Project description:The genus Streptomyces is known for its secondary metabolite biosynthetic capacities. We report here the draft genome sequence of the most extensively studied ?-poly-l-lysine producer, Streptomyces albulus NBRC14147. Bioinformatic analysis of the 9.6-Mb chromosome identified a large number of secondary metabolite biosynthetic gene clusters.
Project description:We determined the complete genome sequence of a soil bacterium, Streptomyces albulus NK660. It can produce ?-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.
Project description:Streptomyces albulus has commercially been used for the production of ?-poly-L-lysine (?-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 ?-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 ?-PL via adaptive evolution or metabolic engineering.
Project description:The biosynthesis of the antibiotic epsilon-poly-lysine (?-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 ?-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 ?-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 ?-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 ?-PL yield improvement and industrial bioprocess optimization.
Project description:Wuyiencin is produced by Streptomyces albulus var. wuyiensis and used widely in agriculture to control a variety of fungal diseases, such as cucumber downy mildew, strawberry powdery mildew, and tomato gray mold. As an industrially-produced biopesticide, reducing production costs is very important for popularization of this approach. To obtain a rapidly growing strain that effectively shortens the fermentation time, we investigated the effects of knockout and overexpression of the wysPII gene, a member of the LuxR regulatory gene family, in S. albulus strain CK-15. The ?wysPII mutant exhibited a reduced rate of growth and sporulation. The time taken to reach the greatest mycelial biomass was approximately 18 h shorter in the ooPII (wysPII overexpressing) strain compared with that of the wild-type (WT) strain. In addition, the time to reach the greatest wuyiencin production was 56 h in the ooPII strain compared with 62 h in the WT strain. Furthermore, wysPII was shown to act as an activator of morphological development without affecting wuyiencin production. Thus, the ooPII strain can be used to reduce costs and increase efficiency in industrial fermentation processes for wuyiencin production.
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 ?-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.
Project description:?-Poly-L-lysine (?-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. ?-PL synthase is the key enzyme responsible for the polymerization of L-lysine to form ?-PL. In this study, the ?-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 ?C31, which resulted in a genetically engineered strain Q-PL2. The titers of ?-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 ?-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 ?-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 ?-PL synthase is one of the rate-limiting enzymes in ?-PL synthesis pathway and lays a foundation for further improving the ?-PL production ability of S. albulus by metabolic engineering.