Enhancing Safe and Controllable Protein Generation via Knowledge Preference Optimization
Authors: Yuhao Wang, Keyan Ding, Kehua Feng, Zeyuan Wang, Ming Qin, Xiaotong Li, Qiang Zhang, Huajun Chen
Accepted at: acl25

Enhancing Safe and Controllable Protein Generation via Knowledge Preference Optimization

Yuhao Wang1,2, Keyan Ding1,2, Kehua Feng1,2, Zeyuan Wang1,2, Ming Qin1,2, Xiaotong Li1,2, Qiang Zhang1,2*, Huajun Chen1,2*
1Zhejiang University, 2ZJU-Hangzhou Global Scientific and Technological Innovation Center {wangyuhao.cs, qiang.zhang.cs, huajunsir}@zju.edu.cn

Abstract

Protein language models have emerged as powerful tools for sequence generation, offering substantial advantages in functional optimization and de novo design. However, these models also present significant risks of generating harmful protein sequences, such as those that enhance viral transmissibility or evade immune responses. These concerns underscore critical biosafety and ethical challenges. To address these issues, we propose a Knowledge-guided Preference Optimization (KPO) framework that integrates prior knowledge via a Protein Safety Knowledge Graph. This framework utilizes an efficient graph pruning strategy to identify preferred sequences and employs reinforcement learning to minimize the risk of generating harmful proteins. Experimental results demonstrate that KPO effectively reduces the likelihood of producing hazardous sequences while maintaining high functionality, offering a robust safety assurance framework for applying generative models in biotechnology.

1 Introduction

Protein language models (PLMs) have significant impact on biological research, providing powerful tools to uncover relationships between protein sequences, structures and functions [Nijkamp et al., 2023]. By leveraging extensive protein sequence datasets, PLMs capture hidden patterns and correlations that are challenging or impossible to discern using traditional methods. For example, in enzyme engineering, pretrained models predict mutations that enhance catalytic efficiency or substrate specificity, significantly accelerating the iterative design process [Madani et al., 2023; Zhou et al., 2024]. Similarly, in antibody discovery, PLMs facilitate the rapid identification of high-affinity candidates, enabling swift responses to emerging pathogens [He et al., 2023; Wang et al., 2024].

Figure 1: Existing PLMs often overlook safety considerations, leading to the generation of potentially harmful protein sequences. In contrast, our KPO framework ensures that PLMs are fine-tuned to prioritize safety.

Despite that PLMs have transformative potential in advancing biological research and biotechnology, they also pose unique and significant safety challenges. Unlike text-based large language models (LLMs), where harmful outputs are typically ethical or social in nature, the biological domain of PLMs entails risks with direct and far-reaching consequences for ecological and human health. The inadvertent generation of harmful sequences—for instance, those enhancing viral transmissibility, evading immune responses, or developing drug resistance—could lead to ecological imbalances, public health crises, or even the creation of bioweapons if disseminated or misused. Current PLMs prioritize functional and generative performance, with little emphasis on safety considerations, as illustrated in Figure 1. Addressing these challenges requires a paradigm shift from traditional performance optimization to safety-aware design. A promising approach involves fine-tuning PLMs to minimize the generation of harmful proteins while retaining their ability to produce functional and beneficial outputs. While current efforts largely focus on introducing safety-enhancing mutations to known protein sequences [Li et al., 2024], they fail to address the higher risks inherent in the generative phase of protein design. This underscores the critical need for rigorous frameworks that proactively mitigate potential hazards during protein genera-
tion while preserving the functional and beneficial capabilities of PLMs.

To address the biosafety challenges of protein language models, we propose Knowledge-guided Preference Optimization (KPO), a novel framework that integrates domain-specific safety knowledge into the generative process of PLMs. Central to KPO is the construction of a Protein Safety Knowledge Graph (PSKG), which encodes critical biochemical properties and intrinsic relationships of both benign and harmful proteins. By leveraging the PSKG, KPO incorporates safety-oriented prior knowledge to impose biologically meaningful constraints during sequence generation, ensuring the production of safer proteins. To manage the computational complexity inherent to the PSKG, we developed a weighted metric-based pruning algorithm, which efficiently trims the graph by retaining key nodes essential for structural and informational integrity. This pruning strategy significantly reduces computational overhead while preserving the graph’s representational capacity. Through this systematic integration of safety knowledge and computational optimization, KPO not only mitigates the risks of generating harmful proteins but also establishes a rigorous and scalable framework for the responsible application of generative models in biotechnology.

The key contributions of this work can be summarized as follows:

• We introduce the first Protein Safety Knowledge Graph (PSKG), a comprehensive resource that encapsulates rich biochemical knowledge on both harmful and benign proteins.

• We develop Knowledge-guided Preference Optimization (KPO), a novel algorithm that seamlessly integrates safety-oriented prior knowledge from the PSKG into protein language models, enabling the generation of safer protein sequences.

• By applying the KPO framework, we significantly enhance the safety of existing generative PLMs, ensuring the production of biologically safe proteins while maintaining their functional efficacy.

2 Related Works

Protein Language Models PLMs [Vig et al., 2020] have emerged as powerful tools in computational biology, leveraging natural language processing methodologies to analyze and predict protein properties. These models have demonstrated their potential in a variety of applications, ranging from structural annotation to functional prediction. For example, the ESM series [Lin et al., 2022; Rives et al., 2021] pioneered the use of masked language modeling for capturing long-range dependencies and inferring structural and functional relationships in protein sequences. MSA-Transformer [Rao et al., 2021] and Co-volution Transformer [Zhang et al., 2021] incorporate evolutionary information through multiple sequence alignments, enhancing functional insights. ProtT5 [Pokharel et al., 2022] adapts the T5 architecture to learn protein representations, while ProtGPT2 [Ferruz et al., 2022] and ProGen2 [Nijkamp et al., 2023] leverage autoregressive objectives to predict subsequent amino acids, making them well-suited for generative tasks such as de novo protein design. Additionally, LM-GVP [Wang et al., 2022] integrates graph representations from protein 3D structures with transformer architectures to capture spatial relationships between residues. Despite these advancements, the potential risks associated with PLM-generated sequences, such as unintended toxic or harmful properties, remain a critical area for exploration.

LLM Safety Ensuring the safety of LLMs has been a primary focus of research, leading to the development of various alignment techniques [Bai et al., 2022]. A key approach is Reinforcement Learning from Human Feedback (RLHF) [Ouyang et al., 2022], which fine-tunes model behavior using human-provided feedback to align outputs with specific objectives. Building on RLHF, other methods, such as Direct Preference Optimization (DPO) [Rafailov et al., 2024], further refine LLM fine-tuning by incorporating ranking information, helping models distinguish the quality of different outputs. Similarly, Reward Ranking-Based Reinforcement Learning (RRHF) [Yuan et al., 2023] enhances alignment by introducing adjusted loss functions that amplify learning signals from ranked feedback. Contrastive learning [Yang et al., 2023] has also shown promise in alignment tasks, improving sample efficiency and model quality by guiding the model towards producing high-quality outputs while discouraging low-quality ones.

LLM unlearning [Kassem et al., 2023; Lu et al., 2022] has emerged as another important technique to enhance model security, enabling models to “for-
get” sensitive or inappropriate data to avoid harmful predictions. Common forgetting techniques include Gradient Ascent methods [Jang et al., 2022], which work by maximizing the prediction loss for forgotten data, and input perturbation methods, which substitute potentially harmful responses with neutral outputs, such as “I don’t know” [Ishibashi and Shimodaira, 2023]. Another approach is retraining, which involves retraining the model from scratch while explicitly excluding the data to be forgotten [Bourtoule et al., 2021]. More recent techniques, such as context-based unlearning [Pawelczyk et al., 2023], use specific instructions to guide models to forget certain knowledge without altering its weights. Despite these advancements, there is currently no method specifically tailored to fine-tuning PLMs to ensure the safety of generated protein sequences, leaving a critical gap in the field.

3 Preliminaries

3.1 Generative PLMs

Generative PLMs learn the language-like structure of protein sequences, similar to how text models predict the next word [Verkuil et al., 2022]. By predicting the next amino acid in a sequence based on previous ones, PLMs capture functional patterns crucial for tasks like generating novel proteins. Given an unlabeled corpus \( D = (p^{(1)}, p^{(2)}, ..., p^{(N)}) \), each sample \( p^{(i)} = (p_1^{(i)}, p_2^{(i)}, ..., p_{W_i}^{(i)}) \) is a protein sequence, and the goal is to train a language model \( \theta \) to maximize the log-likelihood estimate of the training data:

\[
L(\theta) = \sum_{i=1}^N \sum_{w=1}^{W_i} \log P(p_w^{(i)}|p_1^{(i)}, ..., p_{w-1}^{(i)}; \theta),
\]

where \( N \) is the total number of sequences in the corpus, \( W_i \) is the length of the \( i \)-th sequence, and \( P \) is the conditional probability given by PLMs.

3.2 Direct Preference Optimization

DPO fine-tunes language models by directly aligning their output with preference data, such as human feedback. Unlike RLHF, which involves constructing a reward model and training the language model within an RL framework, DPO simplifies this process into a single optimization problem. By bypassing the complexities of reward modeling, DPO offers an efficient and scalable approach to aligning language models with user preferences. Given a set of generated pairs \( \hat{y}_w, \hat{y}_l \) conditioned on an input \( x \), where \( \hat{y}_w \) is the preferred response and \( \hat{y}_l \) is the dispreferred response, DPO maximizes the ratio of probabilities assigned to the preferred responses. The DPO loss is defined as:

\[
L = -\lambda \log \sigma (\log \frac{\pi_\theta(\hat{y}_w|x)}{\pi_{ref}(\hat{y}_w|x)} - \log \frac{\pi_\theta(\hat{y}_l|x)}{\pi_{ref}(\hat{y}_l|x)}).
\]

4 Methodology

To address the critical challenge of aligning PLMs for safety, we introduce a Knowledge-guided Preference Optimization framework, as illustrated in Figure 2. The safety alignment process relies on constructing preference pairs that differentiate benign proteins from harmful ones by capturing their nuanced similarities and differences. To enable the generation of high-quality preference pairs, we construct a Protein Safety Knowledge Graph, which systematically encodes biochemical relationships between harmful and benign proteins. To enhance computational efficiency without compromising the quality of information, we incorporate a weighted metric-based pruning algorithm. This algorithm refines the PSKG by retaining the most informative nodes and edges, thereby reducing computational complexity while preserving its core structure and utility. Finally, we identify benign proteins within the PSKG that share properties with harmful proteins. These proteins are then used to create preference pairs for fine-tuning PLMs. The fine-tuning process aligns the model’s generative capabilities with the safety constraints defined by the PSKG, ensuring the generation of protein sequences that are both biologically relevant and safe.

4.1 Protein Safety Knowledge Graph

Inspired by Ontoprotein[Chen, 2023; Zhang et al., 2022], we construct PSKG to capture the intricate relationships between harmful and benign proteins. Drawing from the Uniprot database, we curate a comprehensive dataset of harmful proteins by retrieving experimentally validated protein sequences annotated with the keywords “toxin” and “antigen”. To collect benign proteins, we filter out harmful proteins from the Swiss-Prot database and select only those proteins verified as benign. A detailed description of the construction process is in Appendix A.1. The biologically meaningful graph offers a robust framework for the interplay analysis between harmful and benign proteins.

Let the set of harmful proteins be denoted as \( P_H = \{p_{H1}^1, p_{H2}^2, ..., p_{Hn}^n\} \), and the set of benign proteins as \( P_B = \{p_{B1}^1, p_{B2}^2, ..., p_{Bm}^m\} \). To capture these
Figure 2: Overview of the proposed KPO framework, which consists of three key stages: (1) PSKG Construction: A Protein Structure Knowledge Graph (PSKG) is built by integrating labeled protein sequence data (benign and harmful proteins) with Gene Ontology (GO) annotations, capturing biochemical relationships between proteins, GO terms, and their interactions. (2) Node Pruning: A weighted pruning algorithm is applied to refine the PSKG. Key nodes and edges are identified based on statistical scores to ensure computational efficiency while retaining critical structural and functional information. (3) PLM Fine-tuning: Preference pairs are generated by identifying benign proteins that are structurally or functionally similar to harmful proteins within graph and latent spaces. These preference data are then used to fine-tune PLMs using methods such as Direct Preference Optimization (DPO).

intricate distinctions, we define indirect relationships between \( P_H \) and \( P_B \) mediated through GO terms. Specifically, a harmful protein \( p_H^i \in P_H \) and a benign protein \( p_B^j \in P_B \) are considered related if they share a common GO term \( g_z \in G = \{g_1, g_2, ..., g_Z\} \), forming an indirect connection represented as \( (p_H^i, g_z, p_B^j) \). The inclusion of hierarchical Gene Ontology relationships enables the PSKG to capture both direct and nuanced associations between harmful and benign proteins. For instance, a broad GO term like “binding activity” may group various harmful and benign proteins, while more specific terms such as “DNA-binding transcription factor activity” can reveal finer distinctions. This integrative framework ensures that the PSKG is not merely a collection of protein annotations but a robust foundational resource capable of supporting downstream analyses. It facilitates the identification of key nodes that represent critical biological properties and uncovers latent patterns that differentiate harmful from harmless proteins. Consequently, the PSKG serves as a pivotal resource for advancing safe and biologically informed protein generation.

4.2 Node Pruning with Weighted Metrics

The constructed PSKG contains hundreds of thousands of protein nodes and requires prolonged computation time for sampling informative proteins for downstream analysis. To address the challenges posed by the large scale and redundant information, we propose a weighted metric-based node pruning method. This method focuses on identifying and retaining the most informative benign protein nodes, \( P_B \), while preserving the essential biological relationships within the graph. For each benign protein node \( p_B^j \in P_B \), we compute a weighted importance score, \( S(p_B^j) \), that balances two critical factors: its connections to high-scoring GO nodes and its degree centrality within the graph. The importance score is defined as:

\[
S(p_B^j) = \alpha \cdot C_{GO}(p_B^j) + \beta \cdot C_{Deg}(p_B^j),
\]

where \( C_{GO}(p_B^j) \) is the GO association factor, \( C_{Deg}(p_B^j) \) is the degree centrality factor, and \( \alpha \) and \( \beta \) are hyperparameters controlling their weights.
GO association factors evaluate the extent to which a benign protein node is indirectly connected to harmful protein nodes, \( P_{\mathrm{H}} \), through high-scoring GO nodes. A GO node \( g_z \in G \) is considered high-scoring if it demonstrates strong bridging properties between harmful and benign proteins, characterized by two key metrics. The first is the harmful-benign bridging degree, \( R(g_z) \), which measures the number of unique \( (p_{\mathrm{H}}^i, p_{\mathrm{B}}^j) \) pairs bridged by \( g_z \), calculated as:

\[
R(g_z) = \sum_{p_{\mathrm{H}}^i \in P_{\mathrm{H}}} \sum_{p_{\mathrm{B}}^j \in P_{\mathrm{B}}} 1((p_{\mathrm{H}}^i, g_z) \in E) \cdot 1((g_z, p_{\mathrm{B}}^j) \in E),
\]

(2)

where \( 1(\cdot) \) is an indicator function that evaluates to 1 if the specified edges exist in the edge set \( E \). The second metric is the neighbor breadth, \( O(g_z) \), which counts the number of benign protein nodes directly connected to \( g_z \), defined as:

\[
O(g_z) = \sum_{p_{\mathrm{B}}^j \in P_{\mathrm{B}}} 1((g_z, p_{\mathrm{B}}^j) \in E).
\]

(3)

The overall significance of a GO node, \( g_z \), is:

\[
C(g_z) = \gamma \cdot R(g_z) + \delta \cdot O(g_z),
\]

(4)

where \( \gamma \) and \( \delta \) are weighting parameters. The GO association factor evaluates the importance of a benign protein node by measuring its connections to the top-\( Q \) of high-scoring GO nodes, denoted as \( G_{\hat{h}} \subset G \). \( G_{\hat{h}} \) are selected based on their overall significance scores \( C(g_z) \), calculated using Eq. (4):

\[
C_{\mathrm{GO}}(p_{\mathrm{B}}^j) = \sum_{g_z \in G_{\hat{h}}} 1((p_{\mathrm{B}}^j, g_z) \in E).
\]

(5)

Degree centrality factors assess the prominence of a benign protein node within the graph based on its degree centrality:

\[
C_{\mathrm{Deg}}(p_{\mathrm{B}}^j) = \sum_{v \in V} 1((p_{\mathrm{B}}^j, v) \in E),
\]

(6)

where \( C_{\mathrm{Deg}}(p_{\mathrm{B}}^j) \) counts the total number of edges connected to \( p_{\mathrm{B}}^j \). Nodes with a higher degree of centrality are more likely to be influential within the graph, as they are involved in more interactions.

Using the computed weighted importance scores, we prune the graph by retaining only the top-\( K \) benign protein nodes with the highest scores. The resulting pruned subgraph, \( G' = (V', E') \), is defined as: \( V' = \{ p_{\mathrm{B}}^j \in P_{\mathrm{B}} \mid S(p_{\mathrm{B}}^j) \text{ is among the top-}K \} \), \( E' = \{ (u, v) \in E \mid u, v \in V' \} \). For specific settings, see Appendix A.3. This pruning strategy significantly reduces the size of the PSKG while preserving its most biologically relevant nodes and edges, thereby accelerating the construction of preference pairs and improving the overall efficiency of the fine-tuning process. By balancing the importance of structural prominence and functional relevance, our method ensures that the retained subgraph continues to provide a rich and meaningful representation of the original graph for safe protein generation. The detailed procedure for node pruning using weighted metrics is provided in Appendix A.2.

4.3 Fine-tuning PLMs with KPO

To enhance PLMs with domain-specific safety knowledge, we incorporate the PSKG into the training process. The PSKG serves as a medium to inject knowledge about protein safety into the model, enabling it to understand and perceive the latent relationships between generated proteins and known harmful proteins. This integration allows the model to recognize similarities between generated proteins and potentially harmful proteins, thereby improving the safety of the generated sequences.

To capture the potential relationships between harmful proteins and benign proteins, we analyze the graph structure at multiple levels. At the structural level, we measure the proximity between harmful and benign proteins using the shortest path distance, denoted as \( \mathrm{dis}(p_{\mathrm{H}}^i, p_{\mathrm{B}}^j) \). A benign protein \( p_{\mathrm{B}}^j \) is considered relevant to a harmful protein \( p_{\mathrm{H}}^i \) if \( \mathrm{dis}(p_{\mathrm{H}}^i, p_{\mathrm{B}}^j) \leq \tau \), where \( \tau \) is a predefined hop threshold. This selection criterion enables us to identify benign proteins that are structurally close to harmful proteins, capturing potential functional or biological similarities.

At the embedding level, we leverage the TransE algorithm to learn low-dimensional representations of nodes in the PSKG. Each node \( v_i \in V' \) is embedded into a vector space as \( e_i \in \mathbb{R}^d \), where \( d \) is the embedding dimension. The TransE algorithm optimizes the following objective function:

\[
L = \sum_{(v_i, r, v_j) \in E} [\|e_i + r - e_j\|^2 + \sum_{(v_i', r, v_j') \notin E} \max(0, \eta - \|e_i' + r - e_j'\|^2)],
\]

(7)

where \( r \in \mathbb{R}^d \) represents the embedding of the edge relation, and \( \eta \) is a margin hyperparameter. This formulation ensures that embeddings reflect the structural relationships in the graph, such that semantically similar nodes are embedded closer
together. By combining structural proximity and embedding-based similarity, we identify benign proteins \( p_B^i \) that are closely related to harmful proteins \( p_H^i \) in both the graph structure and the embedding space. For each harmful protein \( p_H^i \), we select the top-\( M \) benign proteins based on a combined similarity score:

\[
s(p_H^i, p_B^j) = \mu \cdot \frac{1}{\mathrm{dis}(p_H^i, p_B^j)} + (1-\mu) \cdot \cos(e_{p_H^i}, e_{p_B^j}),
\]

where \( \cos(\cdot, \cdot) \) denotes the cosine similarity between embeddings, and \( \mu \in [0, 1] \) is a weighting hyper-parameter.

Using these selected pairs of harmful and benign proteins, we construct preference pairs for fine-tuning the PLM. Each preference pair \( (p_B^j, p_H^i) \) encodes the notion that the model should prefer generating sequences similar to the benign protein \( p_B^j \) over the harmful protein \( p_H^i \). The fine-tuning process is guided by Direct Preference Optimization, which optimizes the following objective:

\[
L_{\text{KPO}} = -\log \sigma \left( \varphi \cdot \left[ \log P_\theta(p_B^j|x) - \log P_\theta(p_H^i|x) \right] \right),
\]

where \( P_\theta(\cdot|x) \) is the probability assigned by the PLM to a sequence given an input prompt or context \( x \), \( \sigma(\cdot) \) is the sigmoid function, and \( \varphi \) is a scaling factor. By fine-tuning the PLM with these preference pairs, the model learns to distinguish subtle differences between harmful and benign proteins. This process integrates the rich biological information in the PSKG into the generative model, significantly enhancing its ability to generate safe and biologically meaningful protein sequences.

5 Experimental Results

5.1 Experimental Settings

Safety Evaluation Metrics In evaluating the harmfulness of generated protein sequences, we employed a comprehensive approach that incorporated sequence similarity analysis, functional domain characterization, and toxicity prediction. This allowed us to rigorously assess the potential risks of the generated proteins, ensuring a robust evaluation of their safety and biological relevance.

For sequence similarity analysis, we utilized BLAST [Madden, 2013] and MMseqs2 [Steinegger and Söding, 2017], two widely adopted tools in bioinformatics. To quantify this similarity, we computed the average alignment score for each generated protein sequence against a test dataset of harmful proteins, normalizing the score by the average sequence length. This normalization allowed for a fair comparison across proteins of varying lengths and ensured that the sequence length did not disproportionately influence the similarity score.

In the functional domain analysis, we turned to Pfam [Finn et al., 2014], a database that provides Hidden Markov Models (HMMs) [Eddy, 1996] for identifying protein domains. Each generated protein sequence and the harmful protein test dataset were analyzed for functional domain content. For domains identified in both the generated sequences and harmful proteins, we employed two distinct strategies to evaluate the significance of these domain matches. The first strategy used dynamic E-value thresholds, where we compared the E-values of functional domains in the generated proteins to those in the harmful protein database. This approach accounts for the unique statistical characteristics of each domain[Mistry et al., 2021]. The second strategy employed a fixed E-value threshold of 0.001[Finn et al., 2014], a commonly accepted cutoff for statistically significant domain matches. Lower E-values indicate a high likelihood of functional similarity to harmful proteins, while higher E-values suggest weaker or no significant functional relationship. See Appendix B for specific reasons for this setting.

In addition, we incorporated ToxinPred3, a machine learning classifier to predict the toxicity of the generated proteins [Rathore et al., 2024]. ToxinPred3 outputs a probability score indicating the likelihood of a protein being toxic. Proteins with toxicity scores above a predefined threshold were classified as toxic. The number of toxic predictions before and after fine-tuning was compared to assess the success of our approach in reducing the generation of potentially harmful proteins.

Functional Evaluation Metrics To assess if the functional capabilities of the pre-trained model remained intact after fine-tuning using the KPO method, we employed a set of well-established protein datasets: GB1 [Wu et al., 2019], PhoQ [Podgornaia and Laub, 2015], UBC9 [Knipscheer et al., 2008], and GFP [Zimmer, 2002]. For each of these datasets, we compared the performance of the pre-trained model and the fine-tuned model by calculating the generation probability score for each mutation. We computed the scores for the top 96 mutations with the highest probabilities gener-
Table 1: Performance of KPO on the top of different base models.

<table>
  <tr>
    <th rowspan="2">Models</th>
    <th colspan="5">Safety Evaluation Metric</th>
    <th colspan="4">Functional Evaluation Metric</th>
  </tr>
  <tr>
    <th>BLAST↓</th>
    <th>MMseq2↓</th>
    <th>Pfam_D↓</th>
    <th>Pfam_E↓</th>
    <th>ToxinPred3↓</th>
    <th>GB1↑</th>
    <th>PhoQ↑</th>
    <th>UBC9↑</th>
    <th>GFP↑</th>
  </tr>
  <tr>
    <td>ProtGPT2</td>
    <td>0.269</td>
    <td>0.325</td>
    <td>0.2933</td>
    <td>0.2701</td>
    <td>0.07</td>
    <td>0.030</td>
    <td>0.015</td>
    <td><b>0.138</b></td>
    <td>1.526</td>
  </tr>
  <tr>
    <td>ProtGPT2+KPO</td>
    <td><b>0.138</b></td>
    <td><b>0.149</b></td>
    <td><b>0.2613</b></td>
    <td><b>0.1819</b></td>
    <td><b>0.024</b></td>
    <td><b>0.041</b></td>
    <td><b>0.315</b></td>
    <td>0.129</td>
    <td><b>2.204</b></td>
  </tr>
  <tr>
    <td>Progen2</td>
    <td>0.155</td>
    <td>0.170</td>
    <td>0.1079</td>
    <td>0.2630</td>
    <td>0.029</td>
    <td><b>0.144</b></td>
    <td><b>0.027</b></td>
    <td>0.068</td>
    <td><b>1.683</b></td>
  </tr>
  <tr>
    <td>Progen2+KPO</td>
    <td><b>0.128</b></td>
    <td><b>0.117</b></td>
    <td><b>0.0922</b></td>
    <td><b>0.0645</b></td>
    <td><b>0.007</b></td>
    <td>0.024</td>
    <td>0.017</td>
    <td><b>0.231</b></td>
    <td>1.562</td>
  </tr>
  <tr>
    <td>InstructProtein</td>
    <td>0.410</td>
    <td>0.285</td>
    <td>0.1662</td>
    <td>0.0835</td>
    <td>0.031</td>
    <td>0.030</td>
    <td>0.016</td>
    <td><b>0.459</b></td>
    <td>1.983</td>
  </tr>
  <tr>
    <td>InstructProtein+KPO</td>
    <td><b>0.086</b></td>
    <td><b>0.079</b></td>
    <td><b>0.1189</b></td>
    <td><b>0.0385</b></td>
    <td><b>0.003</b></td>
    <td><b>0.191</b></td>
    <td><b>0.814</b></td>
    <td>0.451</td>
    <td><b>2.319</b></td>
  </tr>
</table>

ated by both the pre-trained and fine-tuned models. To ensure that fine-tuning did not compromise the model’s ability to generate functionally relevant mutations, we calculated the average fitness of these top 96 mutations. Specific implementation details are provided in the Appendix B.

5.2 Main Results

Performance Comparison The performance of our KPO method, applied to three different base models published in top-tier journals or conferences—ProtGPT2 [Ferruz et al., 2022], Progen2 [Nijkamp et al., 2023] and InstructProtein [Wang et al., 2023]—was evaluated across both safety and functional metrics. The results demonstrate that KPO significantly enhances the safety of generated protein sequences, while preserving or even improving the functional capabilities of the models. This balance between safety and functionality highlights the efficacy of KPO in optimizing protein sequence generation. Our code is available at https://github.com/HICAI-ZJU/KPO.

From Table 1, we can observe that the proposed KPO method consistently reduced the risk of generating harmful proteins across multiple metrics. Additionally, the functional domain-based harmfulness assessment using Pfam also showed improvements. The ToxinPred3 toxicity classifier also showed a sharp reduction in predicted toxicity. One of the reasons for the observed functional improvements after applying KPO is that the fine-tuning process effectively guides the model away from harmful sequence spaces, which can be biologically unproductive, and toward regions of the sequence space that are more likely to produce functional and high-adaptability proteins. By minimizing the generation of harmful proteins, the model avoids the regions that might be associated with harmful or suboptimal structural configurations. As a result, the model can explore the most promising areas of the protein sequence landscape, where the generated proteins exhibit higher fitness and functional relevance. This allows the protein model to focus better on generating sequences that are not only safe but also biologically advantageous, improving its ability to optimize for functions without compromising safety.

Embedding Result Analysis To further investigate the impact of KPO on the generated protein sequences, we analyzed their embeddings using the ESM-2 [Verkuil et al., 2022] model. This analysis aimed to visualize how the fine-tuned models’ generated sequences compared to known harmful protein sequences in the embedding space. Specifically, we selected 100 protein sequences from each of the following categories: harmful proteins, and proteins generated by the KPO fine-tuned models

![The embeddings of proteins generated by the fine-tuned PLM exhibit a clear separation from those of harmful proteins, demonstrating that the KPO framework effectively steers the PLM away from harmful regions in the latent space.](page_1012_1012_410_312.png)

Figure 3: The embeddings of proteins generated by the fine-tuned PLM exhibit a clear separation from those of harmful proteins, demonstrating that the KPO framework effectively steers the PLM away from harmful regions in the latent space.
Figure 4: Ablation study results for three PLMs: ProtGPT2 (left), InstructProtein (middle), and ProGen2 (right). Each plot compares the performance of different optimization methods across five safety evaluation metrics.

(ProtGPT2, Progen2, and InstructProtein). These sequences were then processed through the ESM-2 model to obtain their respective embeddings, which were subsequently visualized using the t-SNE [Van der Maaten and Hinton, 2008] algorithm for dimensionality reduction.

Figure 3 demonstrates a significant divergence between the harmful protein embeddings and those of the generated proteins from the KPO-fine-tuned models. The sequences generated by the KPO-optimized ProtGPT2, Progen2, and InstructProtein models form distinct clusters that are well-separated from the cluster representing the harmful protein embeddings. This separation indicates that the fine-tuning process effectively steered the models away from generating sequences that share structural or functional similarities with known harmful proteins. This shift in embedding space aligns with the improvements observed in safety evaluation metrics, such as BLAST, MMseqs2, and ToxinPred3, further validating the effectiveness of the fine-tuning process.

5.3 Ablation Study

To validate the effectiveness of our PSKG and the proposed graph pruning strategy, we conducted an extensive ablation study. This study compared KPO with three alternative methods: DPO, KPO-random, and KPO-community. Each alternative serves as a variation in the use of PSKG or sampling strategies during the fine-tuning process, enabling a comprehensive evaluation of KPO’s core components and their contributions.

For the DPO baseline, the PSKG was not utilized. Instead, benign proteins were randomly sampled from the Uniprot database to construct the preference dataset, and the standard DPO method was applied for fine-tuning. This evaluates the benefits of incorporating structured domain knowledge into the fine-tuning process. For the KPO-random method, the PSKG was utilized but reduced in scale by randomly pruning nodes and edges. The reduced graph was then used to select benign proteins for fine-tuning with DPO. This method tests the effectiveness of random graph reduction compared to structured pruning. In contrast, the KPO-community method used a community detection algorithm to reduce the scale of the PSKG. By clustering nodes based on their structural relationships, this approach preserved local community structures within the graph while reducing its overall size. Benign proteins were then selected from these communities for DPO fine-tuning. This method evaluates the impact of structural preservation during graph reduction on model performance. The experimental results, summarized in Figure 4, show that the KPO method consistently outperformed the alternative methods across nearly all safety evaluation metrics, including BLAST, MMseqs2, Pfam_D, Pfam_E, and ToxinPred3. The results also highlight the effectiveness of the graph pruning strategy used in KPO. Both KPO-random and KPO-community served as benchmarks to test the validity of our pruning method. The ablation study confirms that the proposed graph pruning algorithm in KPO is not only effective in reducing the graph’s scale but also preserves essential biological relationships.

To further assess the robustness of the KPO framework, we conducted additional experiments and sensitivity analyses using ProtGPT2 under a range of hyperparameter configurations. The hyperparameters \( \alpha, \beta, \gamma, \delta, Q, \) and \( K \) are central to our pruning strategy, and we provide a comprehensive assessment of their influence on model
Table 2: Performance under different \( \alpha, \beta \) combinations.

<table>
  <tr>
    <th>\( \alpha, \beta \)</th>
    <th>BLAST↓</th>
    <th>MMseq2↓</th>
    <th>Pfam_D↓</th>
    <th>Pfam_E↓</th>
    <th>ToxinPred3↓</th>
    <th>GB1↑</th>
    <th>PhoQ↑</th>
    <th>UBC9↑</th>
    <th>GFP↑</th>
  </tr>
  <tr>
    <td>0.1, 0.9</td>
    <td>0.111</td>
    <td>0.125</td>
    <td>0.294</td>
    <td>0.245</td>
    <td>0.017</td>
    <td>0.062</td>
    <td>0.025</td>
    <td>0.137</td>
    <td>1.68</td>
  </tr>
  <tr>
    <td>0.3, 0.7</td>
    <td>0.107</td>
    <td>0.102</td>
    <td>0.318</td>
    <td>0.241</td>
    <td>0.017</td>
    <td>0.033</td>
    <td>0.001</td>
    <td>0.169</td>
    <td>2.12</td>
  </tr>
  <tr>
    <td>0.5, 0.5</td>
    <td>0.138</td>
    <td>0.149</td>
    <td>0.261</td>
    <td>0.182</td>
    <td>0.024</td>
    <td>0.041</td>
    <td>0.315</td>
    <td>0.129</td>
    <td>2.20</td>
  </tr>
  <tr>
    <td>0.7, 0.3</td>
    <td>0.100</td>
    <td>0.100</td>
    <td>0.351</td>
    <td>0.248</td>
    <td>0.010</td>
    <td>0.098</td>
    <td>0.090</td>
    <td>0.099</td>
    <td>2.13</td>
  </tr>
  <tr>
    <td>0.9, 0.1</td>
    <td>0.148</td>
    <td>0.145</td>
    <td>0.181</td>
    <td>0.262</td>
    <td>0.014</td>
    <td>0.046</td>
    <td>0.907</td>
    <td>0.100</td>
    <td>1.66</td>
  </tr>
</table>

Table 3: Performance under different \( \gamma:\sigma \) combinations.

<table>
  <tr>
    <th>\( \gamma:\sigma \)</th>
    <th>BLAST↓</th>
    <th>MMseq2↓</th>
    <th>Pfam_D↓</th>
    <th>Pfam_E↓</th>
    <th>ToxinPred3↓</th>
    <th>GB1↑</th>
    <th>PhoQ↑</th>
    <th>UBC9↑</th>
    <th>GFP↑</th>
  </tr>
  <tr>
    <td>1:1</td>
    <td><b>0.108</b></td>
    <td>0.154</td>
    <td><b>0.171</b></td>
    <td>0.228</td>
    <td><b>0.002</b></td>
    <td><b>0.067</b></td>
    <td>0.116</td>
    <td>0.138</td>
    <td>2.16</td>
  </tr>
  <tr>
    <td>1:2</td>
    <td>0.138</td>
    <td>0.149</td>
    <td>0.261</td>
    <td><b>0.182</b></td>
    <td>0.024</td>
    <td>0.041</td>
    <td>0.315</td>
    <td>0.129</td>
    <td>2.20</td>
  </tr>
  <tr>
    <td>1:3</td>
    <td>0.119</td>
    <td><b>0.111</b></td>
    <td>0.194</td>
    <td>0.263</td>
    <td>0.011</td>
    <td>0.026</td>
    <td><b>0.916</b></td>
    <td>0.145</td>
    <td><b>2.30</b></td>
  </tr>
  <tr>
    <td>2:1</td>
    <td>0.137</td>
    <td>0.125</td>
    <td>0.179</td>
    <td>0.232</td>
    <td>0.026</td>
    <td>0.049</td>
    <td>0.111</td>
    <td>0.114</td>
    <td>1.59</td>
  </tr>
  <tr>
    <td>3:1</td>
    <td>0.153</td>
    <td>0.153</td>
    <td>0.273</td>
    <td>0.274</td>
    <td>0.028</td>
    <td>0.052</td>
    <td>0.001</td>
    <td><b>0.161</b></td>
    <td>1.54</td>
  </tr>
</table>

performance. The parameters \( \alpha \) and \( \beta \) control the trade-off between the connectivity of high-scoring GO nodes (\( \alpha \)) and the degree centrality of benign protein nodes (\( \beta \)). As reported in Table 2, the model demonstrates stable performance across various (\( \alpha, \beta \)) combinations, with safety evaluation metrics (e.g., BLAST, MMseq2) exhibiting only minor variations. Similarly, \( \gamma \) and \( \delta \) balance the harmful-benign bridging degree and the neighborhood breadth, respectively. Table 3 shows that changes in the \( \gamma:\delta \) ratio do not substantially impact model performance. In particular, the configuration \( \gamma:\delta = 1:1 \) yields optimal results for most evaluation metrics, especially in toxicity prediction (ToxinPred3), where the harmfulness score is notably reduced (0.002). These results indicate that the pruning strategy is robust to variations in the balance between structural relevance and functional connectivity. Furthermore, we performed comprehensive sensitivity analyses for \( Q \) and \( K \), with detailed results provided in Appendix B. These findings collectively demonstrate that the KPO framework is robust to variations in key hyperparameters, further supporting the reliability of our proposed approach.

6 Conclusion and Future Work

In this study, we proposed Knowledge-guided Preference Optimization (KPO), a method for fine-tuning protein language models to ensure the safety and functionality of generated sequences. By constructing a comprehensive dataset of Protein Safety Knowledge Graph and integrating it with pruning strategies and reinforcement learning, KPO reduces the risk of generating harmful proteins while maintaining or enhancing functional performance. This work provides a framework for incorporating safety knowledge into PLMs, enabling responsible applications in protein engineering. Future work will focus on extending KPO by integrating structural safety constraints, scaling to larger PLMs, and dynamically updating the PSKG with new safety insights, ensuring adaptability to evolving biological challenges.

Acknowledgements

This work was supported by the “Pioneer” and “Leading Goose” R&D Program of Zhejiang (Grant No. 2025C01097), Hangzhou West Lake Pearl Project Leading Innovative Youth Team Project (TD2023017) and the Ningbo Natural Science Foundation (2024J020).

Limitations

While the KPO framework demonstrates promising results in aligning PLMs with safety constraints, several limitations should be addressed in future work. First, the fine-tuning process in this paper focuses primarily on sequence-level safety constraints. Structural-level constraints, such as ensuring generated proteins avoid toxic 3D conformations, are not directly incorporated and rely on downstream evaluation tools. In addition, while reinforcement learning enables nuanced optimization, the computational overhead for training large
PLMs remains significant, limiting its scalability to larger datasets or more complex safety objectives. Addressing these limitations will be critical for advancing the robustness and applicability of KPO in real-world scenarios, such as therapeutic development and environmental biotechnology.

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A  More Details for Methods

A.1  Construction of PSKG

The PSKG leverages the structured biological knowledge encoded in the Gene Ontology, which provides a hierarchical vocabulary of GO terms describing biological processes, cellular components, and molecular functions. These GO terms \( G = \{g_1, g_2, ..., g_Z\} \) serve as nodes within the graph, connected by GO-GO triples, \((g_i, r, g_j)\), where \(r\) represents relationships such as “is-a” or “part-of”. These triples define hierarchical and associative relationships between terms, forming the backbone of the graph. The PSKG also incorporates associations between proteins and GO terms through Protein-GO triples, \((p, r, g)\), where \(p \in P_H \cup P_B\) represents harmful or benign proteins and \(g \in G\) is the associated GO term. These triples encode functional annotations that link proteins to their biological roles, establishing indirect relationships between \(P_H\) and \(P_B\). The harmful protein \(P_H\) dataset was curated from UniProt by searching for sequences annotated with the keywords “toxin” (approximately 10,000 entries) and “antigen” (approximately 8,000 entries). After removing duplicates, the remaining sequences formed the harmful protein set. The benign protein dataset \(P_B\) was collected from Swiss-Prot by excluding proteins identified as harmful. By integrating these two layers—GO-GO relationships and Protein-GO associations—the PSKG models complex dependencies across the biological network. For instance, harmful proteins \(P_H\) and benign proteins \(P_B\) may share overlapping functional annotations, such as catalytic activity or molecular binding specificity, but harmful proteins often exhibit additional properties that contribute to harmful effects.

A.2  Algorithm definition

We provide the pseudo code of node pruning with weighted metrics as follows so that readers can easily understand the whole process.

Algorithm 1: Graph pruning algorithm based on PSKG

<html>
<table>
<tr><th colspan="2">Algorithm 1: Graph pruning algorithm based on PSKG</th></tr>
<tr><th>Input:</th><td>PSKG \(G = (V, E)\), Set of harmful proteins \(P_H\), benign proteins \(P_B\), Gene Ontology (GO) nodes \(G\), pruning thresholds \(K, Q\), weight parameters \(\alpha, \beta, \gamma, \delta\).</td></tr>
<tr><th>Output:</th><td>Pruned graph \(G' = (V', E')\)</td></tr>
<tr><th colspan="2">foreach \(g_z \in G\) do</th></tr>
<tr><td colspan="2">Compute harmful-benign bridging degree:<br>\(R(g_z) = \sum_{p_{H}^i \in P_H} \sum_{p_{B}^j \in P_B} 1((p_{H}^i, g_z) \in E) \cdot 1((g_z, p_{B}^j) \in E)\)<br>Compute neighbor breadth: \(O(g_z) = \sum_{p_{B}^j \in P_B} 1((g_z, p_{B}^j) \in E)\)<br>Compute GO node importance: \(C(g_z) = \gamma \cdot R(g_z) + \delta \cdot O(g_z)\)</td></tr>
<tr><th colspan="2">Select the top-\(Q\) GO nodes with highest \(C(g_z)\) values: \(G_h \leftarrow\) Top-\(Q\) GO nodes based on \(C(g_z)\)</th></tr>
<tr><th colspan="2">foreach \(p_{B}^j \in P_B\) do</th></tr>
<tr><td colspan="2">Compute GO association factor:\(C_{GO}(p_{B}^j) = \sum_{g_z \in G_h} 1((p_{B}^j, g_z) \in E)\)<br>Compute degree centrality:\(C_{Deg}(p_{B}^j) = \sum_{v \in V} 1((p_{B}^j, v) \in E)\)<br>Compute final weighted importance score: \(S(p_{B}^j) = \alpha \cdot C_{GO}(p_{B}^j) + \beta \cdot C_{Deg}(p_{B}^j)\)</td></tr>
<tr><th colspan="2">Select top-\(K\) benign protein nodes with highest \(S(p_{B}^j)\) values:<br>\(P_B' \leftarrow\) Top-\(K\) benign protein nodes based on \(S(p_{B}^j)\)<br>Define pruned graph: \(V' = P_H \cup P_B' \cup G_h,\quad E' = \{(u, v) \in E \mid u, v \in V'\}\)<br>Return pruned graph \(G' = (V', E')\).</th></tr>
</table>
</html>

A.3  Pruning threshold selection

Figure 5 presents the distribution of importance scores for both GO nodes and protein nodes within the PSKG. The importance scores were computed using the weighted metrics described in the methodology, which integrate structural and functional relevance factors to quantify the significance of each node. The
figure clearly illustrates that the importance scores are highly unevenly distributed, with a significant proportion of nodes exhibiting low scores, while a smaller subset of nodes has substantially higher scores.

To optimize the pruning process, we selected the top-\( Q = 50\% \) of GO nodes and top-\( K = 50\% \) of protein nodes based on their importance scores. This threshold ensures a balance between retaining the most biologically significant nodes and reducing the size of the graph to improve computational efficiency. The decision to use the 50% threshold is grounded in the observed score distributions. For GO nodes, the distribution shows a steep decline in the frequency of nodes with higher scores, indicating that the top-scoring nodes capture the majority of the functional and structural relevance in the graph. Similarly, for protein nodes, the score distribution exhibits a long-tailed pattern, with the top 50% of nodes containing the majority of high-impact nodes based on the computed importance metrics. The choice of the 50% threshold is further justified by the diminishing returns observed in the importance scores beyond this point. Nodes with scores below the median contribute marginally to the biological relationships within the graph, as their connections to key proteins or GO terms are sparse or weak. Retaining these nodes would unnecessarily inflate the graph size without providing meaningful contributions to downstream tasks. The importance scores for GO nodes are calculated using weighting parameters \( \gamma = 1.0 \) and \( \delta = 0.5 \), while the protein node scores are determined using \( \alpha = 0.5 \) and \( \beta = 0.5 \). These values were empirically selected to balance the contributions of key structural and relational factors in the graph.

![Bar charts showing the distribution of importance scores for GO nodes and protein nodes.](page_324_682_1097_377.png)

Figure 5: Visualization of importance scores of GO nodes and protein nodes.

B Experimental setup

Baseline We consider three foundational models and conducted a comprehensive comparative analysis.

• ProtGPT2 ProtGPT2 is a generative PLM based on the GPT-2 architecture, designed specifically for protein sequence generation. The model is pre-trained on a large corpus of protein sequences from databases like UniProt and SwissProt, using an autoregressive objective to predict the next token in a sequence. This approach allows the model to capture sequential dependencies and generate biologically plausible protein sequences with high fidelity. ProtGPT2 excels in creating diverse sequences with well-formed primary structures, making it a widely adopted baseline for protein generation tasks.

• Progen2 Progen2 is a PLM pre-trained using a transformer-based architecture, leveraging a large-scale masked language modeling (MLM) objective. Unlike autoregressive models like ProtGPT2, Progen2 focuses on learning bidirectional contextual representations, making it particularly effective
in capturing long-range dependencies within protein sequences. This capability allows Progen2 to perform well in tasks requiring an understanding of global sequence context, such as predicting functional domains or secondary structures.

• InstructProtein InstructProtein is a specialized PLM designed for instruction-based generation tasks. It incorporates a pre-training strategy that combines supervised fine-tuning with reinforcement learning from human feedback (RLHF), enabling the model to follow specific generation instructions provided during inference. The instruction-based approach enhances the model’s controllability and makes it particularly suited for applications requiring specific functional or structural characteristics in generated proteins.

Evaluation indicators

• Safety Evaluation Metrics. In our evaluation, BLAST (Basic Local Alignment Search Tool) was employed to align the generated protein sequences with a curated testing harmful protein dataset for evaluation. Key metrics such as sequence identity, E-values, and alignment length were analyzed, as high sequence identity and low E-values often indicate strong functional or evolutionary relationships. Such relationships suggest a higher likelihood that the generated protein might share harmful properties with known harmful proteins. Complementing this, MMseqs2 was utilized for high-throughput sequence similarity searches and clustering. MMseqs2 provided additional insights by grouping similar sequences and uncovering potential functional associations between the generated proteins and harmful sequences.

To further assess potential harmfulness, we analyzed domain-level similarities using two approaches within the Pfam database. In the dynamic threshold method, a match was considered significant if the E-value of a domain in the generated protein was lower than the threshold E-value of the corresponding domain in the harmful protein database. This approach accounts for the unique statistical characteristics of each domain, as each hidden Markov model (HMM) in Pfam has its own noise model. By tailoring the significance criteria to individual domains, the dynamic threshold method provides a more precise and domain-specific evaluation of potential harmful associations.

In contrast, the fixed threshold method applied a standardized E-value cutoff across all domains, which we set to 0.001. This method simplifies the evaluation process by enforcing a consistent significance level, ensuring robust and highly significant matches are prioritized. To be considered a match under this method, both the E-value of the domain in the generated protein and the corresponding E-value in the harmful protein database had to fall below the fixed threshold. This ensures consistency while maintaining high statistical confidence in the results, allowing for a systematic evaluation of domain overlap across protein families.

• Functional Evaluation Metrics. The GB1, PhoQ, UBC9, and GFP datasets represent diverse protein families, each with distinct functional roles in biological processes, providing a robust framework for evaluating the fine-tuned model’s performance. By assessing these datasets, we ensured that the fine-tuning process using KPO improved safety by reducing harmful protein generation without compromising the model’s ability to predict biologically relevant and functionally advantageous mutations. The evaluation relied on two key metrics: the generation probability score, which reflects how likely the model is to generate a particular mutation based on its learned patterns, and fitness, which measures how well a mutation preserves or enhances the protein’s intended function. By comparing the average fitness of the top-ranked mutations generated by both the pre-trained and fine-tuned models, we assessed whether KPO fine-tuning shifted the model’s capacity to identify mutations that optimize protein functionality.

The use of the GB1, PhoQ, UBC9, and GFP datasets validated the dual objectives of the KPO method: improving safety while maintaining or enhancing functionality. This comprehensive evaluation framework demonstrated that KPO fine-tuning effectively aligns the model’s outputs with biological safety and functional integrity, ensuring the generation of proteins that are both safe and biologically meaningful.
Implementation Details   Our method was implemented using the PyTorch deep learning framework. To support reinforcement learning during the fine-tuning process, we integrated the TRL [von Werra et al., 2020]. All experiments were conducted on an Ubuntu server with 8 NVIDIA A100 GPUs, each with 40GB of memory. The fine-tuning process was performed on protein language models with varying parameter scales: ProtGPT2 (738M parameters), ProGen2 (764M parameters), and InstructProtein (1.3B parameters). Training each epoch on our A100 GPUs required approximately 2 hours, with the learning rate set around 0.00005 to ensure stable and effective optimization. The dataset of harmful protein sequences, curated from UniProt, was split into training and testing sets in an 8:2 ratio. The training dataset was used to construct the PSKG, forming the harmful protein nodes that guide the fine-tuning process. The testing dataset served as the benchmark for evaluating sequence similarity and other safety-related metrics in the experimental results. Notably, the incorporation of the graph pruning algorithm significantly reduced the time required for generating preference data, with the overall time being halved compared to the baseline. This improvement in computational efficiency is attributed to the pruning process, which reduces the complexity of the knowledge graph by retaining only the most critical nodes and edges, thus accelerating downstream tasks such as preference data generation without compromising the quality of the generated outputs.

Three-dimensional Structure Analysis   To evaluate the impact of the KPO fine-tuning method on the structural characteristics of generated proteins, we conducted a comparative analysis of the three-dimensional structures of proteins generated by the pre-trained and KPO-fine-tuned models. we employed ColabFold [Jumper et al., 2021; Mirdita et al., 2022] to predict the corresponding structural conformations of the generated proteins. For each selected protein, the predicted structure’s confidence was evaluated using the mean predicted Local Distance Difference Test (pLDDT), which serves as a metric to quantify the reliability of the structural predictions.Meanwhile, we assessed the structural similarity between the generated proteins and known harmful proteins using Root Mean Square Deviation (RMSD). Figure 6 illustrates the comparison between harmful proteins and proteins generated by both the KPO-fine-tuned ProtGPT2 model and the pre-trained ProtGPT2 model. Each row of the figure corresponds to one of four harmful proteins with distinct functional and biochemical properties.The first harmful protein analyzed is a beta toxin that binds to sodium channels (Nav) at site-4 in a voltage-independent manner. By shifting the activation voltage to more negative potentials, this toxin enhances spontaneous and repetitive neuronal firing, which is lethal to mammals, including mice. The second beta toxin shares a similar mechanism of action but exhibits a broader specificity, affecting crustaceans and insects while being non-lethal to mammals. It also competitively displaces other beta toxins from mammalian sodium channels at higher concentrations. The third protein is an alpha toxin, binding to site-3 of sodium channels and inhibiting channel inactivation, leading to sustained neuronal activity. This toxin is highly harmful to both insects and mammals, causing convulsions and death upon injection into mice. Lastly, the fourth protein is a potassium channel blocker that targets the pore domain of Kv7 channel family members, disrupting ion transport and inducing severe physiological effects, including acute hypertension, coronary vasospasms, and seizures. In all four cases, the KPO-fine-tuned ProtGPT2 model consistently generated proteins with significantly higher RMSD values compared to the pre-trained ProtGPT2 model. For example, when compared to the first beta toxin, the KPO-generated protein exhibited an RMSD of 7.951 Å, substantially higher than the 1.419 Å RMSD observed for the pre-trained model. Similar trends were observed across the remaining toxins, with RMSD values from the KPO-generated proteins exceeding those of the pre-trained model by approximately 50% in most cases. These results indicate that the structural characteristics of the proteins generated by the KPO model diverge significantly from those of harmful proteins. By integrating safety knowledge from the PSKG, the KPO fine-tuning approach actively learns to generate proteins with sequence and structural features that diverge from those associated with harmfulness. This structural divergence is particularly critical as protein 3D structures are closely linked to their functional and interaction properties; reducing structural similarity to harmful proteins minimizes the risk of generating proteins with harmful biological activities.

In conclusion, the three-dimensional structure analysis provides strong evidence that the KPO fine-tuning approach significantly enhances the safety profile of protein generation models. By producing
Beta-toxin Cb1   pLDDT=93.9
pLDDT=95.1   RMSD=7.951
pLDDT=90.1   RMSD=1.419

Toxin Css39.8   pLDDT=84.9
pLDDT=95   RMSD=4.146
pLDDT=94.7   RMSD=0.128

Toxin Lqh4   pLDDT=97.4
pLDDT=90.1   RMSD=6.533
pLDDT=91.6   RMSD=3.709

Mu-scoloptoxin(15)-Ssm1a   pLDDT=82.3
pLDDT=92.4   RMSD=12.05
pLDDT=90.2   RMSD=3.949

(a) Harmful Protein    (b) ProGPT2+KPO    (c) ProGPT2

Figure 6: Comparison of 3D RMSD Values Between Harmful Proteins and Proteins Generated by Pre-trained and KPO-Fine-Tuned Models.
proteins with greater structural differences from harmful proteins, as evidenced by higher RMSD values, KPO fine-tuning ensures that the generated proteins are not only sequence-safe but also structurally distinct, advancing the biological safety and reliability of protein language models.

Sensitivity Analyses of Hyperparameters  The parameters \( Q \) and \( K \) determine the number of top-ranked GO nodes and benign protein nodes selected based on their importance scores. As \( Q \) and \( K \) decrease, pruning becomes more aggressive, which improves computational efficiency but may compromise safety in protein generation. As shown in Table 4, setting \( Q = 50\% \) and \( K = 50\% \) achieves the best trade-off between computational efficiency and safety, as reflected by improved performance on both safety and functional metrics.

Table 4: Performance under different \( Q, K \) configurations.

<table>
  <tr>
    <th>\( \gamma;\sigma \)</th>
    <th>BLAST↓</th>
    <th>MMseq2↓</th>
    <th>Pfam_D↓</th>
    <th>Pfam_E↓</th>
    <th>ToxinPred3↓</th>
    <th>GB1↑</th>
    <th>PhoQ↑</th>
    <th>UBC9↑</th>
    <th>GFP↑</th>
  </tr>
  <tr>
    <td>50%, 50%</td>
    <td>0.138</td>
    <td>0.149</td>
    <td><b>0.261</b></td>
    <td><b>0.182</b></td>
    <td><b>0.024</b></td>
    <td><b>0.041</b></td>
    <td>0.315</td>
    <td>0.129</td>
    <td><b>2.20</b></td>
  </tr>
  <tr>
    <td>30%, 30%</td>
    <td><b>0.115</b></td>
    <td><b>0.103</b></td>
    <td>0.338</td>
    <td>0.397</td>
    <td>0.045</td>
    <td>0.037</td>
    <td>0.329</td>
    <td>0.142</td>
    <td>1.72</td>
  </tr>
  <tr>
    <td>10%, 10%</td>
    <td>0.116</td>
    <td>0.106</td>
    <td>0.327</td>
    <td>0.366</td>
    <td>0.038</td>
    <td>0.037</td>
    <td><b>0.329</b></td>
    <td><b>0.142</b></td>
    <td>1.72</td>
  </tr>
</table>

C  Potential Risks

The curated dataset of harmful protein sequences, although essential for constructing the PSKG and fine-tuning the model, could potentially be misused if accessed by malicious actors. Such misuse might involve training models specifically designed to generate even more harmful or weaponizable proteins, amplifying the risks to public health and ecological stability. To address these concerns, we have decided to either withhold public access to the harmful protein dataset or release it under strict guidelines and controlled conditions. By limiting its availability, we aim to balance the scientific value of this research with the imperative to minimize potential misuse, ensuring that the advancements introduced by KPO are applied responsibly in biotechnology and synthetic biology.

Reviewer paper summaries: This manuscript proposes the Knowledge-guided Preference Optimization (KPO) framework, which integrates a Protein Safety Knowledge Graph (PSKG) and preference optimization techniques to systematically incorporate biosafety knowledge into protein generation models. The framework not only reduces the risk of generating harmful proteins but also maintains model functionality, offering a novel safety optimization approach for protein engineering and biotechnology.

This paper addresses a critical safety challenge in protein language models (PLMs) - their potential to generate harmful protein sequences that could pose biosafety risks. While PLMs have shown remarkable capabilities in protein design and optimization, they currently lack safety considerations in their generation process, potentially producing proteins that could enhance viral transmissibility, evade immune responses, or develop drug resistance.

The authors propose Knowledge-guided Preference Optimization (KPO), a novel framework that integrates biological safety knowledge into the protein generation process. At the core of KPO is the construction of a Protein Safety Knowledge Graph (PSKG), which systematically captures relationships between harmful and benign proteins through Gene Ontology (GO) terms. The PSKG serves as a comprehensive knowledge base that encodes critical biochemical properties and intrinsic relationships of proteins, helping to distinguish harmful from benign sequences.

To manage computational complexity, the paper introduces a weighted metric-based pruning algorithm that efficiently reduces the size of the PSKG while preserving its most informative components. This pruning strategy considers both GO association factors and degree centrality to identify and retain the most biologically relevant nodes and relationships. The pruned graph maintains its utility for downstream tasks while significantly reducing computational overhead.

The framework uses the processed PSKG to guide the fine-tuning of PLMs through Direct Preference Optimization (DPO). By identifying benign proteins that share certain properties with harmful ones, KPO creates preference pairs that teach the model to generate safer sequences while maintaining desired functional properties. This approach effectively steers the model away from generating potentially harmful proteins while preserving its ability to produce functionally relevant sequences.

The authors validate their approach through comprehensive experiments using three different base models: ProtGPT2, Progen2, and InstructProtein. The evaluation uses multiple safety metrics including BLAST, MMseqs2, Pfam domain analysis, and ToxinPred3 predictions. Results demonstrate that KPO significantly reduces the likelihood of generating harmful sequences across all metrics while maintaining or even improving functional performance on benchmark datasets. Visualization of protein embeddings shows clear separation between KPO-generated proteins and known harmful proteins, further validating the effectiveness of the approach.

The paper acknowledges certain limitations, particularly in addressing structural-level safety constraints and computational overhead for large-scale models. However, it represents a significant step forward in developing safer protein generation methods, providing a framework that could be essential for responsible advancement in protein engineering and biotechnology.
Summary of strengths: 1.The work addresses a critical biosafety challenge in protein generation by proposing a novel framework that effectively reduces the risk of generating harmful proteins while maintaining functional performance.
2.The methodology is comprehensive and theoretically sound. The three-stage approach (PSKG construction, node pruning, and PLM fine-tuning) is logically structured and each component is thoroughly explained with mathematical formulations and clear justifications.
3.The manuscript provides detailed three-dimensional structure analysis using ColabFold, which convincingly shows how KPO-generated proteins structurally diverge from harmful proteins through RMSD comparisons.

This paper presents a novel and timely contribution by introducing the first framework specifically designed to address biosafety concerns in protein language models (PLMs). The proposed Protein Safety Knowledge Graph (PSKG) bridges NLP techniques with biological safety considerations, providing a structured and scalable approach to guiding safe protein generation. Given the growing concerns around responsible AI development and biosafety, this work is particularly relevant. The framework integrates domain-specific constraints to ensure safer generative processes while maintaining functional performance, making it a valuable contribution to both the ACL community and the broader bioinformatics field.

A major strength of the paper lies in its technical innovation and comprehensive evaluation. The authors introduce an efficient weighted metric-based pruning algorithm that balances computational efficiency with information preservation, ensuring the scalability of the approach. Additionally, the adaptation of Direct Preference Optimization (DPO) to protein sequence generation highlights the cross-domain applicability of NLP techniques. The evaluation spans multiple PLMs, including ProtGPT2, ProGen2, and InstructProtein, and employs diverse safety and functionality metrics such as BLAST, MMseqs2, Pfam, and ToxinPred3. Detailed ablation studies and embedding visualizations further demonstrate the model’s ability to generate safe sequences while preserving functionality, reinforcing the practical impact and robustness of the proposed framework.

The practical impact and interdisciplinary relevance of this work are significant, addressing a critical challenge in biotechnology by offering a concrete solution for safer protein generation. The proposed framework provides a scalable methodology that can be extended to other domains requiring safety considerations, such as pharmaceuticals and synthetic biology. Additionally, the paper takes an important step toward responsible AI development by addressing ethical concerns and emphasizing the importance of safety in generative models. With a well-structured methodology, clear explanations, and a transparent discussion of limitations, this work offers valuable insights and a solid foundation for future research in safe and controllable generative AI applications.
Summary of weaknesses: 1. In Table 1, the metrics such as Pfam_D and Pfam_E are not clearly introduced. It would be helpful to explicitly describe in the main text what the highs and lows of these metrics indicate.
2. The manuscript mentions the use of GB1, PhoQ, UBC9, and GFP datasets to evaluate the model's functional optimization capabilities, but it does not provide detailed information on the specific calculation methods or scoring criteria for these metrics.
3. In the "Embedding Result Analysis" section, the manuscript compares the differences between the sequences generated by the fine-tuned models and the known harmful protein sequences in the embedding space by selecting 100 protein sequences from different categories. However, it does not clarify whether these sequences have similar optimization goals or functional backgrounds, or whether they are related in some way. This lack of context may undermine the persuasiveness of the comparison results.

The paper presents a promising approach to improving the safety of protein generation using Protein Language Models (PLMs); however, several areas require further attention to strengthen its contributions. A primary concern is the limited validation of safety claims, as the paper primarily relies on computational similarity and prediction tools without experimental validation through wet lab testing or simulations. Demonstrating tangible safety improvements through case studies and functional assays would enhance credibility. Additionally, the methodological choices, such as the pruning thresholds and hyperparameter selections, lack sufficient justification. A sensitivity analysis of these choices and a clearer explanation of the relationship between graph-space and embedding-space similarity measures would improve the methodological rigor.

The evaluation and scalability aspects also present gaps. The functional evaluation focuses on a limited set of protein families, raising concerns about generalizability to novel proteins. A broader evaluation across diverse families and comparisons with other safety-oriented approaches would provide a clearer picture of the framework’s strengths and weaknesses. The computational efficiency of the pruning strategy is not fully analyzed, and additional insights into time complexity, memory requirements, and scalability to larger models are needed to assess real-world feasibility.

Lastly, the paper would benefit from improved clarity and broader impact considerations. Key methodological details are relegated to appendices, making it challenging to understand the PSKG construction and its impact on safety. Enhanced visualizations and more examples of generated sequences would improve readability. Additionally, a more thorough discussion of ethical considerations, privacy concerns, and failure modes in handling emerging biological threats would strengthen the paper’s societal relevance. Addressing these concerns would make the work more robust, reproducible, and impactful for the research community.
Comments, suggestions and typos: The manuscript mentions Figure 4 before Figure 3. It is recommended that the author adjust the order of the figures or the sequence of their mentions in the text to ensure logical coherence.

To improve the paper’s clarity and organization, it is recommended to restructure Section 4 to distinctly separate PSKG construction, the graph pruning strategy, and the fine-tuning process, making the methodology easier to follow. Including a workflow diagram that visualizes the pipeline from data collection to evaluation would enhance understanding. The paper would also benefit from providing more examples of generated sequences, specific success and failure cases, and visualizations of the pruning process to illustrate its impact effectively.

From a technical perspective, expanding implementation details—such as providing pseudocode for the pruning algorithm, detailed architecture specifications, and training hyperparameters—would improve reproducibility. Clarifying the rationale for the choice of evaluation metrics, functional benchmark datasets, and parameter settings would strengthen the methodological soundness. Additionally, a discussion on updating the PSKG with new knowledge, addressing emerging biological threats, and maintaining system performance over time would enhance the framework’s practicality.

Further analysis is needed to evaluate the method’s efficiency and effectiveness. An ablation study on different pruning thresholds, runtime and memory usage analysis across model sizes, and more quantitative evaluations of graph structure changes and safety-functionality trade-offs would provide deeper insights. Practical considerations such as guidelines for selecting safety thresholds, resource requirements, scaling strategies, and monitoring protocols should also be addressed for real-world deployment.

Minor improvements in writing and presentation include defining acronyms at first use, ensuring consistent notation, improving figure readability, and enhancing table descriptions with standard deviations and statistical significance. Expanding the related work section with recent references and comparisons to alternative safety approaches would provide broader context. Lastly, including a code availability statement, detailed dataset descriptions, and reproduction guidelines would support transparency and adoption. These improvements will strengthen the paper’s clarity, completeness, and overall impact.
Overall assessment scores: 3

3.5