Engineered polyketide synthases: Molecular foundries for commodity chemicals, specialty chemicals, and biofuels

Wednesday, July 19, 2017 Amgen Award Lecture ENGINEERED POLYKETIDE SYNTHASES: MOLECULAR FOUNDRIES FOR COMMODITY CHEMICALS, SPECIALTY CHEMICALS, AND BIOFUELS Jay Keasling, Departments of Chemical Engineering and Bioengineering, University of California, Berkeley, CA 94720 Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720 Joint BioEnergy Institute, Emeryville, CA 94608 [email protected]

Engineered polyketide synthases for production of commodity and specialty chemicals

Engineered modular polyketide synthases (PKSs) have the potential to be an extraordinarily effective retrosynthesis platform. Native PKSs assemble and tailor simple, readily available cellular acyl-CoAs into large, complex, chiral molecules. By successfully rearranging existing polyketide modules and domains, one can exquisitely control chemical structure from DNA sequence alone. As an example of the diverse biosynthetic potential of PKSs, we have concluded that approximately 20 of the roughly 150 commodity chemicals tracked by the petrochemical market information provider ICIS could be produced by mixing and matching naturally occurring PKS domains. To form these chemicals, engineered PKSs load acyl- CoAs, perform a programmed number of extension reactions, and then release products using previously published mechanisms. However, this potential has only just begun to be realized as the compounds that have been made using engineered PKSs represent a small fraction of the potentially accessible chemical space. In my talk, I will highlight work from our laboratory in which we have engineered polyketide synthases to produce a variety of commodity and specialty chemicals and expressed these engineered PKSs in a variety of Streptomyces for production of these molecules from sugars and other inexpensive starting materials

Molecular Characterization of the Opioid Receptors: Design, Development, and Preclinical Evaluation of Salvinorin A-Based Molecular Probes.

The field of salvinorin chemistry represents a novel and emerging field of opioid research. The novelty is derived from the lead pharmacophore: salvinorin A — a neoclerodane diterpenoid natural product isolated from Salvia divinorum. Salvinorin A represents a pharmacologically unique compound in that it is the first known non-nitrogenous KOR subtype-selective agonist, exhibits a comparatively safe physiological profile with no reports of toxicological effects in clinical trials, and, most importantly, has a steadily growing body of literature indicating potentially useful clinical applications (e.g. antinociceptive, anti-addictive, antipruritic, neuroprotective, etc.). This has encouraged the development of analogues as essential molecular probes to elucidate the structure-activity-relationship of the salvinorin-class. In this study, we expand the current field of salvinorin chemistry through the design, development, and preclinical evaluation of a series of C(22)-fused heteroaromatic salvinorin A analogues. Our in vitro models include: opioid receptor competitive radioligand binding affinity and functional [35S]GTP[γS] binding activity assays; while our in vivo models include: antinociceptive, antidepressant, and anxiolytic related assays. This resulted in three analogues exhibiting EC50 sub-200 nM functional activity, of which two displayed antinociceptive activities, with one also demonstrating antidepressant-like activity. As such, this study further supports the importance of the continued development of new salvinorin A analogues as essential research tools to ascertain potential three-dimensional ligand binding requirements, functional activities, and pharmacological consequences mediated through the clinically important opioid receptors

Enhancing Terminal Deoxynucleotidyl Transferase Activity on Substrates with 3' Terminal Structures for Enzymatic De Novo DNA Synthesis.

Enzymatic oligonucleotide synthesis methods based on the template-independent polymerase terminal deoxynucleotidyl transferase (TdT) promise to enable the de novo synthesis of long oligonucleotides under mild, aqueous conditions. Intermediates with a 3' terminal structure (hairpins) will inevitably arise during synthesis, but TdT has poor activity on these structured substrates, limiting its usefulness for oligonucleotide synthesis. Here, we described two parallel efforts to improve the activity of TdT on hairpins: (1) optimization of the concentrations of the divalent cation cofactors and (2) engineering TdT for enhanced thermostability, enabling reactions at elevated temperatures. By combining both of these improvements, we obtained a ~10-fold increase in the elongation rate of a guanine-cytosine hairpin

Selecting RNA aptamers for synthetic biology: investigating magnesium dependence and predicting binding affinity.

The ability to generate RNA aptamers for synthetic biology using in vitro selection depends on the informational complexity (IC) needed to specify functional structures that bind target ligands with desired affinities in physiological concentrations of magnesium. We investigate how selection for high-affinity aptamers is constrained by chemical properties of the ligand and the need to bind in low magnesium. We select two sets of RNA aptamers that bind planar ligands with dissociation constants (K(d)s) ranging from 65 nM to 100 microM in physiological buffer conditions. Aptamers selected to bind the non-proteinogenic amino acid, p-amino phenylalanine (pAF), are larger and more informationally complex (i.e., rarer in a pool of random sequences) than aptamers selected to bind a larger fluorescent dye, tetramethylrhodamine (TMR). Interestingly, tighter binding aptamers show less dependence on magnesium than weaker-binding aptamers. Thus, selection for high-affinity binding may automatically lead to structures that are functional in physiological conditions (1-2.5 mM Mg(2+)). We hypothesize that selection for high-affinity binding in physiological conditions is primarily constrained by ligand characteristics such as molecular weight (MW) and the number of rotatable bonds. We suggest that it may be possible to estimate aptamer-ligand affinities and predict whether a particular aptamer-based design goal is achievable before performing the selection

Rapid metabolic pathway assembly and modification using serine integrase site-specific recombination

Synthetic biology requires effective methods to assemble DNA parts into devices and to modify these devices once made. Here we demonstrate a convenient rapid procedure for DNA fragment assembly using site-specific recombination by ϕC31 integrase. Using six orthogonal attP/attB recombination site pairs with different overlap sequences, we can assemble up to five DNA fragments in a defined order and insert them into a plasmid vector in a single recombination reaction. ϕC31 integrase-mediated assembly is highly efficient, allowing production of large libraries suitable for combinatorial gene assembly strategies. The resultant assemblies contain arrays of DNA cassettes separated by recombination sites, which can be used to manipulate the assembly by further recombination. We illustrate the utility of these procedures to (i) assemble functional metabolic pathways containing three, four or five genes; (ii) optimize productivity of two model metabolic pathways by combinatorial assembly with randomization of gene order or ribosome binding site strength; and (iii) modify an assembled metabolic pathway by gene replacement or addition

CRISPR/Cas9 advances engineering of microbial cell factories

One of the key drivers for successful metabolic engineering in microbes is the efficacy by which genomes can be edited. As such there are many methods to choose from when aiming to modify genomes, especially those of model organisms like yeast and bacteria. In recent years, clustered regularly interspaced palindromic repeats (CRISPR) and its associated proteins (Cas) have become the method of choice for precision genome engineering in many organisms due to their orthogonality, versatility and efficacy. Here we review the strategies adopted for implementation of RNA-guided CRISPR/Cas9 genome editing with special emphasis on their application for metabolic engineering of yeast and bacteria. Also, examples of how nuclease-deficient Cas9 has been applied for RNA-guided transcriptional regulation of target genes will be reviewed, as well as tools available for computer-aided design of guide-RNAs will be highlighted. Finally, this review will provide a perspective on the immediate challenges and opportunities foreseen by the use of CRISPR/Cas9 genome engineering and regulation in the context of metabolic engineering