Tianyuan Su

The regulation of multiple gene expression is pivotal for metabolic engineering. Although CRISPR interference (CRISPRi) has been extensively utilized for multi-gene regulation, the construction of numerous single-guide RNA (sgRNA) expression plasmids for combinatorial regulation remains a significant challenge. In this study, we developed a combinatorial repression system for multiple genes by optimizing the expression of multi-sgRNA with various inducible promoters in Escherichia coli. We designed a modified Golden Gate Assembly method to rapidly construct the sgRNA expression plasmid p3gRNA-LTA. By optimizing both the promoter and the sgRNA handle sequence, we substantially mitigated undesired repression caused by the leaky expression of sgRNA. This method facilitates the rapid assessment of the effects of various inhibitory combinations on three genes by simply adding different inducers. Using the biosynthesis of N-acetylneuraminic acid (NeuAc) as an example, we found that the optimal combinatorial inhibition of the pta, ptsI, and pykA genes resulted in a 2.4-fold increase in NeuAc yield compared to the control. We anticipate that our combinatorial repression system will greatly simplify the regulation of multiple genes and facilitate the fine-tuning of metabolic flow in the engineered strains.

N-Acetylneuraminic acid (NeuAc) performs a variety of biological activities where it is used as a nutraceutical and pharmaceutical intermediate. N-Acetylglucosamine-2-epimerase (AGE) and N-acetylneuraminic lyase (NAL) are the most widely used key enzymes in the industrial production of NeuAc through whole-cell catalytic synthesis. However, both AGE and NAL catalyze reversible reactions, and the equilibrium of these two reactions lies between substrates and products, resulting in a lower conversion rate of NeuAc. In this study, affinity analysis based on the dynamic docking (ADD) strategy was used to rationally design the AGE and NAL to improve enzymes properties. The variant AGE^A172S/C118A showed a 2.19-fold improvement in the catalytic rate. Then, we combinatorially expressed the variant of AGE and NAL in two plasmids for whole cell catalytic synthesis. NeuAc production was 35% higher with the combination of AGE^A172S/C118A and NAL^F252M compared with the wild type. When substrate GlcNAc/Pyruvate was 3:8 and AGE^A172S/C118A and NAL^F252M expressed strains were 1:0.6, the molar conversion rate was 62%. Thus, our modification of AGE and NAL, the key enzymes in producing NeuAc, gave a better AGE variant AGE^A172S/C118A, which could produce 128 g/L NeuAc when using low substrate concentration (0.6 M GlcNAc).

Poly(ethylene terephthalate) (PET) is the most abundant polyester plastic and is causing serious environmental pollution. Rapid biological depolymerization of PET waste at large scale requires powerful engineered enzymes with excellent performance. Here, we designed a computational strategy to analyze the ligand affinity energy of enzymes to PET chains by molecular docking with the dynamic protein conformations, named Affinity analysis based on Dynamic Docking (ADD). After three rounds of protein engineering assisted by ADD, we drastically enhanced the PET-degrading activity of leaf-branch-compost cutinase (LCC). The best variant LCC-A2 degraded >90% of the pretreated, post-consumer PET waste into corresponding monomers within 3.3 h, and over 99% of the products were terminal degradation products (terephthalic acid and ethylene glycol), representing the fastest PET degradation rate reported to date. Structural analysis revealed interesting features that improved the ligand affinity and catalytic performance. In conclusion, the proposed strategy and engineered variants represent a substantial advancement of the biological circular economy for PET.

Synthetic oscillators have become a research hotspot because of their complexity and importance. The construction and stable operation of oscillators in large-scale environments are important and challenging. Here, we introduce a synthetic population-level oscillator in Escherichia coli that operates stably during continuous culture in non-microfluidic environments without the addition of inducers or frequent dilution. Specifically, quorum-sensing components and protease regulating elements are employed, which form delayed negative feedback to trigger oscillation and accomplish the reset of signals through transcriptional and post-translational regulation. We test the circuit in devices with 1 mL, 50 mL, 400 mL of medium, and demonstrate that the circuit could maintain stable population-level oscillations. Finally, we explore potential applications of the circuit in regulating cellular morphology and metabolism. Our work contributes to the design and testing of synthetic biological clocks that function in large populations.

Plastic waste is rapidly accumulating in the environment and becoming a huge global challenge. Many studies have highlighted the role of microbial metabolic engineering for the valorization of polyethylene terephthalate (PET) waste. In this study, we proposed a new conceptual scheme for upcycling of PET. We constructed a multifunctional Pseudomonas putida KT2440 to simultaneously secrete PET hydrolase LCC, a leaf-branch compost cutinase, and synthesize muconic acid (MA) using the PET hydrolysate. The final product MA and extracellular LCC can be separated from the supernatant of the culture by ultrafiltration, and the latter was used for the next round of PET hydrolysis. A total of 0.50 g MA was produced from 1 g PET in each cycle of the whole biological processes, reaching 68% of the theoretical conversion. This new conceptual scheme for the valorization of PET waste should have advantages over existing PET upcycling schemes and provides new ideas for the utilization of other macromolecular resources that are difficult to decompose, such as lignin.

Summary: With the rapid development of synthetic biology, the oleaginous yeast Yarrowia lipolytica has become an attractive microorganism for chemical production. To better optimize and reroute metabolic pathways, we have expanded the CRISPR‐based gene expression toolkit of Y. lipolytica . By sorting the integration sites associated with high expression, new neutral integration sites associated with high expression and high integration efficiency were identified. Diverse genetic components, including promoters and terminators, were also characterized to expand the expression range. We found that in addition to promoters, the newly characterized terminators exhibited large variations in gene expression. These genetic components and integration sites were then used to regulate genes involved in the lycopene biosynthesis pathway, and different levels of lycopene production were achieved. The CRISPR‐based gene expression toolkit developed in this study will facilitate the genetic engineering of Y . lipolytica .