索引
Overview
Engineering of the chassis organisma
Production of a-KB
Further optimization.
Temperature Control
Overview
A-kb is an important molecule with potential for stimulating hair growth, naturally found in the metabolic pathways of various organisms. However, accumulating or extracting a sufficient amount of a-kb from organisms has proven challenging. Current industrial practices for a-kb production primarily rely on chemical or enzymatic methods, which not only incur high costs but also result in the generation of numerous harmful byproducts, negatively impacting the environment.
This year, AIS-China aimed to employ synthetic biology techniques, specifically genetically modifying our Escherichia coli strain AIS, to produce a-kb. Our experiment was conducted in three phases. Firstly, we selectively knocked out three specific genes (rhtA, ilvIH, and ilvBN) in the AIS strain and increased expression of the desired gene ilvA, which is responsible for a-kb production. In the second phase, we sought to optimize the production process by introducing mutations to the ilvA gene (resulting in ilvA*), utilizing vectors with different copy numbers to carry and express ilvA, and enhanced upstream genes. Finally, we successfully tested and implemented two temperature-sensitive repressors. We want replace the need for iPTG induction, thus achieving our goal of thermal-inducible expression in the future.
In our project, we successfully achieved the accumulation of a-kb inside E. coli and attained a production level of 1.8 g/L. We hope for the future mass scale production of a-kb through temperature-induced bacteria!
Engineering of the chassis organism
Threonine serves as the crucial precursor for a-KB production. Thus, we selected the E. coli strain CICC20905 as the chassis organism for a-KB production, which we subsequently referred to as the AIS series, based on extensive research which indicated its ability to generate substantial quantities of threonine. In order to make our chassis organisms more advantageous, we initiated a series of genetic modifications. Initially, we knocked out the rhtA gene to create the AIS-1 strain. This action had the effect of retaining more threonine within the cell, as the rhtA gene, responsible for threonine export, was disrupted. Subsequently, base on the AIS-1 strains, we respectively knocked out ilvIH to construct AIS-2 and knocked out ilvBN to construct AIS-3 strains.this would reduce the outflow of a-KB as downstream byproducts (i.e., 2-acetyl-2-Hydroxybutyrate), ultimately enhancing the accumulation of a-KB.
To accomplish these genetic modifications, we constructed three pTarget plasmids, each carrying a specific gRNA sequence capable of identifying the target gene. We then obtained donor DNA through a two-step PCR process. This donor DNA was employed for homologous recombination with the genomic DNA (refer to Figure 1A). Subsequently, we separately introduced the pTarget plasmids along with their corresponding donor DNA into the host organism, which had already been engineered to carry the pEcCas plasmid. We conducted our knockout experiments according to the experimental procedures previously published by Qi Li, Bingbing Sun, et al. in 2020 (as depicted in Figure 2).
Validation of our genetic modifications was achieved through colony PCR (Figure 1B) and gene sequencing (Figure 1C). These analyses confirmed the successful knockout of the rhtA, ilvIH, and ilvBN genes, allowing us to obtain our first strains: AIS-1 (ΔrhtA), AIS-2 (ΔrhtA and ΔilvIH), and AIS-3 (ΔrhtA and ΔilvBN).Finally, we use three plates carrying different antibiotics to confirm the cure of pTarget and pEcCas plasmid(Figure 1D).
Figure1: Knock out genes in AIS strains. （A）the design of pEcCas、pTarget plasmid and donorDNA for gene knockout. (B)colony PCR to respectively determine the knock-out of rhtA、ilvIH、ilvBN.（C）verified the knock-out result through the sequencing testing. (D)three plates carrying different antibiotics to confirm the cure of pTarget and pEcCas plasmid
Figure 2: Step1: Transformation of the pEcCas plasmid(carry cas9 protein) into E. coli AIS-0 (CICC20905). Step2: construction of the pTarget plasmid and donor DNA. Step3: transformation of pTarget plasmid and donor DNA into the AIS-0 with pEsCas inserted. Step4: Incubation of the transformed strains.
Production of a-KB
In order to produce a-KB, we sought to express the ilvA gene within the AIS strains. The ilvA gene encodes the enzyme Threonine Dehydrase, which is responsible for the removal of water and ammonium molecules from threonine (as depicted in Figure 3A). We intended to use the IPTG-induced pTac promoter system to regulate the expression of the ilvA gene. However, since AIS series strains of E. coli are relatively underexplored. There was uncertainty regarding the functionality of this system in our context.
To ascertain whether the IPTG induction system operates effectively in AIS strains, we conducted a validation experiment using GFP as a signaling protein. This allowed us to assess the efficiency of the IPTG inducer and determine the optimal induction concentration. The results, illustrated in Figure 3B, confirmed that the AIS strain responded positively to the IPTG induction, with concentrations ranging from 0.1 mM to 0.2 mM exhibiting the strong inducing effect.In subsequent experiments, we used 0.3mM iPTG to induce gene expression, which is the most commonly used concentration in the laboratory.
With the IPTG induction system established, we proceeded to construct the PW1-ilvA plasmid for a-KB production. This process began by obtaining the ilvA gene fragment from the E. coli genome through PCR. Subsequently, we integrated this fragment with the PW1 vector using the Golden Gate recombination method. The successful construction of the PW1-ilvA plasmid was verified through colony PCR results (see Figure 3C) and gene sequencing outcomes (as shown in Figure 3D).
figure3: (A) ilvA function and IPTG induced-pTac promotor system.(B)test the induction sysytem by different iPTG concentrations in AIS strains.(C)colony PCR to determine the construction of PW1-ilvA. (D)verified the construction result through sequencing.
We transformed the PW1-ilvA plasmid into strains AIS-0, AIS-1, AIS-2, and AIS-3, each with the aim of producing a-KB. Regrettably, our efforts to insert pw1-ilvA into AIS-3 were unsuccessful. We attribute this outcome to the simultaneous knockout of both the rthA and ilvBN genes, along with the overexpression of the ilvA gene, which appeared to hinder cell growth. We speculate that this inhibition may be due to the cytotoxic effects of a-KB.
For the successfully transformed strains, we conducted HPLC methods to confirm the successful production of a-KB. We acquired a-KB standards from the market, by comparing the peak diagram of the standard a-KB with the supernatant of the AIS fermentation broth, we confirmed the production of a-Kb.Then, we made a standard curve with a-kb concentration of 0-20g/L in order to calculate the production of a-kb in AIS series(figure 4B). Observing the bar charts below, we can see that the production of a-kb increase gradually in AIS-0,AIS-1 and AIS-2, which indicate that the removal of rhtA gene and ilvIH gene can increase the production of a-kb (figure 4C).
Figure 4: (A) HPLC peak diagrams of standard a-KB and AIS supernatant sample. (B) the standard curve about the relationship between peak area and a-KB concentration. (C)the production of a-KB in AIS series with pw1-ilvA plasmid.
Further optimization.
ilvA mutation and Copy number changed
For further improvement on a-kb production levels, we want to optimize threonine's conversion into a-kb, a key step in a-kb production.We tried to use plasmids with different copy numbers to express ilvA to determine the effect on copy number on a-KB production. Thus, we constructed ilvA onto a new plasmid p321-ilvA, which has a low copy number in E. coli (Figure A)
We also mutated ilvA(obtainning ilvA*) to make its expressed enzymes resistant to the inhibition of Isoleucine. Isoleucine is a downstream product of a-kb.Our gene knock out choices do not completely obstruct the pathway continuing downwards of a-kb, thus meaning that Isoleucine is existent in the E.coli cell (Isoleucine inhibits ilvA expressed Threonine Dehydrase, thus negatively affecting Threonine's conversion into a-kb, which is reported in many papers).
We made four base mutations in ilvA (i.e., C1339T, G1341T, C1351G, T1352C), which substitutes the 447th and 451th amino acids (both Leu) into Phe and Ala, respectively (Figure B). After successfully mutating the ilvA gene through PCR with primers coding for the mutated sequences (Figure C), we obtained the strains pw1-ilvA* and p321-ilvA* (ilvA* is the mutated ilvA). The two plasmids, along with pw1-ilvA and p321-ilvA, were transformed into AIS-2, and the a-kb production is measured. The result show that pw1-ilvA* strain was most efficient. Moreover, we found that for both vectors (p321 and pw1), the mutated ilvA* yielded more a-KB compared to the non-mutated ilvA, and no matter whether ilvA is mutated or not, the pw1 plasmid with higher copy number yielded more a-KB than the p321 plasmid with low copy number.
Figure 5: (A) the different copy number of plasmid with ilvA. (B) ilvA*'s resistancy to isoleucine inhibition and codon and amino acid differences between the ilvA and the mutated ilvA*. (C) the design for the mutation of ilvA and the sequencing results of ilvA*. (D)the a-kb production of strain AIS-2 with four different plasmid;
Increase upstream concentrations:
We have also tried modifying the upstream pathway of Threonine for more a-KB by increasing the copy number of gene thrABC in an effort to enhance the conversion of Aspartate into Threonine (Fig.A). For upstream modification, we used pcr to obtain our gene thrABC from DH5a genome, and then constructed two plasmids, p321-thrABC (constitutive expression) and p15a-thrABC (inducible expression). After the indication of a successful construction of plasmid from DNA sequencing (Fig.c), the two plasmids were each transformed into AIS-2, and after fermentation we measured the a-kb production. Unfortuanatley, the a-kb production did not improve, and instead did the contrary. This may be because, instead of an increase in threonine, an inhibition of the thrABC gene in the E.coli genome enhanced, in the end, leading to a decrease in production of a-kb.
Figure 6: (A)the pathway of Aspartate to Threonine in E.coli. (B)Obtain gene fragments thrABC and vectors p321 and p15A. (C) sequencing verified the construction of plasmids p321-thrABC and P15A-thrABC. (D) the a-kb production of AIS-2 with plasmid.
Temperature Control
To enable the practical large-scale implementation of our α-kb production method, we made the strategic decision to transition from using IPTG-induced expression to a more user-friendly and cost-effective temperature-controlled induction approach. To achieve this objective,we tested two temperature sensitive repressors: TcI and TcI40 (TcI40 is a mutated version of TcI, shown in fig.7B).
In comparison to TcI, TcI40 exhibited nonsynonymous mutations at six distinct positions, resulting in alterations to six amino acids: L65S, A67T, K68R, F115L, D126G, and D188G. Additionally, seven synonymous mutations (base is changed but amino acid remains constant) were also present: A50, I69, E128, R129, T152, S160, and L185(fig.7B).
Figure 7: (A)The design of TcI-pR/pL control circuit. (B)The mutation of TcI40 compared to TcI.
We tested the two temperature sensitive repressors in the DH5a strain with RFP as the signaling protein, and the test was carried out in two groups each using one of the stated repressors. The first group's RFP was expressed using a plasimd containing repressor TcI, and the latter group used a plamid containing repressor TcI40. The visible red color of expressed RFP proteins indicates if or if not the repressors loses its inhibitive effects on transcription (and thus expression of genes) at a given temperature.
The results of this test show that at a temperature of 30 degrees Celsius, no visible red color can be seen (fig.8A), indicating that no RFP is expressed. Thus signaling that repressor TcI and TcI40 both show effective inhibition on the transcription process of RFP at such a given temperature. At a temperature of 37 degrees celcius, the transcription inhibition effect of repressor TcI ceases, and RFP expression values reaches its climax. At temperatures of 37, 40 and 42 degrees Celcius, the transcription inhibition effect of repressor TcI40 ceases, while climax values of RFP expression occur at a temperature of 40 degrees Celcius.
Figure 8: Test TcI and TcI40 repressors at different induction temperatures.
We have successfully tested two temperature control systems. By comparing TcI and TcI40 on RFP fluorescence at different induced temperatures, we can find that TcI40 can maximize the suppression of pR/pL promoter, so as to achieve the highest gene expression . In the future industrial production of a-KB, we hope to use TcI40 repressor to induce the production of a-KB to achieve temperature control production in large scale.
reference：
Cheng L, Wang J, Zhao X, et al. An antiphage Escherichia coli mutant for higher production of L-threonine obtained by atmospheric and room temperature plasma mutagenesis. Biotechnol Prog. 2020;36(6):e3058. doi:10.1002/btpr.3058
Li Q, Sun B, Chen J, Zhang Y, Jiang Y, Yang S. A modified pCas/pTargetF system for CRISPR-Cas9-assisted genome editing in Escherichia coli. Acta Biochim Biophys Sin (Shanghai). 2021;53(5):620-627. doi:10.1093/abbs/gmab036
Restrepo-Pineda S, O Pérez N, Valdez-Cruz NA, Trujillo-Roldán MA. Thermoinducible expression system for producing recombinant proteins in Escherichia coli: advances and insights. FEMS Microbiol Rev. 2021;45(6):fuab023. doi:10.1093/femsre/fuab023
Chen L, Chen Z, Zheng P, Sun J, Zeng AP. Study and reengineering of the binding sites and allosteric regulation of biosynthetic threonine deaminase by isoleucine and valine in Escherichia coli. Appl Microbiol Biotechnol. 2013;97(7):2939-2949. doi:10.1007/s00253-012-4176-z
Zhang C, Qi J, Li Y, et al. Production of α-ketobutyrate using engineered Escherichia coli via temperature shift. Biotechnol Bioeng. 2016;113(9):2054-2059. doi:10.1002/bit.25959
Park JH, Oh JE, Lee KH, Kim JY, Lee SY. Rational design of Escherichia coli for L-isoleucine production. ACS Synth Biol. 2012;1(11):532-540. doi:10.1021/sb300071a
Hao R, Wang S, Jin X, Yang X, Qi Q, Liang Q. Dynamic and balanced regulation of the thrABC operon gene for efficient synthesis of L-threonine. Front Bioeng Biotechnol. 2023;11:1118948. Published 2023 Mar 2. doi:10.3389/fbioe.2023.1118948
