Genome Editing for the Production of Natural Products in Escherichia coli

Natural products such as secondary metabolites (e.g., plant terpenoids) are found to be a major source of bioactive compounds. These natural products accumulate as complex mixtures with other related compounds and this chemical complexity adds cost to the downstream recovery and purification of natural products from plant biomass. One aim of synthetic biology and metabolic engineering programmes is to produce such compounds from synthetic gene clusters in heterologous hosts and thereby achieve more targeted and affordable production. Both fungi and bacteria are common hosts for metabolic engineering in industry. Fungal hosts include Penicillium chrysogenum, Saccharomyces cerevisiae, Aspergillus niger and the bacterial hosts Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum. E. coli is often selected as a host given the ease of its genetic manipulation and the long history of using this organism in laboratory‐based bioengineering. The bioengineering of E. coli extends also to feedstock pathways to interface and optimize the production of high value compounds from widely available and inexpensive carbon sources. Genome editing is important in these microbial bioengineering programmes and is needed to isolate stable strains and to optimize production. Herein, this review discusses frequently used methods for genome editing in E. coli in relation to the production of natural compounds and chemicals

Bioproduction of Linalool From Paper Mill Waste

A key challenge in chemicals biomanufacturing is the maintenance of stable, highly productive microbial strains to enable cost-effective fermentation at scale. A “cookie-cutter” approach to microbial engineering is often used to optimize host stability and productivity. This can involve identifying potential limitations in strain characteristics followed by attempts to systematically optimize production strains by targeted engineering. Such targeted approaches however do not always lead to the desired traits. Here, we demonstrate both ‘hit and miss’ outcomes of targeted approaches in attempts to generate a stable Escherichia coli strain for the bioproduction of the monoterpenoid linalool, a fragrance molecule of industrial interest. First, we stabilized linalool production strains by eliminating repetitive sequences responsible for excision of pathway components in plasmid constructs that encode the pathway for linalool production. These optimized pathway constructs were then integrated within the genome of E. coli in three parts to eliminate a need for antibiotics to maintain linalool production. Additional strategies were also employed including: reduction in cytotoxicity of linalool by adaptive laboratory evolution and modification or homologous gene replacement of key bottleneck enzymes GPPS/LinS. Our study highlights that a major factor influencing linalool titres in E. coli is the stability of the genetic construct against excision or similar recombination events. Other factors, such as decreasing linalool cytotoxicity and changing pathway genes, did not lead to improvements in the stability or titres obtained. With the objective of reducing fermentation costs at scale, the use of minimal base medium containing paper mill wastewater secondary paper fiber as sole carbon source was also investigated. This involved simultaneous saccharification and fermentation using either supplemental cellulase blends or by co-expressing secretable cellulases in E. coli containing the stabilized linalool production pathway. Combined, this study has demonstrated a stable method for linalool production using an abundant and low-cost feedstock and improved production strains, providing an important proof-of-concept for chemicals production from paper mill waste streams. For scaled production, optimization will be required, using more holistic approaches that involve further rounds of microbial engineering and fermentation process development

Consolidated Bioprocessing: Synthetic Biology Routes to Fuels and Fine Chemicals

From MDPI via Jisc Publications RouterHistory: accepted 2021-05-14, pub-electronic 2021-05-18Publication status: PublishedFunder: Biotechnology and Biological Sciences Research Council; Grant(s): BB/S011684/1Funder: Engineering and Physical Sciences Research Council; Grant(s): EP/S01778X/1Funder: Office of Naval Research Global; Grant(s): N62909-18-1-2137The long road from emerging biotechnologies to commercial “green” biosynthetic routes for chemical production relies in part on efficient microbial use of sustainable and renewable waste biomass feedstocks. One solution is to apply the consolidated bioprocessing approach, whereby microorganisms convert lignocellulose waste into advanced fuels and other chemicals. As lignocellulose is a highly complex network of polymers, enzymatic degradation or “saccharification” requires a range of cellulolytic enzymes acting synergistically to release the abundant sugars contained within. Complications arise from the need for extracellular localisation of cellulolytic enzymes, whether they be free or cell-associated. This review highlights the current progress in the consolidated bioprocessing approach, whereby microbial chassis are engineered to grow on lignocellulose as sole carbon sources whilst generating commercially useful chemicals. Future perspectives in the emerging biofoundry approach with bacterial hosts are discussed, where solutions to existing bottlenecks could potentially be overcome though the application of high throughput and iterative Design-Build-Test-Learn methodologies. These rapid automated pathway building infrastructures could be adapted for addressing the challenges of increasing cellulolytic capabilities of microorganisms to commercially viable levels

Predictive Engineering of Class I Terpene Synthases Using Experimental and Computational Approaches

From Wiley via Jisc Publications RouterHistory: received 2021-09-14, rev-recd 2021-10-15, pub-electronic 2021-11-03Article version: VoRPublication status: PublishedFunder: Future Biomanufacturing Research Hub; Grant(s): EP/S01778X/1Funder: Engineering and Physical Sciences Research Council (EPSRC)Funder: Biotechnology and Biological Sciences Research Council (BBSRC)Funder: UK Research and Innovation; Id: http://dx.doi.org/10.13039/100014013Abstract: Terpenoids are a highly diverse group of natural products with considerable industrial interest. Increasingly, engineered microbes are used for the production of terpenoids to replace natural extracts and chemical synthesis. Terpene synthases (TSs) show a high level of functional plasticity and are responsible for the vast structural diversity observed in natural terpenoids. Their relatively inert active sites guide intrinsically reactive linear carbocation intermediates along one of many cyclisation paths via exertion of subtle steric and electrostatic control. Due to the absence of a strong protein interaction with these intermediates, there is a remarkable lack of sequence‐function relationship within the TS family, making product‐outcome predictions from sequences alone challenging. This, in combination with the fact that many TSs produce multiple products from a single substrate hampers the design and use of TSs in the biomanufacturing of terpenoids. This review highlights recent advances in genome mining, computational modelling, high‐throughput screening, and machine‐learning that will allow more predictive engineering of these fascinating enzymes in the near future

Combined pulsed electron double resonance EPR and molecular dynamics investigations of calmodulin suggest effects of crowding agents on protein structures

A.M.S. received Early Stage Research Funding from the European Union’s Seventh Framework Programme FP-7-PEOPLE-2013-ITN through the “MAGnetic Innovation in Catalysis” (MAGIC) Initial Training Network (grant agreement no. 606831). Part of this work was also supported by BBSRC grant: BB/M007065/1. J.L. thanks the Royal Society for a University Research Fellowship, the Carnegie Trust (RIG007510), and the Wellcome Trust for a Multi-User Equipment grant (099149/Z/12/Z).Calmodulin (CaM) is a highly dynamic Ca2+-binding protein that exhibits large conformational changes upon binding Ca2+ and target proteins. Although it is accepted that CaM exists in an equilibrium of conformational states in the absence of target protein, the physiological relevance of an elongated helical linker region in the Ca2+-replete form has been highly debated. In this study, we use PELDOR (pulsed electron–electron double resonance) EPR measurements of a doubly spin-labeled CaM variant to assess the conformational states of CaM in the apo-, Ca2+-bound, and Ca2+ plus target peptide-bound states. Our findings are consistent with a three-state conformational model of CaM, showing a semi-open apo-state, a highly extended Ca2+-replete state, and a compact target protein-bound state. Molecular dynamics simulations suggest that the presence of glycerol, and potentially other molecular crowding agents, has a profound effect on the relative stability of the different conformational states. Differing experimental conditions may explain the discrepancies in the literature regarding the observed conformational state(s) of CaM, and our PELDOR measurements show good evidence for an extended conformation of Ca2+-replete CaM similar to the one observed in early X-ray crystal structures.Publisher PDFPeer reviewe

Isopentenol Utilization Pathway for the Production of Linalool in Escherichia coli Using an Improved Bacterial Linalool/Nerolidol Synthase

From Wiley via Jisc Publications RouterHistory: received 2021-03-10, rev-recd 2021-05-02, pub-electronic 2021-05-25Article version: VoRPublication status: PublishedFunder: Future Biomanufacturing Research Hub; Grant(s): EP/S01778X/1Funder: Engineering and Physical Sciences Research Council; Id: http://dx.doi.org/10.13039/501100000266Funder: Biotechnology and Biological Sciences Research Council; Id: http://dx.doi.org/10.13039/501100000268Funder: Office of Naval Research Global; Id: http://dx.doi.org/10.13039/100007297Abstract: Linalool is a monoterpenoid used as a fragrance ingredient, and is a promising source for alternative fuels. Synthetic biology offers attractive alternative production methods compared to extraction from natural sources and chemical synthesis. Linalool/nerolidol synthase (bLinS) from Streptomyces clavuligerus is a bifunctional enzyme, producing linalool as well as the sesquiterpenoid nerolidol when expressed in engineered Escherichia coli harbouring a precursor terpenoid pathway such as the mevalonate (MVA) pathway. Here we identified two residues important for substrate selection by bLinS, L72 and V214, where the introduction of bulkier residues results in variants with reduced nerolidol formation. Terpenoid production using canonical precursor pathways is usually limited by numerous and highly regulated enzymatic steps. Here we compared the canonical MVA pathway to the non‐canonical isopentenol utilization (IU) pathway to produce linalool using the optimised bLinS variant. The IU pathway uses isoprenol and prenol to produce linalool in only five steps. Adjusting substrate, plasmid system, inducer concentration, and cell strain directs the flux towards monoterpenoids. Our integrated approach, combining enzyme engineering with flux control using the artificial IU pathway, resulted in high purity production of the commercially attractive monoterpenoid linalool, and will guide future efforts towards efficient optimisation of terpenoid production in engineered microbes

Renewable and tuneable bio-LPG blends derived from amino acids

From Springer Nature via Jisc Publications RouterHistory: received 2020-05-21, accepted 2020-07-08, registration 2020-07-08, pub-electronic 2020-07-14, online 2020-07-14, collection 2020-12Publication status: PublishedFunder: C3 Biotechnologies LtdFunder: Engineering and Physical Sciences Research Council; doi: http://dx.doi.org/10.13039/501100000266; Grant(s): EP/S01778X/1, EP/J020192/1Funder: Biotechnology and Biological Sciences Research Council; doi: http://dx.doi.org/10.13039/501100000268; Grant(s): BB/M017702/1, BB/L010798/1Funder: Newton-Mosharafa fundAbstract: Background: Microbial biorefinery approaches are beginning to define renewable and sustainable routes to clean-burning and non-fossil fuel-derived gaseous alkanes (known as ‘bio-LPG’). The most promising strategies have used a terminal fatty acid photodecarboxylase, enabling light-driven propane production from externally fed waste butyric acid. Use of Halomonas (a robust extremophile microbial chassis) with these pathways has enabled bio-LPG production under non-sterile conditions and using waste biomass as the carbon source. Here, we describe new engineering approaches to produce next-generation pathways that use amino acids as fuel precursors for bio-LPG production (propane, butane and isobutane blends). Results: Multiple pathways from the amino acids valine, leucine and isoleucine were designed in E. coli for the production of propane, isobutane and butane, respectively. A branched-chain keto acid decarboxylase-dependent pathway utilising fatty acid photodecarboxylase was the most effective route, generating higher alkane gas titres over alternative routes requiring coenzyme A and/or aldehyde deformylating oxygenase. Isobutane was the major gas produced in standard (mixed amino acid) medium, however valine supplementation led to primarily propane production. Transitioning pathways into Halomonas strain TQ10 enabled fermentative production of mixed alkane gases under non-sterile conditions on simple carbon sources. Chromosomal integration of inducible (~ 180 mg/g cells/day) and constitutive (~ 30 mg/g cells/day) pathways into Halomonas generated production strains shown to be stable for up to 7 days. Conclusions: This study highlights new microbial pathways for the production of clean-burning bio-LPG fuels from amino acids. The use of stable Halomonas production strains could lead to gas production in the field under non-sterile conditions following process optimisation