Contribution

In Photorhabdus luminescens, CipA and CipB together form intracellular crystalline structures (Protein Crystalline Inclusions, PCIs), which play a key role in bacterial symbiosis [4]. Although CipA and CipB share low sequence similarity (only 38%), both are rich in hydrophobic amino acids (approximately 50%) and possess the ability to self-assemble into PCIs [2]. Studies show that these crystalline proteins can provide essential nutritional support for symbiotic nematodes, promoting their growth and reproduction [5]. Experiments confirmed that Cip proteins expressed in recombinant Saccharomyces cerevisiae can be utilized by the free-living nematode Panagrellus redivivus, demonstrating a nutritional support role, shortening nematode development time by about 24 hours while enhancing reproductive capacity [5]. This nutritional function suggests that Cip proteins likely play a similar role in the symbiosis of entomopathogenic nematodes.

Research has found that in Photorhabdus luminescens, the cipB promoter possesses high activity and can drive strong expression of the reporter gene mCherry in promoter trapping screens [6]. This indicates that the expression of cip genes may be regulated by host environmental signals, further supporting their function in host adaptation and nutrient supply.

In the Photorhabdus-nematode symbiotic system, the crystalline proteins formed by CipA function as nutritional reserves [4]. Studies indicate that these proteins form highly ordered crystalline structures within the bacterial cell, potentially providing essential amino acids and energy sources for the symbiont during periods of nutrient scarcity. After the nematode infects an insect host, Photorhabdus is released from the nematode's intestine into the insect hemolymph and begins producing Cip proteins. These proteins are subsequently utilized by the symbiotic nematode to support its growth and reproduction [5]. This nutrient transfer mechanism represents an efficient symbiotic adaptation strategy, enabling the nematode-bacterium symbiotic complex to survive and reproduce in variable environments.

Experiments with cipA and cipB mutants constructed by insertional inactivation further confirm the nutritional function of these crystalline proteins. Bintrim and Ensign found that cip mutants could not support nematode growth and reproduction, indicating that Cip proteins are essential for maintaining the symbiotic relationship [1]. Furthermore, You et al. expressed the cipA and cipB genes in E. coli and used them to feed axenic J1 larvae of Steinernema nematodes; they found that only E. coli expressing Cip proteins could support the nematodes' development into durable juveniles (DJs), whereas the control group without Cip expression could not complete this developmental process [5].

CipA and CipB possess the unique ability to act as protein scaffolds, efficiently organizing foreign proteins into PCIs in both prokaryotic and eukaryotic systems [2]. For example, fusion expression of CipA with GFP or LacZ can form crystalline inclusions with fluorescence or enzyme activity in E. coli. These PCIs are easily separable, highly stable, and maintain structural integrity under high temperature and extreme pH conditions [2]. These characteristics make them ideal tools for enzyme immobilization and spatial organization of metabolic pathways.

The most attractive feature of CipA for synthetic biologists is its ability to serve as a molecular scaffold for organizing multi-enzyme complexes. This property can be leveraged to enhance the efficiency of metabolic pathways. For instance, in Escherichia coli, CipA-mediated enzyme self-assembly significantly enhanced the biosynthesis efficiency of pyrogallol [7]. This strategy co-localizes multiple enzymes in a metabolic pathway, reducing the diffusion loss of intermediates and increasing metabolic flux.

Wang et al. (2017) [2] successfully used the CipA scaffold to co-assemble multiple enzymes (VioA-E) from the violacein biosynthesis pathway into PCIs, significantly increasing violacein yield and reducing byproduct formation. This strategy is also applicable to multi-enzyme cascade reactions, such as co-assembling GFP and LacZ into mixed crystals with dual functionality [2].

In principle, this strategy mimics the natural metabolic channeling mechanism and offers the following advantages: (1) reduced diffusion loss of intermediates; (2) avoidance of interference from competitive reactions; (3) reduced cellular damage from toxic intermediates; (4) balancing reaction rates of different enzymes. Utilizing the modular structure of CipA, customized scaffold systems for specific metabolic pathways can be designed to optimize the efficiency of the entire biosynthetic process.

Fusion expression of CipA with a target protein can form insoluble PCIs, enabling one-step purification via simple centrifugation with purity exceeding 90% [2]. This system has been successfully used for the immobilization of tyrosinase TyrVs; the constructed TyrVs-CipA could be reused more than 7 times in catalyzing the DOPA modification of hydrolyzed silk fibroin peptide (HSF), with a single modification degree exceeding 70% [3]. Similarly, Chen et al. developed a novel purification strategy exploiting this property by fusing CipA with the self-cleaving intein Ssp DnaB, enabling highly efficient purification of tag-free target proteins through simple centrifugation and cleavage steps[8]; Tang et al. employed this strategy by fusing the antimicrobial peptides bovine lactoferricin and lactoferrampin (LFC-LFA) with CipB in a Photorhabdus luminescens host. This approach effectively neutralized the peptides' toxicity to the expression host, allowing high-yield production. The fusion protein was easily purified by pH-dependent solubility shift and the active peptides were subsequently released by pepsin cleavage [9].