Optimal Fermentation Research Articles

Postbiotic emissaries: a comprehensive review on the bioprospecting and production of bioactive compounds by Enterococcus species

SummaryEnterococcus species have been acknowledged for their diverse metabolic capabilities, resulting in a wide array of bioactive compounds that show promise for therapeutic use. This article presents a thorough examination of the exploration and synthesis of bioactive compounds by Enterococcus species. It encompasses the taxonomy, natural habitat, and significance in the biotechnology of Enterococcus, emphasising the various bioactive compounds it produces, including antibacterial, immunomodulatory, and antioxidant agents. Strategies for exploring potential bioactivities, encompassing isolation methodologies and screening techniques, are discussed. The biotechnological synthesis of bioactive compounds derived from Enterococcus via fermentation processes, optimisation approaches, and scaling methodologies is expounded. Moreover, the utilisation of these bioactive compounds in functional foods, pharmaceuticals, and agriculture, alongside considerations of safety and regulatory aspects, is examined. The review concludes with future perspectives and challenges in the field of Enterococcus‐derived bioactive compounds.

Identification, fermentation optimization, and biocontrol efficacy of actinomycete YG-5 for the prevention of Alternaria leaf spot disease in star anise

Star anise (Illicium verum), a valuable spice tree, faces significant threats from fungal diseases, particularly Alternaria leaf spot. This study investigates the potential of a soil-derived actinomycete strain, YG-5, as a biocontrol agent against Alternaria tenuissima, the causative pathogen on Alternaria leaf spot in star anise. Through comprehensive morphology, physiology, biochemistry, and genetic analyses, we identified the isolate as Streptomyces sp. YG-5. The strain exhibited broad-spectrum antimicrobial activity against several plant pathogens, with inhibition rates ranging between 36.47 to 80.34%. We systematically optimized the fermentation conditions for YG-5, including medium composition and cultivation parameters. The optimized process resulted in an 89.56% inhibition rate against A. tenuissima, a 14.72% improvement over non-optimized conditions. Notably, the antimicrobial compounds produced by YG-5 demonstrated stability across various temperatures, pH levels, and UV irradiation. In vivo efficacy trials showed promising results, with YG-5 fermentation broth reducing Alternaria leaf spot incidence on star anise leaves by 56.95%. These findings suggest that Streptomyces sp. YG-5 holds significant potential as a biocontrol agent against Alternaria leaf spot in star anise cultivation, offering a sustainable approach to disease management in this valuable crop.

Nitrogen Availability and Utilisation of Oligopeptides by Yeast in Industrial Scotch Grain Whisky Fermentation

Scotch grain whisky is produced with a substantial proportion of unmalted grains, which can result in nitrogen deficiency for yeast in the fermentable grain mash. This study examined nitrogen source availability and utilisation by three commercial whisky strains (Saccharomyces cerevisiae) during Scotch grain whisky fermentation, focusing on oligopeptides. Peptide uptake kinetics in synthetic whisky mash with defined peptides showed that oligopeptides of up to nine amino acids were taken up by the strains, albeit with some variability between the strains. The study found that peptides with appropriate molecular weights could replace free amino acids without negatively affecting fermentation kinetics. Moreover, fermentation performance improved when additional nitrogen was provided via peptides rather than diammonium phosphate. Analysis of industrial grain mash indicated that despite low initial yeast assimilable nitrogen, residual proteolytic activity from malt increased nitrogen availability during fermentation. Approximately 30% of the nitrogen consumed by yeast during grain mash fermentation was derived from peptides. LC-HRMS peptide analysis revealed complex dynamics of peptide formation, degradation, and utilisation. This study highlights the importance of oligopeptides in ensuring optimal fermentation efficiency in Scotch grain mash and similar substrates.

Conjugated Linoleic Acid Production in Pine Nut Oil: A Lactiplantibacillus plantarum Lp-01 Fermentation Approach.

Conjugated linoleic acid (CLA) is a class of bioactive fatty acids that exhibit various physiological activities such as anti-cancer, anti-atherosclerosis, and lipid-lowering. It is an essential fatty acid that cannot be synthesized by the human body and must be derived from dietary sources. The natural sources of CLA are limited, predominantly relying on chemical and enzymatic syntheses methods. Microbial biosynthesis represents an environmentally benign approach for CLA production. Pine nut oil, containing 40-60% linoleic acid, serves as a promising substrate for CLA enrichment. In the present study, we developed a novel method for the production of CLA from pine nut oil using Lactiplantibacillus plantarum (L. plantarum) Lp-01, which harbors a linoleic acid isomerase. The optimal fermentation parameters for CLA production were determined using a combination of single-factor and response surface methodologies: an inoculum size of 2%, a fermentation temperature of 36 °C, a fermentation time of 20 h, and a pine nut oil concentration of 11%. Under these optimized conditions, the resultant CLA yield was 33.47 μg/mL. Gas chromatography analysis revealed that the fermentation process yielded a mixture of c9, t11CLA and t10, c12 CLA isomers, representing 4.91% and 4.86% of the total fatty acid content, respectively.

Optimization and Stability Assessment of Monochamus alternatus Antimicrobial Peptide MaltAtt-1 in Komagataella phaffii GS115 for the Control of Pine Wood Nematode.

MaltAtt-1 is an antimicrobial peptide isolated from Monochamus alternatus with nematocidal activity against pine wood nematode. In this study, a eukaryotic expression system based on Komagataella phaffii GS115 was established, and its secretory expression of MaltAtt-1 was realized. The basic properties and secondary and tertiary structures of the antimicrobial peptide MaltAtt-1 were identified by bioinformatics analysis. MaltAtt-1 is a hydrophilic stable protein, mainly composed of an α-helix (Hh), β-folds (Ee), and irregular curls (Cc). The optimal fermentation conditions for MaltAtt-1 were determined by a single-factor test and the Box-Behnken response surface method, including an induction time of 72 h, induction temperature of 30 °C, culture medium of pH 7.6, methanol volume fraction of 2.0%, and an initial glycerol concentration of 1%. The stability of MaltAtt-1 indicated its resistant to UV irradiation and repeated freezing and thawing, but the antibacterial activity decreased significantly under the influence of high temperature and a strong acid and base, and it decreased significantly to 1.1 cm and 0.83 cm at pH 2.0 and pH 10.0, respectively. The corrected mortality of B. xylophilus achieved 71.94% in 3 h at a concentration of 300 mg·L-1 MaltAtt-1 exposure. The results provide a theoretical basis for the antimicrobial peptide MaltAtt-1 to become a new green and efficient nematicide.

Hybrid Modeling for On-Line Fermentation Optimization and Scale-Up: A Review

Modeling is a crucial tool in the biomanufacturing industry, namely in fermentation processes. This work discusses both mechanistic and data-driven models, each with unique benefits and application potential. It discusses semi-parametric hybrid modeling, a growing field that combines these two types of models for more accurate and easy result extrapolation. The characteristics and structure of such hybrid models will be examined. Moreover, its versatility will be highlighted, showing its usefulness in various stages of process development, including real-time monitoring and optimization. Scale-up remains one of the most relevant topics in fermentation processes, as it is important to have reproducible critical quality attributes, such as titer and yield, on larger scales. Furthermore, the process still relies on empirical correlations and iterative optimization. For these reasons, it is important to improve scale-up predictions, through e.g., the use of digital tools. Perspectives will be presented on the potential that hybrid modeling has by predicting performance across different process scales. This could provide more efficient and reliable biomanufacturing processes that require less resource consumption through experimentation.

Characterization and subcellular localization of selenium in Limosilactobacillus fermentum Ln-9 obtained by intense pulsed light-ultraviolet combined mutagenesis

Lactic acid bacteria (LAB) can convert inorganic selenium (Se) to organic Se and elemental forms with low toxicity and high bioavailability, but a comprehensive Se analysis of Se-enriched LAB is lacking. In this study, Limosilactobacillus fermentum Ln-9 was obtained by intense pulsed light-ultraviolet combined mutagenesis, and its characteristics and subcellular localization of Se were analyzed. The results displayed that Ln-9 accumulated 3.03 times Se that of the original strain. Under optimal fermentation conditions, the total Se content of Se-enriched Ln-9 (SeLn-9) reached 12.16 mg/g with 96.34% contained in Se nanoparticles (SeNPs), which was much higher than that of organic macromolecules. Furthermore, SeNPs were mainly localized outside the cell, Se-proteins were in the membrane and cytoplasmic fractions, and Se-polysaccharides were in the membrane fraction. Besides, SeLn-9 maintained a good morphology and gastrointestinal tolerance and had an enhanced antioxidant capacity. These findings make Ln-9 promising for applications in the food industry.

Comparative transcriptomics analysis-guided metabolic engineering of Yarrowia lipolytica for improved erythritol and fructooligosaccharides production

Currently, fructooligosaccharides (FOS) are converted from sucrose by purified enzymes or fungal cells, but these methods are costly and time-consuming. Here, the optimal fermentation conditions for strain E326 were determined through fermentation optimization: initial glucose 200 g/L, NaCl 25 g/L, inoculum volume 20 %, dissolved oxygen 20–30 %, pH 3, and glucose feeding concentration 100 g/L, which increased erythritol titer by 1.5 times. The co-expression of HGT1 and APC11 genes alleviated the erythritol synthesis stagnation, shorten the fermentation time by 16.7 %, and increased the erythritol productivity by 17.2 %. The episomal plasmids based on yeast mitochondrial replication origins (mtORIs) were constructed to surface display fructosyltransferase, effectively utilizing waste yeast cells generated during erythritol fermentation. Under the conditions of 60℃ and pH 6, the FOS yield reached 65 %, which to our best of knowledge is so-far the highest yield of FOS obtained. These findings will contribute to the industrial production of erythritol and FOS.

Enterobacter cloacae Rs-2 inoculum replaces fertiliser application by half in the field and modifies microbial community structure

Using microbial inoculant partial substitution synthetic fertilizer is a new type of environmentally friendly way to significantly improve the corps production and ecological environment. The effects of supplementing Enterobacter cloacae Rs-2 with synthetic fertilizer on the maize growth and soil microbial community diversity in field were investigated in this paper. The optimal fermentation conditions were determined as 5 g·L−1 industrial glucose, 30 g·L−1 industrial peptone, 1 g·L−1 MgSO4 and 0.5 g·L−1 KCl firstly. Field experiment showed that the fresh weight of shoots and roots was increased by 39.69% and 32.46% when half synthetic fertilizer was replaced by microbial inoculant. The soil physical and chemical properties were also greatly improved, especially the contents of available phosphorus, water-soluble calcium and alkali-hydrolyzed nitrogen were significantly increased in T4 (Rs-2 and half chemical fertilizer) and full chemical fertilizer treatment. This result was accompanied by increased the relative abundance of genes related to phospholipid metabolism, phosphotransferase system (PTS) and oxidative phosphorylation metabolism, indicating that Rs-2 may play an important role in the transformation and utilization of phosphorus in soil. The richness, dominance of Proteobacteria, Enterobacteriaceae in the microbial communities were further improved when the microbial inoculant applied. Function prediction PICRUSt analysis indicated that amines and amino acids were the most representative of the total carbon source utilization by the soil microbial communities. It was concluded that the application of 50% of the synthetic fertilizer supplemented with 30 mL microbial inoculant enhanced both the functional diversity of soil microbial communities and maize yield.

A Preliminary Study on the Effect of Adding Sugarcane Syrup on the Flavor of Barley Lager Fermentation.

This study focuses on the diversified utilization of the sugarcane industry, and sugarcane syrup, as a by-product of the sugarcane industry, is a good raw material for fermentation. Bringing sugarcane syrup into beer is conducive to the enrichment of the sugar industry, and it can improve the flavor of beer and make it more aromatic. This study determined the optimal fermentation process for beer. By analyzing the consumption rate of the carbon and nitrogen sources of raw materials, the nutrient utilization of yeast, and the causes of differences in flavor substances, the flavor composition and flavor stability of beer were determined by SPME-HS-GC-MS technology. The results showed that beer brewed with sugarcane syrup as an auxiliary raw material met the basic specifications of beer. The addition of sugarcane syrup to the wort base increased the utilization of amino acids by the yeast, and LS (lager with added cane syrup) increased the nine flavor compounds of the beer, which constituted the basic flavor of the beer, bringing new flavor compounds compared with the normal all-barley beer. Forced aging experiments showed that LS produced fewer aging compounds than OWBL. Various experiments have shown that it is feasible to ferment beer with sugarcane syrup instead of partial wort.

Study on Optimization of Liquid Fermentation Medium and Antitumor Activity of the Mycelium on Phyllopora lonicerae.

Phylloporia lonicerae is an annual fungus that specifically parasitizes living Lonicera plants, offering significant potential for developing new resource food and medicine. However, wild resources and mycelium production of this fungus is limited, and its anti-tumor active ingredients and mechanisms remain unclear, hampering the development of this fungus. Thus, we optimized the fermentation medium of P. lonicerae and studied the anti-tumor activity of its mycelium. The results indicated that the optimum fermentation medium consisted of 2% sucrose, 0.2% peptone, 0.1% KH2PO4, 0.05% MgSO4·7H2O, 0.16% Lonicera japonica petals, 0.18% P fungal elicitor, and 0.21% L. japonica stem. The biomass reached 7.82 ± 0.41 g/l after 15 days of cultivation in the optimized medium, a 142% increase compared with the potato dextrose broth medium, with a 64% reduction in cultivation time. The intracellular alcohol extract had a higher inhibitory effect on A549 and Eca-109 cells than the intracellular water extract, with half-maximal inhibitory concentration values of 2.42 and 2.92 mg/ml, respectively. Graded extraction of the alcohol extract yielded petroleum ether phase, chloroform phase, ethyl acetate phase, and n-butanol phase. Among them, the petroleum ether phase exhibited a better effect than the positive control, with a half-maximal inhibitory concentration of 113.3 μg/ml. Flow cytometry analysis indicated that petroleum ether components could induce apoptosis of Eca-109 cells, suggesting that this extracted component can be utilized as an anticancer agent in functional foods. This study offers valuable technical support and a theoretical foundation for promoting the comprehensive development and efficient utilization of P. lonicerae.

Optimization Co-Culture of Monascus purpureus and Saccharomyces cerevisiae on Selenium-Enriched Lentinus edodes for Increased Monacolin K Production.

Selenium-enriched Lentinus edodes (SL) is a kind of edible fungi rich in organic selenium and nutrients. Monascus purpureus with high monacolin K (MK) production and Saccharomyces cerevisiae were selected as the fermentation strains. A single-factor experiment and response surface methodology were conducted to optimize the production conditions for MK with higher contents from selenium-enriched Lentinus edodes fermentation (SLF). Furthermore, we investigated the nutritional components, antioxidant capacities, and volatile organic compounds (VOCs) of SLF. The MK content in the fermentation was 2.42 mg/g under optimal fermentation conditions. The organic selenium content of SLF was 7.22 mg/kg, accounting for 98% of the total selenium content. Moreover, the contents of total sugars, proteins, amino acids, reducing sugars, crude fiber, fat, and ash in SLF were increased by 9%, 23%, 23%, 94%, 38%, 44%, and 25%, respectively. The antioxidant test results demonstrated that 1.0 mg/mL of SLF exhibited scavenging capacities of 40%, 70%, and 79% for DPPH, ABTS, and hydroxyl radicals, respectively. Using gas chromatography-ion mobility spectrometry technology, 34 unique VOCs were identified in SLF, with esters, alcohols, and ketones being the main components of its aroma. This study showed that fungal fermentation provides a theoretical reference for enhancing the nutritional value of SL.

Balancing pH and yield: exploring itaconic acid production in Ustilago cynodontis from an economic perspective

BackgroundItaconic acid is a promising bio-based building block for the synthesis of polymers, plastics, fibers and other materials. In recent years, Ustilago cynodontis has emerged as an additional itaconate producing non-conventional yeast, mainly due to its high acid tolerance, which significantly reduces saline waste coproduction during fermentation and downstream processing. As a result, this could likely improve the economic viability of the itaconic acid production process with Ustilaginaceae.ResultsIn this study, we characterized a previously engineered itaconate hyper-producing Ustilago cynodontis strain in controlled fed-batch fermentations to determine the minimal and optimal pH for itaconate production. Under optimal fermentation conditions, the hyper-producing strain can achieve the theoretical maximal itaconate yield during the production phase in a fermentation at pH 3.6, but at the expense of considerable base addition. Base consumption is strongly reduced at the pH of 2.8, but at cost of production yield, titer, and rate. A techno-economic analysis based on the entire process demonstrated that savings due to an additional decrease in pH control reagents and saline waste costs cannot compensate the yield loss observed at the highly acidic pH value 2.8.ConclusionsOverall, this work provides novel data regarding the balancing of yield, titer, and rate in the context of pH, thereby contributing to a better understanding of the itaconic acid production process with Ustilago cynodontis, especially from an economic perspective.

Fermentation of DaiDai fruit and its biological activity.

DaiDai fruit, a medicinal and edible plant fruit, is abundant in biologically active compounds and has a long history of use in traditional Chinese medicine. This research focuses on utilizing fermentation to develop a functional DaiDai fruit fermentation broth. Lactobacillus, Bacillus subtilis and Saccharomyces cerevisiae were employed in the fermentation process. By conducting screenings of bacterial strains, single factor experiments, and response surface methodology, the total flavonoids, polysaccharides, polyphenols, and 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) free radical scavenging rate were used as the index for selection, ultimately identifying Lactobacillus L-13 as the optimal fermentation strain. The optimal fermentation conditions were determined to be a time of 108 h, a temperature of 43.6°C, and a solid-liquid ratio of 1:15.157 (w/v). Under these conditions, the total flavonoid content reached 412.01 mg/g, representing a 36.71% increase compared to conventional extraction methods. The contents of polysaccharides and polyphenols and the DPPH scavenging rate were also increased. The fermentation broth of DaiDai fruit exhibited inhibitory effects on tyrosinase and melanin production in mouse melanoma cells B16-F10 induced by α-MSH and anti-inflammatory properties in a zebrafish inflammation model. These indicate that the DaiDai fruit fermentation broth possesses anti-melanoma, whitening, and anti-inflammatory properties, showcasing significant potential for applications in medicine, cosmetics, and other industries.

Aspergillus niger as an efficient biological agent for separator sludge remediation: two-level factorial design for optimal fermentation.

The booming palm oil industry is in line with the growing population worldwide and surge in demand. This leads to a massive generation of palm oil mill effluent (POME). POME is composed of sterilizer condensate (SC), separator sludge (SS), and hydro-cyclone wastewater (HCW). Comparatively, SS exhibits the highest organic content, resulting in various environmental impacts. However, past studies mainly focused on treating the final effluent. Therefore, this pioneering research investigated the optimization of pollutant removal in SS via different aspects of bioremediation, including experimental conditions, treatment efficiencies, mechanisms, and degradation pathways. A two-level factorial design was employed to optimize the removal of chemical oxygen demand (COD) and turbidity using Aspergillus niger. Bioremediation of SS was performed through submerged fermentation (SmF) under several independent variables, including temperature (20-40°C), agitation speed (100-200 RPM), fermentation duration (72-240 h), and initial sample concentration (20-100%). The characteristics of the treated SS were then compared to that of raw sludge. Optimal COD and turbidity removal were achieved at 37°C 100 RPM, 156 h, and 100% sludge. The analysis of variance (ANOVA) revealed a significant effect of selective individual and interacting variables (p<0.05). The highest COD and turbidity removal were 97.43% and 95.11%, respectively, with less than 5% error from the predicted values. Remarkably, the selected optimized conditions also reduced other polluting attributes, namely, biological oxygen demand (BOD), oil and grease (OG), color, and carbon content. In short, this study demonstrated the effectiveness of A.niger in treating SS through the application of a two-level factorial design.

Polygonati rhizoma fermentation by Monascus ruber and evaluation of fermentation products in vitro

This study investigated the conditions for polygonati rhizoma (PR) fermentation by Monascus ruber and the lipid-lowering activity of the resulting product. Monascus ruber was used for the solid-state fermentation of PR, and the response surface method was used to optimize the fermentation culture medium parameters. The optimal fermentation system yielded a maximum monacolin K concentration of 21.82 ± 0.34 mg/g. The fermentation conditions were as follows: 12.5 mmol/kg of L-glutamic acid, 0.64 mmol/kg of cyclic adenosine monophosphate, 3.0 wt% of yeast extract powder, a fermentation period of 15 days, and a fermentation temperature of 30 ℃. The binding capacities of unfermented PR, “Nine-Steam-Nine-Bask” processed Polygonati rhizoma (NPR), and Monascus ruber solid-state fermented polygonati rhizoma (MPR) to sodium cholate, sodium taurocholate, and sodium glycolate were determined. MPR showed a stronger bile acid-binding capacity than unfermented PR. In addition, MPR exhibited the highest cholesterol inhibitory capacity of 51.43±1.41 %. Thus, MPR can be used as a potential antilipemic agent with potent hypolipidemic effects.

Effects of Bacillus altitudinis inoculants on cigar tobacco leaf fermentation.

Microbial succession and metabolic adjustment during cigar tobacco leaf (CTL) fermentation are key factors to improve the quality and flavor of CTLs. However, the interactions in the above processes remain to be further elucidated. Bacillus altitudinis inoculants were added to the CTLs, and metagenomics and metabolomics were used to analyze the effects of the inoculants on regulating microbial succession, metabolic shift, and aroma production during fermentation. The addition of the inoculants reinforced the CTL macromolecule transformation and facilitated the aroma production efficiently, and the total aroma production was increased by 43% compared with natural fermentation. The omics analysis showed that Staphylococcus was a main contributor to fatty acid degradation, inositol phosphate metabolism, energy supply (oxidative phosphorylation), nutrient transport (ABC transporter and phosphotransferase system [PTS]), and aroma production (terpenoid backbone biosynthesis, phenylalanine metabolism, and degradation of aromatic compounds). Furthermore, Staphylococcus was positively correlated with TCA cycle intermediates (citric acid, fumaric acid, and aconitic acid), cell wall components, peptidoglycan intermediates (GlcNAc-1-P and UDP-GlcNAc), and phytic acid degradation products (inositol). The characteristics collectively showed Staphylococcus to be the most dominant in the microbial community at the genus level during microflora succession. The addition of the inoculants supplemented the nutritional components of the CTLs, enhanced the metabolic activity and diversity of bacteria such as Corynebacterium, improved their competitive advantages in the microflora succession, and facilitated the richness of microbial communities. Additionally, a metabolic shift in nicotine degradation and NAD + anabolism from Staphylococcus to Corynebacterium in fermentation with inoculants was first observed. Meanwhile, the significantly correlative differential metabolites with Staphylococcus and Corynebacterium were a metabolic complement, thus forming a completely dynamic fermentation ecosystem. The results provided evidence for CTL fermentation optimization.

Optimization of enzymatic hydrolysis and fermentation of bleached garlic straw for bioethanol production

Optimization of enzymatic hydrolysis and fermentation of bleached garlic straw for bioethanol production

Enhancing the nutritional value and antioxidant properties of foxtail millet by solid‐state fermentation with edible fungi

AbstractFoxtail millet is typically dehulled before consumption or processing. However, foxtail millet bran also contains abundant phenolic compounds and other nutrients. Edible fungi have rich extracellular enzyme systems; are environmentally friendly and safe for consumption; and have been shown to effectively degrade lignin and cellulose. This study aimed to screen edible fungi that can effectively ferment undehusked foxtail millet, improving its nutritional value and antioxidant properties through solid‐state fermentation (SSF). The results demonstrated that fermentation utilizing Pleurotus geesteranus exhibited significant improvements in both the phenolic compound content and antioxidant properties of foxtail millet, with the optimal fermentation period determined to be 30 days. The physical and functional properties of fermented undehusked foxtail millet (FFM) flour were effectively improved, increasing crude protein, vitamin C, and crude polysaccharide contents by 11.46%, 27.78%, and 54.17%, respectively. In vitro scavenging activities of FFM were 73.19%, 93.86%, and 63.75% for 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH), 2,2′‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulfonic acid) (ABTS·+), and superoxide anion radicals (O2−), respectively. The total antioxidant capability (T‐AOC) and superoxide dismutase (SOD) activity of FFM were 1.01 mM Trolox equivalents (TE)/g and 89.05 U/g, respectively. Additionally, T‐AOC, SOD, and glutathione peroxidase (GSH‐Px) activities increased, whereas malondialdehyde (MDA) levels decreased in the heart, liver, and kidneys of mice treated with FFM flour, indicating enhanced antioxidant capacity. Therefore, fermentation with edible fungi is suitable for improving the nutritional composition and antioxidant properties of foxtail millet.

Rational construction of a high-quality and high-efficiency biosynthetic system and fermentation optimization for A82846B based on combinatorial strategies in Amycolatopsis orientalis

BackgroundOritavancin is a new generation of semi-synthetic glycopeptide antibiotics against Gram-positive bacteria, which served as the first and only antibiotic with a single-dose therapeutic regimen to treat ABSSSI. A naturally occurring glycopeptide A82846B is the direct precursor of oritavancin. However, its application has been hampered by low yields and homologous impurities. This study established a multi-step combinatorial strategy to rationally construct a high-quality and high-efficiency biosynthesis system for A82846B and systematically optimize its fermentation process to break through the bottleneck of microbial fermentation production.ResultsFirstly, based on the genome sequencing and analysis, we deleted putative competitive pathways and constructed a better A82846B-producing strain with a cleaner metabolic background, increasing A82846B production from 92 to 174 mg/L. Subsequently, the PhiC31 integrase system was introduced based on the CRISPR-Cas12a system. Then, the fermentation level of A82846B was improved to 226 mg/L by over-expressing the pathway-specific regulator StrR via the constructed PhiC31 system. Furthermore, overexpressing glycosyl-synthesis gene evaE enhanced the production to 332 mg/L due to the great conversion of the intermediate to target product. Finally, the scale-up production of A82846B reached 725 mg/L in a 15 L fermenter under fermentation optimization, which is the highest reported yield of A82846B without the generation of homologous impurities.ConclusionUnder approaches including blocking competitive pathways, inserting site-specific recombination system, overexpressing regulator, overexpressing glycosyl-synthesis gene and optimizing fermentation process, a multi-step combinatorial strategy for the high-level production of A82846B was developed, constructing a high-producing strain AO-6. The combinatorial strategies employed here can be widely applied to improve the fermentation level of other microbial secondary metabolites, providing a reference for constructing an efficient microbial cell factory for high-value natural products.