue light responsive protein is selected from the YF1-FixJ dual protein system; the red light responsive protein is selected from the KillerRed protein.

[0019] Preferably, the blue light responsive protein YF1-FixJ system can be replaced by any light responsive inducible transcriptional expression system.

[0020] Further preferably, the KillerRed protein that generates reactive oxygen species induced by red light can be replaced by any protein that generates reactive oxygen species in response to light or other light-inducible systems such as light-induced expression of lethal genes.

[0021] More preferably, the above-mentioned blue-red light response system is replaceable in response to light of different wavelengths.

[0022] More preferably, the above-mentioned dual-plasmid expression system (plasmid pSUPAR and plasmid pJN105) can be replaced by any two plasmid systems capable of simultaneous expression.

[0023] The present invention also discloses the above-mentioned optogenetics-assisted continuous evolution screening method for engineering strains, comprising the following steps:

[0024] S1: The engineered strain was plated in a single layer and cultured in a normal medium. Inducers were added to induce the expression of the fusion mutant protein.

[0025] S2: Identify the distribution of target and non-target strains by microscopic imaging;

[0026] S3: Based on the distribution, blue light is applied to the target strain and antibiotics are added to the culture medium to keep the target bacteria alive and kill non-target bacteria. At the same time, red light is applied to the non-target strain to induce the lethal proteins in the non-target bacteria to produce reactive oxygen species and kill the non-target bacteria.

[0027] S4: Repeat the operations of S1 to S3 for the surviving target bacteria to perform the next round of screening until the target bacteria are screened.

[0028] Preferably, the inducer is a chemical inducer or a photoinducer.

[0029] Further preferably, the inducer can be replaced by any chemical inducer or photoinducer.

[0030] The present invention also discloses the application of the above-mentioned optogenetics-assisted continuous evolution screening engineered strain in directed evolution screening.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] The optogenetics-assisted continuous evolution screening engineered strain disclosed in the present invention includes a mutation unit, a reporter unit, and a lethal screening unit. The mutation unit is for introducing a large number of point mutations in the target protein region downstream of the promoter. The reporter unit can change the fluorescence characteristics of the target protein or the downstream reporter protein regulated by the target protein. The lethal screening unit is a double lethal design that can complete a double response, one of which is to synthesize antibiotics in response to light to protect the target bacteria, and the other is to produce reactive oxygen species in response to light to kill non-target bacteria. The structural design of the engineered strain is reasonable, and it can meet the screening design requirements of directed evolution through mutation induction and lethal screening, thereby solving the problem of a large number of independent (high-throughput) optogenetic tests in biology. Its overall structure is simple, the applicability is wide, and the actual operation is convenient. It is of great significance to improving the development efficiency of high-throughput optogenetic gene circuits and studying the basic principles of light-sensitive genes. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Schematic diagram of the mutation-reporter unit of the invented engineered strain;

[0034] Figure 2 This is a schematic diagram of the engineered strain lethality-screening unit of the present invention;

[0035] Figure 3 Flow chart of the optogenetics-assisted continuous directed evolution screening method of the present invention;

[0036] Figure 4 This is the growth curve of the engineered strain's blue light-induced survival and antibiotic bactericidal screening;

[0037] Figure 5 This figure shows the effect of arabinose concentration on induced mutations. DETAILED DESCRIPTION

[0038] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts should fall within the scope of protection of the present invention.

[0039] It should be noted that the terms "first", "second", etc. in the description and claims of the present invention and the above-mentioned drawings are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that the numbers used in this way can be interchanged where appropriate, so that the embodiments of the present invention described herein can be implemented in an order other than those illustrated or described herein. In addition, the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions. For example, a process, method, system, product or device that includes a series of steps or units is not necessarily limited to those steps or units clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0040] The present invention is described in further detail below with reference to the accompanying drawings:

[0041] Example 1

[0042] This embodiment uses the selected mutant protein AID732 as an example to illustrate the scheme of the present invention.

[0043] The optogenetics-assisted continuous evolution screening engineered strain designed in this embodiment includes two units: a mutation-reporter unit and a lethality-screening unit.

[0044] See also Figure 1 , a mutation-reporter unit, was chosen for expression using the plasmid pJN105. This plasmid carries an arabinose promoter, controlling gene expression by the sugar arabinose. It expresses a T7 polymerase-mutant fusion protein, which can be replaced by any protein capable of inducing single-base mutations. This unit is used to introduce a large number of point mutations into a specific region, generating material for directed evolution.

[0045] See also Figure 2 , which is a lethal-screening unit, was expressed using the pSUPAR plasmid. The lethal unit was a double-lethal design, in which the light-responsive proteins YF1 and FixJ responded to blue light to activate the expression of the downstream accC1 gentamicin-resistance gene. Blue light can be used to screen specific target bacteria to enable them to survive under antibiotic conditions. In addition, the expressed KillerRed protein responded to red light and could produce a large amount of ROS to kill bacteria. Red light can be used to kill non-target bacteria while allowing target bacteria to survive.

[0046] See also Figure 3 The present invention provides a method for continuous evolution screening of engineered strains based on the above-mentioned optogenetics-assisted continuous evolution screening as follows:

[0047] Using ordinary culture medium, an inducer is added to induce the expression of the fused mutant protein. The fused mutant protein mutates at the target sequence, which directly affects the fluorescence intensity of the target protein or indirectly affects the fluorescence intensity of the fluorescent reporter system. Microscopic imaging and image recognition are used to analyze the distribution of target and non-target strains. Based on this distribution, blue light is applied to the target strain and the corresponding antibiotic is added to the culture medium to allow the target bacteria to survive and the non-target bacteria to die. At the same time, red light is applied to the non-target bacteria to induce the lethal protein in the non-target bacteria to produce reactive oxygen species, killing the non-target bacteria. The surviving target bacteria can then proceed to the next round of screening.

[0048] Specifically, the engineered strain was spread in a monolayer in a device for culturing a monolayer of bacteria (the culture device disclosed in application number CN202111405686.9). According to the characteristics of the engineered strain (taking Escherichia coli MG1655 as an example), LB medium and corresponding double antibiotics (corresponding to tetracycline and chloramphenicol) were given for culture. The inducer was added at a final concentration of 0.4% arabinose to induce the expression of the fusion mutant protein (AID732-RNAP). At the same time, the microscopic imaging and image recognition system collected fluorescence images of the monolayer of engineered bacteria, and the fluorescence intensity was analyzed to confirm the target bacteria and non-target bacteria.

[0049] Using an optogenetic control system and control method (such as the control system and method disclosed in application number CN202210935805.X), the target bacteria are illuminated with blue light to induce expression of the resistance gene accC1. Gentamycin is then added to the culture medium at a final concentration of 15 μg / mL 0.5 hours later to kill non-target bacteria while retaining the target bacteria. At the same time, red light is applied to the non-target bacteria to induce the production of reactive oxygen species, killing them. The surviving bacteria then re-grow and the antibiotics in the culture medium are stripped away. The next round of screening is then performed after the bacteria re-fill the device.

[0050] The testing of the engineered strain and directed evolution screening method of the present invention is mainly divided into two parts. The first part is to retain the target strain under blue light illumination, and the second part is to induce mutation by arabinose.

[0051] First, under the condition of protection from light, the engineered strain was revived on LB medium agar plate containing 10 μg / mL tetracycline and 37 μg / mL chloramphenicol. The revived engineering strain was resuspended in the same medium and placed in a transparent shaking tube. The culture was shaken at 37 degrees Celsius and 250 rpm. After shaking for 2 hours and 15 minutes, gentamicin was added at a final concentration of 30 μg / mL (twice the conventional culture concentration, or 15 μg / mL final concentration). The strain was divided into a blue light illumination group and a dark protection group and continued to be cultured. The OD600 value was detected regularly. The growth curve obtained is as follows: Figure 4As shown, the engineered strain in the blue light illumination group grew normally, while the engineered strain in the light-avoidance group quickly stopped growing and gradually lysed.

[0052] Secondly, the engineered strain was cultured overnight under dark conditions, and then expanded at 1:100. At the same time, different final concentrations of arabinose were added to induce the expression of the mutant protein. After 2 hours of induction culture, the target gene region on the genome of the engineered strain was amplified by high-fidelity PCR. The obtained PCR product was purified and sequenced and compared with the target region. The number of mutations in a single reading frame (about 700 bp) was counted for data analysis, such as Figure 5 As shown, it can be seen that adding arabinose at an induction concentration of 0.2% to 0.5% can induce more mutations, while the control group without arabinose induction produces fewer mutations.

[0053] The above content is only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. A method for continuous evolution screening of engineered strains based on optogenetics-assisted continuous evolution screening, characterized in that: The following steps are involved: S1: The engineered strain is plated in a single layer and cultured in a normal medium. An inducer is added to induce the expression of the fused mutant protein. The fused mutant protein mutates at the target sequence. The resulting mutation directly affects the fluorescence intensity of the target protein or indirectly affects the fluorescence intensity of the fluorescent reporter system. S2: Analyze the distribution of target and non-target strains through microscopic imaging and image recognition; S3: Based on the distribution, the target strain is irradiated with blue light and antibiotics are added to the culture medium, allowing the target bacteria to survive and killing non-target bacteria. At the same time, light is applied to the non-target strains to induce the lethal proteins in the non-target bacteria to produce reactive oxygen species, killing the non-target bacteria. S4: Repeat the steps S1 to S3 for the surviving target bacteria to perform the next round of screening until the target bacteria are obtained; Wherein, the engineered strain comprises two units: A mutation-reporter unit is used to introduce a point mutation in the promoter target protein region and to change the fluorescence properties of the target protein or the downstream reporter protein regulated by the target protein; The lethal-selection unit is used to protect target bacteria by synthesizing antibiotics in response to light, while simultaneously generating reactive oxygen species in response to light to kill non-target bacteria.

2. The method for continuous evolution screening of engineered strains based on optogenetics-assisted continuous evolution screening according to claim 1, characterized in that: The inducer is a chemical inducer or a light inducer.

3. The method for continuous evolution screening of engineered strains based on optogenetics-assisted continuous evolution screening according to claim 1, characterized in that: The mutation-reporter unit selects one plasmid for expression, and the lethal-screening unit selects one plasmid for expression, forming a dual-plasmid expression system capable of simultaneous expression.

4. The method for continuous evolution screening of engineered strains based on optogenetics-assisted continuous evolution screening according to claim 3, characterized in that: The mutation-reporter unit selects plasmid pJN105 for expression; the lethality-screening unit selects plasmid pSUPAR for expression.

5. The method for continuous evolution screening of engineered strains based on optogenetics-assisted continuous evolution screening according to claim 4, characterized in that: The plasmid pJN105 carries an arabinose promoter for expressing a mutant protein AID732 fused with T7 polymerase RNAP.

6. The method for continuous evolution screening of engineered strains based on optogenetics-assisted continuous evolution screening according to claim 5, characterized in that: The mutant protein AID732 fused to T7 polymerase RNAP is replaced by any single-base mutant protein, or by expressing multiple single-base mutant proteins simultaneously.

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