Fermentation

Research

Jump to: Review

13 pages, 3685 KiB

Open AccessArticle

Improved Fermentation Yield of Doramectin from Streptomyces avermitilis N72 by Strain Selection and Glucose Supplementation Strategies

by Xiaojun Pan and Jun Cai

Fermentation 2023, 9(2), 121; https://doi.org/10.3390/fermentation9020121 - 26 Jan 2023

Cited by 3 | Viewed by 1779

Abstract

Doramectin is a macrolide antiparasitic that is widely used in the treatment of mammalian parasitic diseases. Doramectin is usually produced by Streptomyces avermitilis fermentation using cyclohexanecarboxylic acid (CHC) as a precursor; however, the growth of S. avermitilis is usually inhibited by CHC, resulting [...] Read more.

Doramectin is a macrolide antiparasitic that is widely used in the treatment of mammalian parasitic diseases. Doramectin is usually produced by Streptomyces avermitilis fermentation using cyclohexanecarboxylic acid (CHC) as a precursor; however, the growth of S. avermitilis is usually inhibited by CHC, resulting in a low fermentation yield of doramectin. In this study, a high-yielding strain XY-62 was obtained using the S. avermitilis mutant strain S. avermitilis N72 as the starting strain, then combined with a CHC tolerance screening strategy using ultraviolet and nitrosoguanidine mutagenesis, and a 96 microtiter plate solid-state fermentation primary sieving and shake flask fermentation rescreening method. Compared with S. avermitilis N72, the doramectin fermentation yield increased by more than 1.3 times, and it was more adaptable to temperature, pH, and CHC concentration of the culture; additionally, the viability of the mycelial growth was enhanced. In addition, further studies on the high-yielding strain XY-62 revealed that the accumulation of doramectin could be further increased by glucose supplementation during the fermentation process, and the yield of doramectin reached 1068 μg/mL by scaling up the culture in 50 L fermenters; this has the potential for industrial production. Therefore, mutagenesis combined with CHC tolerance screening is an effective way to enhance the fermentation production of doramectin by S. avermitilis. Our strategy and findings can help to improve the production of doramectin in industrial strains of S. avermitilis. Full article

(This article belongs to the Special Issue Applied Microorganisms and Industrial/Food Enzymes)

► Show Figures

Figure 1

12 pages, 3167 KiB

Open AccessArticle

Insight into the Substrate Specificity of Lactobacillus paracasei Aspartate Ammonia-Lyase

by Yi-Hao Huang, Weir-Chiang You, Yung-Ju Chen, Jhih-Ying Ciou and Lu-Sheng Hsieh

Fermentation 2023, 9(1), 49; https://doi.org/10.3390/fermentation9010049 - 06 Jan 2023

Cited by 5 | Viewed by 1671

Abstract

Aspartate ammonia-lyase (AAL) catalyzes the reversible conversion reactions of aspartate to fumaric acid and ammonia. In this work, Lactobacillus paracasei LpAAL gene was heterologously expressed in Escherichia coli. As well as a recombinant His-tagged LpAAL protein, a maltose-binding protein (MBP) fused LpAAL protein [...] Read more.

Aspartate ammonia-lyase (AAL) catalyzes the reversible conversion reactions of aspartate to fumaric acid and ammonia. In this work, Lactobacillus paracasei LpAAL gene was heterologously expressed in Escherichia coli. As well as a recombinant His-tagged LpAAL protein, a maltose-binding protein (MBP) fused LpAAL protein was used to enhance its protein solubility and expression level. Both recombinant proteins showed broad substrate specificity, catalyzing aspartic acid, fumaric acid, phenylalanine, and tyrosine to produce fumaric acid, aspartic acid, trans-cinnamic acid, and p-coumaric acid, respectively. The optimum reaction pH and temperature of LpAAL protein for four substrates were measured at 8.0 and 40 °C, respectively. The K_m values of LpAAL protein for aspartic acid, fumaric acid, phenylalanine, and tyrosine as substrates were 5.7, 8.5, 4.4, and 1.2 mM, respectively. The k_cat values of LpAAL protein for aspartic acid, fumaric acid, phenylalanine, and tyrosine as substrates were 6.7, 0.45, 4.96, and 0.02 s⁻¹, respectively. Therefore, aspartic acid, fumaric acid, phenylalanine, and tyrosine are bona fide substrates for LpAAL enzyme. Full article

(This article belongs to the Special Issue Applied Microorganisms and Industrial/Food Enzymes)

► Show Figures

Figure 1

14 pages, 2694 KiB

Open AccessArticle

Galactitol Transport Factor GatA Relieves ATP Supply Restriction to Enhance Acid Tolerance of Escherichia coli in the Two-Stage Fermentation Production of D-Lactate

by Jinhua Yang, Zheng Peng, Xiaomei Ji, Juan Zhang and Guocheng Du

Fermentation 2022, 8(12), 665; https://doi.org/10.3390/fermentation8120665 - 23 Nov 2022

Viewed by 1360

Abstract

Escherichia coli is a major contributor to the industrial production of organic acids, but its production capacity and cost are limited by its acid sensitivity. Enhancing acid resistance in E. coli is essential for improving cell performance and production value. Here, we propose [...] Read more.

Escherichia coli is a major contributor to the industrial production of organic acids, but its production capacity and cost are limited by its acid sensitivity. Enhancing acid resistance in E. coli is essential for improving cell performance and production value. Here, we propose a feasible strategy for improving cellular acid tolerance by reducing ATP supply restriction. Transcriptome assays of acid-tolerant evolved strains revealed that the galactitol phosphotransferase system transporter protein GatA is an acid-tolerance factor that assists E. coli in improving its resistance to a variety of organic acids. Enhanced GatA expression increased cell survival under conditions of lethal stress due to D-lactic acid, itaconic acid and succinic acid by 101.8-fold, 29.4-fold and 41.6-fold, respectively. In addition, fermentation patterns for aerobic growth and oxygen-limited production of D-lactic acid were identified, and suitable transition and induction stages were evaluated. GatA effectively compensated for the lack of cellular energy during oxygen limitation and enabled the D-lactic acid producing strain to exhibit more sustainable productivity in acidic fermentation environments with a 55.7% increase in D-lactic acid titer from 9.5 g·L⁻¹ to 14.8 g·L⁻¹ and reduced generation of by-product. Thus, this study developed a method to improve the acid resistance of E. coli cells by compensating for the energy gap without affecting normal cell metabolism while reducing the cost of organic acid production. Full article

(This article belongs to the Special Issue Applied Microorganisms and Industrial/Food Enzymes)

► Show Figures

Figure 1

13 pages, 2050 KiB

Open AccessArticle

Changes of Physicochemical Properties in Black Garlic during Fermentation

by Xinyu Yuan, Zhuochen Wang, Lanhua Liu, Dongdong Mu, Junfeng Wu, Xingjiang Li and Xuefeng Wu

Fermentation 2022, 8(11), 653; https://doi.org/10.3390/fermentation8110653 - 20 Nov 2022

Cited by 1 | Viewed by 2053

Abstract

To investigate the changes of the main ingredients in black garlic (BG) during fermentation, the contents of moisture, total acids and reducing sugars were determined. Allicin, 5-Hydroxymethylfurfural (5-HMF), and total phenols were also determined as bioactive substances. DPPH scavenging capacity was determined to [...] Read more.

To investigate the changes of the main ingredients in black garlic (BG) during fermentation, the contents of moisture, total acids and reducing sugars were determined. Allicin, 5-Hydroxymethylfurfural (5-HMF), and total phenols were also determined as bioactive substances. DPPH scavenging capacity was determined to indicate the antioxidant activity of BG. The changes in hardness and color were detected as well. The results showed that the moisture content decreased from 66.13% to 25.8% during the fermentation. The content of total acids, total phenols, and reducing sugars increased from 0.03 g/g to 0.29 g/g, from 0.045 μg/g to 0.117 μg/g, and from 0.016 g/g to 0.406 g/g, respectively. The content of 5-HMF increased from 0 to 4.12 μg/mL continuously, while the content of allicin increased from 0.09 mmol/100 g to 0.30 mmol/100 g and then decayed to 0.00 mmol/100 g. The L*, a*, and b* values of BG were 23.65 ± 0.44, 0.64 ± 0.06, and 0.85 ± 0.05, respectively. There was a higher intensity of dark color in BG than that in fresh garlic. The hardness values decreased first and then increased in later fermentation from 465.47 g to 27,292.38 g. Principal component analysis (PCA) showed that the samples were divided into three clusters, including cluster1 (fresh garlic, S0), cluster2 (S1), and cluster3 (S3−S9). This research effectively clarified the various stage of the BG fermentation process, and it is expected to supply references for reducing production time in industrial BG fermentation. Full article

(This article belongs to the Special Issue Applied Microorganisms and Industrial/Food Enzymes)

► Show Figures

Figure 1

15 pages, 2736 KiB

Open AccessArticle

Biocontrol of Geosmin Production by Inoculation of Native Microbiota during the Daqu-Making Process

by Hai Du, Junlin Wei, Xitong Zhang and Yan Xu

Fermentation 2022, 8(11), 588; https://doi.org/10.3390/fermentation8110588 - 30 Oct 2022

Cited by 1 | Viewed by 1394

Abstract

Geosmin produced by Streptomyces can cause an earthy off-flavor at trace levels, seriously deteriorating the quality of Chinese liquor. Geosmin was detected during the Daqu (Chinese liquor fermentation starter)-making process, which is a multi-species fermentation process in an open system. Here, biocontrol, using [...] Read more.

Geosmin produced by Streptomyces can cause an earthy off-flavor at trace levels, seriously deteriorating the quality of Chinese liquor. Geosmin was detected during the Daqu (Chinese liquor fermentation starter)-making process, which is a multi-species fermentation process in an open system. Here, biocontrol, using the native microbiota present in Daqu making, was used to control the geosmin contamination. Six native strains were obtained according to their inhibitory effects on Streptomyces and then were inoculated into the Daqu fermentation. After inoculation, the content of geosmin decreased by 34.40% (from 7.18 ± 0.13 μg/kg to 4.71 ± 0.30 μg/kg) in the early stage and by 55.20% (from 8.86 ± 1.54 μg/kg to 3.97 ± 0.78 μg/kg) in the late stage. High-throughput sequencing combined with an interaction network revealed that the fungal community played an important role in the early stage and the correlation between Pichia and Streptomyces changed from the original indirect promotion to direct inhibition after inoculation. This study provides an effective strategy for controlling geosmin contamination in Daqu via precisely regulating microbial communities, as well as highlights the potential of biocontrol for controlling off-flavor chemicals at trace levels in complex fermentation systems. Full article

(This article belongs to the Special Issue Applied Microorganisms and Industrial/Food Enzymes)

► Show Figures

Figure 1

12 pages, 1834 KiB

Open AccessArticle

Comparison of Trichoderma longibrachiatum Xyloglucanase Production Using Tamarind (Tamarindus indica) and Jatoba (Hymenaea courbaril) Seeds: Factorial Design and Immobilization on Ionic Supports

by Alex Graça Contato, Ana Claudia Vici, Vanessa Elisa Pinheiro, Tássio Brito de Oliveira, Emanuelle Neiverth de Freitas, Guilherme Mauro Aranha, Almir Luiz Aparecido Valvassora Junior, Carem Gledes Vargas Rechia, Marcos Silveira Buckeridge and Maria de Lourdes Teixeira de Moraes Polizeli

Fermentation 2022, 8(10), 510; https://doi.org/10.3390/fermentation8100510 - 02 Oct 2022

Cited by 2 | Viewed by 1716

Abstract

Xyloglucan (XG) is the predominant hemicellulose in the primary cell wall of superior plants. It has a fundamental role in controlling the stretching and expansion of the plant cell wall. There are five types of enzymes known to cleave the linear chain of [...] Read more.

Xyloglucan (XG) is the predominant hemicellulose in the primary cell wall of superior plants. It has a fundamental role in controlling the stretching and expansion of the plant cell wall. There are five types of enzymes known to cleave the linear chain of xyloglucan, and the most well-known is xyloglucanase (XEG). The immobilization process can be used to solve problems related to stability, besides the economic benefits brought by the possibility of its repeated use and recovery. Therefore, this study aims at the optimization of the xyloglucanase production of Trichoderma longibrachiatum using a central composite rotatable design (CCRD) with tamarind and jatoba seeds as carbon sources, as well as XEG immobilization on ionic supports, such as MANAE (monoamine-N-aminoethyl), DEAE (diethylaminoethyl)-cellulose, CM (carboxymethyl)-cellulose, and PEI (polyethyleneimine). High concentrations of carbon sources (1.705%), at a temperature of 30 °C and under agitation for 72 h, were the most favorable conditions for the XEG activity from T. longibrachiatum with respect to both carbon sources. However, the tamarind seeds showed 23.5% higher activity compared to the jatoba seeds. Therefore, this carbon source was chosen to continue the experiments. The scaling up from Erlenmeyer flasks to the bioreactor increased the XEG activity 1.27-fold (1.040 ± 0.088 U/mL). Regarding the biochemical characterization of the crude extract, the optimal temperature range was 50–55 °C, and the optimal pH was 5.0. Regarding the stabilities with respect to pH and temperature, XEG was not stable for prolonged periods, which was crucial to immobilizing it on ionic resins. XEG showed the best immobilization efficiency on CM-cellulose and DEAE-cellulose, with activities of 1.16 and 0.89 U/g of the derivative (enzyme plus support), respectively. This study describes, for the first time in the literature, the immobilization of a fungal xyloglucanase using these supports. Full article

(This article belongs to the Special Issue Applied Microorganisms and Industrial/Food Enzymes)

► Show Figures

Figure 1

13 pages, 3763 KiB

Open AccessArticle

Heterologous Expression of Thermotolerant α-Glucosidase in Bacillus subtilis 168 and Improving Its Thermal Stability by Constructing Cyclized Proteins

by Zhi Wang, Mengkai Hu, Ming Fang, Qiang Wang, Ruiqi Lu, Hengwei Zhang, Meijuan Xu, Xian Zhang and Zhiming Rao

Fermentation 2022, 8(10), 498; https://doi.org/10.3390/fermentation8100498 - 29 Sep 2022

Viewed by 1564

Abstract

α-glucosidase is an essential enzyme for the production of isomaltooligosaccharides (IMOs). Allowing α-glucosidase to operate at higher temperatures (above 60 °C) has many advantages, including reducing the viscosity of the reaction solution, enhancing the catalytic reaction rate, and achieving continuous production of IMOs. [...] Read more.

α-glucosidase is an essential enzyme for the production of isomaltooligosaccharides (IMOs). Allowing α-glucosidase to operate at higher temperatures (above 60 °C) has many advantages, including reducing the viscosity of the reaction solution, enhancing the catalytic reaction rate, and achieving continuous production of IMOs. In the present study, the thermal stability of α-glucosidase was significantly improved by constructing cyclized proteins. We screened a thermotolerant α-glucosidase (AGL) with high transglycosylation activity from Thermoanaerobacter ethanolicus JW200 and heterologously expressed it in Bacillus subtilis 168. After forming the cyclized α-glucosidase by different isopeptide bonds (SpyTag/SpyCatcher, SnoopTag/SnoopCatcher, SdyTag/SdyCatcher, RIAD/RIDD), we determined the enzymatic properties of cyclized AGL. The optimal temperature of all cyclized AGL was increased by 5 °C, and their thermal stability was generally improved, with SpyTag-AGL-SpyCatcher having a 1.74-fold increase compared to the wild-type. The results of molecular dynamics simulations showed that the RMSF values of cyclized AGL decreased, indicating that the rigidity of the cyclized protein increased. This study provides an efficient method for improving the thermal stability of α-glucosidase. Full article

(This article belongs to the Special Issue Applied Microorganisms and Industrial/Food Enzymes)

► Show Figures

Figure 1

15 pages, 4645 KiB

Open AccessArticle

Optimized Recombinant Expression and Characterization of Collagenase in Bacillus subtilis WB600

by Yaqing Zhu, Linlin Wang, Kaixuan Zheng, Ping Liu, Wenkang Li, Jian Lin, Wenjing Liu, Shoushui Shan, Liqin Sun and Hailing Zhang

Fermentation 2022, 8(9), 449; https://doi.org/10.3390/fermentation8090449 - 09 Sep 2022

Cited by 1 | Viewed by 2575

Abstract

Background: The collagenase encoding gene col was cloned into a pP43NMK vector and amplified in Escherichia coli JM109 cells. The shuttle vector pP43NMK was used to sub-clone the col gene to obtain the vector pP43NMK-col for the expression of collagenase in Bacillus [...] Read more.

Background: The collagenase encoding gene col was cloned into a pP43NMK vector and amplified in Escherichia coli JM109 cells. The shuttle vector pP43NMK was used to sub-clone the col gene to obtain the vector pP43NMK-col for the expression of collagenase in Bacillus subtilis WB600. The enzyme was characterized and the composition of the expression medium and culture conditions were optimized. Methods: The expressed recombinant enzyme was purified by ammonium sulfate, ultrafiltration, and through a nickel column. The purified collagenase had an activity of 9405.54 U/mg. Results: The recombinant enzyme exhibited optimal activity at pH 9.0 and 50 °C. Catalytic efficiency of the recombinant collagenase was inhibited by Fe³⁺ and Cu²⁺, but stimulated by Co²⁺, Ca²⁺, Zn²⁺, and Mg²⁺. The optimal conditions for its growth were at pH 7.0 and 35 °C, using 15 g/L of fructose and 36 g/L of yeast powder and peptone mixture (2:1) at 260 rpm with 11% inoculation. The maximal extracellular activity of the recombinant collagenase reached 2746.7 U/mL after optimization of culture conditions, which was 2.4-fold higher than that before optimization. Conclusions: This study is a first attempt to recombinantly express collagenase in B. subtilis WB600 and optimize its expression conditions, its production conditions, and possible scale-up. Full article

(This article belongs to the Special Issue Applied Microorganisms and Industrial/Food Enzymes)

► Show Figures

Figure 1

14 pages, 2649 KiB

Open AccessArticle

A Novel Salt-Tolerant L-Glutaminase: Efficient Functional Expression, Computer-Aided Design, and Application

by Hengwei Zhang, Mengkai Hu, Qing Wang, Fei Liu, Meijuan Xu, Xian Zhang and Zhiming Rao

Fermentation 2022, 8(9), 444; https://doi.org/10.3390/fermentation8090444 - 06 Sep 2022

Cited by 6 | Viewed by 1858

Abstract

The low productivity in long fermentation duration and high-salt working conditions limit the application of L-glutaminase in soy sauce brewing. In this study, a novel L-glutaminase (LreuglsA) with eminent salt tolerance was mined and achieved more than 70% activity with 30% NaCl. To [...] Read more.

The low productivity in long fermentation duration and high-salt working conditions limit the application of L-glutaminase in soy sauce brewing. In this study, a novel L-glutaminase (LreuglsA) with eminent salt tolerance was mined and achieved more than 70% activity with 30% NaCl. To improve the robustness of the enzyme at different fermentation strategies, mutation LreuglsA^H105K was built by a computer-aided design, and the recombinant protein expression level, an essential parameter in industrial applications, was increased 5.61-fold with the synthetic biology strategy by improving the mRNA stability. Finally, the LreuglsA^H105K functional expression box was contributed to Bacillus subtilis 168 by auxotrophic complementation, and the production in a 5-L bioreactor was improved to 2516.78 ± 20.83 U mL⁻¹, the highest production ever reported. When the immobilized cells were applied to high-salt dilute-state soy sauce brewing, the L-glutamate level was increased by 45.9%. This work provides insight into the salt-tolerant enzyme for improving the efficiency of industrial applications. Full article

(This article belongs to the Special Issue Applied Microorganisms and Industrial/Food Enzymes)

► Show Figures

Figure 1

10 pages, 1787 KiB

Open AccessArticle

Design of 5′-UTR to Enhance Keratinase Activity in Bacillus subtilis

by Jun Fang, Guanyu Zhou, Xiaomei Ji, Guoqiang Zhang, Zheng Peng and Juan Zhang

Fermentation 2022, 8(9), 426; https://doi.org/10.3390/fermentation8090426 - 27 Aug 2022

Cited by 5 | Viewed by 1824

Abstract

Keratinase is an important industrial enzyme, but its application performance is limited by its low activity. A rational design of 5′-UTRs that increases translation efficiency is an important approach to enhance protein expression. Herein, we optimized the 5′-UTR of the recombinant keratinase KerZ1 [...] Read more.

Keratinase is an important industrial enzyme, but its application performance is limited by its low activity. A rational design of 5′-UTRs that increases translation efficiency is an important approach to enhance protein expression. Herein, we optimized the 5′-UTR of the recombinant keratinase KerZ1 expression element to enhance its secretory activity in Bacillus subtilis WB600 through Spacer design, RBS screening, and sequence simplification. First, the A/U content in Spacer was increased by the site-directed saturation mutation of G/C bases, and the activity of keratinase secreted by mutant strain B. subtilis WB600-SP was 7.94 times higher than that of KerZ1. Subsequently, the keratinase activity secreted by the mutant strain B. subtilis WB600-SP-R was further increased to 13.45 times that of KerZ1 based on the prediction of RBS translation efficiency and the multi-site saturation mutation screening. Finally, the keratinase activity secreted by the mutant strain B. subtilis WB600-SP-R-D reached 204.44 KU mL⁻¹ by reducing the length of the 5′ end of the 5′-UTR, which was 19.70 times that of KerZ1. In a 5 L fermenter, the keratinase activity secreted by B. subtilis WB600-SP-R-D after 25 h fermentation was 797.05 KU mL⁻¹, which indicated its high production intensity. Overall, the strategy of this study and the obtained keratinase mutants will provide a good reference for the expression regulation of keratinase and other industrial enzymes. Full article

(This article belongs to the Special Issue Applied Microorganisms and Industrial/Food Enzymes)

► Show Figures

Figure 1

21 pages, 3495 KiB

Open AccessArticle

A High-Throughput Absolute Abundance Quantification Method for the Characterisation of Daqu Core Fungal Communities

by Hai Du, Jia Sun, Tianci Zhou and Yan Xu

Fermentation 2022, 8(8), 345; https://doi.org/10.3390/fermentation8080345 - 22 Jul 2022

Cited by 2 | Viewed by 1571

Abstract

An inherent issue in high-throughput sequencing applications is that they provide compositional data for relative abundance. This often obscures the true biomass and potential functions of fungi in the community. Therefore, we presented a high-throughput absolute quantification (HAQ) method to quantitatively estimate the [...] Read more.

An inherent issue in high-throughput sequencing applications is that they provide compositional data for relative abundance. This often obscures the true biomass and potential functions of fungi in the community. Therefore, we presented a high-throughput absolute quantification (HAQ) method to quantitatively estimate the fungal abundance in Daqu. In this study, five internal standard plasmids (ISPs) were designed for the fungal ITS2 subregion with high length variations. Five ISPs were then utilised to establish standard curves with a quantitative concentration range of 10³–10⁷ cells/g, and this was used to quantify the core fungi, including Basidiomycota, Ascomycota, and Mucoromycota. Using three types of mature Daqu from different regions, we demonstrated that the HAQ method yielded community profiles substantially different from those derived using relative abundances. Then, the HAQ method was applied to the Daqu during fermentation. The initial formation of the Daqu surface occurred in the fourth stage, which was mainly driven by moisture. The key fungi that caused the initial formation of the Daqu surface included Hyphopichia burtonii, Saccharomycopsis fibuligera, and Pichia kudriavzevii. The initial formation of the Daqu core occurred in the fifth stage, which was mainly affected by moisture and reducing the sugar content. The key fungi that cause the initial formation of the Daqu core included S. fibuligera and Paecilomyces verrucosus. We conclude that the HAQ method, when applied to ITS2 gene fungal community profiling, is quantitative and that its use will greatly improve our understanding of the fungal ecosystem in Daqu. Full article

(This article belongs to the Special Issue Applied Microorganisms and Industrial/Food Enzymes)

► Show Figures

Figure 1

13 pages, 2965 KiB

Open AccessArticle

Rational Metabolic Engineering Combined with Biosensor-Mediated Adaptive Laboratory Evolution for l-Cysteine Overproduction from Glycerol in Escherichia coli

by Xiaomei Zhang, Zhenhang Sun, Jinyu Bian, Yujie Gao, Dong Zhang, Guoqiang Xu, Xiaojuan Zhang, Hui Li, Jinsong Shi and Zhenghong Xu

Fermentation 2022, 8(7), 299; https://doi.org/10.3390/fermentation8070299 - 25 Jun 2022

Cited by 4 | Viewed by 2256

Abstract

l-Cysteine is an important sulfur-containing amino acid with numerous applications in the pharmaceutical and cosmetic industries. The microbial production of l-cysteine has received substantial attention, and the supply of the precursor l-serine is important in l-cysteine biosynthesis. In this [...] Read more.

l-Cysteine is an important sulfur-containing amino acid with numerous applications in the pharmaceutical and cosmetic industries. The microbial production of l-cysteine has received substantial attention, and the supply of the precursor l-serine is important in l-cysteine biosynthesis. In this study, to achieve l-cysteine overproduction, we first increased l-serine production by deleting genes involved in the pathway of l-serine degradation to glycine (serine hydroxymethyl transferase, SHMT, encoded by glyA genes) in strain 4W (with l-serine titer of 1.1 g/L), thus resulting in strain 4WG with l-serine titer of 2.01 g/L. Second, the serine-biosensor based on the transcriptional regulator NCgl0581 of C. glutamicum was constructed in E. coli, and the validity and sensitivity of the biosensor were demonstrated in E. coli. Then 4WG was further evolved through adaptive laboratory evolution (ALE) combined with serine-biosensor, thus yielding the strain 4WGX with 4.13 g/L l-serine production. Moreover, the whole genome of the evolved strain 4WGX was sequenced, and ten non-synonymous mutations were found in the genome of strain 4WGX compared with strain 4W. Finally, 4WGX was used as the starting strain, and deletion of the l-cysteine desulfhydrases (encoded by tnaA), overexpression of serine acetyltransferase (encoded by cysE) and the key enzyme of transport pathway (encoded by ydeD) were performed in strain 4WGX. The recombinant strain 4WGX-∆tnaA-cysE-ydeD can produce 313.4 mg/L of l-cysteine using glycerol as the carbon source. This work provides an efficient method for the biosynthesis of value-added commodity products associated with glycerol conversion. Full article

(This article belongs to the Special Issue Applied Microorganisms and Industrial/Food Enzymes)

► Show Figures

Figure 1

20 pages, 8663 KiB

Open AccessArticle

Application of Ultrafiltration and Ion Exchange Separation Technology for Lysozyme Separation and Extraction

by Shanshan Chen, Yaqing Tan, Yaqing Zhu, Liqin Sun, Jian Lin and Hailing Zhang

Fermentation 2022, 8(7), 297; https://doi.org/10.3390/fermentation8070297 - 24 Jun 2022

Cited by 1 | Viewed by 2057

Abstract

In this study, the fermentation broth of the recombinant Pichia pastoris strain ncy-2 was studied. After pretreatment, separation, and purification, lysozyme was optimized using biofilm and ion exchange separation. Finally, lysozyme dry enzyme powder was prepared by concentrating and vacuum drying. The removal [...] Read more.

In this study, the fermentation broth of the recombinant Pichia pastoris strain ncy-2 was studied. After pretreatment, separation, and purification, lysozyme was optimized using biofilm and ion exchange separation. Finally, lysozyme dry enzyme powder was prepared by concentrating and vacuum drying. The removal rate of bacterial cells was 99.99% when the fermentation broth was centrifuged at low temperature. The optimum conditions were: transmembrane pressure of 0.20 MPa, pH 6.5, 96.6% yield of lysozyme, enzyme activity of 2612.1 u/mg, which was 1.78 times higher than that of the original enzyme; D152 resin was used for adsorption and elution. Process conditions were optimized: the volume ratio of resin to liquid was 15%; the adsorption time was 4 h; the concentration of NaCl was 1.0 mol/L; the recovery rate of lysozyme activity was 95.67%; the enzyme activity was 3879.6 u/mL; and the purification multiple was 0.5, 3.1 times of the original enzyme activity. The enzyme activity of lysozyme dry enzyme powder was 12,573.6 u/mg, which had an inhibitory effect on microsphere lysozyme. Its enzymatic properties were almost the same as those of natural lysozyme, which demonstrated good application prospects and production potential. Full article

(This article belongs to the Special Issue Applied Microorganisms and Industrial/Food Enzymes)

► Show Figures

Figure 1

15 pages, 2034 KiB

Open AccessArticle

Breeding of a High-Nisin-Yielding Bacterial Strain and Multiomics Analysis

by Leshan Han, Xiaomeng Liu, Chongchuan Wang, Jianhang Liu, Qinglong Wang, Shuo Peng, Xidong Ren, Deqiang Zhu and Xinli Liu

Fermentation 2022, 8(6), 255; https://doi.org/10.3390/fermentation8060255 - 27 May 2022

Viewed by 2414

Abstract

Nisin is a green, safe and natural food preservative. With the expansion of nisin application, the demand for nisin has gradually increased, which equates to increased requirements for nisin production. In this study, Lactococcus lactis subsp. lactis lxl was used as the original [...] Read more.

Nisin is a green, safe and natural food preservative. With the expansion of nisin application, the demand for nisin has gradually increased, which equates to increased requirements for nisin production. In this study, Lactococcus lactis subsp. lactis lxl was used as the original strain, and the compound mutation method was applied to induce mutations. A high-yielding and genetically stable strain (Lactobacillus lactis A32) was identified, with the nisin titre raised by 332.2% up to 5089.29 IU/mL. Genome and transcriptome sequencing was used to analyse A32 and compare it with the original lxl strain. The comparative genomics results show that 107 genes in the A32 genome had mutations and most base mutations were not located in the four well-researched nisin-related operons, nisABTCIPRK, nisI, nisRK and nisFEG: 39 single-nucleotide polymorphisms (SNPs), 34 insertion mutations and 34 deletion mutations. The transcription results show that the expression of 92 genes changed significantly, with 27 of these differentially expressed genes upregulated, while 65 were downregulated. Our findings suggest that the output of nisin increased in L. lactis strain A32, which was accompanied by changes in the DNA replication-related gene dnaG, the ABC-ATPase transport-related genes patM and tcyC, the cysteine thiometabolism-related gene cysS, and the purine metabolism-related gene purL. Our study provides new insights into the traditional genetic mechanisms involved nisin production in L. lactis, which could provide clues for a more efficient metabolic engineering process. Full article

(This article belongs to the Special Issue Applied Microorganisms and Industrial/Food Enzymes)

► Show Figures

Figure 1

11 pages, 964 KiB

Open AccessArticle

Dynamic Regulation of Transporter Expression to Increase L-Threonine Production Using L-Threonine Biosensors

by Sumeng Wang, Ruxin Hao, Xin Jin, Xiaomeng Li, Qingsheng Qi and Quanfeng Liang

Fermentation 2022, 8(6), 250; https://doi.org/10.3390/fermentation8060250 - 26 May 2022

Cited by 3 | Viewed by 2143

Abstract

The cytotoxicity of overexpressed transporters limits their application in biochemical production. To overcome this problem, we developed a feedback circuit for L-threonine production that uses a biosensor to regulate transporter expression. First, we used IPTG-induced rhtA regulation, L-threonine exporter, to simulate dynamic regulation [...] Read more.

The cytotoxicity of overexpressed transporters limits their application in biochemical production. To overcome this problem, we developed a feedback circuit for L-threonine production that uses a biosensor to regulate transporter expression. First, we used IPTG-induced rhtA regulation, L-threonine exporter, to simulate dynamic regulation for improving L-threonine production, and the results show that it had significant advantages compared with the constitutive overexpression of rhtA. To further construct a feedback circuit for rhtA auto-regulation, three L-threonine sensing promoters, P_cysJ, P_cysD, and P_cysJH, were characterized with gradually decreasing strength. The dynamic expression of rhtA with a threonine-activated promoter considerably increased L-threonine production (21.19 g/L) beyond that attainable by the constitutive expression of rhtA (8.55 g/L). Finally, the autoregulation method was used in regulating rhtB and rhtC to improve L-threonine production and achieve a high titer of 26.78 g/L (a 161.01% increase), a yield of 0.627 g/g glucose, and a productivity of 0.743 g/L/h in shake-flask fermentation. This study analyzed in detail the influence of dynamic regulation and the constitutive expression of transporters on L-threonine production. For the first time, we confirmed that dynamically regulating transporter levels can efficiently promote L-threonine production by using the end-product biosensor. Full article

(This article belongs to the Special Issue Applied Microorganisms and Industrial/Food Enzymes)

► Show Figures

Figure 1

15 pages, 3714 KiB

Open AccessArticle

Regulation of β-Disaccharide Accumulation by β-Glucosidase Inhibitors to Enhance Cellulase Production in Trichoderma reesei

by Tingting Long, Peng Zhang, Jingze Yu, Yushan Gao, Xiaoqin Ran and Yonghao Li

Fermentation 2022, 8(5), 232; https://doi.org/10.3390/fermentation8050232 - 17 May 2022

Cited by 6 | Viewed by 2473

Abstract

Trichoderma reesei is a high-yield producer of cellulase for applications in lignocellulosic biomass conversion, but its cellulase production requires induction. A mixture of glucose and β-disaccharide has been demonstrated to achieve high-level cellulase production. However, as inducers, β-disaccharides are prone to be hydrolyzed [...] Read more.

Trichoderma reesei is a high-yield producer of cellulase for applications in lignocellulosic biomass conversion, but its cellulase production requires induction. A mixture of glucose and β-disaccharide has been demonstrated to achieve high-level cellulase production. However, as inducers, β-disaccharides are prone to be hydrolyzed by β-glucosidase (BGL) during fermentation, therefore β-disaccharides need to be supplemented through feeding to overcome this problem. Here, miglitol, an α-glucosidase inhibitor, was investigated as a BGL inhibitor, and exhibited an IC₅₀ value of 2.93 μg/mL. The cellulase titer was more than two-fold when miglitol was added to the fermentation medium of T. reesei. This method was similar to the prokaryotic expression system using unmetabolized isopropyl-β-D-thiogalactopyranoside (IPTG) as the inducer instead of lactose to continuously induce gene expression. However, cellulase activity was not enhanced with BGL inhibition when lactose or cellulose was used as an inducer, which demonstrated that the transglycosidase activity of BGL is important for the inducible activity of lactose and cellulose. This novel method demonstrates potential in stimulating cellulase production and provides a promising system for T. reesei protein expression. Full article

(This article belongs to the Special Issue Applied Microorganisms and Industrial/Food Enzymes)

► Show Figures

Figure 1

14 pages, 4013 KiB

Open AccessArticle

Medium Optimization for GA4 Production by Gibberella fujikuroi Using Response Surface Methodology

by Bingxuan Wang, Kainan Yin, Choufei Wu, Liang Wang, Lianghong Yin and Haiping Lin

Fermentation 2022, 8(5), 230; https://doi.org/10.3390/fermentation8050230 - 17 May 2022

Cited by 4 | Viewed by 2089

Abstract

Gibberellin is an important plant growth regulator that has been widely used in agricultural production with great market prospects. However, the low yield from Gibberella fujikuroi restricts its application. To improve the production of gibberellin A4 (GA4), the response surface methodology was used [...] Read more.

Gibberellin is an important plant growth regulator that has been widely used in agricultural production with great market prospects. However, the low yield from Gibberella fujikuroi restricts its application. To improve the production of gibberellin A4 (GA4), the response surface methodology was used in this study to explore the effect of different types and concentrations of vegetable oil and precursors on the production of GA4. Based on a single factor experiment, the Behnken box and central composite designs were used to establish the fermentation condition model, and the response surface method was used for analysis. The results indicated that the optimum formula was 0.55% palm oil, 0.60% cottonseed oil, 0.64% sesame oil, 0.19 g/L pyruvic acid, 0.21 g/L oxaloacetic acid, and 0.21 g/L citric acid for 48 h, which produced a yield 4.32 times higher than that without optimization. This suggests that the mathematical model is valid for predicting GA4 production in Gibberella fujikuroi QJGA4-1. Full article

(This article belongs to the Special Issue Applied Microorganisms and Industrial/Food Enzymes)

► Show Figures

Figure 1

11 pages, 1786 KiB

Open AccessArticle

Construction of L-Asparaginase Stable Mutation for the Application in Food Acrylamide Mitigation

by Bing Yuan, Pengfei Ma, Yuxuan Fan, Bo Guan, Youzhen Hu, Yan Zhang, Wenli Yan, Xu Li and Yongqing Ni

Fermentation 2022, 8(5), 218; https://doi.org/10.3390/fermentation8050218 - 11 May 2022

Cited by 2 | Viewed by 2281

Abstract

Acrylamide, a II A carcinogen, widely exists in fried and baked foods. L-asparaginase can inhibit acrylamide formation in foods, and enzymatic stability is the key to its application. In this study, the Escherichia coli L-asparaginase (ECA) stable variant, D60W/L211R/L310R, was obtained with molecular [...] Read more.

Acrylamide, a II A carcinogen, widely exists in fried and baked foods. L-asparaginase can inhibit acrylamide formation in foods, and enzymatic stability is the key to its application. In this study, the Escherichia coli L-asparaginase (ECA) stable variant, D60W/L211R/L310R, was obtained with molecular dynamics (MD) simulation, saturation mutation, and combinatorial mutation, the half-life of which increased to 110 min from 60 min at 50 °C. Furthermore, the working temperature (maintaining the activity above 80%) of mutation expanded from 31 °C–43 °C to 35 °C–55 °C, and the relative activity of mutation increased to 82% from 65% at a pH range of 6–10. On treating 60 U/mL and 100 U/g flour L-asparaginase stable mutant (D60W/L211R/L310R) under uncontrolled temperature and pH, the acrylamide content of potato chips and bread was reduced by 66.9% and 51.7%, which was 27% and 49.9% higher than that of the wild type, respectively. These results demonstrated that the mutation could be of great potential to reduce food acrylamide formation in practical applications. Full article

(This article belongs to the Special Issue Applied Microorganisms and Industrial/Food Enzymes)

► Show Figures

Figure 1

17 pages, 3900 KiB

Open AccessArticle

Fermented Myriophyllum aquaticum and Lactobacillus plantarum Affect the Distribution of Intestinal Microbial Communities and Metabolic Profile in Mice

by Yueyang Li, Yuxi Ling, Jia Liu, Michael Zhang, Zuming Li, Zhihui Bai, Zhenlong Wu, Ran Xia, Zhichao Wu, Yingxin Wan and Qiyun Zhou

Fermentation 2022, 8(5), 210; https://doi.org/10.3390/fermentation8050210 - 05 May 2022

Viewed by 1951

Abstract

This research explores the effects of fermented Myriophyllum aquaticum (F) and Lactobacillus plantarum BW2013 (G) as new feed additives on the gut microbiota composition and metabolic profile of mice. Crude protein (p = 0.045), lipid (p = 0.000), and ash ( [...] Read more.

This research explores the effects of fermented Myriophyllum aquaticum (F) and Lactobacillus plantarum BW2013 (G) as new feed additives on the gut microbiota composition and metabolic profile of mice. Crude protein (p = 0.045), lipid (p = 0.000), and ash (p = 0.006) contents in Myriophyllum aquaticum (N) were improved, whereas raw fiber (p = 0.031) content was decreased after solid-state fermentation by G. Mice were fed with no additive control (CK), 10%N (N), 10%N + G (NG), 10%F (F), and 10%F + G (FG). High-throughput sequencing results showed that, compared with the CK group, Parabacteroides goldsteinii was increased in treatment groups and that Lactobacillus delbrueckii, Bacteroides vulgatus, and Bacteroides coprocola were increased in the F and FG groups. Bacteroides vulgatus and Bacteroides coprocola were increased in the F group compared with the N group. Metabolomic results showed that vitamin A, myricetin, gallic acid, and luteolin were increased in the F group compared with the N group. Reduction in LPG 18:1 concentration in the N and F groups could be attenuated or even abolished by supplementation with G. Furthermore, 9-oxo-ODA was upregulated in the FG group compared with the F group. Collectively, N, F, and G have beneficial effects on gut microbiota and metabolic profile in mice, especially intake of FG. Full article

(This article belongs to the Special Issue Applied Microorganisms and Industrial/Food Enzymes)

► Show Figures

Figure 1

17 pages, 733 KiB

Open AccessArticle

Assessing Hydrolyzed Gluten Content in Dietary Enzyme Supplements Following Fermentation

by Ekaterina Khokhlova, Pyeongsug Kim, Joan Colom, Shaila Bhat, Aoife M. Curran, Najla Jouini, Kieran Rea, Christopher Phipps and John Deaton

Fermentation 2022, 8(5), 203; https://doi.org/10.3390/fermentation8050203 - 29 Apr 2022

Viewed by 2282

Abstract

Partially digested gluten fragments from grains including wheat, rye, spelt and barley are responsible for triggering an inflammatory response in the intestinal tract of Celiac Disease (CD) and Non-Celiac Gluten Sensitive (NCGS) individuals. Fermentation is an effective method to metabolize gluten, with enzymes [...] Read more.

Partially digested gluten fragments from grains including wheat, rye, spelt and barley are responsible for triggering an inflammatory response in the intestinal tract of Celiac Disease (CD) and Non-Celiac Gluten Sensitive (NCGS) individuals. Fermentation is an effective method to metabolize gluten, with enzymes from bacterial or fungal species being released to help in this process. However, the levels of gluten in commercially available enzymes, including those involved in gluten fermentation, are unknown. In this study we investigated gluten levels in commercially available dietary enzymes combined with assessing their effect on inflammatory response in human cell culture assays. Using antibodies that recognize different gluten epitopes (G12, R5, 2D4, MloBS and Skerritt), we employed ELISA and immunoblotting methodologies to determine gluten content in crude gluten, crude gliadin, pepsin-trypsin digested gluten and a selection of commercially available enzymes. We further investigated the effect of these compounds on inflammatory response in immortalized immune and intestinal human cell lines, as well as in peripheral blood mononuclear cells (PBMCs) from coeliac individuals. All tested supplemental enzyme products reported a gluten concentration that was equivalent to or below 20 parts per million (ppm) as compared with an intact wheat reference standard and a pepsin-trypsin digested standard. Similarly, the inflammatory response to IL-8 and TNF-α inflammatory cytokines in mammalian cell lines and PBMCs from coeliac individuals to the commercial enzymes was not significantly different to 20 ppm of crude gluten, crude gliadin or pepsin-trypsin digested gluten. This combined approach provides insight into the extent of gluten breakdown in the fermentation process and the safety of these products to gluten-sensitive individuals. Full article

(This article belongs to the Special Issue Applied Microorganisms and Industrial/Food Enzymes)

► Show Figures

Figure 1

15 pages, 2119 KiB

Open AccessArticle

Correlation Analysis of Microbiota and Volatile Flavor Compounds of Caishiji Soybean Paste

by Jing Cai, Yueting Han, Wei Wu, Xuefeng Wu, Dongdong Mu, Shaotong Jiang and Xingjiang Li

Fermentation 2022, 8(5), 196; https://doi.org/10.3390/fermentation8050196 - 27 Apr 2022

Cited by 1 | Viewed by 1698

Abstract

Microbial diversity plays a crucial part in the fermentation of Caishiji soybean paste (CSP). In the current study, the microbiota and volatile flavor compounds (VFCs) in CSP were identified through Illumina MiSeq sequencing and headspace gas chromatography–mass spectrometry. Five bacterial (Bacillus, [...] Read more.

Microbial diversity plays a crucial part in the fermentation of Caishiji soybean paste (CSP). In the current study, the microbiota and volatile flavor compounds (VFCs) in CSP were identified through Illumina MiSeq sequencing and headspace gas chromatography–mass spectrometry. Five bacterial (Bacillus, Tetragenococcus, Salinivibrio, Halomonas, and Staphylococcus) and four fungal genera (Aspergillus, Debaryomyces, Nigrospora, and Curvularia) were revealed as dominant among the entire microbiome of CSP. More than 70 VFCs, including 8 acids, 15 esters, 8 alcohols, 14 aldehydes, 4 ketones, 5 phenols, and 20 miscellaneous VFCs were detected during the fermentation process. A total of 12 kinds of VFCs were identified in the odor activity value (OAV) analysis. The results of the correlation analysis between microbiota and VFCs indicated that Bacillus, Tetragenococcus, Staphylococcus, and Aspergillus were the main microbiota affecting the flavor of CSP. These results may serve as a reference for enhancing the quality of CSP. Full article

(This article belongs to the Special Issue Applied Microorganisms and Industrial/Food Enzymes)

► Show Figures

Figure 1

11 pages, 2002 KiB

Open AccessArticle

Controlling the Formation of Foams in Broth to Promote the Co-Production of Microbial Oil and Exopolysaccharide in Fed-Batch Fermentation

by Yan-Feng Guo, Meng-Qi Wang, Yi-Lei Wang, Hong-Tao Wang and Jian-Zhong Xu

Fermentation 2022, 8(2), 68; https://doi.org/10.3390/fermentation8020068 - 07 Feb 2022

Cited by 3 | Viewed by 5518

Abstract

A large amount of foam is generated in the production of microbial oil and exopolysaccharide (EPS) by Sporidiobolus pararoseus JD-2, which causes low efficiency in fermentation. In this study, we aimed to reduce the negative effects of foams on the co-production of oil [...] Read more.

A large amount of foam is generated in the production of microbial oil and exopolysaccharide (EPS) by Sporidiobolus pararoseus JD-2, which causes low efficiency in fermentation. In this study, we aimed to reduce the negative effects of foams on the co-production of oil and EPS by controlling the formation of foams in broth. As we have found, the formation of foams is positively associated with cell growth state, air entrapment, and properties of broth. The efficient foam-control method of adding 0.03% (v/v) of the emulsified polyoxyethylene polyoxypropylene pentaerythritol ether (PPE) and feeding corn steep liquor (CSL) at 8–24 h with speed of 0.02 L/h considerably improved the fermentation performance of S. pararoseus JD-2, and significantly increased the oil and EPS concentrations by 8.7% and 12.9%, respectively. The biomass, oil, and EPS concentrations were further increased using a foam backflow device combined with adding 0.03% (v/v) of the emulsified PPE and feeding CSL at 8–24 h, which reached to 62.3 ± 1.8 g/L, 31.2 ± 0.8 g/L, and 10.9 ± 0.4 g/L, respectively. The effective strategy for controlling the formation of foams in fermentation broth reported here could be used as a technical reference for producing frothing products in fed-batch fermentation. Full article

(This article belongs to the Special Issue Applied Microorganisms and Industrial/Food Enzymes)

► Show Figures

Figure 1

Review

Jump to: Research

16 pages, 1277 KiB

Open AccessReview

Research Progress on the Effect of Autolysis to Bacillus subtilis Fermentation Bioprocess

by Kexin Ren, Qiang Wang, Mengkai Hu, Yan Chen, Rufan Xing, Jiajia You, Meijuan Xu, Xian Zhang and Zhiming Rao

Fermentation 2022, 8(12), 685; https://doi.org/10.3390/fermentation8120685 - 28 Nov 2022

Cited by 3 | Viewed by 4343

Abstract

Bacillus subtilis is a gram-positive bacterium, a promising microorganism due to its strong extracellular protein secretion ability, non-toxic, and relatively mature industrial fermentation technology. However, cell autolysis during fermentation restricts the industrial application of B. subtilis. With the fast advancement of molecular [...] Read more.

Bacillus subtilis is a gram-positive bacterium, a promising microorganism due to its strong extracellular protein secretion ability, non-toxic, and relatively mature industrial fermentation technology. However, cell autolysis during fermentation restricts the industrial application of B. subtilis. With the fast advancement of molecular biology and genetic engineering technology, various advanced procedures and gene editing tools have been used to successfully construct autolysis-resistant B. subtilis chassis cells to manufacture various biological products. This paper first analyses the causes of autolysis in B. subtilis from a mechanistic perspective and outlines various strategies to address autolysis in B. subtilis. Finally, potential strategies for solving the autolysis problem of B. subtilis are foreseen. Full article

(This article belongs to the Special Issue Applied Microorganisms and Industrial/Food Enzymes)

► Show Figures

Figure 1

Journal Menu

Journal Browser

Applied Microorganisms and Industrial/Food Enzymes

Share This Special Issue

Special Issue Editors

Special Issue Information

Keywords

Published Papers (23 papers)

Research

Review

Further Information

Guidelines

MDPI Initiatives

Follow MDPI