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

Deadline for manuscript submissions: 30 June 2025 | Viewed by 7618

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Dr. Libang Zhou

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Guest Editor

College of Food Science & Technology, Nanjing Agricultural University, Nanjing, China
Interests: microbial synthetic biology and metabolic engineering

Special Issue Information

Dear Colleagues,

Metabolic engineering in microbial synthesis represents a vanguard discipline within the biotechnological landscape, fundamentally transforming the way valuable compounds are produced through targeted manipulation of microbial metabolic pathways. This pioneering research endeavors to optimize microbial cell factories, rendering them highly proficient in the biosynthesis of biofuels, pharmaceuticals, chemicals, and a myriad of other economically and environmentally significant products. Pivotal advancements in synthetic biology, systems biology, and computational modeling have bestowed upon researchers unparalleled precision and control in the fine-tuning of microbial metabolism. These interdisciplinary synergies have engendered marked enhancements in product yields, substantial reductions in production costs, and the advent of inherently sustainable bioprocess configurations. Consequently, metabolic engineering in microbial synthesis stands as a cornerstone of modern biotechnology, driving continuous innovation in the quest for efficient, cost-effective, and eco-friendly manufacturing of high-value chemicals and materials from renewable biological resources.

The Special Issue invites submissions of original research papers, comprehensive reviews, and insightful perspectives that delve into a broad range of themes within the metabolic engineering of industrial microorganisms, encompassing, but not restricted to, the following areas:

Genetic Manipulation and Synthetic Biology for strain improvement
Pathway Optimization and Flux Balancing for boosting microbial productivity
Metabolite Sensing and Feedback Control for biosynthetic processes
Microbial Consortia and Community Engineering for enhancing overall productivity
Bioprocess Development and Scale-up for successful translation from the laboratory to industry
Novel Tools and Techniques for robustness, stability, and scalability

We aim to stimulate interdisciplinary cooperation and propel the progression of metabolic engineering in the context of microbial synthesis. We extend a warm invitation to researchers and experts in the field to share their groundbreaking contributions within this Special Issue, thereby catalyzing innovation and facilitating the dissemination of knowledge within the rapidly evolving domain of metabolic engineering.

Dr. Libang Zhou
Guest Editor

Manuscript Submission Information

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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 2100 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

metabolic engineering
synthetic biology
systems biology
pathway engineering
microbial cell factories
fermentation
industrial biotechnology

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Published Papers (7 papers)

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Research

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16 pages, 3144 KiB

Open AccessArticle

Improving Organic Acid Secretion of Aspergillus niger by Overexpression C4-Dicarboxylic Acid Transporters

by Yiyang Tan, Shutong Liu, Sheng Wu, Xiaolu Wang, Depei Wang and Xianli Xue

Fermentation 2025, 11(3), 156; https://doi.org/10.3390/fermentation11030156 - 20 Mar 2025

Viewed by 474

Abstract

C4-dicarboxylic acids are essential organic compounds characterized by a four-carbon structure and two carboxyl groups. Their export from cells is mediated by specialized transporter proteins known as C4-dicarboxylic acid transporters (DCTs). The objective of this study was to investigate the specificity of six DCTs (DCT1-5 and C4t318) from Aspergillus niger or Aspergillus oryzae, focusing on their role in different production strategies for C4-dicarboxylic acids. The results indicate that compared to the WT strain, overexpressing dct2 or dct3 in A. niger CGMCC NO. 40550 specifically enhances the production of succinic acid, increasing its yield from 5.69 g/L to 6.28 g/L, and L-malic acid, increasing its yield from 11.02 g/L to 12.11 g/L. Additionally, dct5 appears to be involved in the transport of both succinic acid (6.19 g/L) and L-malic acid (16.33 g/L). The total acid yields of T-D3-7, which lacks the oxaloacetate hydrolase gene, were improved to 27.75 g/L, compared to 25.19 g/L for T-D3-26, due to blocking the branch of oxaloacetate metabolism. Furthermore, the heterologous expression of A. oryzae C4T318 in A. niger increased the production of L-malic acid by approximately 22.5%. Furthermore, the best results were observed when the strains T-D3-7 and T-D5-16 were scaled up in a 30 L bioreactor for 84 h. The succinic acid and L-malic acid yields of T-D3-7 and T-D5-16 reached 14.51 g/L and 70.79 g/L or 41.59 g/L and 81.08 g/L, respectively. Moreover, the purity of L-malic acid produced by T-D3-7 reached 71%. This work further clarifies the specificity of C4-dicarboxylic acid transporters and provides valuable insights for optimizing organic acid production. Full article

(This article belongs to the Special Issue Metabolic Engineering in Microbial Synthesis)

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20 pages, 6097 KiB

Open AccessArticle

Transcriptome Analysis of Sclerotium rolfsii: Unraveling Impact of Glycolytic Pathway on Substrate Utilization and Microbial Polysaccharide Production

by Jia Song, Junfeng Li, Chenrui Zhen, Juan Du, Rui Zhao, Bingqian Fan, Jiayi Hou, Bingning Gao, Yu Zheng, Linna Tu and Min Wang

Fermentation 2025, 11(3), 143; https://doi.org/10.3390/fermentation11030143 - 13 Mar 2025

Viewed by 588

Abstract

Scleroglucan is the extracellular polysaccharide (EPS) produced by Sclerotium rolfsii (S. rolfsii). The low EPS titer and limited substrate utilization of S. rolfsii present significant challenges in the fermentation process, restricting industrial applications of scleroglucan. In this study, we performed a transcriptomic analysis on the mycelium of S. rolfsii fermented with different carbon sources. The key genes involved in polysaccharide biosynthesis (6-phosphofructokinase 1 (PFK1), pyruvate decarboxylase (PDC), aldehyde dehydrogenase (NAD (P)+) (ALDH3), and acetyl-CoA synthase (ACS)) were identified and their roles in the process were investigated. The supplementation of specific precursors—fructose-6-phosphate, pyruvate, aldehydes, and acetate—was shown to enhance both the polysaccharide titer and substrate utilization. By adding precursors, the titer of SEPS produced in a 5 L fermentation tank reached 48.69 ± 3.8 g/L. Notably, the addition of these precursors increased the titer of EPS fermented with sucrose (SEPS) by 65.63% and substrate utilization by 119.3%, while the titer of EPS fermented with lactose (LEPS) rose by 80.29% and substrate utilization rose by 47.08%. These findings suggest that precursor supplementation can effectively improve polysaccharide production and substrate efficiency, thereby minimizing resource waste and environmental impact. Full article

(This article belongs to the Special Issue Metabolic Engineering in Microbial Synthesis)

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14 pages, 1971 KiB

Open AccessArticle

Metabolic Engineering of Zymomonas mobilis for Xylonic Acid Production from Lignocellulosic Hydrolysate

by Banrui Ruan, Xiongying Yan, Zhaoqing He, Qiaoning He and Shihui Yang

Fermentation 2025, 11(3), 141; https://doi.org/10.3390/fermentation11030141 - 13 Mar 2025

Viewed by 613

Abstract

Bio-based xylonic acid produced from inexpensive lignocellulosic biomass has enormous market potential and enhances the overall economic benefits of biorefinery processes. In this study, the introduction of genes encoding xylose dehydrogenase driven by the promoter Ppdc into Z. mobilis using a plasmid vector resulted in the accumulation of xylonic acid at a titer of 16.8 ± 1.6 g/L. To achieve stable xylonic acid production, a gene cassette for xylonic acid production was integrated into the genome at the chromosomal locus of ZMO0038 and ZMO1650 using the endogenous type I-F CRISPR-Cas system. The titer of the resulting recombinant strain XA3 reduced to 12.2 ± 0.56 g/L, which could be the copy number difference between the plasmid and chromosomal integration. Oxygen content was then identified to be the key factor for xylonic acid production. To further increase xylonic acid production capability, a recombinant strain, XA9, with five copies of a gene cassette for xylonic acid production was constructed by integrating the gene cassette into the genome at the chromosomal locus of ZMO1094, ZMO1547, and ZMO1577 on the basis of XA3. The titer of xylonic acid increased to 51.9 ± 0.1 g/L with a maximum yield of 1.10 g/g, which is close to the theoretical yield in a pure sugar medium. In addition, the recombinant strain XA9 is genetically stable and can produce 16.2 ± 0.14 g/L of xylonic acid with a yield of 1.03 ± 0.01 g/g in the lignocellulosic hydrolysate. Our study thus constructed a recombinant strain, XA9, of Z. mobilis for xylonic acid production from lignocellulosic hydrolysate, demonstrating the capability of Z. mobilis as a biorefinery chassis for economic lignocellulosic biochemical production. Full article

(This article belongs to the Special Issue Metabolic Engineering in Microbial Synthesis)

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13 pages, 1269 KiB

Open AccessArticle

Co-Cultivations of Beauveria bassiana, Metarhizium anisopliae, and Trichoderma harzianum to Produce Bioactive Compounds for Application in Agriculture

by Pauline Flores da Silva, Maicon Sérgio Nascimento dos Santos, Beatriz de Andrade Araújo, Bruno Douglas Kerber, Heloisa Alves Pinto de Oliveira, Jerson Vanderlei Carús Guedes, Marcio Antonio Mazutti, Marcus Vinícius Tres and Giovani Leone Zabot

Fermentation 2025, 11(1), 30; https://doi.org/10.3390/fermentation11010030 - 14 Jan 2025

Cited by 1 | Viewed by 1513

Abstract

Regenerative agriculture aims to improve soil quality and restore soil biodiversity, re-establishing natural systems in agricultural areas. Among some strategies, it is important to reduce the use of chemical pesticides that affect the productive capacity of the soil and cause problems to the environment. Based on this necessity, we present a strategy of producing a single product with bioinsecticidal and biofungicidal effects by submerged co-cultivations and paired cultivations of Beauveria bassiana, Metarhizium anisopliae, and Trichoderma harzianum using different concentrations of glucose, sucrose, hydrolyzed animal protein (HAP), soybean meal hydrolysate plus organic phosphorus (SMH), and hydrolyzed feathers (HF) as renewable nutrients. The single cultivations and double and triple co-cultivations were carried out for 7 days at 28 °C in orbital agitation at 120 rpm. Most of the highest values of conidia were obtained in the treatments at the central point, in which (g L⁻¹) glucose (20), sucrose (10), HAP (7.5), SMH (2.5), and HF (2.5) were used. The fermented broths were applied to the backs of adult bugs (Euschistus heros), which mostly provided 66–88% mortality. Beauveria bassiana + Metarhizium anisopliae showed approximately 70% inhibition against Sclerotinia sclerotiorum and Macrophomina phaseolina. As a way forward, this product demonstrated integrated bioactivities as insecticide and fungicide and can be optimized to substitute chemical pesticides that have negative impacts on the environment. Full article

(This article belongs to the Special Issue Metabolic Engineering in Microbial Synthesis)

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16 pages, 2528 KiB

Open AccessArticle

Continuous Production of Chitin Oligosaccharides Utilizing an Optimized Enzyme Production-Adsorption-Enzymolysis-Product Separation (EAES) System

by Xiuling Zhou, Yang Huang, Yuying Liu, Delong Pan and Yang Zhang

Fermentation 2024, 10(12), 634; https://doi.org/10.3390/fermentation10120634 - 12 Dec 2024

Viewed by 1012

Abstract

Chitin oligosaccharide (CHOS) is a chitin derivative with excellent biological activities. Enzymatic hydrolysis of chitin-rich biomass into CHOS is a hot topic in research on the high-value utilization of chitin resources. The disadvantages of complex preparation and purification processes and the high cost of chitin-degrading enzymes limit large-scale enzymatic production and application of CHOS. In this study, the activity of chitinase increased from 1.8 U/mL to 3.52 U/mL by 94.4% after optimizing the carbon and nitrogen source of Chitiniphilus sp. LZ32 fermentation. An enzyme production-adsorption-enzymolysis-product separation (EAES) system was constructed using fermentation, an adsorption purification module, and a product ultrafiltration module of a chitin-degrading enzyme. CHOS production by continuous enzymatic hydrolysis was performed in an EAES system using housefly larval powder (HLP) as the substrate. After the C. sp. LZ32 fermentation broth was circulated in the adsorption module for 90 min, the adsorption rate of the chitin-degrading enzyme reached more than 90%. The ultrafiltration module effectively separated CHOS at an operating pressure of 2 bar. Four batches of CHOS were produced in the EAES system using repeated batch fermentation. The running time of a single batch decreased from 115 h in the first batch to 48 h, and the CHOS output of each batch gradually increased. The total CHOS output was 61 g, and the production efficiency was 0.24 g/h. The CHOS produced by the EAES system (ECHOS) has high antioxidant activity. In this study, the EAES system was used to simplify the purification and separation steps of CHOS preparation, and the continuous production of CHOS was realized, which has potential application prospects in the field of green CHOS production. Full article

(This article belongs to the Special Issue Metabolic Engineering in Microbial Synthesis)

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16 pages, 2462 KiB

Open AccessArticle

Optogenetic Fine-Tuning of Sus scrofa Basic Fibroblast Growth Factor Expression in Escherichia coli

by Fanqiang Meng, Zhimin Xu, Xia Fan, Zhisheng Wang and Libang Zhou

Fermentation 2024, 10(12), 612; https://doi.org/10.3390/fermentation10120612 - 29 Nov 2024

Viewed by 955

Abstract

Basic fibroblast growth factor (bFGF) is a crucial protein with diverse applications in biotechnology and medicine. This study aims to investigate the use of EL222-based optogenetic control systems to fine-tune the expression of porcine (Sus scrofa) bFGF in Escherichia coli. The bioactivity and the productivity of blue light-induced bFGF were demonstrated to be comparable to those achieved using a conventional T7-expression system. Secondly, through systematic optimization of regulatory elements, optimal expression of bFGF was achieved using a medium-strength promoter for EL222 expression, a strong RBS upstream of the bFGF gene, and an optimized C120 configuration within the blue light-inducible promoter. Moreover, various parameters of blue light illumination during fermentation were investigated, including initial cell density, light intensity, illumination duration, and pulsed illumination patterns. The results identified optimal conditions for maximizing bFGF yield in E. coli, specifically an initial OD₆₀₀ of 0.6, 800 lux blue light intensity, and 8 h total illumination in a 2 h on/off pattern. Overall, this successful implementation of optogenetically controlled bFGF expression in E. coli serves as a proof-of-concept for light-responsive systems in industrial biotechnology, highlighting the potential of optogenetic control for biologically active protein production. Full article

(This article belongs to the Special Issue Metabolic Engineering in Microbial Synthesis)

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Review

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26 pages, 3878 KiB

Open AccessReview

Clavulanic Acid Overproduction: A Review of Environmental Conditions, Metabolic Fluxes, and Strain Engineering in Streptomyces clavuligerus

by David Gómez-Ríos, Luisa María Gómez-Gaona and Howard Ramírez-Malule

Fermentation 2024, 10(10), 526; https://doi.org/10.3390/fermentation10100526 - 16 Oct 2024

Viewed by 1806

Abstract

Clavulanic acid is a potent β-lactamase inhibitor produced by Streptomyces clavuligerus, widely used in combination with β-lactam antibiotics to combat antimicrobial resistance. This systematic review analyzes the most successful methodologies for clavulanic acid overproduction, focusing on the highest yields reported in bench-scale and bioreactor-scale fermentations. Studies have demonstrated that glycerol is the preferred carbon source for clavulanic acid production over other sources like starch and dextrins. The optimization of feeding strategies, especially in fed-batch operations, has improved glycerol utilization and extended the clavulanic acid production phase. Organic nitrogen sources, particularly soybean protein isolates and amino acid supplements such as L-arginine, L-threonine, and L-glutamate, have been proven effective at increasing CA yields both in batch and fed-batch cultures, especially when balanced with appropriate carbon sources. Strain engineering approaches, including mutagenesis and targeted genetic modifications, have allowed for the obtainment of overproducer S. clavuligerus strains. Specifically, engineering efforts that overexpress key regulatory genes such as ccaR and claR, or that disrupt competing pathways, redirect the metabolic flux towards CA biosynthesis, leading to high clavulanic acid titers. The fed-batch operation at the bioreactor scale emerges as the most feasible alternative for prolonged clavulanic acid production with both wild-type and mutant strains, allowing for the attainment of high titers during cultivations. Full article

(This article belongs to the Special Issue Metabolic Engineering in Microbial Synthesis)

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