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  • Pathway design of the secretion pathway in Bacillus subtilis
  • Pathway design of a FADS-based ultra-high-throughput evolution of PBAT-degrading enzyme
  • Engineered KT2440 Strain Construction and ALE-Derived Mutant Isolation for TPA and BDO Utilization
  • Reference
    • Pathway design of the secretion pathway in Bacillus subtilis

    We first established an efficient secretion system within Bacillus subtilis. The signal peptide AprE was fused to the N-terminus of the enzymes to enable them to be effectively secreted out of the cells via the Sec pathway (Figure 1a). In B. subtilis, chaperones help the signal peptide to fold initially, and then SRP (signal recognition particle) combines with the signal peptide to form a complex. Chaperones, FtsY, and SecA together lead the enzyme to the Sec transport proteins. After the precursor protein traversing the membrane, the signal peptide is cleaved and degraded, and the extracellular chaperones assist the protein to form a stable conformation[1, 2]. Then we performed Prolonged Overlap Extension PCR (POE-PCR) to separately polymerize the sequences of the three enzymes and the linearized pHCM12M vector into polymers (Figure 1b)[3], which were then transformed into Bacillus subtilis. To verify whether the enzymes were secreted out of the cells, we cultivated the bacterium on the plates with polycaprolactone (PCL), which can be hydrolyzed by lipases, esterases, keratinases and so on, commonly serving as a model substrate for assessing the extracellular secretion of hydrolases[4]. The observation of hydrolysis zones on the plates indicated successful expression and secretion of the enzymes.

    Figure 1. (a) An efficient secretion pathway in Bacillus subtilis. In the pathway there is the Sec signal peptide AprE separately with the genes of interest (GOI) requiring secretion downstream of it. GOI include IsPETase, BsLipA, and Lipase1028. Downstream of the GOI is the 6×His tag for the purification of the protein; (b) Flowchart of the prolonged overlap extension PCR (POE-PCR) process for the transformation of GOI into Bacillus subtilis SCK6. We obtain the linearized vector and DNA fragment with homologous arms by PCR reaction. During POE-PCR, GOI and the vector fragments are used simultaneously as primers and templates to obtain linear multimeric DNA, which is transformed into the super competent Bacillus subtilis SCK6 and circularized later to form recombinant plasmids. Compared to plasmid, multimeric DNA exhibits higher transformation efficiency when transformed into Bacillus subtilis[3].
    Figure 2. Construction and screening strategies for mutation libraries.
    Pathway design of a FADS-based ultra-high-throughput evolution of PBAT-degrading enzyme

    The method of screening by observing hydrolysis zones is common, straightforward, cost-effective, and simple to implement. However, its efficiency is limited a lot because of following drawbacks: (1) its throughput is low (~104)[5] ; (2) human bias when selecting colonies may happen; (3) the measurement of hydrolysis zone size may lead to errors that affect the accuracy of the screening process; (4) the hydrolysis zones may overlap, making it challenging to accurately measure the size of each colony. Therefore, a high-throughput and precise screening method for PBAT-degrading enzymes in B.subtilis is needed.

    A biosensor system for sensing TPA has been developed (Figure 3a)[6]. In this system, carboxylic acid reductase (CARMm) from Mycobacterium marinum and the luciferase LuxAB from Photorhabdus luminescense have been transformed into Bacillus subtilis. TPA can enter the cells and be reduced to aldehyde by CARMm. Then, LuxAB utilizes the aldehyde as a substrate to produce the corresponding acid, generating bioluminescence. By measuring the bioluminescence intensity at 482 nm, semi-quantitative detection of TPA can be achieved[6]. Our goal is to combine this system with FADS, which couples the activity of each variants with the fluorescence intensity, and finally facilitates high-throughput screening (~107) (Figure 3b) [5, 7].

    Figure 3. Biosensor-based TPA detection coupled with bioluminescent FADS screening.(a) An enzyme-coupled biosensor for detecting TPA produced by PBAT degradation. (1) PBAT enzymes degrade PBAT, releasing monomer molecules including TPA, BDO, and AA. The structure of the PBAT hydrolase is derived from our evolutionarily screened ND. (BDO and AA are not shown). (2) TPA can be reduced by CARMm to the corresponding dialdehyde and monoaldehyde (ligand molecule PPTNi not shown). These aldehydes serve as substrates for LuxAB to generate corresponding acids and emit fluorescence, achieving bioluminescence. The excitation wavelength of the LuxAB reaction is 482 nm, which belongs to the visible cyan light spectrum. (b) Schematic diagram of FADS screening process. Bacillus subtilis is encapsulated in microfluidic droplets and incubated to allow cells to fully express PBAT hydrolase, CARMm, and LuxAB. After a period of incubation, PBAT is injected into the microfluidic droplets to generate a fluorescent signal. Fluorescence signal detection and sorting are then performed to separate target droplets from non-target droplets. Droplets with strong fluorescence signals typically contain the target enzymes or cells with specific biochemical reaction products, while droplets with weaker fluorescence signals are considered non-target droplets. A highly active PBAT enzyme can degrade more TPA and achieve a higher level of luminescence.
    Engineered KT2440 Strain Construction and ALE-Derived Mutant Isolation for TPA and BDO Utilization

    To upcycle PBAT, a multifunctional P. putida KT2440 strain was engineered to secrete PET hydrolase and degrade PBAT hydrolysates TPA and BDO (Figure 4a). The engineering involved: (1) transforming the tph gene cluster into KT2440 for constitutive expression; (2) adaptive laboratory evolution (ALE) of KT2440-tph to utilize TPA and 1,4-butanediol; (3) transforming evolved mutants into KT2440-tph. The tph gene cluster from Pseudomonas stutzeri TPA3 was electroporated into KT2440, enabling TPA degradation (Figure 3b and 3c). ALE experiments with wild-type and engineered strains in mineral salt medium supplemented with 1,4-butanediol and TPA showed increased growth rates, indicating enhanced utilization of BDO and TPA. Further ALE is planned to improve degradation efficiency.

    Figure 4. (a) The construction process for engineered KT2440.The engineering modification of Pseudomonas putida KT2440 mainly consists of three parts: (1) transforming the tph gene cluster into KT2440 to obtain KT2440-tph and expressing the gene cluster constitutively; (2) adaptive laboratory evolution (ALE) of KT2440-tph to enable the utilization of TPA as carbon sources and a certain concentration of 1,4-butanediol; (3) transforming the mutants into the evolved KT2440-tph. (b) An engineered P. putida strain with abilities of PBAT depolymerization and co-degradation of TPA and BDO. Schematic of a single engineered strain Pseudomonas putida KT2440 capable of degrading PBAT and co-degrading its degradation products TPA and BDO. The entire tph gene cluster was transformed into KT2440 and constitutively expressed to achieve co-degradation of TPA and BDO. (c) The feature of tph cluster. A tph cluster containing genes encoding the transcriptional regulator (tphR), tpa transporter (tpaK), tpa 1, 2-dioxygenase (tphA), and 1, 2-dihydroxy-3, 5-cyclohexadiene-1, 4-dicarboxylate dehydrogenase (tphB).
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