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

Engineering of the PETNR active site to accommodate novel α/β substituted enone substrates

Experiments facilitating the engineering of the PETNR active site to accommodate a range of non natural enone substrates with substituents localised on the α and β carbons of the unsaturated bond are described. In order to facilitate the high throughput purification of PETNR libraries poly histidine (PETNRHis) and biotin (PETNRBio) tagged PETNR variants were generated. High throughput protocols were developed for the automated generation, purification and screening of libraries in a 96 well format. Protocols were optimised and trialled using blocks consisting of PETNRHis WT only and characterised in terms of intra block variation. A range of single site saturation mutagenic libraries were generated at positions in the active site consisting of T26, Y68, W102, H181, H184, Y186, Q241 and Y351. Sequencing results indicated randomised libraries with the occasional instance of bias evident. Expression and purification in a 96 well format was monitored by SDS PAGE and protein quantitation. Library activity was quantified and demonstrated to retain varying degrees of activity with the model substrate 2-cyclohexenone. Following this verification of the experimental protocol libraries were screened against a range of substrates analogous to substrates demonstrated to be active with PETNRWT but incorporating substituents at the α and β carbons. 'Hits' generated from these screening reactions were studied further by the determination of the specific activity and quantitation of substrate/product from biotransformation reactions. From these screening experiments totalling 3,600 individual reactions, 35 were identified as potential hits, of these 8 proved to be genuinely improved variants. Substituents at the β carbon were demonstrated to compromise the activity of the WT enzyme most severely. Positions 68, 102, and 351 were demonstrated to play an important role in the accommodation of substituents at the α carbon whilst residues 26 and 351 are important for the β carbon. The best variants demonstrated up to 9 fold improvements in poor substrates which represented rates in excess of those observed for model substrates.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Biochemical and structural studies of human methionine synthase reductase

Human methionine synthase reductase (MSR) is a 78-kDa diflavin enzyme involved in folate and methionine metabolism. It regenerates the cofactor of methionine synthase (MS), cob(II), to reduce inactive MS. MSR and one of its the FAD/NADPH binding domain were cloned as GST-tagged fusion proteins for expression and purification in Escherichia coli. And a 1.9 Å Crystals of the FAD/NADPH binding domain of MSR with and without NADP+ were produced and carried out X-ray diffraction experiment and the structure of the crystal was solved by molecule replacement method. The activation domain of human MS was also expressed and purified in Escherichia coli and crystallization conditions determined. A new expression vector for full-length MSR, which contains a N-terminal GST tag, and C-terminal 6× His tag, was constructed and validated by sequencing, restriction enzymes digestion and successfully expressed in E. coli and Yeast Pichia pastoris. Based on the structure information, site-directed mutagenesis on the two positions Asp652 and trpytophan697 of MSR were designed and completed. The variants D652A, D652R, D652N of the FAD/NADPH binding domain of MSR and the variants D652A,D652R,D652N, W696A,W697H of the full-length MSR were cloned and expressed in BL21 (E. coli). The proteins of these mutants were purified by affinity chromatography, anion exchange chromatography and gel filtration chromatography. And the kinetic studies on these variants of MSR were investigated in steady state kinetic study, steady state inhibition studies, stopped-flow pre steady-state kinetic and redox potential studies. Compared with the data of the wild type MSR, the turnover number of variants all decreased, the catalytic ability become lower and the midpoint potential of cofactor FAD occurred positive shift. Both 2'5-ADP and NADP+ were competitive inhibitors for variants of MSR. However, 2'5'-ADP was relative strong inhibitor than NADP+. All the data on variants of MSR suggested the Asp652 and tryptophan697 were two important structural and function determinant of MSR. To investigate the dynamic properties of EPR, ENDOR and ESMME are used to investigate the existence of the semiquinone flavin cofactors, FAD and FMN, and the hyperfine coupling arising from the interaction of some nuclei with the unpaired electron spin. ELDOR spectroscopy was applied to measure the distance between the FAD and FMN in MSR under the binding of 2', 5'-ADP, NADP and the activation domain of MS to further check the conformational change of MSR.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

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

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