Organism Engineering for the Bioproduction of the Triaminotrinitrobenzene (TATB) Precursor Phloroglucinol (PG).

Organism engineering requires the selection of an appropriate chassis, editing its genome, combining traits from different source species, and controlling genes with synthetic circuits. When a strain is needed for a new target objective, for example, to produce a chemical-of-need, the best strains, genes, techniques, software, and expertise may be distributed across laboratories. Here, we report a project where we were assigned phloroglucinol (PG) as a target, and then combined unique capabilities across the United States Army, Navy, and Air Force service laboratories with the shared goal of designing an organism to produce this molecule. In addition to the laboratory strain Escherichia coli, organisms were screened from soil and seawater. Putative PG-producing enzymes were mined from a strain bank of bacteria isolated from aircraft and fuel depots. The best enzyme was introduced into the ocean strain Marinobacter atlanticus CP1 with its genome edited to redirect carbon flux from natural fatty acid ester (FAE) production. PG production was also attempted in Bacillus subtilis and Clostridium acetobutylicum. A genetic circuit was constructed in E. coli that responds to PG accumulation, which was then ported to an in vitro paper-based system that could serve as a platform for future low-cost strain screening or for in-field sensing. Collectively, these efforts show how distributed biotechnology laboratories with domain-specific expertise can be marshalled to quickly provide a solution for a targeted organism engineering project, and highlights data and material sharing protocols needed to accelerate future efforts.

A Pressure Test to Make 10 Molecules in 90 Days: External Evaluation of Methods to Engineer Biology.

Centralized facilities for genetic engineering, or "biofoundries", offer the potential to design organisms to address emerging needs in medicine, agriculture, industry, and defense. The field has seen rapid advances in technology, but it is difficult to gauge current capabilities or identify gaps across projects. To this end, our foundry was assessed via a timed "pressure test", in which 3 months were given to build organisms to produce 10 molecules unknown to us in advance. By applying a diversity of new approaches, we produced the desired molecule or a closely related one for six out of 10 targets during the performance period and made advances toward production of the others as well. Specifically, we increased the titers of 1-hexadecanol, pyrrolnitrin, and pacidamycin D, found novel routes to the enediyne warhead underlying powerful antimicrobials, established a cell-free system for monoterpene production, produced an intermediate toward vincristine biosynthesis, and encoded 7802 individually retrievable pathways to 540 bisindoles in a DNA pool. Pathways to tetrahydrofuran and barbamide were designed and constructed, but toxicity or analytical tools inhibited further progress. In sum, we constructed 1.2 Mb DNA, built 215 strains spanning five species ( Saccharomyces cerevisiae, Escherichia coli, Streptomyces albidoflavus, Streptomyces coelicolor, and Streptomyces albovinaceus), established two cell-free systems, and performed 690 assays developed in-house for the molecules.

Spatially distinct functions of Clb2 in the DNA damage response.

In budding yeast four mitotic cyclins (Clb1-4) cooperate in a partially redundant manner to bring about M-phase specific events, including the apical isotropic switch that ends polarized bud growth initiated at bud emergence. How exactly this morphogenetic transition is regulated by mitotic CDKs remains poorly understood. We have taken advantage of the isotropic bud growth that prevails in cells responding to DNA damage to unravel the contribution of mitotic cyclins in this cellular context. We find that clb2∆, in contrast to the other mitotic cyclin mutants, inappropriately respond to the presence of DNA damage. This aberrant response is characterized by a Cdc42- and Bni1-dependent but Cln-independent resumption of polarized bud growth after a brief period of actin depolarization. Biochemical and genetic evidence is presented that formally excludes the possibility of indirect effects due for instance to unrestrained APC activity, untimely mitotic exit or Swe1-mediated CDK inhibition. Importantly, our data demonstrate that in order to maintain the characteristic dumbbell arrest phenotype upon checkpoint activation Clb2 needs to be efficiently exported into the cytoplasm. We propose that the inhibition of mitotic cyclin destruction by the DNA damage checkpoint pathway leads to a buildup of Clb2 in the cytoplasm where this cyclin can stabilize the apical isotropic switch throughout a G 2/M checkpoint arrest. Our study also unveils an essential role of nuclear Clb2 in both survival and adaptation to the DNA damage checkpoint, illustrating a spatially distinct dual function of this mitotic cyclin in the response to DNA damage.

Cdk and the anillin homolog Bud4 define a new pathway regulating septin organization in yeast.

In budding yeast, the cortical structure formed by the septins is remodeled at the onset of mitotic exit and delineates a specialized compartment dedicated to cytokinesis. How this septin function is spatially and timely regulated remains poorly understood. In this study, we report a role of the anillin-like protein Bud4 in the formation and the disassembly of the double ring structure formed by the septins at the time of cytokinesis. Bud4 acts with Bud3 in this pathway and in parallel with septin phosphorylation by the p21-activated kinase Cla4 and the septin-dependent kinase Gin4. In addition, we show that the function of Bud4 is regulated by the cyclin-dependent protein kinase Cdk1, the master regulator of cell cycle progression. This result suggests that the Cdks, or a locally specific pool of the kinase, may have a role past mitotic exit.

Compartmentalization of the functions and regulation of the mitotic cyclin Clb2 in S. cerevisiae.

Orderly progression through the eukaryotic cell cycle is a complex process involving both regulation of cyclin dependent kinase activity and control of specific substrate-Cdk interactions. In Saccharomyces cerevisiae, the mitotic cyclin Clb2 has a central role in regulating the onset of anaphase and in maintaining the cellular shape of the bud by inhibiting growth polarization induced in G1. However, how Clb2 and the partially redundant cyclin Clb1 confer specificity to Cdk1 in these processes still remains unclear. Here, we show that Clb2 mutants impaired in nuclear import or export are differentially affected for subsets of Clb2 functions while remaining fully functional for others. Our data support a direct role of the cytoplasmic pool of Clb1,2-Cdk1 in terminating cytoskeleton and growth polarization, independently of G1 cyclin transcriptional regulation. By contrast, the nuclear form of the cyclin is required for timely initiation of anaphase. Clb2 localization influences its stage-specific degradation as well. We report that Clb2 trapped in the cytoplasm is stabilized during anaphase but not at the time of mitotic exit. Altogether, our results demonstrate that the subcellular localization of the mitotic cyclin Clb2 is one of the key determinants of its biological function.