News Source& Photographer: School of Life Science

Editor: Xiao Wen

Translator: Huang Yuxuan, News Agency of BIT

Recently, Professor Huo Yixin's team of BIT published an important review (https://doi.org/10.1016/j.tibtech.2023.06.009) in the field of protein self-assembly. The review was published in Trends in Biotechnology (impact factor: 17.2996). It was carried out with BIT as the first communication unit, with Associate Researcher Chen Zhenya as the first author, Professor Huo Yixin as the corresponding author, doctoral students Wu Tong and Yu Shengzhu, as well as master students Li Min and Fan Xuanhe as participating authors.

Enzyme self-assembly refers to the aggregation of target enzymes, facilitated by protein self-assembly scaffolds, to form ordered macromolecular structures (Figure 1). In the field of metabolic engineering, self-assembly strategies have been employed to aggregate multiple enzymes within the same pathway to improve the sequential catalytic efficiency of these enzymes. This, in turn, increases the metabolic flux of the entire pathway, improves the efficiency of target product synthesis, and enables high-level production. The performance of protein self-assembly scaffolds is critical for the construction of efficient and stable multi-enzyme assembly systems. This paper begins by analyzing the challenges faced in current-stage metabolic engineering, followed by an explanation of the advantages of using self-assembled scaffolds in solving these problems. The existing protein self-assembly scaffolds employed in the field of metabolic engineering are then classified, and the assembly methods of different scaffolds are comprehensively analyzed. Then, the article elaborates on the application scenarios of self-assembled scaffolds in different modules within the field of metabolic engineering. These applications include enhancing the catalytic efficiency of individual enzymes, improving the sequential catalytic efficiency of multi-enzyme cascade, reducing side reactions, and enhancing the supply of cofactors. In addition, this paper examines the limitations of existing self-assembly scaffolds, expounds the assembly performance of different scaffolds, and proposes performance improvement strategies for different scaffolds, which provides new ideas for the construction of efficient microbial cell factories.

Figure 1 Enzyme self-assembly and self-assembly-mediated metabolic engineering production

The above review is a summary and analysis of the development and application of protein self-assembly scaffolds in metabolic flow regulation by our team in recent years. The relevant work is as follows:

1. A new protein immobilization method was constructed by using self-assembled scaffolds

In vitro biosynthesis has gained attention as an attractive method for producing high-value compounds due to its controllability and high conversion efficiency. Enzyme reuse is crucial  in the process of in vitro biosynthesis to save costs and enhance synthesis efficiency. Enzyme immobilization is a simple and direct method to realize enzyme reuse. The development of a convenient and efficient ezyme immobilization method enables rapid immobilization of enzymes. Based on this, the researchers focused on the in vitro biosynthesis of isobutyraldeyde, an important precursor for food production, as an example, and established a one-step self-assembly immobilization strategy based on the self-assembly characteristics of CipA. By using this strategy, the immobilization of LeuDH and KivD related enzymes in the synthesis pathway from valine to isobutyral is realized. The structural simulation results showed that the immobilization of enzymes mediated by CIPA did not affect the structure and catalytic mechanism of the target enzyme (Figure 2). Compared to free enzymes, immobilized enzymes exhibited higher conversion capacity and thermal stability. In addition, batch conversion experiments demonstrated that the immobilized enzymes recovered after multiple rounds maintained similar conversion efficiency to that of the immobilized enzymes in the first round reaction. Subsequently, the continuous production of isobutyral was realized in vitro by setting up a continuous production unit and loading the immobilized enzyme into the continuous production unit. This work not only expands the application range of the self-assembly system, but also provides guidance for the production of high-value compounds in vitro. Related research results were published in the Journal of Agricultural and Food Chemistry (Impact factors: 6.1004) In vitro biosynthesis of isobutyraldehyde through the establishment of a one-step self-assembly based immobilization strategy.). Associate Researcher Chen Zhenya and Master's student Zhao Luyao are co-first authors, and Professor Huo Yixin and Associate Researcher Chen Zhenya are co-corresponding authors.

Figure 2. CipA-KivD-LeuDH complex model with substrate

2.Redirecting cellular metabolic flow using self-assembled scaffolds

Cathol is a phenolic compound with a variety of physiological functions and pharmaceutical value. The applicant successfully constructed an unnatural biosynthesis pathway of pyrogallol using glucose as the initial carbon source in Escherichia coli. In the biosynthesis process, a key precursor, 4-hydroxybenzoic acid, was first catalyzed by Y385F/T294A PobA to generate 3,4-dihydroxybenzoic acid. Subsequently, 3,4-dihydroxybenzoic acid was further catalyzed by Y385F/T294A PobA to generate gallic acid. Finally, the decarboxylase PDC converted gallic acid to pyrogallol. However, the decarboxylase PDC also results in a decarboxylation reaction to the intermediate 3,4-dihydroxybenzoic acid to produce catechols as a byproduct. After assembling the complete pathway and conducting fermentation in host bacteria, it was found that the yield of catechol was very low while the yield of catechol was high. This indicated that the catalytic process of 3,4-dihydroxybenzoic acid to gallic acid by Y385F/T294A PobA was not efficient enough, resulting in a significant portion of the carbon sources being directed towards the production of catechol. To address this issue, the applicant utilized the self-assembly characteristics of CipA and fused CipA with Y385F/T294A PobA, a key rate-limiting enzyme in the synthesis pathway, to form a fusion protein (Y385F/T294A PobA-CipA or CipA-Y385F/T294A PobA), which then aggregated intracellular to form inclusion bodies (Figure 3). The aggregation of Y385F/T294A PobA within these inclusion bodies accelerated the two-step conversion of 4-hydroxybenzoic acid to gallic acid, while reducing the catalytic activity of decarboxylase PDC to 3,4-dihydroxybenzoic acid. As a result, the yield of pyrogallol, the target product, was increased. The findings were published in the journal Applied Microbiology and Biotechnology (impact factor: 5.0002) (CipA-mediating enzyme self-assembly to enhance the biosynthesis of pyrogallol in Escherichia coli). Associate Professor Chen Zhenya is the corresponding author.

Figure 3 Self-assembled aggregation of Y385F/T294A PobA

3. Construction of a novel protein purification method based on self-assembled scaffolds

The commonly used traditional protein isolation and purification method is affinity purification, which relies on the use of affinity chromatography columns. Affinity purification is costly and unsuitable for large-scale production. Additionally, these methods often result in purified proteins carrying purification tags that can affect the activity of the target protein. Therefore, in order to save the cost of protein purification, simplify the purification steps, and maintain the original characteristics of the target protein, the applicant designed and developed a self-assembled protein purification method, which relies on a bifunctional purification tag CipA-DnaB, which has both self-assembly and self-cleavage functions, and the tag can be fused with the target protein to purify the tag-free target protein, and the target protein can maintain the original activity. The CipA-DnaB tag is obtained by fusing the self-assembled protein CipA and the peptide protein Ssp DnaB.  CipA can spontaneously assemble into protein inclusion bodies, and Ssp DnaB is a short endopeptide that can undergo self-cleavage at the C-terminal under weak acid conditions. After fusing this bifunctional tag to the target protein, the target protein is self-assembled under the guidance of CipA (Figure 4). Subsequently, rapid purification of the soluble, tag-free target protein can be achieved through centrifugation and self-lysis steps. Then, in order to improve the protein purification efficiency, the ligent peptide and self-cleavage conditions between CipA and Ssp DnaB were optimized, and the results showed that the addition of EDTA to the lysate with flexible linker peptide could significantly improve the protein purification efficiency. To demonstrate the universality of this self-assembled protein purification method, MBP, KivD and AdhP were purified by this method. The purified KivD and AdhP enzyme activities were detected, and the results showed that the purified KivD and AdhP still had high specific enzyme activity. The novel self-assembly purification method established by this work provides a low-cost and effective option for industrial protein purification. The findings were published in the Journal of Biotechnology (impact factor: 4.0998). (A novel protein purification strategy mediated by the combination of CipA and Ssp DnaB intein.) Associate researcher Zhenya Chen and master student Luyao Zhao are co-first authors, and Professor Yixin Huo and Associate Professor Zhenya Chen are co-corresponding authors.

Figure 4 CipA-DnaB assembles eGFP in E. coli

This work was supported by the National Natural Science Foundation of China and the special fund of Basic scientific Research operation of central universities, and also thanks to the support of the Public Experimental Center of Biology and Medical Engineering of BIT.