Pathway engineering for high-yield production of lutein in Escherichia coli
Ontology highlight
ABSTRACT:
SUBMITTER: Takemura M
PROVIDER: S-EPMC8546607 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Plant polyphenols have been the subject of several recent scientific investigations since many of the molecules in this class have been found to be highly active in the human body, with a plethora of health-promoting activities against a variety of diseases, including heart disease, diabetes, and cancer, and with even the potential to slow aging. Further development of these potent natural therapeutics hinges on the formation of robust industrial production platforms designed using specifically selected as well as engineered protein sources along with the construction of optimal expression platforms. In this work, we first report the investigation of various stilbene synthases from an array of plant species considering structure-activity relationships, their expression efficiency in microorganisms, and their ability to synthesize resveratrol. Second, we looked into the construct environment of recombinantly expressed stilbene synthases, including different promoters, construct designs, and host strains, to create an Escherichia coli strain capable of producing superior resveratrol titers sufficient for commercial usage. Further improvement of metabolic capabilities of the recombinant strain aimed at improving the intracellular malonyl-coenzyme A pool, a resveratrol precursor, resulted in a final improved titer of 2.3 g/liter resveratrol.
| S-EPMC3126431 | biostudies-literature
Project description:BackgroundMethyl methacrylate (MMA) is a key precursor of polymethyl methacrylate, extensively used as a transparent thermoplastic in various industries. Conventional MMA production poses health and environmental risks; hence, citramalate serves as an alternative bacterial compound precursor for MMA production. The highest citramalate titer was previously achieved by Escherichia coli BW25113. However, studies on further improving citramalate production through metabolic engineering are limited, and phage contamination is a persistent problem in E. coli fermentation.ResultsThis study aimed to construct a phage-resistant E. coli BW25113 strain capable of producing high citramalate titers from glucose. First, promoters and heterologous cimA genes were screened, and an effective biosynthetic pathway for citramalate was established by overexpressing MjcimA3.7, a mutated cimA gene from Methanococcus jannaschii, regulated by the BBa_J23100 promoter in E. coli. Subsequently, a phage-resistant E. coli strain was engineered by integrating the Ssp defense system into the genome and mutating key components of the phage infection cycle. Then, the strain was engineered to include the non-oxidative glycolysis pathway while removing the acetate synthesis pathway to enhance the supply of acetyl-CoA. Furthermore, glucose utilization by the strain improved, thereby increasing citramalate production. Ultimately, 110.2 g/L of citramalate was obtained after 80 h fed-batch fermentation. The citramalate yield from glucose and productivity were 0.4 g/g glucose and 1.4 g/(L·h), respectively.ConclusionThis is the highest reported citramalate titer and productivity in E. coli without the addition of expensive yeast extract and additional induction in fed-bath fermentation, emphasizing its potential for practical applications in producing citramalate and its derivatives.
| S-EPMC11340173 | biostudies-literature
Project description:BackgroundGeraniol is an acyclic monoterpene alcohol, which exhibits good prospect as a gasoline alternative. Geraniol is naturally encountered in plants at low concentrations and an attractive target for microbial engineering. Geraniol has been heterologously produced in Escherichia coli, but the low titer hinders its industrial applications. Moreover, bioconversion of geraniol by E. coli remains largely unknown.ResultsRecombinant overexpression of Ocimum basilicum geraniol synthase, Abies grandis geranyl diphosphate synthase, and a heterotic mevalonate pathway in E. coli BL21 (DE3) enabled the production of up to 68.6 ± 3 mg/L geraniol in shake flasks. Initial fed-batch fermentation only increased geraniol production to 78.8 mg/L. To further improve the production yield, the fermentation conditions were optimized. Firstly, 81.4 % of volatile geraniol was lost during the first 5 h of fermentation in a solvent-free system. Hence, isopropyl myristate was added to the culture medium to form an aqueous-organic two-phase culture system, which effectively prevented volatilization of geraniol. Secondly, most of geraniol was eventually biotransformed into geranyl acetate by E. coli, thus decreasing geraniol production. For the first time, we revealed the role of acetylesterase (Aes, EC 3.1.1.6) from E. coli in hydrolyzing geranyl acetate to geraniol, and production of geraniol was successfully increased to 2.0 g/L under controlled fermentation conditions.ConclusionsAn efficient geraniol production platform was established by overexpressing several key pathway proteins in engineered E. coli strain combined with a controlled fermentation system. About 2.0 g/L geraniol was obtained using our controllable aqueous-organic two-phase fermentation system, which is the highest yield to date. In addition, the interconversion between geraniol and geranyl acetate by E. coli was first elucidated. This study provided a new and promising strategy for geraniol biosynthesis, which laid a basis for large-scale industrial application.
| S-EPMC4787002 | biostudies-literature
Project description:BackgroundL(-)-carnitine production has been widely studied because of its beneficial properties on various diseases and dysfunctions. Enterobacteria possess a specific biotransformation pathway which can be used for the enantioselective production of L(-)-carnitine. Although bioprocesses catalyzed by enzymes or whole cells can overcome the lack of enantioselectivity of chemical methods, current processes for L(-)-carnitine production still have severe disadvantages, such as the low yields, side reactions and the need of high catalyst concentrations and anaerobic conditions for proper expression of the biotransformation pathway. Additionally, genetically engineered strains so far constructed for L(-)-carnitine production are based on plasmids and, therefore, suffer from segregational unstability.ResultsIn this work, a stable, high yielding strain for L(-)-carnitine production from low cost substrates was constructed. A metabolic engineering strategy was implemented in a multiple mutant for use in both growing and resting cells systems. The effect of mutations on gene expression and metabolism was analyzed to characterize the productivity constraints of the wild type and the overproducer strains. Precise deletion of genes which encode proteins of central and carnitine metabolisms were performed. Specifically, flux through the TCA cycle was increased by deletion of aceK (which encodes a bifunctional kinase/phosphatase which inhibits isocitrate dehydrogenase activity) and the synthesis of the by-product ?-butyrobetaine was prevented by deletion of caiA (which encodes a crotonobetainyl-CoA reductase). Both mutations led to improve the L(-)-carnitine production by 20 and 42%, respectively. Moreover, the highly regulated promoter of the cai operon was substituted by a constitutive artificial promoter increasing the biotransformation rate, even under aerobic conditions. Resting cells of the BW ?aceK ?caiA p37cai strain produced 59.6 mmol l(-1)?·?h(-1) of L(-)-carnitine, doubling the productivity of the wild type strain. In addition, almost total conversion was attained in less than two hours without concomitant production of the side product ?-butyrobetaine.ConclusionsL(-)-carnitine production has been enhanced by strain engineering. Metabolic engineering strategies herein implemented allowed obtaining a robust and high yielding E. coli strain. The new overproducer strain attained almost complete conversion of crotonobetaine into L(-)-carnitine with growing and resting cells, and even under aerobic conditions, overcoming the main environmental restriction to carnitine metabolism expression. So far, this is the best performing L(-)-carnitine production E. coli strain described.
| S-EPMC3680233 | biostudies-literature
Project description:BackgroundXanthophylls are a large class of carotenoids that are found in a variety of organisms and play particularly important roles in the light-harvesting and photoprotection processes of plants and algae. Violaxanthin is an important plant-derived xanthophyll with wide potential applications in medicines, foods, and cosmetics because of its antioxidant activity and bright yellow color. To date, however, violaxanthins have not been produced using metabolically engineered microbes on a commercial scale. Metabolic engineering for microbial production of violaxanthin is hindered by inefficient synthesis pathway in the heterologous host. We systematically optimized the carotenoid chassis and improved the functional expression of key enzymes of violaxanthin biosynthesis in Escherichia coli.ResultsCo-overexpression of crtY (encoding lycopene β-cyclase), crtZ (encoding β-carotene 3-hydroxylase), and ZEP (encoding zeaxanthin epoxidase) had a notable impact on their functions, resulting in the accumulation of intermediate products, specifically lycopene and β-carotene. A chassis strain that did not accumulate the intermediate was optimized by several approaches. A promoter library was used to optimize the expression of crtY and crtZ. The resulting strain DZ12 produced zeaxanthin without intermediates. The expression of ZEP was further systematically optimized by using DZ12 as the chassis host. By using a low copy number plasmid and a modified dithiol/disulfide system, and by co-expressing a full electron transport chain, we generated a strain producing violaxanthin at about 25.28 ± 3.94 mg/g dry cell weight with decreased byproduct accumulation.ConclusionWe developed an efficient metabolically engineered Escherichia coli strain capable of producing a large amount of violaxanthin. This is the first report of a metabolically engineered microbial platform that could be used for the commercial production of violaxanthin.
| S-EPMC10283192 | biostudies-literature
Project description:Glutarate is a key monomer in polyester and polyamide production. The low efficiency of the current biosynthetic pathways hampers its production by microbial cell factories. Herein, through metabolic simulation, a lysine-overproducing E. coli strain Lys5 is engineered, achieving titer, yield, and productivity of 195.9 g/L, 0.67 g/g glucose, and 5.4 g/L·h, respectively. Subsequently, the pathway involving aromatic aldehyde synthase, monoamine oxidase, and aldehyde dehydrogenase (AMA pathway) is introduced into E. coli Lys5 to produce glutarate from glucose. To enhance the pathway's efficiency, rational mutagenesis on the aldehyde dehydrogenase is performed, resulting in the development of variant Mu5 with a 50-fold increase in catalytic efficiency. Finally, a glutarate tolerance gene cbpA is identified and genomically overexpressed to enhance glutarate productivity. With enzyme expression optimization, the glutarate titer, yield, and productivity of E. coli AMA06 reach 88.4 g/L, 0.42 g/g glucose, and 1.8 g/L·h, respectively. These findings hold implications for improving glutarate biosynthesis efficiency in microbial cell factories.
| S-EPMC10838341 | biostudies-literature
Project description:The identification of optimal genotypes that result in improved production of recombinant metabolites remains an engineering conundrum. In the present work, various strategies to reengineer central metabolism in Escherichia coli were explored for robust synthesis of flavanones, the common precursors of plant flavonoid secondary metabolites. Augmentation of the intracellular malonyl coenzyme A (malonyl-CoA) pool through the coordinated overexpression of four acetyl-CoA carboxylase (ACC) subunits from Photorhabdus luminescens (PlACC) under a constitutive promoter resulted in an increase in flavanone production up to 576%. Exploration of macromolecule complexes to optimize metabolic efficiency demonstrated that auxiliary expression of PlACC with biotin ligase from the same species (BirAPl) further elevated flavanone synthesis up to 1,166%. However, the coexpression of PlACC with Escherichia coli BirA (BirAEc) caused a marked decrease in flavanone production. Activity improvement was reconstituted with the coexpression of PlACC with a chimeric BirA consisting of the N terminus of BirAEc and the C terminus of BirAPl. In another approach, high levels of flavanone synthesis were achieved through the amplification of acetate assimilation pathways combined with the overexpression of ACC. Overall, the metabolic engineering of central metabolic pathways described in the present work increased the production of pinocembrin, naringenin, and eriodictyol in 36 h up to 1,379%, 183%, and 373%, respectively, over production with the strains expressing only the flavonoid pathway, which corresponded to 429 mg/liter, 119 mg/liter, and 52 mg/liter, respectively.
| S-EPMC1932724 | biostudies-literature
Project description:Salicin is a notable phenolic glycoside derived from plants including Salix and Populus genus and has multiple biological activities such as anti-inflammatory and antiarthritic, anticancer, and antiaging effects. In this work, we engineered production of salicin from cheap renewable carbon resources in Escherichia coli (E. coli) by extending the shikimate pathway. We first investigated enzymes synthesizing salicylate from chorismate. Subsequently, carboxylic acid reductases (CARs) from different resources were screened to achieve efficient reduction of salicylate. Third, glucosyltransferases from different sources were selected for constructing cell factories of salicin. The enzymes including salicylate synthase AmS from Amycolatopsis methanolica, carboxylic acid reductase CARse from Segniliparus rotundus, and glucosyltransferase UGT71L1 from Populous trichocarpa were overexpressed in a modified E. coli strain MG1655-U7. The engineered strain produced 912.3 ± 12.7 mg/L salicin in 72 h of fermentation. These results demonstrated the production of salicin in a microorganism and laid significant foundation for its commercialization for pharmaceutical and nutraceutical applications.
| S-EPMC9494424 | biostudies-literature
Project description:Branched five carbon (C5) alcohols are attractive targets for microbial production due to their desirable fuel properties and importance as platform chemicals. In this study, we engineered a heterologous isoprenoid pathway in E. coli for the high-yield production of 3-methyl-3-buten-1-ol, 3-methyl-2-buten-1-ol, and 3-methyl-1-butanol, three C5 alcohols that serve as potential biofuels. We first constructed a pathway for 3-methyl-3-buten-1-ol, where metabolite profiling identified NudB, a promiscuous phosphatase, as a likely pathway bottleneck. We achieved a 60% increase in the yield of 3-methyl-3-buten-1-ol by engineering the Shine-Dalgarno sequence of nudB, which increased protein levels by 9-fold and reduced isopentenyl diphosphate (IPP) accumulation by 4-fold. To further optimize the pathway, we adjusted mevalonate kinase (MK) expression and investigated MK enzymes from alternative microbes such as Methanosarcina mazei. Next, we expressed a fusion protein of IPP isomerase and the phosphatase (Idi1~NudB) along with a reductase (NemA) to diversify production to 3-methyl-2-buten-1-ol and 3-methyl-1-butanol. Finally, we used an oleyl alcohol overlay to improve alcohol recovery, achieving final titers of 2.23 g/L of 3-methyl-3-buten-1-ol (~70% of pathway-dependent theoretical yield), 150 mg/L of 3-methyl-2-buten-1-ol, and 300 mg/L of 3-methyl-1-butanol.
| S-EPMC4459108 | biostudies-literature
Project description:Cross-linking mass spectrometry (XL-MS) is an emerging technique to obtain structural information of biomacromolecules and their complexes in cells and in vitro. In particular, certain photo-reactive amino acids (pA) such as photo-leucine (pLeu) and photo-methionine can provide unique short-distance information on structural core regions of proteins. Here, we present a protocol for high-yield incorporation of pLeu in proteins recombinantly expressed in Escherichia coli. The protein is expressed at high cell densities, which reduces the required amount of the photo-reactive amino acid by a factor of 10 compared to standard protocols, while maintaining high incorporation rates. For the two chaperones Trigger Factor and SecB, up to 3 mg of photo-leucine labeled protein were thus obtained from 100 ml of cell culture, with label incorporation rates of up to 34%. For Trigger factor, UV-induced cross-linking lead to the identification of 12 cross-links that are in agreement with published three-dimensional structures. The accessibility of milligram amounts of photo-leucine-labeled proteins at low costs will be highly useful to address structural biology questions.
2020-05-26 | PXD017584 | Pride