Functional screening and in vitro analysis reveals thioesterases with enhanced substrate specificity profiles that improve short-chain fatty acid production in Escherichia coli

Short-chain fatty acid (SCFAs) biosynthesis is pertinent to production of biofuels, industrial compounds, and pharmaceuticals from renewable resources. To expand on Escherichia coli SCFA products, we previously implemented a coenzyme A (CoA)-dependent pathway that condenses acetyl-CoA to a diverse group of short chain fatty acyl-CoAs. To increase product titers and reduce premature pathway termination products, we describe in vivo and in vitro analyses to understand and improve the specificity of the acyl-CoA thioestera enzyme, which releases fatty acids from CoA. A total of 62 putative bacterial thioesterases, including from the cow rumen microbiome, were inserted into a pathway that condenses acetyl-CoA to an acyl-CoAmolecule derived from exogenously provided propionic or isobutyric acid. Functional screening revealed thioesterases that increase production of saturated (valerate), unsaturated (trans-2-pentenoate) and branched (4-methylvalerate) SCFAs compared to overexpression of E. coli thioesterase tesB or native expression of endogenous thioesterases. To determine if altered thioesterase acyl-CoA substrate specificity caused the increase in product titers, six of the most promising enzymes were analyzed in vitro. Biochemical assays revealed that the most productive thioesterases rely on promiscuous activity, but have greater specificity for product-associated acyl-CoAs than for precursor acyl-CoAs. Here we introduce novel thioesterases with improved specificity for saturated, branched and unsaturated short-chain acyl-CoAs, thereby expanding the diversity of potential fatty acid products while increasing titers of current products. The growing uncertainty associated with protein database annotations denotes this study as a model for isolating functional biochemical pathway enzymes in situations where experimental evidence of enzyme function is absent.United States. Army Research Office (Institute for Collaborative Biotechnologies, grant W911NF-09-0001

Scarless Cas9 Assisted Recombineering (no‐SCAR) in Escherichia coli, an Easy‐to‐Use System for Genome Editing

The discovery and development of genome editing systems that leverage the site‐specific DNA endonuclease system CRISPR/Cas9 has fundamentally changed the ease and speed of genome editing in many organisms. In eukaryotes, the CRISPR/Cas9 system utilizes a “guide” RNA to enable the Cas9 nuclease to make a double‐strand break at a particular genome locus, which is repaired by non‐homologous end joining (NHEJ) repair enzymes, often generating random mutations in the process. A specific alteration of the target genome can also be generated by supplying a DNA template in vivo with a desired mutation, which is incorporated by homology‐directed repair. However, E. coli lacks robust systems for double‐strand break repair. Thus, in contrast to eukaryotes, targeting E. coli chromosomal DNA with Cas9 causes cell death. However, Cas9‐mediated killing of bacteria can be exploited to select against cells with a specified genotype within a mixed population. In combination with the well described λ‐Red system for recombination in E. coli, we created a highly efficient system for marker‐free and scarless genome editing.National Institute of Food and Agriculture (U.S.) (Award 2013-67012-21022)United States. Army Research Office (Grant W911NF-09-0001

The zero-sum game of pathway optimization: Emerging paradigms for tuning gene expression

With increasing price volatility and growing awareness of the lack of sustainability of traditional chemical synthesis, microbial chemical production has been tapped as a promising renewable alternative for the generation of diverse, stereospecific compounds. Nonetheless, many attempts to generate them are not yet economically viable. Due to the zero-sum nature of microbial resources, traditional strategies of pathway optimization are attaining minimal returns. This result is in part a consequence of the gross changes in host physiology resulting from such efforts and underscores the need for more precise and subtle forms of gene modulation. In this review, we describe alternative strategies and emerging paradigms to address this problem and highlight potential solutions from the emerging field of synthetic biology.National Science Foundation (U.S.) (Synthetic Biology Engineering Research Center (SynBERC), grant number EEC-0540879)National Science Foundation (U.S.) (NSF CAREER Award (grant number CBET-0954986))Natural Sciences and Engineering Research Council of Canada (Fellowship

Dynamic metabolic engineering: New strategies for developing responsive cell factories

Metabolic engineering strategies have enabled improvements in yield and titer for a variety of valuable small molecules produced naturally in microorganisms, as well as those produced via heterologous pathways. Typically, the approaches have been focused on up- and downregulation of genes to redistribute steady-state pathway fluxes, but more recently a number of groups have developed strategies for dynamic regulation, which allows rebalancing of fluxes according to changing conditions in the cell or the fermentation medium. This review highlights some of the recently published work related to dynamic metabolic engineering strategies and explores how advances in high-throughput screening and synthetic biology can support development of new dynamic systems. Dynamic gene expression profiles allow trade-offs between growth and production to be better managed and can help avoid build-up of undesired intermediates. The implementation is more complex relative to static control, but advances in screening techniques and DNA synthesis will continue to drive innovation in this field.National Science Foundation (U.S.) (CBET-0954986)United States. National Institutes of Health (T32GM008334

Microbial Engineering for Aldehyde Synthesis

Aldehydes are a class of chemicals with many industrial uses. Several aldehydes are responsible for flavors and fragrances present in plants, but aldehydes are not known to accumulate in most natural microorganisms. In many cases, microbial production of aldehydes presents an attractive alternative to extraction from plants or chemical synthesis. During the past 2 decades, a variety of aldehyde biosynthetic enzymes have undergone detailed characterization. Although metabolic pathways that result in alcohol synthesis via aldehyde intermediates were long known, only recent investigations in model microbes such as Escherichia coli have succeeded in minimizing the rapid endogenous conversion of aldehydes into their corresponding alcohols. Such efforts have provided a foundation for microbial aldehyde synthesis and broader utilization of aldehydes as intermediates for other synthetically challenging biochemical classes. However, aldehyde toxicity imposes a practical limit on achievable aldehyde titers and remains an issue of academic and commercial interest. In this minireview, we summarize published efforts of microbial engineering for aldehyde synthesis, with an emphasis on de novo synthesis, engineered aldehyde accumulation in E. coli, and the challenge of aldehyde toxicity.MIT Synthetic Biology Engineering Research Center (Grant EEC-0540879)National Science Foundation (U.S.). Graduate Research Fellowshi

Improvement of DNA minicircle production by optimization of the secondary structure of the 5′-UTR of ParA resolvase

The use of minicircles in gene therapy applications is dependent on the availability of high-producer cell systems. In order to improve the performance of minicircle production in Escherichia coli by ParA resolvase-mediated in vivo recombination, we focus on the 5′ untranslated region (5′-UTR) of parA messenger RNA (mRNA). The arabinose-inducible P[subscript BAD]/araC promoter controls ParA expression and strains with improved arabinose uptake are used. The 27-nucleotide-long 5′-UTR of parA mRNA was optimized using a predictive thermodynamic model. An analysis of original and optimized mRNA subsequences predicted a decrease of 8.6–14.9 kcal/mol in the change in Gibbs free energy upon assembly of the 30S ribosome complex with the mRNA subsequences, indicating a more stable mRNA-rRNA complex and enabling a higher (48–817-fold) translation initiation rate. No effect of the 5′-UTR was detected when ParA was expressed from a low-copy number plasmid (∼14 copies/cell), with full recombination obtained within 2 h. However, when the parA gene was inserted in the bacterial chromosome, a faster and more effective recombination was obtained with the optimized 5′-UTR. Interestingly, the amount of this transcript was 2.6–3-fold higher when compared with the transcript generated from the original sequence, highlighting that 5′-UTR affects the level of the transcript. A Western blot analysis confirmed that E. coli synthesized higher amounts of ParA with the new 5′-UTR (∼1.8 ± 0.7-fold). Overall, these results show that the improvements made in the 5′-UTR can lead to a more efficient translation and hence to faster and more efficient minicircle generation.MIT-Portugal ProgramFundação para a Ciência e a Tecnologia (PhD grant SFRH/BD/33786/2009

The no-SCAR (Scarless Cas9 Assisted Recombineering) system for genome editing in Escherichia coli

Genome engineering methods in E. coli allow for easy to perform manipulations of the chromosome in vivo with the assistance of the λ-Red recombinase system. These methods generally rely on the insertion of an antibiotic resistance cassette followed by removal of the same cassette, resulting in a two-step procedure for genomic manipulations. Here we describe a method and plasmid system that can edit the genome of E. coli without chromosomal markers. This system, known as Scarless Cas9 Assisted Recombineering (no-SCAR), uses λ-Red to facilitate genomic integration of donor DNA and double stranded DNA cleavage by Cas9 to counterselect against wild-type cells. We show that point mutations, gene deletions, and short sequence insertions were efficiently performed in several genomic loci in a single-step with regards to the chromosome and did not leave behind scar sites. The single-guide RNA encoding plasmid can be easily cured due to its temperature sensitive origin of replication, allowing for iterative chromosomal manipulations of the same strain, as is often required in metabolic engineering. In addition, we demonstrate the ability to efficiently cure the second plasmid in the system by targeting with Cas9, leaving the cells plasmid-free.Shell Global Solutions (US)National Institute of Food and Agriculture (U.S.) (Postdoctoral Fellowship 2013-67012-21022

Biosynthesis of chiral 3-hydroxyvalerate from single propionate-unrelated carbon sources in metabolically engineered E. coli

Background The ability to synthesize chiral building block molecules with high optical purity is of considerable importance to the fine chemical and pharmaceutical industries. Production of one such compound, 3-hydroxyvalerate (3HV), has previously been studied with respect to the in vivo or in vitro enzymatic depolymerization of biologically-derived co-polymers of poly(3-hydroxybutyrate-co-3-hydroxyvalerate). However, production of this biopolymeric precursor typically necessitates the supplementation of a secondary carbon source (e.g., propionate) into the culture medium. In addition, previous approaches for producing 3HV have not focused on its enantiopure synthesis, and thus suffer from increased costs for product purification. Results Here, we report the selective biosynthesis of each 3HV stereoisomer from a single, renewable carbon source using synthetic metabolic pathways in recombinant strains of Escherichia coli. The product chirality was controlled by utilizing two reductases of opposing stereoselectivity. Improvement of the biosynthetic pathway activity and host background was carried out to elevate both the 3HV titers and 3HV/3HB ratios. Overall, shake-flask titers as high as 0.31 g/L and 0.50 g/L of (S)-3HV and (R)-3HV, respectively, were achieved in glucose-fed cultures, whereas glycerol-fed cultures yielded up to 0.19 g/L and 0.96 g/L of (S)-3HV and (R)-3HV, respectively. Conclusions Our work represents the first report of direct microbial production of enantiomerically pure 3HV from a single carbon source. Continued engineering of host strains and pathway enzymes will ultimately lead to more economical production of chiral 3HV.Synthetic Biology Engineering Research CenterNational Science Foundation (Grant EEC-0540879)Massachusetts Institute of Technology. Energy InitiativeShell Oil Compan

Retro-biosynthetic screening of a modular pathway design achieves selective route for microbial synthesis of 4-methyl-pentanol

Increasingly complex metabolic pathways have been engineered by modifying natural pathways and establishing de novo pathways with enzymes from a variety of organisms. Here we apply retro-biosynthetic screening to a modular pathway design to identify a redox neutral, theoretically high yielding route to a branched C6 alcohol. Enzymes capable of converting natural E. coli metabolites into 4-methyl-pentanol (4MP) via coenzyme A (CoA)-dependent chemistry were taken from nine different organisms to form a ten-step de novo pathway. Selectivity for 4MP is enhanced through the use of key enzymes acting on acyl-CoA intermediates, a carboxylic acid reductase from Nocardia iowensis and an alcohol dehydrogenase from Leifsonia sp. strain S749. One implementation of the full pathway from glucose demonstrates selective carbon chain extension and acid reduction with 4MP constituting 81% (90±7 mg l⁻¹) of the observed alcohol products. The highest observed 4MP titre is 192±23 mg l⁻¹. These results demonstrate the ability of modular pathway screening to facilitate de novo pathway engineering.United States. Army Research Office (W911NF-09-0001

Exploring the Mechanism of Biocatalyst Inhibition in Microbial Desulfurization

Microbial desulfurization, or biodesulfurization (BDS), of fuels is a promising technology because it can desulfurize compounds that are recalcitrant to the current standard technology in the oil industry. One of the obstacles to the commercialization of BDS is the reduction in biocatalyst activity concomitant with the accumulation of the end product, 2-hydroxybiphenyl (HBP), during the process. BDS experiments were performed by incubating Rhodococcus erythropolis IGTS8 resting-cell suspensions with hexadecane at 0.50 (vol/vol) containing 10 mM dibenzothiophene. The resin Dowex Optipore SD-2 was added to the BDS experiments at resin concentrations of 0, 10, or 50 g resin/liter total volume. The HBP concentration within the cytoplasm was estimated to decrease from 1,100 to 260 μM with increasing resin concentration. Despite this finding, productivity did not increase with the resin concentration. This led us to focus on the susceptibility of the desulfurization enzymes toward HBP. Dose-response experiments were performed to identify major inhibitory interactions in the most common BDS pathway, the 4S pathway. HBP was responsible for three of the four major inhibitory interactions identified. The concentrations of HBP that led to a 50% reduction in the enzymes' activities (IC[subscript 50]s) for DszA, DszB, and DszC were measured to be 60 ± 5 μM, 110 ± 10 μM, and 50 ± 5 μM, respectively. The fact that the IC[subscript 50]s for HBP are all significantly lower than the cytoplasmic HBP concentration suggests that the inhibition of the desulfurization enzymes by HBP is responsible for the observed reduction in biocatalyst activity concomitant with HBP generation.National Institutes of Health (U.S.). Biotechnology Training Progra