An Engineered Microbial Consortium Provides Precursors for Fengycin Production by Bacillus subtilis.

Fengycin has great potential for applications in biological control because of its biosafety and degradability. In this study, the addition of exogenous precursors increased fengycin production by Bacillus subtilis. Corynebacterium glutamicum was engineered to produce high levels of precursors (Thr, Pro, Val, and Ile) to promote the biosynthesis of fengycin. Furthermore, recombinant C. glutamicum and Yarrowia lipolytica providing amino acid and fatty acid precursors were co-cultured to improve fengycin production by B. subtilis in a three-strain artificial consortium, in which fengycin production was 2100 mg·L-1. In addition, fengycin production by the consortium in a 5 L bioreactor reached 3290 mg·L-1. Fengycin had a significant antifungal effect on Rhizoctonia solani, which illustrates its potential as a food preservative. Taken together, this work provides a new strategy for improving fengycin production by a microbial consortium and metabolic engineering.

Enhanced Iturin A Production of Engineered Bacillus amyloliquefaciens by Knockout of Endogenous Plasmid and Rap Phosphatase Genes.

Iturin A biosynthesis has garnered considerable interest, yet bottlenecks persist in its low productivity in wild strains and the ability to engineer Bacillus amyloliquefaciens producers. This study reveals that deleting the endogenous plasmid, plas1, from the wild-type B. amyloliquefaciens HM618 notably enhances iturin A synthesis, likely related to the effect of the Rap phosphatase gene within plas1. Furthermore, inactivating Rap phosphatase-related genes (rapC, rapF, and rapH) in the genome of the strain also improved the iturin A level and specific productivity while reducing cell growth. Strategic rap genes and plasmid elimination achieved a synergistic balance between cell growth and iturin A production. Engineered strain HM-DR13 exhibited an increase in iturin A level to 849.9 mg/L within 48 h, significantly shortening the production period. These insights underscore the critical roles of endogenous plasmids and Rap phosphatases in iturin A biosynthesis, presenting a novel engineering strategy to optimize iturin A production in B. amyloliquefaciens.

Integrated Biofilm Modification and Transcriptional Analysis for Improving Fengycin Production in Bacillus amyloliquefaciens.

Although fengycin exhibits broad-spectrum antifungal properties, its application is hindered due to its low biosynthesis level and the co-existence of iturin A and surfactin in Bacillus amyloliquefaciens HM618, a probiotic strain. In this study, transcriptome analysis and gene editing were used to explore the potential mechanisms regulating fengycin production in B. amyloliquefaciens. The fengycin level of B. amyloliquefacien HM-3 (∆itu-ΔsrfAA) was 88.41 mg/L after simultaneously inhibiting the biosyntheses of iturin A and surfactin. The knockout of gene eps associated with biofilm formation significantly increased the fengycin level of the strain HM618, whereas the fengycin level decreased 32.05% after knocking out sinI, a regulator of biofilm formation. Transcriptome analysis revealed that the differentially expressed genes, involved in pathways of amino acid and fatty acid syntheses, were significantly down-regulated in the recombinant strains, which is likely associated with a decrease of fengycin production. The knockout of gene comQXPA and subsequent transcriptome analysis revealed that the ComQXPA quorum sensing system played a positive regulatory role in fengycin production. Through targeted genetic modifications and fermentation optimization, the fengycin production of the engineered strain HM-12 (∆itu-ΔsrfAA-ΔyvbJ) in a 5-L fermenter reached 1.172 g/L, a 12.26-fold increase compared to the fengycin level in the strain HM-3 (∆itu-ΔsrfAA) in the Erlenmeyer flask. Taken together, these results reveal the underlying metabolic mechanisms associated with fengycin synthesis and provide a potential strategy for improving fengycin production in B. amyloliquefaciens.

Mutualistic microbial community of Bacillus amyloliquefaciens and recombinant Yarrowia lipolytica co-produced lipopeptides and fatty acids from food waste.

Bioconversion is an important method for transforming food waste (FW) into high value-added products, rendering it harmless, and recycling resources. An artificial microbial consortium (AMC) was constructed to produce FW-based lipopeptides in order to investigate the strategy of FW bioconversion into value-added products. Exogenous fatty acids as a precursor significantly improved the lipopeptide production of Bacillus amyloliquefaciens HM618. To enhance fatty acid synthesis and efflux in AMC, the recombinant Yarrowia lipolytica YL21 (strain YL21) was constructed by screening 12 target genes related to fatty acids to replace exogenous fatty acids in order to improve lipopeptide production. The levels of fengycin, surfactin, and iturin A in the AMC of strains HM618 and YL21 reached 76.19, 192.80, and 31.32 mg L-1, increasing 7.24-, 12.13-, and 3.23-fold compared to the results from the pure culture of strain HM618 in flask with Landy medium, respectively. Furthermore, free fatty acids were almost undetectable in the co-culture of strains HM618 and YL21, although its level was around 1.25 g L-1 in the pure culture of strain YL21 with Landy medium. Interestingly, 470.24 mg L-1 of lipopeptides and 18.11 g L-1 of fatty acids were co-produced in this AMC in a bioreactor with FW medium. To our knowledge, it is the first report of FW biotransformation into co-produce of lipopeptides and fatty acids in the AMC of B. amyloliquefaciens and Y. lipolytica. These results provide new insights into the biotransformation potential of FW for value-added co-products by AMC.

Enhancing fengycin production in the co-culture of Bacillus subtilis and Corynebacterium glutamicum by engineering proline transporter.

Fengycin possesses antifungal activity but has limited application due to its low yields. Amino acid precursors play a crucial role in fengycin synthesis. Herein, the overexpression of alanine, isoleucine, and threonine transporter-related genes in Bacillus subtilis increased fengycin production by 34.06%, 46.66%, and 7.83%, respectively. Particularly, fengycin production in B. subtilis reached 871.86 mg/L with the addition of 8.0 g/L exogenous proline after enhancing the expression of the proline transport-related gene opuE. To overcome the metabolic burden caused by excessive enhancement of gene expression for supplying precursors, B. subtilis and Corynebacterium glutamicum which produced proline, were co-cultured, which further improved fengycin production. Fengycin production in the co-culture of B. subtilis and C. glutamicum in shake flasks reached 1554.74 mg/L after optimizing the inoculation time and ratio. The fengycin level in the fed-batch co-culture was 2309.96 mg/L in a 5.0-L bioreactor. These findings provide a new strategy for improving fengycin production.

Improved the lipopeptide production of Bacillus amyloliquefaciens HM618 under co-culture with the recombinant Corynebacterium glutamicum producing high-level proline.

The application of antibacterial lipopeptides is limited by high cost and low yield. Herein, the exogenous L-proline significantly improved lipopeptide production by Bacillus amyloliquefaciens HM618. A recombinant Corynebacterium glutamicum producing high levels of proline using genetically modifying proB and putA was used to establish consortium, to improve lipopeptide production of strain HM618. Compared to a pure culture, the levels of iturin A, fengycin, and surfactin in consortium reached 67.75, 39.32, and 37.25 mg L-1, respectively, an increase of 3.19-, 2.05-, and 1.63-fold over that produced by co-cultures of B. amyloliquefaciens and recombinant C. glutamicum with normal medium. Commercial amylase and recombinant Pichia pastoris with a heterologous amylase gene were used to hydrolyze kitchen waste. A three-strain consortium with recombinant P. pastoris and C. glutamicum increased the lipopeptide production of strain HM618 in medium containing KW. This work provides new strategies to improve lipopeptide production by B. amyloliquefaciens.

Artificial microbial consortium producing oxidases enhanced the biotransformation efficiencies of multi-antibiotics.

Antibiotic mixtures in the environment result in the development of bacterial strains with resistance against multiple antibiotics. Oxidases are versatile that can bio-remove antibiotics. Various laccases (LACs), manganese peroxidases (MNPs), and versatile peroxidase (VP) were reconstructed in Pichia pastoris. For the single antibiotics, over 95.0% sulfamethoxazole within 48 h, tetracycline, oxytetracycline, and norfloxacin within 96 h were bio-removed by recombinant VP with α-signal peptide, respectively. In a mixture of the four antibiotics, 80.2% tetracycline and 95.6% oxytetracycline were bio-removed by recombinant MNP2 with native signal peptide (NSP) within 8 h, whereas < 80.0% sulfamethoxazole was bio-removed within 72 h, indicating that signal peptides significantly impacted removal efficiencies of antibiotic mixtures. Regarding mediators for LACs, 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) resulted in better removal efficiencies of multi-antibiotic mixtures than 1-hydroxybenzotriazole or syringaldehyde. Furthermore, artificial microbial consortia (AMC) producing LAC2 and MNP2 with NSP significantly improved bio-removal efficiency of sulfamethoxazole (95.5%) in four-antibiotic mixtures within 48 h. Tetracycline and oxytetracycline were completely bio-removed by AMC within 48 and 72 h, respectively, indicating that AMC accelerated sulfamethoxazole, tetracycline, and oxytetracycline bio-removals. Additionally, transformation pathways of each antibiotic by recombinant oxidases were proposed. Taken together, this work provides a new strategy to simultaneously remove antibiotic mixtures by AMC.

Improved Production of Fengycin in Bacillus subtilis by Integrated Strain Engineering Strategy.

Fengycin is a lipopeptide with broad-spectrum antifungal activity. However, its low yield limits its commercial application. Therefore, we iteratively edited multiple target genes associated with fengycin synthesis by combinatorial metabolic engineering. The ability of Bacillus subtilis 168 to manufacture lipopeptides was restored, and the fengycin titer was 1.81 mg/L. Fengycin production was further increased to 174.63 mg/L after knocking out pathways associated with surfactin and bacillaene synthesis and replacing the native promoter (PppsA) with the Pveg promoter. Subsequently, fengycin levels were elevated to 258.52 mg/L by upregulating the expression of relevant genes involved in the fatty acid pathway. After blocking spore and biofilm formation, fengycin production reached 302.51 mg/L. Finally, fengycin production was increased to approximately 885.37 mg/L after adding threonine in the optimized culture medium, which was 488-fold higher compared with that of the initial strain. Integrated strain engineering provides a strategy to construct a system for improving fengycin production.