Streptomyces bacteria, known for their proficiency in producing secondary metabolites, have emerged as promising hosts for heterologous protein expression. In the realm of biotechnology, the heterologous production of recombinant proteins is gaining traction due to its advantages in terms of scalability, productivity, and versatility in applications. Streptomyces species offer a unique set of characteristics that make them attractive hosts for this purpose. While they excel in expressing various secondary metabolite biosynthetic gene clusters and secretory enzymes, challenges such as low yield, genetic manipulation difficulties, and complex cellular features have hindered their widespread use. In this comprehensive review, we delve into the rational engineering strategies employed to optimize the heterologous production of secondary metabolites and recombinant proteins in Streptomyces species, focusing on genetic tool development and chassis construction. By exploring the design-build-test-learn cycle in systems, we propose strategies to enhance the efficacy of Streptomyces as a chassis for heterologous protein expression.
Streptomyces as a Host for Heterologous Protein Expression
When it comes to industrial-scale protein production, the selection of a suitable heterologous expression host is paramount. Various hosts like E. coli, Bacillus subtilis, Saccharomyces cerevisiae, and Chinese hamster ovary (CHO) cells have been extensively used due to their distinct advantages. Streptomyces, a soil-derived Gram-positive bacterium, stands out as an attractive microbial host for heterologous protein expression. Its robust and scalable growth, coupled with efficient protein secretion systems, makes Streptomyces a favorable candidate for recombinant protein production. The extracellular milieu of Streptomyces provides an oxidizing environment conducive to proper protein folding, a crucial aspect often impeded in the reducing cytoplasmic environment of other bacteria. Moreover, the absence of lipopolysaccharides (LPSs) in Streptomyces simplifies downstream purification processes by ensuring minimal contamination with intracellular proteins.
Streptomyces as a Host for Secondary Metabolite Biosynthetic Gene Clusters
Diving deeper into Streptomyces’ potential as a chassis for heterologous expression, we uncover its proficiency in hosting secondary metabolite biosynthetic gene clusters (BGCs). With an average of 36.5 BGCs per genome, Streptomyces species boast a rich repertoire of biosynthetic enzymes, essential for synthesizing diverse secondary metabolites. The complex structures of these enzymes necessitate a conducive cellular environment to maintain their functionality, a requirement well-met by Streptomyces hosts. Unlike some other microorganisms, Streptomyces does not necessitate additional codon optimization for efficient translation of GC-rich BGC sequences. The redox state of Streptomyces’ cytoplasm aligns well with the native host, fostering correct disulfide bond formation critical for enzyme functionality. Additionally, the presence of chaperones further enhances the functionality of biosynthetic enzymes, underscoring Streptomyces’ competency as a host for BGC expression.
Rational Engineering Strategies for Heterologous Expression in Streptomyces
To harness Streptomyces’ potential as a chassis for heterologous protein expression, rational engineering strategies play a pivotal role. Genetic tools tailored for BGC heterologous expression in Streptomyces have been developed to streamline the cloning and assembly processes. Various cloning strategies, both in vivo and in vitro, have been devised to efficiently capture large BGCs into vectors for expression in Streptomyces hosts. The advent of tools like plasmid Streptomyces bacterial artificial chromosome (pSBAC), linear-to-circular homologous recombination (LCHR), and transformation-associated recombination (TAR) cloning has revolutionized the efficiency and fidelity of BGC cloning in Streptomyces.
Genetic Toolkits and Chassis Development for Streptomyces
In the realm of genetic toolkits, the choice of vectors, promoters, terminators, and riboswitches plays a crucial role in optimizing gene expression in Streptomyces hosts. Replicative vectors based on SCP2 replicon and integrative vectors utilizing ΦC31 and ΦBT1 integrases have been instrumental in facilitating stable gene expression in Streptomyces. Moreover, the strategic selection of promoters like ermE series, kasOp*, and synthetic promoters has enhanced gene expression efficiency in Streptomyces hosts. The inclusion of terminators like TD1 and synthetic bi-directional transcriptional terminator B (ttsbiB) further ensures precise gene regulation in Streptomyces.
Conclusion
In conclusion, Streptomyces bacteria present a compelling microbial chassis for heterologous protein expression and secondary metabolite biosynthetic gene cluster hostings due to their robust growth, efficient secretion systems, and genetic manipulability. By leveraging rational engineering approaches and sophisticated genetic toolkits, the challenges associated with low yield and genetic manipulation in Streptomyces hosts can be effectively addressed. Through continuous advancements in genetic tools and chassis development, Streptomyces is poised to play a pivotal role in the realm of biotechnology and synthetic biology, offering a versatile platform for diverse applications.
Key Takeaways:
- Streptomyces bacteria offer a promising chassis for heterologous protein expression and secondary metabolite biosynthetic gene cluster hosting.
- Rational engineering strategies and advanced genetic toolkits have enhanced the efficiency of gene cloning and expression in Streptomyces hosts.
- Streptomyces’ unique characteristics, including robust growth, efficient secretion systems, and genetic manipulability, position it as a versatile platform for biotechnological applications.
- Continuous advancements in genetic tool development and chassis optimization further elevate Streptomyces’ potential as a preferred host for recombinant protein production and secondary metabolite synthesis.
Tags: automation, transcriptomics, codon optimization, genome editing, protein folding, secretion, regulatory, synthetic biology, chaperones, protein purification
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