Rational search of genetic design space for a heterologous terpene metabolic pathway in Streptomyces.

Modern tools in DNA synthesis and assembly give genetic engineers control over the nucleotide-level design of complex, multi-gene systems. Systematic approaches to explore genetic design space and optimize the performance of genetic constructs are lacking. Here we explore the application of a five-level Plackett-Burman fractional factorial design to improve the titer of a heterologous terpene biosynthetic pathway in Streptomyces. A library of 125 engineered gene clusters encoding the production of diterpenoid ent-atiserenoic acid (eAA) via the methylerythritol phosphate pathway was constructed and introduced into Streptomyces albidoflavus J1047 for heterologous expression. The eAA production titer varied within the library by over two orders of magnitude and host strains showed unexpected and reproducible colony morphology phenotypes. Analysis of Plackett-Burman design identified expression of dxs, the gene encoding the first and the flux-controlling enzyme, having the strongest impact on eAA titer, but with a counter-intuitive negative correlation between dxs expression and eAA production. Finally, simulation modeling was performed to determine how several plausible sources of experimental error/noise and non-linearity impact the utility of Plackett-Burman analyses.

Complete genome sequences of Streptomyces spp. isolated from disease-suppressive soils.

BACKGROUND: Bacteria within the genus Streptomyces remain a major source of new natural product discovery and as soil inoculants in agriculture where they promote plant growth and protect from disease. Recently, Streptomyces spp. have been implicated as important members of naturally disease-suppressive soils. To shine more light on the ecology and evolution of disease-suppressive microbial communities, we have sequenced the genome of three Streptomyces strains isolated from disease-suppressive soils and compared them to previously sequenced isolates. Strains selected for sequencing had previously showed strong phenotypes in competition or signaling assays. RESULTS: Here we present the de novo sequencing of three strains of the genus Streptomyces isolated from disease-suppressive soils to produce high-quality complete genomes. Streptomyces sp. GS93-23, Streptomyces sp. 3211-3, and Streptomyces sp. S3-4 were found to have linear chromosomes of 8.24 Mb, 8.23 Mb, and greater than 7.5 Mb, respectively. In addition, two of the strains were found to have large, linear plasmids. Each strain harbors between 26 and 38 natural product biosynthetic gene clusters, on par with previously sequenced Streptomyces spp. We compared these newly sequenced genomes with those of previously sequenced organisms. We see substantial natural product biosynthetic diversity between closely related strains, with the gain/loss of episomal DNA elements being a primary driver of genome evolution. CONCLUSIONS: Long read sequencing data facilitates large contig assembly for high-GC Streptomyces genomes. While the sample number is too small for a definitive conclusion, we do not see evidence that disease suppressive soil isolates are particularly privileged in terms of numbers of biosynthetic gene clusters. The strong sequence similarity between GS93-23 and previously isolated Streptomyces lydicus suggests that species recruitment may contribute to the evolution of disease-suppressive microbial communities.

Semisynthesis of the Neuroprotective Metabolite, Serofendic Acid.

Serofendic acid is a natural neuroprotective molecule found in fetal calf serum. It is able to protect neurons against mechanisms of cell death associated with neurodegenerative disease. Because only trace quantities are present in fetal calf serum and complete chemical syntheses are long and inefficient, its development as a therapeutic agent has been slow. We engineered a heterologous metabolic pathway in Streptomyces to produce a late-stage synthetic intermediate, ent-atiserenoic acid, at high titers. We completed the total synthesis of serofendic acid from this intermediate in four steps.

Designing and Implementing Algorithmic DNA Assembly Pipelines for Multi-Gene Systems.

Advances in DNA synthesis and assembly technology allow for the high-throughput fabrication of hundreds to thousands of multi-part genetic constructs in a short time. This allows for rapid hypothesis-testing and genetic optimization in multi-gene biological systems. Here, we discuss key considerations to design and implement an algorithmic DNA assembly pipeline that provides the freedom to change nearly any design variable in a multi-gene system. In addition to considerations for pipeline design, we describe protocols for three useful molecular biology techniques in plasmid construction.

Genome Sequences for Streptomyces spp. Isolated from Disease-Suppressive Soils and Long-Term Ecological Research Sites.

We report here the high-quality genome sequences of three Streptomyces spp. isolated as part of a long-term study of microbial soil ecology. Streptomyces sp. strain GS93-23 was isolated from naturally disease-suppressive soil (DSS) in Grand Rapids, MN, and Streptomyces sp. strains S3-4 and 3211-3 were isolated from experimental plots in the Cedar Creek Ecosystem Science Reserve (CCESR).

Encapsulation of multiple cargo proteins within recombinant Eut nanocompartments.

Spatial organization via encapsulation of enzymes within recombinant nanocompartments may increase efficiency in multienzyme cascades. Previously, we reported the encapsulation of single cargo proteins within nanocompartments in the heterologous host Escherichia coli. This was achieved by coexpression of the Salmonella enterica LT2 ethanolamine utilization bacterial microcompartment shell proteins EutS or EutSMNLK, with a signal sequence EutC1-19 cargo protein fusion. Optimization of this system, leading to the targeting of more than one cargo protein, requires an understanding of the encapsulation mechanism. In this work, we report that the signal sequence EutC1-19 targets cargo to the interior of nanocompartments via a hydrophobic interaction with a helix on shell protein EutS. We confirm that EutC1-19 does not interact with other Eut BMC shell proteins, EutMNLK. Furthermore, we show that a second signal sequence EutE1-21 interacts specifically with the same helix on EutS. Both signal sequences appear to compete for the same EutS helix to simultaneously colocalize two cargo proteins to the interior of recombinant nanocompartments. This work offers the first insights into signal sequence-shell protein interactions required for cargo sequestration within Eut BMCs. It also provides a basis for the future engineering of Eut nanocompartments as a platform for the potential colocalization of multienzyme cascades for synthetic biology applications.

Engineering formation of multiple recombinant Eut protein nanocompartments in E. coli.

Compartmentalization of designed metabolic pathways within protein based nanocompartments has the potential to increase reaction efficiency in multi-step biosynthetic reactions. We previously demonstrated proof-of-concept of this aim by targeting a functional enzyme to single cellular protein nanocompartments, which were formed upon recombinant expression of the Salmonella enterica LT2 ethanolamine utilization bacterial microcompartment shell proteins EutS or EutSMNLK in Escherichia coli. To optimize this system, increasing overall encapsulated enzyme reaction efficiency, factor(s) required for the production of more than one nanocompartment per cell must be identified. In this work we report that the cupin domain protein EutQ is required for assembly of more than one nanocompartment per cell. Overexpression of EutQ results in multiple nanocompartment assembly in our recombinant system. EutQ specifically interacts with the shell protein EutM in vitro via electrostatic interactions with the putative cytosolic face of EutM. These findings lead to the theory that EutQ could facilitate multiple nanocompartment biogenesis by serving as an assembly hub for shell proteins. This work offers insights into the biogenesis of Eut bacterial microcompartments, and also provides an improved platform for the production of protein based nanocompartments for targeted encapsulation of enzyme pathways.