The power of synthetic biology for bioproduction, remediation and pollution control

The agenda of the UN's Sustainable Development Goals (SDGs) 1 challenges the synthetic biology community—and the life sciences as a whole—to develop transformative technologies that help to protect, even expand our planet's habitability. While modern tools for genome editing already benefit applications in health and agriculture, sustainability also asks for a dramatic transformation of our use of natural resources. The challenge is not just to limit and, wherever possible revert emissions of pollutants and greenhouse gases, but also to replace environmentally costly processes based on fossil fuels with bio‐based sustainable alternatives. This task is not exclusively a scientific and technical one but will also require guidelines and regulations for the development and large‐scale deployment of this new type of bio‐based production. Some recent advances that can (or soon could) enable us to make progress in these areas—and several possible governance principles—need to be addressed

Coupling carboxylic acid reductase to inorganic pyrophosphatase enhances cell-free in vitro aldehyde biosynthesis

© 2015 Elsevier B.V. Carboxylic acid reductases (CARs) have been harnessed in metabolic pathways to produce aldehydes in engineered organisms. However, desired aldehyde products inhibit cell growth and limit product titers currently achievable from fermentative processes. Aldehyde toxicity can be entirely circumvented by performing aldehyde biosynthesis in non-cellular systems. Use of purified CARs for preparative-scale aldehyde synthesis has been limited by in vitro turnover of model CARs, such as Car. Ni from Nocardia iowensis, despite robust conversion of substrates associated with expression in heterologous hosts such as E. coli and yeast. In this study, we report that in vitro activity of Car. Ni is inhibited by formation of the co-product pyrophosphate, and that pairing of an inorganic pyrophosphatase (Ppa. Ec) with Car. Ni substantially improves the rate and yield of aldehyde biosynthesis. We demonstrate that, in the presence of Ppa. Ec, Michaelis-Menten kinetic models based on initial rate measurements accurately predict Car. Ni kinetics within an in vitro pathway over longer timescales. We rationalize our novel observations for Car. Ni by examining previously posed arguments for pyrophosphate hydrolysis made in the context of other adenylate-forming enzymes. Overall, our findings may aid in increasing adoption of CARs for cell-free in vitro aldehyde biosynthetic processes

A dynamic metabolite valve for the control of central carbon metabolism

Successful redirection of endogenous resources into heterologous pathways is a central tenet in the creation of efficient microbial cell factories. This redirection, however, may come at a price of poor biomass accumulation, reduced cofactor regeneration and low recombinant enzyme expression. In this study, we propose a metabolite valve to mitigate these issues by dynamically tuning endogenous processes to balance the demands of cell health and pathway efficiency. A control node of glucose utilization, glucokinase (Glk), was exogenously manipulated through either engineered antisense RNA or an inverting gene circuit. Using these techniques, we were able to directly control glycolytic flux, reducing the specific growth rate of engineered Escherichia coli by up to 50% without altering final biomass accumulation. This modulation was accompanied by successful redirection of glucose into a model pathway leading to an increase in the pathway yield and reduced carbon waste to acetate. This work represents one of the first examples of the dynamic redirection of glucose away from central carbon metabolism and enables the creation of novel, efficient intracellular pathways with glucose used directly as a substrate. © 2012 Elsevier Inc

A Robust CRISPR Interference Gene Repression System in Pseudomonas

© 2018 American Society for Microbiology. Pseudomonas spp. are widely used model organisms in different areas of research. Despite the relevance of Pseudomonas in many applications, the use of protein depletion tools in this host remains limited. Here, we developed the CRISPR interference system for gene repression in Pseudomonas spp. using a nuclease-null Streptococcus pasteurianus Cas9 variant (dead Cas9, or dCas9). We demonstrate a robust and titratable gene depletion system with up to 100-fold repression in β-galactosidase activity in P. aeruginosa and 300-fold repression in pyoverdine production in Pseudomonas putida. This inducible system enables the study of essential genes, as shown by ftsZ depletions in P. aeruginosa, P. putida, and Pseudomonas fluorescens that led to phenotypic changes consistent with depletion of the targeted gene. Additionally, we performed the first in vivo characterization of protospacer adjacent motif (PAM) site preferences of S. pasteurianus dCas9 and identified NNGCGA as a functional PAM site that resulted in repression efficiencies comparable to the consensus NNGTGA sequence. This discovery significantly expands the potential genomic targets of S. pasteurianus dCas9, especially in GC-rich organisms

Transcription Factor Allosteric Regulation Through Substrate Coordination to Zinc

The development of new synthetic biology circuits for biotechnology and medicine requires deeper mechanistic insight on allosteric transcription factors (aTFs). Here we studied the aTF UxuR, which is a dimer, with each monomer consisting of two structured domains connected by a highly flexible linker region. In order to explore how ligand binding to UxuR affects protein dynamics we performed molecular dynamics simulations in the free protein and the aTF bound to the inducer D-fructuronate or the structural isomer D-glucuronate. We then validated our results by constructing a sensor plasmid for D-fructuronate in E. coli and performed site-directed mutagenesis. Our results show that zinc coordination is necessary for UxuR function, since mutation to alanines prevents expression de-repression by D-fructuronate. Analyzing the different complexes, we found that the disordered linker regions allow the N-terminal domains to display fast and large movements. When the inducer is bound, UxuR is able to sample an open conformation with a more pronounced negative charge at the surface of the N-terminal DNA binding domains. In opposition, in the free and D-glucuronate bond forms the protein samples closed conformations, with a more positive character at the surface of the DNA binding regions. These molecular insights provide a new basis to better harness these systems for biological systems engineering

Rational design of thiolase substrate specificity for metabolic engineering applications

© 2018 Wiley Periodicals, Inc. Metabolic engineering efforts require enzymes that are both highly active and specific toward the synthesis of a desired output product to be commercially feasible. The 3-hydroxyacid (3HA) pathway, also known as the reverse β-oxidation or coenzyme-A-dependent chain-elongation pathway, can allow for the synthesis of dozens of useful compounds of various chain lengths and functionalities. However, this pathway suffers from byproduct formation, which lowers the yields of the desired longer chain products, as well as increases downstream separation costs. The thiolase enzyme catalyzes the first reaction in this pathway, and its substrate specificity at each of its two catalytic steps sets the chain length and composition of the chemical scaffold upon which the other downstream enzymes act. However, there have been few attempts reported in the literature to rationally engineer thiolase substrate specificity. In this study, we present a model-guided, rational design study of ordered substrate binding applied to two biosynthetic thiolases, with the goal of increasing the ratio of C6/C4 products formed by the 3HA pathway, 3-hydroxy-hexanoic acid and 3-hydroxybutyric acid. We identify thiolase mutants that result in nearly 10-fold increases in C6/C4 selectivity. Our findings can extend to other pathways that employ the thiolase for chain elongation, as well as expand our knowledge of sequence–structure–function relationship for this important class of enzymes

Analyzing the effects of metabolic state and perturbations on genetic device performance by metabolomics and 13C-labeled metabolic flux analysis

Characterization of engineered cellular systems is required for understanding the metabolic state and associated metabolic burden that cells endure from the implementation of genetic devices and circuits. Competition for shared resources, such as cellular macromolecules, in genetic circuits can impair essential cellular functions and thus limits the robustness of engineered cellular systems to be implemented in therapeutic and medicinally relevant synthetic biology applications. To characterize the metabolic state and burden of genetic circuits at the systems-level, metabolomics and 13C-labeled metabolic flux analysis (13CMFA), with a focus on bioenergetics and central carbon metabolism, can be performed. Changes in the concentrations of key metabolites, such as G6P and ATP, measured by GC-MS and LC-MS/MS and analyzed in metabolomics, point directly to the metabolic state of a cell. Similarly, 13CMFA estimates the intracellular metabolic fluxes using the measured concentrations of key metabolites obtained through cellular metabolism and by feeding cells isotopically labeled substrates. The estimated changes in metabolic fluxes in response to competition for shared resources can shed light on the complex interplay among gene expression, protein expression, and the environment. Genetic variants designed to probe the tolerance of central endogenous genes to metabolic perturbation are being tested and analyzed using both metabolomics and 13C-MFA. Finally, strategies to engineer autoregulation on shared resources are being investigated for minimizing metabolic burden.</div

Transcription factor allosteric regulation through substrate coordination to zinc

Abstract The development of new synthetic biology circuits for biotechnology and medicine requires deeper mechanistic insight into allosteric transcription factors (aTFs). Here we studied the aTF UxuR, a homodimer of two domains connected by a highly flexible linker region. To explore how ligand binding to UxuR affects protein dynamics we performed molecular dynamics simulations in the free protein, the aTF bound to the inducer D-fructuronate or the structural isomer D-glucuronate. We then validated our results by constructing a sensor plasmid for D-fructuronate in Escherichia coli and performed site-directed mutagenesis. Our results show that zinc coordination is necessary for UxuR function since mutation to alanines prevents expression de-repression by D-fructuronate. Analyzing the different complexes, we found that the disordered linker regions allow the N-terminal domains to display fast and large movements. When the inducer is bound, UxuR can sample an open conformation with a more pronounced negative charge at the surface of the N-terminal DNA binding domains. In opposition, in the free and D-glucuronate bond forms the protein samples closed conformations, with a more positive character at the surface of the DNA binding regions. These molecular insights provide a new basis to harness these systems for biological systems engineering.</jats:p

The Importance and Future of Biochemical Engineering

© 2020 Wiley Periodicals LLC Today's Biochemical Engineer may contribute to advances in a wide range of technical areas. The recent Biochemical and Molecular Engineering XXI conference focused on “The Next Generation of Biochemical and Molecular Engineering: The role of emerging technologies in tomorrow's products and processes”. On the basis of topical discussions at this conference, this perspective synthesizes one vision on where investment in research areas is needed for biotechnology to continue contributing to some of the world's grand challenges