Metabolic Engineering Communications最新文献

5-Deoxy(iso)flavonoids are structural representatives of phenylpropanoid-derived compounds and play critical roles in plant ecophysiology. Recently, 5-deoxy(iso)flavonoids gained significant interest due to their potential applications as pharmaceuticals, nutraceuticals, and food additives. Given the difficulties in their isolation from native plant sources, engineered biosynthesis of 5-deoxy(iso)flavonoids in a microbial host is a highly promising alternative approach. However, the production of 5-deoxy(iso)flavonoids is hindered by metabolic flux imbalances that result in a product profile predominated by non-reduced analogues. In this study, GmCHS7 (chalcone synthase from Glycine max) and GuCHR (chalcone reductase from Glycyrrhizza uralensis) were preliminarily utilized to improve the CHR ratio (CHR product to total CHS product). The use of this enzyme combination improved the final CHR ratio from 39.7% to 50.3%. For further optimization, a protein-protein interaction strategy was employed, basing on the spatial adhesion of GmCHS7:PDZ and GuCHR:PDZlig. This strategy further increased the ratio towards the CHR-derived product (54.7%), suggesting partial success of redirecting metabolic flux towards the reduced branch. To further increase the total carbon metabolic flux, 15 protein scaffolds were programmed with stoichiometric arrangement of the three sequential catalysts GmCHS7, GuCHR and MsCHI (chalcone isomerase from Medicago sativa), resulting in a 1.4-fold increase in total flavanone production, from 69.4 mg/L to 97.0 mg/L in shake flasks. The protein self-assembly strategy also improved the production and direction of the lineage-specific compounds 7,4′-dihydroxyflavone and daidzein in Escherichia coli. This study presents a significant advancement of 5-deoxy(iso)flavonoid production and provides the foundation for production of value-added 5-deoxy(iso)flavonoids in microbial hosts.

Trans-regulating elements such as noncoding RNAs are crucial in modifying cells, and has shown broad application in synthetic biology, metabolic engineering and RNA therapies. Although effective, titration of the regulatory levels of such elements is less explored. Encouraged by the need of fine-tuning cellular functions, we studied key parameters of the antisense RNA design including oligonucleotide length, targeting region and relative dosage to achieve differentiated inhibition. We determined a 30-nucleotide configuration that renders efficient and robust inhibition. We found that by targeting the core RBS region proportionally, quantifiable inhibition levels can be rationally obtained. A mathematic model was established accordingly with refined energy terms and successfully validated by depicting the inhibition levels for genomic targets. Additionally, we applied this fine-tuning approach for 4-hydroxycoumarin biosynthesis by simultaneous and quantifiable knockdown of multiple targets, resulting in a 3.58-fold increase in titer of the engineered strain comparing to that of the non-regulated. We believe the developed tool is broadly compatible and provides an extra layer of control in modifying living systems.

Cell-free systems present a significant opportunity to harness the metabolic potential of diverse organisms. Removing the cellular context provides the ability to produce biological products without the need to maintain cell viability and enables metabolic engineers to explore novel chemical transformation systems. Crude extracts maintain much of a cell’s capabilities. However, only limited tools are available for engineering the contents of the extracts used for cell-free systems. Thus, our ability to take full advantage of the potential of crude extracts for cell-free metabolic engineering is constrained. Here, we employ Multiplex Automated Genomic Engineering (MAGE) to tag proteins for selective depletion from crude extracts so as to specifically direct chemical production. Specific edits to central metabolism are possible without significantly impacting cell growth. Selective removal of pyruvate degrading enzymes resulted in engineered crude lysates that are capable of up to 40-fold increases in pyruvate production when compared to the non-engineered extract. The described approach melds the tools of systems and synthetic biology to showcase the effectiveness of cell-free metabolic engineering for applications like bioprototyping and bioproduction.

Docosadienoic acid (DDA, 22:2–13,16) and docosatrienoic acid (DTA, 22:3–13,16,19) are two very long chain polyunsaturated fatty acids (VLCPUFAs) that are recently shown to possess strong anti-inflammatory and antitumor properties. An ELO type elongase (EhELO1) from wild plant Eranthis hyemalis can synthesize the two fatty acids by sequential elongation of linoleic acid and alpha-linolenic acid, respectively. Seed-specific expression of this gene in oilseed crop Brassica carinata produced a considerable amount of DDA and DTA in transgenic seeds. However, these fatty acids were excluded from the sn-2 position of triacylglycerols (TAGs). To improve the production level and nutrition value of the VLCPUFAs in the transgenic oilseed crop, a cytoplasmic lysophosphatidic acid acyltransferase (EhLPAAT2) for the incorporation of the two fatty acids into the sn-2 position of triacylglycerols was identified from E. hyemalis. RT-PCR analysis showed that it was preferentially expressed in developing seeds where EhELO1 was exclusively expressed in E. hyemalis. Seed specific expression of EhLPAAT2 along with EhELO1 in B. carinata resulted in the effective incorporation of DDA and DTA at the sn-2 position of TAGs, thereby increasing the total amount of DDA and DTA in transgenic seeds. To our knowledge, this is the first plant LPAAT that can incorporate VLCPUFAs into TAGs. Improved production of DDA and DTA in the oilseed crop using EhLPAAT2 and EhELO1 provides a real commercial opportunity for high value agriculture products for nutraceutical uses.

Cyanobacteria can be utilized as a platform for direct phototrophic conversion of CO₂ to produce several types of carbon-neutral biofuels. One promising compound to be produced photobiologically in cyanobacteria is isobutene. As a volatile compound, isobutene will quickly escape the cells without building up to toxic levels in growth medium or get caught in the membranes. Unlike liquid biofuels, gaseous isobutene may be collected from the headspace and thus avoid the costly extraction of a chemical from culture medium or from cells. Here we investigate a putative synthetic pathway for isobutene production suitable for a photoautotrophic host. First, we expressed α-ketoisocaproate dioxygenase from Rattus norvegicus (RnKICD) in Escherichia coli. We discovered isobutene formation with the purified RnKICD with the rate of 104.6 ​± ​9 ​ng (mg protein)^-1 min^-1 using α-ketoisocaproate as a substrate. We further demonstrate isobutene production in the cyanobacterium Synechocystis sp. PCC 6803 by introducing the RnKICD enzyme. Synechocystis strain heterologously expressing the RnKICD produced 91 ​ng ​l⁻¹ OD₇₅₀⁻¹ ​h⁻¹. Thus, we demonstrate a novel sustainable platform for cyanobacterial production of an important building block chemical, isobutene. These results indicate that RnKICD can be used to further optimize the synthetic isobutene pathway by protein and metabolic engineering efforts.

Increasing understanding of metabolic and regulatory networks underlying microbial physiology has enabled creation of progressively more complex synthetic biological systems for biochemical, biomedical, agricultural, and environmental applications. However, despite best efforts, confounding phenotypes still emerge from unforeseen interplay between biological parts, and the design of robust and modular biological systems remains elusive. Such interactions are difficult to predict when designing synthetic systems and may manifest during experimental testing as inefficiencies that need to be overcome. Transforming organisms such as Escherichia coli into microbial factories is achieved via several engineering strategies, used individually or in combination, with the goal of maximizing the production of chosen target compounds. One technique relies on suppressing or overexpressing selected genes; another involves introducing heterologous enzymes into a microbial host. These modifications steer mass flux towards the set of desired metabolites but may create unexpected interactions. In this work, we develop a computational method, termed Metabolic Disruption Workflow (MDFlow), for discovering interactions and network disruptions arising from enzyme promiscuity – the ability of enzymes to act on a wide range of molecules that are structurally similar to their native substrates. We apply MDFlow to two experimentally verified cases where strains with essential genes knocked out are rescued by interactions resulting from overexpression of one or more other genes. We demonstrate how enzyme promiscuity may aid cells in adapting to disruptions of essential metabolic functions. We then apply MDFlow to predict and evaluate a number of putative promiscuous reactions that can interfere with two heterologous pathways designed for 3-hydroxypropionic acid (3-HP) production. Using MDFlow, we can identify putative enzyme promiscuity and the subsequent formation of unintended and undesirable byproducts that are not only disruptive to the host metabolism but also to the intended end-objective of high biosynthetic productivity and yield. As we demonstrate, MDFlow provides an innovative workflow to systematically identify incompatibilities between the native metabolism of the host and its engineered modifications due to enzyme promiscuity.

Terpenoids are a large and diverse group of natural products with commercial applications. Microbial production of terpenes is considered as a feasible approach for the stable supply of these complex hydrocarbons. Cyanobacteria, photosynthetic prokaryotes, are attractive hosts for sustainable bioproduction, because these autotrophs require only light and CO₂ for growth. Despite cyanobacteria having been engineered to produce a variety of compounds, their productivities of terpenes are generally low. Further research is needed to determine the bottleneck reactions for enhancing terpene production in cyanobacteria. In this study, we engineered the fast-growing cyanobacterium Synechococcus elongatus UTEX 2973 to produce a commercially-used terpenoid, limonene. We identified a beneficial mutation in the gene encoding geranylgeranyl pyrophosphate synthase crtE, leading to a 2.5-fold increase in limonene production. The engineered strain produced 16.4 ​mg ​L⁻¹ of limonene at a rate of 8.2 ​mg ​L⁻¹ day⁻¹, which is 8-fold higher than limonene productivities previously reported in other cyanobacterial species. Furthermore, we employed a combinatorial metabolic engineering approach to optimize genes involved in the upstream pathway of limonene biosynthesis. By modulating the expression of genes encoding the enzymes in the MEP pathway and the geranyl pyrophosphate synthase, we showed that optimization of the expression level is critical to enhance limonene production in cyanobacteria.

Surfactin, a quorum sensing signal molecule, is correlated with carbon metabolism in Bacillus amyloliquefaciens. In the present work, we found that mutation of srfA (ΔsrfA) led to an obviously changed carbon metabolism in B. amyloliquefaciens. Firstly, the PTS-glucose system was significantly increased as a feedback to glucose exhaustion. Secondly, the basic carbon metabolism such as glycolysis and TCA cycle was obviously weakened in ΔsrfA. Thirdly, the global regulator of CcpA (carbon catabolite protein A) and P ~ Ser₄₆-HPr (seryl-phosphorylated form of histidine-containing protein) to mediate the CcpA-dependent CCR (carbon catabolite repression) were not increased, but the ability to use extracellular non- and less-preferred carbon sources was down-regulated in ΔsrfA. Fourthly, the carbon overflow metabolism such as biosynthesis of acetate was enhanced while biosynthesis of acetoin/2,3-butanediol and branched-chain amino acids were weakened in ΔsrfA. Finally, ΔsrfA could use most of non- and less-preferred carbon sources except for fatty acids, branched chain amino acids, and some organic acids (e.g. pyruvate, citrate and glutamate) after glucose exhaustion. Collectively, surfactin showed a global influence on carbon metabolism in B. amyloliquefaciens. Our studies highlighted a way to correlate quorum sensing with carbon metabolism via surfactin in Bacillus species.