In recent years, diseases caused by antibiotic-resistant pathogens have imposed a severe medical burden and significant economic losses. Many antibiotics have lost their original efficacy against these superbugs.

This is why we have identified a suitable alternative: antimicrobial peptides (AMPs). AMPs, typically composed of 10-50 amino acids, offer advantages such as broad-spectrum antibacterial activity and low propensity for inducing resistance.

However, current AMP production methods suffer from low yield, resulting in high costs. In our project, we successfully introduced four different promoters (FDH1, CAT1, AOX1, AOX713) and four signal peptides (0030, SP4, SP14, α-factor) into an engineered yeast strain GS115 for producing the antimicrobial peptide Plectasin NZ2114. We compared their expression levels using protein electrophoresis and the Oxford cup assay. At the same time, we conducted tests on the antibacterial curve, antibacterial activity, and antibacterial morphology of the optimal combined engineered bacterial strains obtained through screening after fermentation, focusing on the activity of plectasin NZ2114.

The main gains and achievements of the project:

(1) Obtained construction vectors for different expression regulatory elements and engineered Pichia pastoris strains

a) Promoters: AOX1, FDH1, CAT1, AOX713

b) Signal peptides: Alpha-factor, SP4, SP14, 0030

Experimental verification revealed that the combination of the AOX1 promoter and the α-factor signal peptide yielded the highest production. Consequently, we constructed a highly productive engineered yeast strain using this AOX1 promoter and α-factor signal peptide combination, effectively enhancing the yield of the antimicrobial peptide.

(2) Conducted fermentation tests around the final high-yield strain and evaluated the functional activity of the products.

(3) Conducted educational and human practical activities centered around antibiotic resistance and antimicrobial peptides, while also exploring commercialization pathways related to the products.

Antibiotics have been used widely in food, medical treatment and other industries. As time goes by, however, its overuse has caused the serious consequence of antimicrobial resistance. As a result, it cannot meet our expectation to achieve its original purpose in treating microbial infection and diseases. One of the examples is MRSA (Methicillin-resistant Staphylococcus aureus), which can cause symptoms like infections, fever, abscesses and bleeding, resulting in 12 thousand deaths per year.

Methicillin-resistant Staphylococcus aureus (MRSA) infections are caused by a type of staphylococcus that has developed resistance to many antibiotics commonly used for treating regular staphylococcal infections. The emergence of MRSA is a consequence of the long-term misuse of antibiotics in unnecessary situations. In everyday life, antibiotics have often been used to treat colds, influenza, and other viral infections that have no specific effective treatment. Excessive use of antibiotics increases selective pressure on bacteria, causing them to live in a rapidly changing external environment, leading to advantageous bacteria being able to withstand this frequent antibiotic pressure and survive with resistance [1].

Fig. 1 Symptoms related to MRSA, definition of resistance, and challenges in hospitalization [2].

According to the report of the World Health Organization (WHO), the disease caused by pathogens with antimicrobial resistance now becomes one of the top global public health threats, causing 1.27 million global mortality cases directly and 4.95 million indirectly in 2019 alone [3]. According to the scientists, it is predicted that 10 million people might die from the consequence of antimicrobial resistance annually starting from 2050 [4].

Fig. 2 Death rater per 100 thousand caused by AMR, all ages, data of 2021 (B) and predicted 2050 (C)

Moreover, pathogens with antimicrobial resistance also resulted in some economic losses. Around 1 trillion additional dollars will be spent on the field of health care in the world by 2050, in that more expensive methods or additional medicine are needed to counteract the effects of antibiotic resistance [5].

For this problem, antimicrobial peptides (AMPs) now become a promising solution. Widely found in plants, animals, and microbes, AMPs are short peptides made up of 10 to 50 amino acids. Unlike antibiotics, AMPs achieve the same results through stimulating the immune system to take action against pathogens or direct killing, which, given the existing resistance to antibiotics, is an ideal substitution [6]. However, people still have the difficulty in producing AMPs economically and efficiently. The yields of both synthesizing AMPs artificially and extracting AMPs from natural materials are quite low, not to mention the high cost associated with them. So the curative potential of AMPs has not yet been fully discovered in medical settings. Specifically, the cost of production of antibiotics is 0.80 dollars per gram, while for amino acids that make up AMPs, that is 50-400 dollars per gram using the method of solid phase synthesis, a costly chemical method [7]. The actual cost of producing AMPs is even higher. So cheaper and more efficient methods are needed to produce AMPs, which is the topic we are currently working on.

Nowadays, numerous approaches have been developed to deal with the issue of bacterial resistance, each with its own set of advantages and drawbacks. The table below provides an illustration and description of part of these methods [8, 9].

Table. 1 Summary of the current solution for AMR.

As mentioned in the previous text, antimicrobial peptides are an ideal solution to address antibacterial resistance. However, due to their high cost and low production efficiency, this solution has not been widely adopted. Our project is precisely aimed at solving these two problems: 1) antimicrobial resistance; 2) highly production of antimicrobial peptide plectasin NZ2114.

The specific solutions are as follows: by means of molecular biology, we will explore highly expressed promoters and signal peptides in Pichia pastoris to provide more and better expression elements for Pichia pastoris. We will test the ability of recombinant strains to express plectasin NZ2114 and enhance the ability of Pichia pastoris to synthesize plectasin NZ2114. Through these efforts, the production yield of antimicrobial peptides will be increased and their cost will be reduced [10,11].

Why we choose Plectasin NZ2114 [12]

• NZ2114 exhibits strong activity against Gram-positive bacteria.
• NZ2114 shows no toxicity to primary human cells at the minimum inhibitory concentration (MIC99) of 6.1 μM.
• NZ2114 can retain its anti-mycobacterial ability at the minimum inhibitory concentration (MIC99) of 6.1 μM.
• NZ2114 has good stability and is resistant to serum degradation.
• The activity of NZ2114 against methicillin-sensitive Staphylococcus aureus and MRSA is two to three times that of plectasin.
• NZ2114 demonstrates good thermal stability at 20~80℃.
• NZ2114 has a post-antibiotic effect.

Our project aims to maximize antimicrobial peptide (AMP) production by constructing high-yield yeast cells through the combinatorial optimization of the most productive promoters and signal peptides.

Traditional approaches to treating diseases caused by antibiotic-resistant pathogens—such as vaccines and novel antibiotic development—suffer from high costs and a fundamental limitation: pathogens inevitably develop resistance over time. In contrast, our engineered antimicrobial peptide NZ2114 offers inherent advantages. Its mechanism of action enables direct killing of diverse pathogens with a significantly lower probability of inducing resistance. Furthermore, production in yeast ensures lower host protein interference and enhanced safety compared to alternative expression systems.

We began by constructing yeast strains containing individual high-performance promoters and signal peptides, identifying the combinations yielding the highest protein expression. These optimal genetic elements were then combinatorially assembled and introduced into yeast to create our high-yield production strain.

The resulting NZ2114 antimicrobial peptide delivers broad societal benefits across multiple sectors:

Medical Applications: Compared to conventional antibiotics, NZ2114 offers superior efficacy against bacterial infections, reduces inflammation, and promotes wound healing. This has the potential to save lives and reduce healthcare expenditures.

Animal Husbandry: NZ2114 can decrease livestock morbidity rates.

Food Industry: Its low toxicity and broad-spectrum antibacterial properties make it an effective natural preservative.