Metabolic engineering of Bacillus amyloliquefaciens for efficient production of α-glucosidase inhibitor1-deoxynojirimycin
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SUBMITTER: Li X
PROVIDER: S-EPMC10485785 | biostudies-literature | 2023 Jun
REPOSITORIES: biostudies-literature
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Project description:BackgroundIturins, which belong to antibiotic cyclic lipopeptides mainly produced by Bacillus sp., have the potential for application in biomedicine and biocontrol because of their hemolytic and antifungal properties. Bacillus amyloliquefaciens LL3, isolated previously by our lab, possesses a complete iturin A biosynthetic pathway as shown by genomic analysis. Nevertheless, iturin A could not be synthesized by strain LL3, possibly resulting from low transcription level of the itu operon.ResultsIn this work, enhanced transcription of the iturin A biosynthetic genes was implemented by inserting a strong constitutive promoter C2up into upstream of the itu operon, leading to the production of iturin A with a titer of 37.35 mg l-1. Liquid chromatography-mass spectrometry analyses demonstrated that the strain produced four iturin A homologs with molecular ion peaks at m/z 1044, 1058, 1072 and 1086 corresponding to [C14 + 2H]2+, [C15 + 2H]2+, [C16 + 2H]2+ and [C17 + 2H]2+. The iturin A extract exhibited strong inhibitory activity against several common plant pathogens. The yield of iturin A was improved to 99.73 mg l-1 by the optimization of the fermentation conditions using a response surface methodology. Furthermore, the yield of iturin A was increased to 113.1 mg l-1 by overexpression of a pleiotropic regulator DegQ.ConclusionsTo our knowledge, this is the first report on simultaneous production of four iturin A homologs (C14-C17) by a Bacillus strain. In addition, this study suggests that metabolic engineering in combination with culture conditions optimization may be a feasible method for enhanced production of bacterial secondary metabolites.
| S-EPMC6457013 | biostudies-literature
Project description:BackgroundGenome reduction and metabolic engineering have emerged as intensive research hotspots for constructing the promising functional chassis and various microbial cell factories. Surfactin, a lipopeptide-type biosurfactant with broad spectrum antibiotic activity, has wide application prospects in anticancer therapy, biocontrol and bioremediation. Bacillus amyloliquefaciens LL3, previously isolated by our lab, contains an intact srfA operon in the genome for surfactin biosynthesis.ResultsIn this study, a genome-reduced strain GR167 lacking ~ 4.18% of the B. amyloliquefaciens LL3 genome was constructed by deleting some unnecessary genomic regions. Compared with the strain NK-1 (LL3 derivative, ΔuppΔpMC1), GR167 exhibited faster growth rate, higher transformation efficiency, increased intracellular reducing power level and higher heterologous protein expression capacity. Furthermore, the chassis strain GR167 was engineered for enhanced surfactin production. Firstly, the iturin and fengycin biosynthetic gene clusters were deleted from GR167 to generate GR167ID. Subsequently, two promoters PRsuc and PRtpxi from LL3 were obtained by RNA-seq and promoter strength characterization, and then they were individually substituted for the native srfA promoter in GR167ID to generate GR167IDS and GR167IDT. The best mutant GR167IDS showed a 678-fold improvement in the transcriptional level of the srfA operon relative to GR167ID, and it produced 311.35 mg/L surfactin, with a 10.4-fold increase relative to GR167.ConclusionsThe genome-reduced strain GR167 was advantageous over the parental strain in several industrially relevant physiological traits assessed and it was highlighted as a chassis strain for further genetic modification. In future studies, further reduction of the LL3 genome can be expected to create high-performance chassis for synthetic biology applications.
| S-EPMC7720510 | biostudies-literature
Project description:Poly-γ-glutamic acid (γ-PGA) is a biocompatible and biodegradable polypeptide with wide-ranging applications in foods, cosmetics, medicine, agriculture and wastewater treatment. Bacillus amyloliquefaciens LL3 can produce γ-PGA from sucrose that can be obtained easily from sugarcane and sugar beet. In our previous work, it was found that low intracellular glutamate concentration was the limiting factor for γ-PGA production by LL3. In this study, the γ-PGA synthesis by strain LL3 was enhanced by chromosomally engineering its glutamate metabolism-relevant networks. First, the downstream metabolic pathways were partly blocked by deleting fadR, lysC, aspB, pckA, proAB, rocG and gudB. The resulting strain NK-A6 synthesized 4.84 g l-1 γ-PGA, with a 31.5% increase compared with strain LL3. Second, a strong promoter PC 2up was inserted into the upstream of icd gene, to generate strain NK-A7, which further led to a 33.5% improvement in the γ-PGA titre, achieving 6.46 g l-1 . The NADPH level was improved by regulating the expression of pgi and gndA. Third, metabolic evolution was carried out to generate strain NK-A9E, which showed a comparable γ-PGA titre with strain NK-A7. Finally, the srf and itu operons were deleted respectively, from the original strains NK-A7 and NK-A9E. The resulting strain NK-A11 exhibited the highest γ-PGA titre (7.53 g l-1 ), with a 2.05-fold improvement compared with LL3. The results demonstrated that the approaches described here efficiently enhanced γ-PGA production in B. amyloliquefaciens fermentation.
| S-EPMC6680638 | biostudies-literature
Project description:1-deoxynojirimycin (DNJ) is a well-known α-glucosidase inhibitor. A series of phenyltriazole-deoxynojirimycin hybrids containing C4 and C6 (4 and 6 methylenes, respectively) linkers were synthesized. These novel compounds were assessed for preliminary glucosidase inhibition and cytotoxicity tests in vitro. Among them, compounds 12-14 and 16-20 (IC50: 105 ± 9-11 ± 1 μM) were more active than deoxynojirimycin (DNJ, IC50 = 155 ± 15 μM). The kinetics of enzyme inhibition measured by using Lineweaver-Burk plots indicated that compounds 18 and 19 were competitive inhibitors. In addition, a molecular docking study of α-glucosidase revealed that the interaction modes and the orientations of compound 18 and DNJ were clearly different. Furthermore, in tissue culture, HL60 cell compounds showed no cytotoxicity at low concentrations. When the concentration reached 50 µM, only compound 20 exhibited cytotoxicity. The structure-activity relationships exhibit that the length of the linker and the nature of 4-position substituents on the phenyl have a significant effect on the inhibitory potency of glucosidases and cytotoxicity.
| S-EPMC11547804 | biostudies-literature
Project description:Bacillus amyloliquefaciens is the dominant strain used to produce γ-polyglutamic acid from inulin, a non-grain raw material. B. amyloliquefaciens has a highly efficient tricarboxylic acid cycle metabolic flux and glutamate synthesis ability. These features confer great potential for the synthesis of glutamate derivatives. However, it is challenging to efficiently convert high levels of glutamate to a particular glutamate derivative. Here, we conducted a systematic study on the biosynthesis of L-ornithine by B. amyloliquefaciens using inulin. First, the polyglutamate synthase gene pgsBCA of B. amyloliquefaciens NB was knocked out to hinder polyglutamate synthesis, resulting in the accumulation of intracellular glutamate and ATP. Second, a modular engineering strategy was applied to coordinate the degradation pathway, precursor competition pathway, and L-ornithine synthesis pathway to prompt high levels of intracellular precursor glutamate for l-ornithine synthesis. In addition, the high-efficiency L-ornithine transporter was further screened and overexpressed to reduce the feedback inhibition of L-ornithine on the synthesis pathway. Combining these strategies with further fermentation optimizations, we achieved a final L-ornithine titer of 31.3 g/L from inulin. Overall, these strategies hold great potential for strengthening microbial synthesis of high value-added products derived from glutamate.
| S-EPMC9214239 | biostudies-literature
Project description:Terpenoids are the largest group of small-molecule natural products, with more than 60,000 compounds made from isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP). As the most diverse group of small-molecule natural products, terpenoids play an important role in the pharmaceutical, food, and cosmetic industries. For decades, Escherichia coli (E. coli) and Saccharomyces cerevisiae (S. cerevisiae) were extensively studied to biosynthesize terpenoids, because they are both fully amenable to genetic modifications and have vast molecular resources. On the other hand, our literature survey (20 years) revealed that terpenoids are naturally more widespread in Bacillales. In the mid-1990s, an inherent methylerythritol phosphate (MEP) pathway was discovered in Bacillus subtilis (B. subtilis). Since B. subtilis is a generally recognized as safe (GRAS) organism and has long been used for the industrial production of proteins, attempts to biosynthesize terpenoids in this bacterium have aroused much interest in the scientific community. This review discusses metabolic engineering of B. subtilis for terpenoid production, and encountered challenges will be discussed. We will summarize some major advances and outline future directions for exploiting the potential of B. subtilis as a desired "cell factory" to produce terpenoids.
| S-EPMC4628092 | biostudies-literature
Project description:Acetoin is a potential platform compound for a variety of chemicals. Bacillus licheniformis MW3, a thermophilic and generally regarded as safe (GRAS) microorganism, can produce 2,3-butanediol with a high concentration, yield, and productivity. In this study, B. licheniformis MW3 was metabolic engineered for acetoin production. After deleting two 2,3-butanediol dehydrogenases encoding genes budC and gdh, an engineered strain B. licheniformis MW3 (ΔbudCΔgdh) was constructed. Using fed-batch fermentation of B. licheniformis MW3 (ΔbudCΔgdh), 64.2 g/L acetoin was produced at a productivity of 2.378 g/[L h] and a yield of 0.412 g/g from 156 g/L glucose in 27 h. The fermentation process exhibited rather high productivity and yield of acetoin, indicating that B. licheniformis MW3 (ΔbudCΔgdh) might be a promising acetoin producer.
| S-EPMC7047894 | biostudies-literature
Project description:Commercial carotenoid production is dominated by chemical synthesis and plant extraction, both of which are unsustainable and can be detrimental to the environment. A promising alternative for the mass production of carotenoids from both an ecological and commercial perspective is microbial synthesis. To date, C30 carotenoid production in Bacillus subtilis has been achieved using plasmid systems for the overexpression of biosynthetic enzymes. In the present study, we employed a clustered regularly interspaced short palindromic repeat-Cas9 (CRISPR-Cas9) system to develop an efficient, safe, and stable C30 carotenoid-producing B. subtilis strain, devoid of plasmids and antibiotic selection markers. To this end, the expression levels of crtM (dehydrosqualene synthase) and crtN (dehydrosqualene desaturase) genes from Staphylococcus aureus were upregulated by the insertion of three gene copies into the chromosome of B. subtilis. Subsequently, the supply of the C30 carotenoid precursor farnesyl diphosphate (FPP), which is the substrate for CrtMN enzymes, was enhanced by expressing chromosomally integrated Bacillus megaterium-derived farnesyl diphosphate synthase (FPPS), a key enzyme in the FPP pathway, and abolishing the expression of farnesyl diphosphate phosphatase (YisP), an enzyme responsible for the undesired conversion of FPP to farnesol. The consecutive combination of these features resulted in a stepwise increased production of C30 carotenoids. For the first time, a B. subtilis strain that can endogenously produce C30 carotenoids has been constructed, which we anticipate will serve as a chassis for further metabolic engineering and fermentation optimization aimed at developing a commercial scale bioproduction process.
| S-EPMC10148934 | biostudies-literature
Project description:The biosynthesis of antifungal lipopeptides iturin and fengycin has attracted broad interest; however, there is a bottleneck in its low yield in wild strains. Because the key metabolic mechanisms in the lipopeptides synthesis pathway remain unclear, genetic engineering approaches are all ending up with a single or a few gene modifications. The aim of this study is to develop a systematic engineering approach to improve the antifungal activity and biosynthesis of iturin and fengycin in Bacillus amyloliquefaciens. First, blocking the carbon overflow metabolic pathway to increase precursor supply of the branched-chain amino acids by knockout of bdh, disrupting sporulation to extend the stage for producing antifungal lipopeptides by deletion of kinA, blocking of siderophore synthesis to enhance the availability of amino acids and fatty acids by deletion of dhbF, and increasing Spo0A∼P by deletion of rapA, could improve the antifungal activity by 24%, 10%, 13% and 18%, respectively. Second, the double knockout strain ΔbdhΔkinA, triple knockout strain ΔbdhΔkinAΔdhbF and quadruple knockout strain ΔkinAΔbdhΔdhbFΔrapA could improve the antifungal activity by 38%, 44% and 53%, respectively. Finally, overexpression of sfp in ΔkinAΔbdhΔdhbFΔrapA further increased the antifungal activity by 65%. After purifying iturin and fengycin as standards for quantitative analysis of lipopeptides, we found the iturin titer was 17.0 mg/L in the final engineered strain, which was 3.2-fold of the original strain. After fermentation optimization, the titer of iturin and fengycin reached 31.1 mg/L and 175.3 mg/L in flask, and 123.5 mg/L and 1200.8 mg/L in bioreactor. Compared to the original strain, the iturin and fengycin titer in bioreactor increased by 22.8-fold and 15.9-fold in the final engineered strain, respectively. This study may pave the way for the commercial production of green antifungal lipopeptides, and is also favorable for understanding the regulatory and biosynthetic mechanism of iturin and fengycin.
| S-EPMC9490133 | biostudies-literature
Project description:Alkaline protease has been widely applied in food, medicine, environmental protection and other industrial fields. However, the current activity and yield of alkaline protease cannot meet the demand. Therefore, it is important to identify new alkaline proteases with high activity. In this study, we cloned a potential alkaline protease gene bsp-1 from a Bacillus subtilis strain isolated in our laboratory. BSP-1 shows the highest sequence similarity to subtilisin NAT (S51909) from B. subtilis natto. Then, we expressed BSP-1 in Bacillus amyloliquefaciens BAX-9 and analyzed the protein expression level under a collection of promoters. The results show that the P43 promoter resulted in the highest transcription level, protein level and enzyme activity. Finally, we obtained a maximum activity of 524.12 U/mL using the P43 promoter after fermentation medium optimization. In conclusion, this study identified an alkaline protease gene bsp-1 from B. subtilis and provided a new method for high-efficiency alkaline protease expression in B. amyloliquefaciens.
| S-EPMC9468883 | biostudies-literature