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Fermentation
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Research on Microbial Protein Synthesis
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Research on Microbial Protein Synthesis

A special issue of Fermentation (ISSN 2311-5637). This special issue belongs to the section "Microbial Metabolism, Physiology & Genetics".

Deadline for manuscript submissions: 30 April 2024 | Viewed by 13536

Special Issue Editor

Prof. Dr. Dong Liu

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Guest Editor
College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing 211816, China
Interests: continuous fermentation; microbiological physiology; metabolic engineering; cofactor engineering; protein secretion; biofilm; cell morphology; edible fungi; plant cell culture
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

Protein is among the most important kinds of biomolecules to have evolved in the process of sustaining life. These days, with the unleashing of their functional potential, proteins are becoming increasingly important to our world, acting as industrial biocatalysts, food and nutrients, therapeutic agents, biomaterial, and finding a variety of other uses. Additionally, more and more protein-derived products are being developed for new application. To cope with this emerging trend, efficient and low-cost synthesis of protein is in high demanding. Microbial synthesis is a prevalent way to produce proteins of wide interest and with many advantages. Microbes can synthesize a great variety of natural or unnatural proteins with complicated structures and long chains beyond chemical synthesis. Microbial cells are also generally easier to grow at low cost compared to other cells such as the animal cells. Despite these factors, microbial protein synthesis still encounters certain challenges and continuous efforts have been made to improve the production efficiency and reduce production cost, whether by fine-tuning the translation machinery, enhancing protein transport or secretion pathways, the applying dynamic control to global metabolic fluxes, and developing cost-effective fermentation process.

The goal of this Special Issue is to publish recent research results as well as review papers on synthesis machinery, fermentation production, function characterization, and the product development of various proteins (e.g., enzyme, pharmaceutical protein, food ingredient, protein material) from microbial systems (e.g., bacterium, yeast, filamentous fungus, mushroom culture). The synthesis of protein through cell-free extracts and the engineering of protein behaviours (e.g., phase separation, aggregation, self-assembly) in microbial cells are also of interest.

Dr. Dong Liu
Guest Editor

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Fermentation is an international peer-reviewed open access monthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2600 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • protein expression
  • protein secretion
  • molecular chaperone
  • recombinant protein
  • heterologous expression
  • alternative protein
  • precision fermentation
  • continuous fermentation
  • microbial cell factories

Published Papers (7 papers)

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Research

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14 pages, 4834 KiB  
Article
Continuous Secretion of Human Epidermal Growth Factor Based on Escherichia coli Biofilm
by Chong Zhang, Jinglin Liao, Yuancong Li, Shuli Liu, Mengting Li, Di Zhang, Zhenyu Wang, Dong Liu and Hanjie Ying
Fermentation 2024, 10(4), 202; https://doi.org/10.3390/fermentation10040202 - 09 Apr 2024
Viewed by 559
Abstract
Human epidermal growth factor (hEGF) holds significant importance in the fields of medicine and cosmetics. Therefore, it becomes imperative to develop a highly efficient fermentation system for hEGF production. In this study, a stable hEGF-secreting expression strain was created by integrating the hEGF [...] Read more.
Human epidermal growth factor (hEGF) holds significant importance in the fields of medicine and cosmetics. Therefore, it becomes imperative to develop a highly efficient fermentation system for hEGF production. In this study, a stable hEGF-secreting expression strain was created by integrating the hEGF gene into the genome of Escherichia coli (E. coli) BL21, and an immobilized fermentation system was developed based on biofilm to facilitate continuous hEGF production. After optimization of fermentation conditions and gene dosage, the production of hEGF was increased from 13.9 mg/L to 52.4 mg/L in free-cell fermentation. Moreover, genetic modifications targeting dgcC, csgD, bcsA, and bcsB proved to enhance biofilm formation. When the bcsB was overexpressed in BL21-hEGF-C5, the biofilm-forming ability was enhanced by 91.1% and the production of hEGF was increased by 28% in biofilm-immobilized continuous fermentation. In conclusion, this study successfully confirms the feasibility of continuous hEGF production through the biofilm system of E. coli, providing valuable insights for the development of other proteins in the field of continuous biomanufacturing. Full article
(This article belongs to the Special Issue Research on Microbial Protein Synthesis)
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15 pages, 13495 KiB  
Article
High-Level Secretory Production of Recombinant E2-Spy Antigen Protein via Combined Strategy in Pichia pastoris
by Bingkun Li, Yiheng Zheng, Shida Zhao, Yaohan Zhang and Ding Li
Fermentation 2024, 10(2), 99; https://doi.org/10.3390/fermentation10020099 - 08 Feb 2024
Viewed by 1175
Abstract
E2-Spy (abbreviated as ES) plays a vital role as a component in the Bacterial-Like Particles (BLPs) vaccine against classical swine fever virus (CSFV). This vaccine demonstrates remarkable immunoprotection, highlighting the importance of augmenting ES production in the development of CSFV subunit vaccines. In [...] Read more.
E2-Spy (abbreviated as ES) plays a vital role as a component in the Bacterial-Like Particles (BLPs) vaccine against classical swine fever virus (CSFV). This vaccine demonstrates remarkable immunoprotection, highlighting the importance of augmenting ES production in the development of CSFV subunit vaccines. In this study, a Pichia pastoris strain capable of high-yield secretory production of ES was developed through signal peptide engineering, gene dosage optimization and co-expression of molecular chaperones. Initially, a hybrid signal peptide cSP3 was engineered, leading to a 3.38-fold increase in ES production when compared to the control strain 1-α-ES. Subsequently, cSP3 was evaluated for its expression efficiency alongside different commonly used signal peptides under multicopy conditions. SDS-PAGE analysis revealed that 2-αd14-ES exhibited the highest ES production, displaying a 4.38-fold increase in comparison to 1-α-ES. Afterwards, SSA1, YDJ1, BIP, LHS1, and their combinations were integrated into 2-αd14-ES, resulting in a 1.92-fold rise in ES production compared to 2-αd14-ES (equivalent to a 6.18-fold increase compared to 1-α-ES). The final yield of ES was evaluated as 168.3 mg/L through comparison with serially diluted BSA protein bands. Full article
(This article belongs to the Special Issue Research on Microbial Protein Synthesis)
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14 pages, 1184 KiB  
Article
Effect of Glucose Concentration on the Production of Proteolytic Extract by Different Strains of Aspergillus under Solid-State Fermentation
by Liliana Londoño-Hernández, María de Jesús García-Gómez, Sergio Huerta-Ochoa, Anna María Polanía-Rivera, Cristóbal Noé Aguilar and Lilia Arely Prado-Barragán
Fermentation 2024, 10(2), 97; https://doi.org/10.3390/fermentation10020097 - 07 Feb 2024
Viewed by 1241
Abstract
Proteases are important enzymes because of their extended uses in several industries, such as food, beverages, pharmacy, detergents, and many others. Aspergillus is one of the most used fungi strains for enzyme production by solid-state fermentation (SSF). Disponibility of the carbon source is [...] Read more.
Proteases are important enzymes because of their extended uses in several industries, such as food, beverages, pharmacy, detergents, and many others. Aspergillus is one of the most used fungi strains for enzyme production by solid-state fermentation (SSF). Disponibility of the carbon source is a key factor for protease production. In addition, the selection of solid support has great importance, as it must provide suitable airflow through the packed bed and nutrient diffusion inside the fermentable mass. Six Aspergillus strains and two inert supports (Agrolite (AL) and Polyurethane (PUF)) were tested for protease production from fish flour (FF) at different glucose concentrations (0, 5, 10, 15%) by SSF. The FF/PUF mixture at 70/30 (w/w) ratio, with 75.39% moisture, and a critical moisture point of 0.11 gH2O/g, presented a texture that allowed heat and mass transfer and provided enough moisture to make free water available as required for microorganism growth during the fermentation process. Aspergillus oryzae 2095 produced higher amounts of neutral and alkaline proteases with the addition of 5% glucose to the growth medium. Kinetics studies reveal that protease production is partially associated with growth. The extracts obtained can be used in different industries, and especially to prepare fish high-value by-product hydrolysates. Full article
(This article belongs to the Special Issue Research on Microbial Protein Synthesis)
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18 pages, 7638 KiB  
Article
A Universal Strategy for the Efficient Expression of Nanobodies in Pichia pastoris
by Yiheng Zheng, Bingkun Li, Shida Zhao, Jiawei Liu and Ding Li
Fermentation 2024, 10(1), 37; https://doi.org/10.3390/fermentation10010037 - 02 Jan 2024
Viewed by 1970
Abstract
In recent years, nanobodies have played an increasingly crucial role in virus neutralization, ELISA detection, and medical imaging. This study aimed to explore a universal expression strategy in Pichia pastoris using three nanobodies, denoted Va, Vb, and Vc, as model proteins. Initially, plasmids [...] Read more.
In recent years, nanobodies have played an increasingly crucial role in virus neutralization, ELISA detection, and medical imaging. This study aimed to explore a universal expression strategy in Pichia pastoris using three nanobodies, denoted Va, Vb, and Vc, as model proteins. Initially, plasmids pLD-AOXα and pLD-AOX were engineered to minimize the risk of antibiotic resistance gene drift. Optimization of promoters and signal peptides resulted in a 1.38-fold and 1.89-fold increase in Va production. Further optimization of gene dosage led to an additional 1.39-fold enhancement in Va yield. Subsequently, 25 molecular chaperones were co-expressed with Va under the control of the wild-type AOX1 promoter, with HAC1 further increasing Va yield by 1.5-fold. By fine-tuning the promoter strength for HAC1, Va production was increased by 2.41-fold under the control of the 55p promoter. Finally, through high-density fermentation, the Va yield reached 2.13 g/L, representing a 49.8-fold increase compared to the initial strain 1-AOXα-Va in shake-flask culture. Integration of pLD-55p-HAC1 into the GS115 genome resulted in the H55 host, and the transformation of multicopy plasmids into this host led to a 1.98-fold increase in Vb yield and a 2.34-fold increase in Vc yield, respectively. The engineering of antibiotic-free parental plasmids, modification of expression components, gene dosage optimization, and the H55 host are regarded as a composite strategy which will pave the way for efficient expression of nanobodies in the future. Full article
(This article belongs to the Special Issue Research on Microbial Protein Synthesis)
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18 pages, 12743 KiB  
Article
Effect of Replacing Fish Meal Using Fermented Soybean Meal on Growth Performance, Intestine Bacterial Diversity, and Key Gene Expression of Largemouth Bass (Micropterus salmoides)
by Longfei Weng, Zhi Wang, Wei Zhuang, Tiezhu Yang, Xinxin Xu, Jinle Liu, Jixiang Liu, Zhengzhong Xu, Ruitao Chen, Qi Wang, Shilei Wang, Yafan Cai and Hanjie Ying
Fermentation 2023, 9(6), 520; https://doi.org/10.3390/fermentation9060520 - 27 May 2023
Cited by 4 | Viewed by 1377
Abstract
In China, aquaculture costs have increased because of the increase in fish meal (FM) prices. Plant proteins, such as soybean meal, have the potential to replace FM partially and thus reduce the cost of aquatic feed. In this study, soybean meal (SBM) was [...] Read more.
In China, aquaculture costs have increased because of the increase in fish meal (FM) prices. Plant proteins, such as soybean meal, have the potential to replace FM partially and thus reduce the cost of aquatic feed. In this study, soybean meal (SBM) was fermented using compound microorganisms (lactic acid bacteria, yeast, and Bacillus). Fermented soybean meal (FSBM) replaced FM in the diet. The effect of replacing FM on largemouth bass was comprehensively evaluated at three levels: macro (growth performance), microbial (bacterial diversity and metabolic), and gene (key gene expression) levels. The results showed that FSBM increased the crude protein content by 7.45% and decreased the phytic acid concentration by 48.66% compared with original SBM. Compared with the control, the weight gain rate and feed conversion ratio showed no significant difference (p > 0.05) when the replacement amount of FM was less than 30%. Compared with the 50% FSBM treatment, the 10% and 30% treatments showed more steatosis in the liver slices. Although the replacement of FSBM with less than 30% did not have a significant (p < 0.05) negative impact on body weight, FSBM substitution adversely affected the height and width of the intestinal villi. The expression levels of Hepcidin-1, Hepcidin-2, TGF-β1, and IL-10 in 30% and 50% FSBM treatments were lower than those in the control, whereas the expression levels of SOD1 and SOD2 in 30% FSBM treatment were higher than those in the control group, and the expression level of SOD3a in 30% FSBM treatment was equal to the control. In addition, FSBM substitution could affect the composition of intestinal microorganisms and thus influence metabolic pathways, especially the biosynthesis of amino acids and plant secondary metabolites. Full article
(This article belongs to the Special Issue Research on Microbial Protein Synthesis)
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17 pages, 6102 KiB  
Article
Heterologous Biosynthesis of Hyaluronic Acid Using a New Hyaluronic Acid Synthase Derived from the Probiotic Streptococcus thermophilus
by Qian Zhong, Yanqin Ma, Delei Xu, Peng Lei, Sha Li, Hong Xu and Yibin Qiu
Fermentation 2023, 9(6), 510; https://doi.org/10.3390/fermentation9060510 - 25 May 2023
Cited by 1 | Viewed by 1536
Abstract
Hyaluronic acid (HA) is a natural linear polysaccharide extensively used in many fields, including the food, medicine, and cosmetics industries. Currently, species that produce HA synthetase (HAS) from microbial sources are relatively small and mainly pathogenic, such as Streptococcus pyogenes and Pasteurella multicide [...] Read more.
Hyaluronic acid (HA) is a natural linear polysaccharide extensively used in many fields, including the food, medicine, and cosmetics industries. Currently, species that produce HA synthetase (HAS) from microbial sources are relatively small and mainly pathogenic, such as Streptococcus pyogenes and Pasteurella multicide. Moreover, there is limited research on the safe microbial sources of HAS. Thus, we characterized SthasA, a HAS derived from the probiotic Streptococcus thermophilus, and used it for the de novo synthesis of HA in a chassis strain of Bacillus amyloliquefaciens. Metabolic engineering of the precursor supply modules suggested that hasB (encoding UDPG dehydrogenase), which was derived from Corynebacterium glutamicum ATCC 13032, effectively promoted the accumulation of HA products. Furthermore, by combining the expression of the global regulatory factor CcpA, HA yield from the recombinant strain reached 3.20 g/L. Finally, we obtained a yield of 5.57 g/L HA with a molecular weight of 1.7 × 106 Da using various process optimization strategies in a 5 L bioreactor. This study enriches our understanding of obtaining HAS from non-pathogenic bacteria and provides a safe and effective process for producing HA, which has the potential to promote the industrial applications of HA further. Full article
(This article belongs to the Special Issue Research on Microbial Protein Synthesis)
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18 pages, 2792 KiB  
Review
Nattokinase: Insights into Biological Activity, Therapeutic Applications, and the Influence of Microbial Fermentation
by Mudannan Fang, Beichen Yuan, Meng Wang, Junfeng Liu and Zheng Wang
Fermentation 2023, 9(11), 950; https://doi.org/10.3390/fermentation9110950 - 02 Nov 2023
Viewed by 5143
Abstract
Nattokinase, a serine protease that originates from the traditional food natto, has garnered widespread attention due to its pharmacological functions and therapeutic potential. This review aims to delve into the major advancements of nattokinase across various domains, particularly its emerging roles in Alzheimer’s [...] Read more.
Nattokinase, a serine protease that originates from the traditional food natto, has garnered widespread attention due to its pharmacological functions and therapeutic potential. This review aims to delve into the major advancements of nattokinase across various domains, particularly its emerging roles in Alzheimer’s disease prevention and the treatment of retinal diseases, thereby seeking to usher in a newfound hope in the fields of neurology and ophthalmology. However, the production and preservation of nattokinase present a multitude of challenges, including issues of unstable yield and enzyme activity loss. To address these challenges, we explore potential solutions such as the heterologous expression of the nattokinase gene, the optimization of microbial fermentation strategies, and innovative purification methods. Furthermore, we focus on enhancing the stability and protection of nattokinase through encapsulation and immobilization techniques, thus ensuring its sustainability across a wide array of applications. This review provides readers with the opportunity to gain an in-depth understanding of the diverse prospects for nattokinase applications. Future research directions will encompass a deeper exploration of its biological mechanisms, the development of novel nattokinase derivatives, and the extension of its applications into a broader spectrum of disease treatments and health maintenance. Full article
(This article belongs to the Special Issue Research on Microbial Protein Synthesis)
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