Genome Sequence of the ?-Poly-l-Lysine-Producing Strain Streptomyces albulus NK660, Isolated from Soil in Gutian, Fujian Province, China.
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SUBMITTER: Gu Y
PROVIDER: S-EPMC4056293 | biostudies-literature | 2014 Jun
REPOSITORIES: biostudies-literature
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Genome Sequence of the ε-Poly-l-Lysine-Producing Strain Streptomyces albulus NK660, Isolated from Soil in Gutian, Fujian Province, China.
Gu Yanyan Y Yang Chao C Wang Xiaomeng X Geng Weitao W Sun Yang Y Feng Jun J Wang Yuanyuan Y Quan Yufen Y Che You Y Zhang Chi C Gong Ting T Zhang Wei W Gao Weixia W Zuo Zhenqiang Z Song Cunjiang C Wang Shufang S
Genome announcements 20140612 3
We determined the complete genome sequence of a soil bacterium, Streptomyces albulus NK660. It can produce ε-poly-l-lysine, which has antimicrobial activity against a spectrum of microorganisms. The genome of S. albulus NK660 contains a 9,360,281-bp linear chromosome and a 12,120-bp linear plasmid. ...[more]
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Project description:ε-Poly-L-lysine (ε-PL), showing a wide range of antimicrobial activity, is now industrially produced as a food additive by a fermentation process. A new strain capable of producing ε-PL was isolated from a soil sample collected from Gutian, Fujian Province, China. Based on its morphological and biochemical features and phylogenetic similarity with 16S rRNA gene, the strain was identified as Streptomyces albulus and named NK660. The yield of ε-PL in 30 l fed-batch fermentation with pH control was 4.2 g l(-1) when using glycerol as the carbon source. The structure of ε-PL was determined by nuclear magnetic resonance (NMR) and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). Previous studies have shown that the antimicrobial activity of ε-PL is dependent on its molecular size. In this study, the polymerization degree of the ε-PL produced by strain NK660 ranged from 19 to 33 L-lysine monomers, with the main component consisting of 24-30 L-lysine monomers, which implied that the ε-PL might have higher antimicrobial activity. Furthermore, the ε-PL synthetase gene (pls) was cloned from strain NK660 by genome walking. The pls gene with its native promoter was heterologously expressed in Streptomyces lividans ZX7, and the recombinant strain was capable of synthesizing ε-PL. Here, we demonstrated for the first time heterologous expression of the pls gene in S. lividans. The heterologous expression of pls gene in S. lividans will open new avenues for elucidating the molecular mechanisms of ε-PL synthesis.
| S-EPMC3937719 | biostudies-literature
Project description:BackgroundAdpA is a global regulator of morphological differentiation and secondary metabolism in Streptomyces, but the regulatory roles of the Streptomyces AdpA family on the biosynthesis of the natural product ε-poly-L-lysine (ε-PL) remain unidentified, and few studies have focused on increasing the production of ε-PL by manipulating transcription factors in Streptomyces.ResultsIn this study, we revealed the regulatory roles of different AdpA homologs in ε-PL biosynthesis and morphological differentiation and effectively promoted ε-PL production and sporulation in Streptomyces albulus NK660 by heterologously expressing adpA from S. neyagawaensis NRRLB-3092 (adpASn). First, we identified a novel AdpA homolog named AdpASa in S. albulus NK660 and characterized its function as an activator of ε-PL biosynthesis and morphological differentiation. Subsequently, four heterologous AdpA homologs were selected to investigate their phylogenetic relationships and regulatory roles in S. albulus, and AdpASn was demonstrated to have the strongest ability to promote both ε-PL production and sporulation among these five AdpA proteins. The ε-PL yield of S. albulus heterologously expressing adpASn was approximately 3.6-fold higher than that of the control strain. Finally, we clarified the mechanism of AdpASn in enhancing ε-PL biosynthesis and its effect on ε-PL polymerization degree using real-time quantitative PCR, microscale thermophoresis and MALDI-TOF-MS. AdpASn was purified, and its seven direct targets, zwf, tal, pyk2, pta, ack, pepc and a transketolase gene (DC74_2409), were identified, suggesting that AdpASn may cause the redistribution of metabolic flux in central metabolism pathways, which subsequently provides more carbon skeletons and ATP for ε-PL biosynthesis in S. albulus.ConclusionsHere, we characterized the positive regulatory roles of Streptomyces AdpA homologs in ε-PL biosynthesis and their effects on morphological differentiation and reported for the first time that AdpASn promotes ε-PL biosynthesis by affecting the transcription of its target genes in central metabolism pathways. These findings supply valuable insights into the regulatory roles of the Streptomyces AdpA family on ε-PL biosynthesis and morphological differentiation and suggest that AdpASn may be an effective global regulator for enhanced production of ε-PL and other valuable secondary metabolites in Streptomyces.
| S-EPMC8994273 | biostudies-literature
Project description:ε-Poly-L-lysine (ε-PL) is a natural amino acid polymer produced by microbial fermentation. It has been mainly used as a preservative in the food and cosmetics industries, as a drug carrier in medicines, and as a gene carrier in gene therapy. ε-PL synthase is the key enzyme responsible for the polymerization of L-lysine to form ε-PL. In this study, the ε-PL synthase gene was overexpressed in Streptomyces albulus CICC 11022 by using the kasOp∗ promoter and the ribosome binding site from the capsid protein of phage ϕC31, which resulted in a genetically engineered strain Q-PL2. The titers of ε-PL produced by Q-PL2 were 88.2% ± 8.3% higher than that produced by the wild strain in shake flask fermentation. With the synergistic effect of 2 g/L sodium citrate, the titers of ε-PL produced by Q-PL2 were 211.2% ± 17.4% higher than that produced by the wild strain. In fed-batch fermentations, 20.1 ± 1.3 g/L of ε-PL was produced by S. albulus Q-PL2 in 72 h with a productivity of 6.7 ± 0.4 g/L/day, which was 3.2 ± 0.3-fold of that produced by the wild strain. These results indicate that ε-PL synthase is one of the rate-limiting enzymes in ε-PL synthesis pathway and lays a foundation for further improving the ε-PL production ability of S. albulus by metabolic engineering.
| S-EPMC7188835 | biostudies-literature
Project description:Hyaluronic acid (HA) is used in a wide range of medical applications, where its performance and therapeutic efficacy are highly dependent on its molecular weight. In the microbial production of HA, it has been suggested that a high level of intracellular ATP enhances the productivity and molecular weight of HA. Here, we report on heterologous HA production in an ε-poly-l-lysine producer, Streptomyces albulus, which has the potential to generate ATP at high level. The hasA gene from Streptococcus zooepidemicus, which encodes HA synthase, was refactored and expressed under the control of a late-log growth phase-operating promoter. The expression of the refactored hasA gene, along with genes coding for UDP-glucose dehydrogenase, UDP-N-acetylglucosamine pyrophosphorylase, and UDP-glucose pyrophosphorylase, which are involved in HA precursor sugar biosynthesis, resulted in efficient production of HA in the 2.0 MDa range, which is greater than typical bacterial HA, demonstrating that a sufficient amount of ATP was provided to support the biosynthesis of the precursor sugars, which in turn promoted HA production. In addition, unlike in the case of streptococcal HA, S. albulus-derived HA was not cell associated. Based on these findings, our heterologous production system appears to have several advantages for practical HA production. We propose that the present system could be applicable to the heterologous production of a wide variety of molecules other than HA in the case their biosynthesis pathways require ATP in vivo.
| S-EPMC4421038 | biostudies-literature
Project description:The biosynthesis of the antibiotic epsilon-poly-lysine (ε-PL) in Streptomyces albulus is performed by polylysine synthase (pls); however, the regulatory mechanism of this process is still unknown. Here, we first obtained the complete genome sequence of S. albulus ZPM, which consists of 9,784,577 bp and has a GC content of 72.2%. The genome houses 44 gene clusters for secondary metabolite biosynthesis, in which 20 gene clusters are involved in the biosynthesis of polyketides and nonribosomally synthesized peptides. High-throughput sequencing was further performed, and genetic variants were identified from pooled libraries consisting of the 30 highest-yield mutants or 30 lowest-yield mutants. More than 350 genetic variants associated with ε-PL yield have been identified. One hundred sixty-two affected proteins, from important metabolic enzymes to novel transcriptional regulators, were identified as being related to ε-PL synthesis. HrdD, one of the affected genes, is a sigma factor that shows the most sensitive response to pH change and contains a non-synonymous mutation (A132V) in mutant strains with lower ε-PL yields. Electrophoretic mobility shift assays showed that the pls gene is likely regulated by transcriptional activator HrdD. The data obtained in this study will facilitate future studies on ε-PL yield improvement and industrial bioprocess optimization.
| S-EPMC4361855 | biostudies-literature
Project description:Streptomyces albulus has commercially been used for the production of ε-poly-L-lysine (ε-PL), a natural food preservative, where acid stress is inevitably encountered in the biosynthesis process. To elucidate the acid tolerance response (ATR), a comparative physiology and transcriptomic analysis of S. albulus M-Z18 at different environmental pH (5.0, 4.0, and 3.0) was carried out. In response to acid stress, cell envelope regulated the membrane fatty acid composition and chain length to reduce damage. Moreover, intracellular pH homeostasis was maintained by increasing H+-ATPase activity and intracellular ATP and amino acid (mainly arginine, glutamate, aspartate and lysine) concentrations. Transcriptional analysis based on RNA-sequencing indicated that acid stress aroused global changes and the differentially expressed genes involved in transcriptional regulation, stress-response protein, transporter, cell envelope, secondary metabolite biosynthesis, DNA and RNA metabolism and ribosome subunit. Consequently, the ATR of S. albulus was preliminarily proposed. Notably, it is indicated that the biosynthesis of ε-PL is also a response mechanism for S. albulus to combat acid stress. These results provide new insights into the ATR of S. albulus and will contribute to the production of ε-PL via adaptive evolution or metabolic engineering.
| S-EPMC7317143 | biostudies-literature
Project description:Introductionε-poly-L-lysine (ε-PL) is a high value, widely used natural antimicrobial peptide additive for foods and cosmetic products that is mainly produced by Streptomyces albulus. In previous work, we developed the high-yield industrial strain S. albulus WG-608 through successive rounds of engineering.MethodsHere, we use integrated physiological, transcriptomic, and proteomics association analysis to resolve the complex mechanisms underlying high ε-PL production by comparing WG-608 with the progenitor strain M-Z18.ResultsOur results show that key genes in the glycolysis, pentose phosphate pathway, glyoxylate pathway, oxidative phosphorylation, and L-lysine biosynthesis pathways are differentially upregulated in WG-608, while genes in the biosynthetic pathways for fatty acids, various branched amino acids, and secondary metabolite by-products are downregulated. This regulatory pattern results in the introduction of more carbon atoms into L-lysine biosynthesis and ε-PL production. In addition, significant changes in the regulation of DNA replication, transcription, and translation, two component systems, and quorum sensing may facilitate the adaptability to environmental pressure and the biosynthesis of ε-PL. Overexpression of ppk gene and addition of polyP6 further enhanced the ε-PL production.DiscussionThis study enables comprehensive understanding of the biosynthetic mechanisms of ε-PL in S. albulus WG-608, while providing some genetic modification and fermentation strategies to further improve the ε-PL production.
| S-EPMC10157215 | biostudies-literature
Project description:ε-Poly-L-lysine (ε-PL) is a food additive produced by Streptomyces and is widely used in many countries. Working with Streptomyces albulus FEEL-1, we established a method to activate ε-PL synthesis by successive introduction of multiple antibiotic-resistance mutations. Sextuple mutant R6 was finally developed by screening for resistance to six antibiotics and produced 4.41 g/L of ε-PL in a shake flask, which is 2.75-fold higher than the level produced by the parent strain. In a previous study, we constructed a double-resistance mutant, SG-31, with high ε-PL production of 3.83 g/L and 59.50 g/L in a shake flask and 5-L bioreactor, respectively. However, we found that R6 did not show obvious advantages in fed-batch fermentation when compared with SG-31. For further activation of ε-PL synthesis ability, we optimized the fermentation process by using an effective acidic pH shock strategy, by which R6 synthetized 70.3 g/L of ε-PL, 2.79-fold and 1.18-fold greater than that synthetized by FEEL-1 and SG-31, respectively. To the best of our knowledge, this is the highest reported ε-PL production to date. This ε-PL overproduction may be due to the result of R99P and Q856H mutations in ribosomal protein S12 and RNA polymerase, respectively, which may be responsible for the increased transcription of the ε-poly-lysine synthetase gene (pls) and key enzyme activities in the Lys synthesis metabolic pathway. Consequently, ε-PL synthetase activity, intracellular ATP, and Lys concentrations were improved and directly contributed to ε-PL overproduction. This study combined ribosome engineering, high-throughput screening, and targeted strategy optimization to accelerate ε-PL production and probe the fermentation characteristics of hyperyield mutants. The information presented here may be useful for other natural products produced by Streptomyces.
| S-EPMC6528598 | biostudies-literature
Project description:Purpose: Streptomyces albulus is an industrial producer of ε-poly-L-lysine, an antimicrobial cationic homo poly-amino acid used practically as a natural food preservative. Here, we present RNA sequencing data set unveiling differentially expressed transcripts during ε-poly-L-lysine production in the most extensively studied poly-L-Lys producer, S. albulus NBRC14147. Methods: During the poly-L-Lys fermentation, cells grown for 8 hours and 35 hours were harvested as growth phase cells and production phase cells, respectively, and total RNA were extracted individually. A 100-bp paired-end mRNA sequencing was performed for each sample on the Illumina HiSeq 2500 system. Result: Using an optimized data analysis workflow, we were able to map more than 44 million sequence reads per sample to the reference genome (GenBank accession number ASM385166v1). Differential gene expression analysis was performed using the edgeR. The RNA-seq data revealed that a total of 2449 genes were considered to be differentially expressed during poly-L-Lys production using a fold change cutoff of log2 less than -1 and greater than 1 (equivalent to a ±2-fold change). Conclusion: Our data will serve as a primary source for investigating the regulatory mechanism which govern poly-L-Lys production in S. albulus NBRC14147.
2022-01-19 | GSE192684 | GEO
Project description:ε-Poly-L-lysine (ε-PL), a natural food preservative, has recently gained interest and mainly produced by Streptomyces albulus. Lacking of efficient breeding methods limit ε-PL production improving, knockout byproducts and increase of main product flux strategies as a logical solution to increase yield. However, removing byproduct formation and improving main product synthesis has seen limited success due to the genetic background of ε-PL producing organism is not clear. To overcome this limitation, random mutagenesis continues to be the best way towards improving strains for ε-PL production. Recent advances in Illumina sequencing opened new avenues to understand improved strains. In this work, we used genome shuffling on strains obtained by ribosome engineering to generate a better ε-PL producing strain. The mutant strain SG-86 produced 144.7% more ε-PL than the parent strain M-Z18. Except that SG-86 displayed obvious differences in morphology and ATP compared to parent strain M-Z18. Using Illumina sequencing, we mapped the genomic changes leading to the improved phenotype. Sequencing two strains showed that the genome of the mutant strain was about 2.1 M less than that of the parent strain, including a large number of metabolic pathways, secondary metabolic gene clusters, and gene deletions. In addition, there are many SNPs (single nucleotide polymorphisms) and InDels (insertions and deletions) in the mutant strain. Based on the results of data analysis, a mechanism of ε-PL overproduction in S. albulus SG-86 was preliminarily proposed. This study is of great significance for improving the fermentation performance and providing theoretical guidance for the metabolic engineering construction of ε-PL producing strains.
| S-EPMC9195005 | biostudies-literature