Metabolic engineering for the production of isoprene and isopentenol by Escherichia coli.
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ABSTRACT:
SUBMITTER: Li M
PROVIDER: S-EPMC6132537 | biostudies-literature | 2018 Sep
REPOSITORIES: biostudies-literature
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Metabolic engineering for the production of isoprene and isopentenol by Escherichia coli.
Li Meijie M Nian Rui R Xian Mo M Zhang Haibo H
Applied microbiology and biotechnology 20180714 18
The biotechnological production of isoprene and isopentenol has recently been studied. Isoprene, which is currently made mainly from petroleum, is an important platform chemical for synthesizing pesticides, medicines, oil additives, fragrances, and more and is especially important in the rubber production industry. Isopentenols, which have better combustion properties than well-known biofuels (ethanol), have recently received more attention. Supplies of petroleum, the conventional source of isop ...[more]
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Project description:BACKGROUND: Riboflavin (vitamin B2), the precursor of the flavin cofactors flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), is used commercially as an animal feed supplement and food colorant. E. coli is a robust host for various genetic manipulations and has been employed for efficient production of biofuels, polymers, amino acids, and bulk chemicals. Thus, the aim of this study was to understand the metabolic capacity of E. coli for the riboflavin production by modification of central metabolism, riboflavin biosynthesis pathway and optimization of the fermentation conditions. RESULTS: The basic producer RF01S, in which the riboflavin biosynthesis genes ribABDEC from E. coli were overexpressed under the control of the inducible trc promoter, could accumulate 229.1 mg/L of riboflavin. Further engineering was performed by examining the impact of expression of zwf (encodes glucose 6-phosphate dehydrogenase) and gnd (encodes 6-phosphogluconate dehydrogenase) from Corynebacterium glutamicum and pgl (encodes 6-phosphogluconolactonase) from E. coli on riboflavin production. Deleting pgi (encodes glucose-6-phosphate isomerase) and genes of Entner-Doudoroff (ED) pathway successfully redirected the carbon flux into the oxidative pentose phosphate pathway, and overexpressing the acs (encodes acetyl-CoA synthetase) reduced the acetate accumulation. These modifications increased riboflavin production to 585.2 mg/L. By further modulating the expression of ribF (encodes riboflavin kinase) for reducing the conversion of riboflavin to FMN in RF05S, the final engineering strain RF05S-M40 could produce 1036.1 mg/L riboflavin in LB medium at 37°C. After optimizing the fermentation conditions, strain RF05S-M40 produced 2702.8 mg/L riboflavin in the optimized semi-defined medium, which was a value nearly 12-fold higher than that of RF01S, with a yield of 137.5 mg riboflavin/g glucose. CONCLUSIONS: The engineered strain RF05S-M40 has the highest yield among all reported riboflavin production strains in shake flask culture. This work collectively demonstrates that E. coli has a potential to be a microbial cell factory for riboflavin bioproduction.
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Project description:Amino-acid producers have traditionally been developed by repeated random mutagenesis owing to the difficulty in rationally engineering the complex and highly regulated metabolic network. Here, we report the development of the genetically defined L-threonine overproducing Escherichia coli strain by systems metabolic engineering. Feedback inhibitions of aspartokinase I and III (encoded by thrA and lysC, respectively) and transcriptional attenuation regulations (located in thrL) were removed. Pathways for Thr degradation were removed by deleting tdh and mutating ilvA. The metA and lysA genes were deleted to make more precursors available for Thr biosynthesis. Further target genes to be engineered were identified by transcriptome profiling combined with in silico flux response analysis, and their expression levels were manipulated accordingly. The final engineered E. coli strain was able to produce Thr with a high yield of 0.393 g per gram of glucose, and 82.4 g/l Thr by fed-batch culture. The systems metabolic engineering strategy reported here may be broadly employed for developing genetically defined organisms for the efficient production of various bioproducts.
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Project description:Xylonate is a valuable chemical for versatile applications. Although the chemical synthesis route and microbial conversion pathway were established decades ago, no commercial production of xylonate has been obtained so far. In this study, the industrially important microorganism Escherichia coli was engineered to produce xylonate from xylose. Through the coexpression of a xylose dehydrogenase (xdh) and a xylonolactonase (xylC) from Caulobacter crescentus, the recombinant strain could convert 1 g/L xylose to 0.84 g/L xylonate and 0.10 g/L xylonolactone after being induced for 12 h. Furthermore, the competitive pathway for xylose catabolism in E. coli was blocked by disrupting two genes (xylA and xylB) encoding xylose isomerase and xylulose kinase. Under fed-batch conditions, the finally engineered strain produced up to 27.3 g/L xylonate and 1.7 g/L xylonolactone from 30 g/L xylose, about 88% of the theoretical yield. These results suggest that the engineered E. coli strain has a promising perspective for large-scale production of xylonate.
| S-EPMC3702539 | biostudies-literature
Project description:BackgroundL-malate is one of the most important platform chemicals widely used in food, metal cleaning, textile finishing, pharmaceuticals, and synthesis of various fine chemicals. Recently, the development of biotechnological routes to produce L-malate from renewable resources has attracted significant attention.ResultsA potential L-malate producing strain E. coli BA040 was obtained by inactivating the genes of fumB, frdABCD, ldhA and pflB. After co-overexpression of mdh and pck, BA063 achieved 18 g/L glucose consumption, leading to an increase in L-malate titer and yield of 13.14 g/L and 0.73 g/g, respectively. Meantime, NADH/NAD+ ratio decreased to 0.72 with the total NAD(H) of 38.85 µmol/g DCW, and ATP concentration reached 715.79 nmol/g DCW. During fermentation in 5L fermentor with BA063, 41.50 g/L glucose was consumed within 67 h with the final L-malate concentration and yield of 28.50 g/L, 0.69 g/g when heterologous CO2 source was supplied.ConclusionsThe availability of NAD(H) was correlated positively with the glucose utilization rate and cellular metabolism capacities, and lower NADH/NAD+ ratio was beneficial for the accumulation of L-malate under anaerobic conditions. Enhanced ATP level could significantly enlarge the intracellular NAD(H) pool under anaerobic condition. Moreover, there might be an inflection point, that is, the increase of NAD(H) pool before the inflection point is followed by the improvement of metabolic performance, while the increase of NAD(H) pool after the inflection point has no significant impacts and NADH/NAD+ ratio would dominate the metabolic flux. This study is a typical case of anaerobic organic acid fermentation, and demonstrated that ATP level, NAD(H) pool and NADH/NAD+ ratio are three important regulatory parameters during the anaerobic production of L-malate.
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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.
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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:BackgroundIcariside D2 is a plant-derived natural glycoside with pharmacological activities of inhibiting angiotensin-converting enzyme and killing leukemia cancer cells. Production of icariside D2 by plant extraction and chemical synthesis is inefficient and environmentally unfriendly. Microbial cell factory offers an attractive route for economical production of icariside D2 from renewable and sustainable bioresources.ResultsWe metabolically constructed the biosynthetic pathway of icariside D2 in engineered Escherichia coli. We screened the uridine diphosphate glycosyltransferases (UGTs) and obtained an active RrUGT3 that regio-specifically glycosylated tyrosol at phenolic position to exclusively synthesize icariside D2. We put heterologous genes in E. coli cell for the de novo biosynthesis of icariside D2. By fine-tuning promoter and copy number as well as balancing gene expression pattern to decrease metabolic burden, the BMD10 monoculture was constructed. Parallelly, for balancing pathway strength, we established the BMT23-BMD12 coculture by distributing the icariside D2 biosynthetic genes to two E. coli strains BMT23 and BMD12, responsible for biosynthesis of tyrosol from preferential xylose and icariside D2 from glucose, respectively. Under the optimal conditions in fed-batch shake-flask fermentation, the BMD10 monoculture produced 3.80 g/L of icariside D2 using glucose as sole carbon source, and the BMT23-BMD12 coculture produced 2.92 g/L of icariside D2 using glucose-xylose mixture.ConclusionsWe for the first time reported the engineered E. coli for the de novo efficient production of icariside D2 with gram titer. It would be potent and sustainable approach for microbial production of icariside D2 from renewable carbon sources. E. coli-E. coli coculture approach is not limited to glycoside production, but could also be applied to other bioproducts.
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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.
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Project description:Most flavonoids exist as sugar conjugates. Naturally occurring flavonoid sugar conjugates include glucose, galactose, glucuronide, rhamnose, xylose, and arabinose. These flavonoid glycosides have diverse physiological activities, depending on the type of sugar attached. To synthesize an unnatural flavonoid glycoside, Actinobacillus actinomycetemcomitans gene tll (encoding dTDP-6-deoxy-L-lyxo-4-hexulose reductase, which converts the endogenous nucleotide sugar dTDP-4-dehydro-6-deoxy-L-mannose to dTDP-6-deoxytalose) was introduced into Escherichia coli. In addition, nucleotide-sugar dependent glycosyltransferases (UGTs) were screened to find a UGT that could use dTDP-6-deoxytalose. Supplementation of this engineered strain of E. coli with quercetin resulted in the production of quercetin-3-O-(6-deoxytalose). To increase the production of quercetin 3-O-(6-deoxytalose) by increasing the supplement of dTDP-6-deoxytalose in E. coli, we engineered nucleotide biosynthetic genes of E. coli, such as galU (UTP-glucose 1-phosphate uridyltransferase), rffA (dTDP-4-oxo-6-deoxy-d-glucose transaminase), and/or rfbD (dTDP-4-dehydrorahmnose reductase). The engineered E. coli strain produced approximately 98 mg of quercetin 3-O-(6-deoxytalose)/liter, which is 7-fold more than that produced by the wild-type strain, and the by-products, quercetin 3-O-glucose and quercetin 3-O-rhamnose, were also significantly reduced.
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