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The role of microorganisms in effectively terminating harmful algal blooms (HABs) is crucial for maintaining environmental stability. Recent studies have placed increased emphasis on bio-agents capable of inhibiting HABs. The bacterium Pseudoalteromonas sp. strain FDHY-MZ2 has exhibited impressive algicidal abilities against Karenia mikimotoi, a notorious global HAB-forming species. To augment this capability, cultures were progressively scaled from shake flask conditions to small-scale (5 L) and pilot-scale (50 L) fermentation. By employing a specifically tailored culture medium (2216E basal medium with 1.5% soluble starch and 0.5% peptone), under precise conditions (66 h, 20 °C, 450 rpm, 30 L/min ventilation, 3% seeding, and constant starch flow), a notable increase in algicidal bacterial biomass was observed; the bacterial dosage required to entirely wipe out K. mikimotoi within a day decreased from 1% to 0.025%. Compared to an unoptimized shake flask group, the optimized fermentation culture caused significant reductions in algal chlorophyll and protein levels (21.85% and 78.3%, respectively). Co-culturing induced increases in algal malondialdehyde and H2O2 by 5.98 and 5.38 times, respectively, leading to further disruption of algal photosynthesis. This study underscores the unexplored potential of systematically utilized microbial agents in mitigating HABs, providing a pathway for their wider application.

Background The GRAS and oleaginous yeast Yarrowia lipolytica ( Y. lipolytica ) is an attractive cell factory for the production of chemicals and biofuels. The production of many natural products of commercial interest have been investigated in this cell factory by introducing heterologous biosynthetic pathways and by modifying the endogenous pathways. However, since natural products anabolism involves long pathways and complex regulation, re-channelling carbon into the product of target compounds is still a cumbersome work, and often resulting in low production performance. Results In this work, the carotenogenic genes contained carB and bi-functional carRP from Mucor circinelloides and carotenoid cleavage dioxygenase 1 ( CCD1 ) from Petunia hybrida were introduced to Y. lipolytica and led to the low production of β-ionone of 3.5 mg/L. To further improve the β-ionone synthesis, we implemented a modular engineering strategy for the construction and optimization of a biosynthetic pathway for the overproduction of β-ionone in Y. lipolytica . The strategy involved the enhancement of the cytosolic acetyl-CoA supply and the increase of MVA pathway flux, yielding a β-ionone titer of 358 mg/L in shake-flask fermentation and approximately 1 g/L (~ 280-fold higher than the baseline strain) in fed-batch fermentation. Conclusions An efficient β-ionone producing GRAS Y. lipolytica platform was constructed by combining integrated overexpressed of heterologous and native genes. A modular engineering strategy involved the optimization pathway and fermentation condition was investigated in the engineered strain and the highest β-ionone titer reported to date by a cell factory was achieved. This effective strategy can be adapted to enhance the biosynthesis of other terpenoids in Y. lipolytica .

Background Limonene is an important biologically active natural product widely used in the food, cosmetic, nutraceutical and pharmaceutical industries. However, the low abundance of limonene in plants renders their isolation from plant sources non-economically viable. Therefore, engineering microbes into microbial factories for producing limonene is fast becoming an attractive alternative approach that can overcome the aforementioned bottleneck to meet the needs of industries and make limonene production more sustainable and environmentally friendly. Results In this proof-of-principle study, the oleaginous yeast Yarrowia lipolytica was successfully engineered to produce both d-limonene and l-limonene by introducing the heterologous d-limonene synthase from Citrus limon and l-limonene synthase from Mentha spicata, respectively. However, only 0.124 mg/L d-limonene and 0.126 mg/L l-limonene were produced. To improve the limonene production by the engineered yeast Y. lipolytica strain, ten genes involved in the mevalonate-dependent isoprenoid pathway were overexpressed individually to investigate their effects on limonene titer. Hydroxymethylglutaryl-CoA reductase (HMGR) was found to be the key rate-limiting enzyme in the mevalonate (MVA) pathway for the improving limonene synthesis in Y. lipolytica. Through the overexpression of HMGR gene, the titers of d-limonene and l-limonene were increased to 0.256 mg/L and 0.316 mg/L, respectively. Subsequently, the fermentation conditions were optimized to maximize limonene production by the engineered Y. lipolytica strains from glucose, and the final titers of d-limonene and l-limonene were improved to 2.369 mg/L and 2.471 mg/L, respectively. Furthermore, fed-batch fermentation of the engineered strains Po1g KdHR and Po1g KlHR was used to enhance limonene production in shake flasks and the titers achieved for d-limonene and l-limonene were 11.705 mg/L (0.443 mg/g) and 11.088 mg/L (0.385 mg/g), respectively. Finally, the potential of using waste cooking oil as a carbon source for limonene biosynthesis from the engineered Y. lipolytica strains was investigated. We showed that d-limonene and l-limonene were successfully produced at the respective titers of 2.514 mg/L and 2.723 mg/L under the optimal cultivation condition, where 70% of waste cooking oil was added as the carbon source, representing a 20-fold increase in limonene titer compared to that before strain and fermentation optimization. Conclusions This study represents the first report on the development of a new and efficient process to convert waste cooking oil into d-limonene and l-limonene by exploiting metabolically engineered Y. lipolytica strains for fermentation. The results obtained in this study lay the foundation for more future applications of Y. lipolytica in converting waste cooking oil into various industrially valuable products.

In this study, a new isolated strain YJY-12 with high yield of spinosad was isolated and its fermentation conditions for spinosad production was optimized. It was preliminary identified by morphological and molecular biology analysis to be S. spinosa YJY-12, and it demonstrated stable high-yield trait, with a variation of less than 5% in fermentation levels across five generations. Subsequently, its fermentation condition for producing spinosad was optimized in shake flask experiments. The process involved single-factor tests, Plackett–Burman (PB) experiments, steepest ascent experiments, and central composite design (CCD) methodology. After optimization, the spinosad fermentation level by strain YJY-12 reached up to 4.38 g/L. The fermentation process was further scaled up to a 30-L fermentor by fed-batch fermentation, achieving a spinosad fermentation level of 6.22 ± 0.12 g/L after 16-day. Finally, the efficacy of spinosad in controlling Eggplant Thrips was investigated and the technical spinosad produced by strain YJY-12 exhibited superior efficacy of thrips control. Overall, this study provided a methodological foundation for enhancing spinosad fermentation efficiency and offers guidance for future spinosad fermentation optimization research.

Fidaxomicin, a macrolide antibiotic, is widely used to treat Clostridioides difficile infection (CDI). It demonstrats significantly higher clinical efficacy than vancomycin and metronidazole. However, the large-scale industrial production of it remains a significant challenge because of the low fermentation yields. In this study, we chosen the strain OE-R1/WT as the starting strain, in which a pathway-specific positive regulatory factor fadR1 was overexpressed. By using the kanR/gusA dual-reporter system and ARTP mutagenesis, we screened a high-yield strain, PA-13, which produced 757.34 mg/L of fidaxomicin, representing a 5.5-fold increase over OE-R1/WT and having enhanced genetic stability. Furthermore, by overexpressing two methyltransferases within the biosynthetic cluster and supplementing with exogenous DMSO, we further increased the production of fidaxomicin to 929.17 mg/L, while reducing the accumulation of the major by-product to 20.9 %. Finally, through the optimization of fermentation strategies at both the shake flask and 15 L fermenter levels, we achieved a final yield of 3949.05 mg/L in the 15 L fermenter, which represents the highest yield up to date. Our study represents the first successful enhancement of fidaxomicin production in Actinoplanes deccanensis to over 3.9 g/L in a 15 L fermenter, establishing a robust foundation for industrial-scale fermentation. Additionally, it provides significant insights for the development of high-yield strains in other actinomycetes.

L-tryptophan (L-trp), produced through bio-manufacturing, is widely used in the pharmaceutical and food industries. Based on the previously developed L-trp-producing strain, this study significantly improved the titer and yield of L-trp, through metabolic engineering of the shikimate pathway and the L-tryptophan branch. First, the rate-limiting steps in the shikimate pathway were investigated and deciphered, revealing that the combined overexpression of the genes aroE and aroD increased L-trp production. Then, L-trp synthesis was further enhanced at the shaking flask level by improving the intracellular availability of L-glutamine (L-gln) and L-serine (L-ser). In addition, the transport system and the competing pathway of L-trp were also modified, indicating that elimination of the gene TnaB contributed to the extracellular accumulation of L-trp. Through optimizing formulas, the robustness and production efficiency of engineered strains were enhanced at the level of the 30 L fermenter. After 42 h of fed-batch fermentation, the resultant strain produced 53.65 g/L of L-trp, with a yield of 0.238 g/g glucose. In this study, the high-efficiency L-trp-producing strains were created in order to establish a basis for further development of more strains for the production of other highly valuable aromatic compounds or their derivatives.

In this study, a new isolated strain YJY-12 with high yield of spinosad was isolated and its fermentation conditions for spinosad production was optimized. It was preliminary identified by morphological and molecular biology analysis to be S. spinosa YJY-12, and it demonstrated stable high-yield trait, with a variation of less than 5% in fermentation levels across five generations. Subsequently, its fermentation condition for producing spinosad was optimized in shake flask experiments. The process involved single-factor tests, Plackett-Burman (PB) experiments, steepest ascent experiments, and central composite design (CCD) methodology. After optimization, the spinosad fermentation level by strain YJY-12 reached up to 4.38 g/L. The fermentation process was further scaled up to a 30-L fermentor by fed-batch fermentation, achieving a spinosad fermentation level of 6.22 ± 0.12 g/L after 16-day. Finally, the efficacy of spinosad in controlling Eggplant Thrips was investigated and the technical spinosad produced by strain YJY-12 exhibited superior efficacy of thrips control. Overall, this study provided a methodological foundation for enhancing spinosad fermentation efficiency and offers guidance for future spinosad fermentation optimization research.

Cyclodextrins (CDs) are cyclic oligosaccharides composed of α(1 → 4) linked glucose units, which are widely used as solubilizers and stabilizers in the food, pharmaceutical and cosmetic industries. Among the CDs, γ-CD has attracted much attention due to its larger hydrophobic cavity and higher solubility. However, the industrial production of γ-CD is limited by lack of suitable enzymes and production process shortcomings. In this study, various strategies of improving heterologous enzyme production and optimization of the starch conversion process were applied to increase the production of γ-CD. A γ-cyclodextrin glucanotransferase with good product specificity from Bacillus sp. FJAT-44876 (BFγ-CGTase) and a liquefying β-CGTase from Bacillus sp. 1011 (Bsβ-CGTase) were successfully secreted by Pichia pastoris. After codon optimization and using the one-factor-at-a-time (OFAT) principle to improve the fermentation, the yield of recombinant BFγ-CGTase was increased 13.3 times to 463 U/L. Next a process was established involving Bsβ-CGTase-assisted starch liquefaction and simultaneous pullulanase debranching and BFγ-CGTase production of γ-CD. The yield of γ-CD increased by 17.67% via optimizing the amounts of BFγ-CGTase and BtPul used for the reaction. Overall, combination of the various improvements provided a new process for efficient preparation of γ-CD.

Background Microbial engineering aims to enhance the ability of bacteria to produce valuable products, including vitamin B 6 for various applications. Numerous microorganisms naturally produce vitamin B 6 , yet the metabolic pathways involved are rigorously controlled. This regulation by the accumulation of vitamin B 6 poses a challenge in constructing an efficient cell factory. Results In this study, we conducted transcriptome and metabolome analyses to investigate the effects of the accumulation of pyridoxine, which is the major commercial form of vitamin B 6 , on cellular processes in Escherichia coli . Our omics analysis revealed associations between pyridoxine and amino acids, as well as the tricarboxylic acid (TCA) cycle. Based on these findings, we identified potential targets for fermentation optimization, including succinate, amino acids, and the carbon-to-nitrogen (C/N) ratio. Through targeted modifications, we achieved pyridoxine titers of approximately 514 mg/L in shake flasks and 1.95 g/L in fed-batch fermentation. Conclusion Our results provide insights into pyridoxine biosynthesis within the cellular metabolic network for the first time. Our comprehensive analysis revealed that the fermentation process resulted in a remarkable final yield of 1.95 g/L pyridoxine, the highest reported yield to date. This work lays a foundation for the green industrial production of vitamin B 6 in the future.

Background Raw starch-degrading α-amylase (RSDA) can hydrolyze raw starch at moderate temperatures, thus contributing to savings in starch processing costs. However, the low production level of RSDA limits its industrial application. Therefore, improving the extracellular expression of RSDA in Bacillus subtilis , a commonly used industrial expression host, has great value. Results In this study, the extracellular production level of Pontibacillus sp. ZY raw starch-degrading α-amylase (AmyZ1) in B. subtilis was enhanced by expression regulatory element modification and fermentation optimization. As an important regulatory element of gene expression, the promoter, signal peptide, and ribosome binding site (RBS) sequences upstream of the amyZ1 gene were sequentially optimized. Initially, based on five single promoters, the dual-promoter P veg -P ylB was constructed by tandem promoter engineering. Afterward, the optimal signal peptide SP NucB was obtained by screening 173 B. subtilis signal peptides. Then, the RBS sequence was optimized using the RBS Calculator to obtain the optimal RBS1. The resulting recombinant strain WBZ-VY-B-R1 showed an extracellular AmyZ1 activity of 4824.2 and 41251.3 U/mL during shake-flask cultivation and 3-L fermenter fermentation, which were 2.6- and 2.5-fold greater than those of the original strain WBZ-Y, respectively. Finally, the extracellular AmyZ1 activity of WBZ-VY-B-R1 was increased to 5733.5 U/mL in shake flask by optimizing the type and concentration of carbon source, nitrogen source, and metal ions in the fermentation medium. On this basis, its extracellular AmyZ1 activity was increased to 49082.1 U/mL in 3-L fermenter by optimizing the basic medium components as well as the ratio of carbon and nitrogen sources in the feed solution. This is the highest production level reported to date for recombinant RSDA production. Conclusions This study represents a report on the extracellular production of AmyZ1 using B. subtilis as a host strain, and achieved the current highest expression level. The results of this study will lay a foundation for the industrial application of RSDA. In addition, the strategies employed here also provide a promising way for improving other protein production in B. subtilis .