Manipulation of the precursor supply for high-level production of longifolene by metabolically engineered Escherichia coli.
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ABSTRACT:
SUBMITTER: Cao Y
PROVIDER: S-EPMC6331559 | biostudies-literature | 2019 Jan
REPOSITORIES: biostudies-literature
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Project description:Escherichia coli strains (KJ060 and KJ073) that were previously developed for succinate production have now been modified for malate production. Many unexpected changes were observed during this investigation. The initial strategy of deleting fumarase isoenzymes was ineffective, and succinate continued to accumulate. Surprisingly, a mutation in fumarate reductase alone was sufficient to redirect carbon flow into malate even in the presence of fumarase. Further deletions were needed to inactivate malic enzymes (typically gluconeogenic) and prevent conversion to pyruvate. However, deletion of these genes (sfcA and maeB) resulted in the unexpected accumulation of D-lactate despite the prior deletion of mgsA and ldhA and the absence of apparent lactate dehydrogenase activity. Although the metabolic source of this D-lactate was not identified, lactate accumulation was increased by supplementation with pyruvate and decreased by the deletion of either pyruvate kinase gene (pykA or pykF) to reduce the supply of pyruvate. Many of the gene deletions adversely affected growth and cell yield in minimal medium under anaerobic conditions, and volumetric rates of malate production remained low. The final strain (XZ658) produced 163 mM malate, with a yield of 1.0 mol (mol glucose(-1)), half of the theoretical maximum. Using a two-stage process (aerobic cell growth and anaerobic malate production), this engineered strain produced 253 mM malate (34 g liter(-1)) within 72 h, with a higher yield (1.42 mol mol(-1)) and productivity (0.47 g liter(-1) h(-1)). This malate yield and productivity are equal to or better than those of other known biocatalysts.
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Project description:Salidroside (1) is the most important bioactive component of Rhodiola (also called as "Tibetan Ginseng"), which is a valuable medicinal herb exhibiting several adaptogenic properties. Due to the inefficiency of plant extraction and chemical synthesis, the supply of salidroside (1) is currently limited. Herein, we achieved unprecedented biosynthesis of salidroside (1) from glucose in a microorganism. First, the pyruvate decarboxylase ARO10 and endogenous alcohol dehydrogenases were recruited to convert 4-hydroxyphenylpyruvate (2), an intermediate of L-tyrosine pathway, to tyrosol (3) in Escherichia coli. Subsequently, tyrosol production was improved by overexpressing the pathway genes, and by eliminating competing pathways and feedback inhibition. Finally, by introducing Rhodiola-derived glycosyltransferase UGT73B6 into the above-mentioned recombinant strain, salidroside (1) was produced with a titer of 56.9 mg/L. Interestingly, the Rhodiola-derived glycosyltransferase, UGT73B6, also catalyzed the attachment of glucose to the phenol position of tyrosol (3) to form icariside D2 (4), which was not reported in any previous literatures.
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Project description:There has been much interest in producing natural colorants to replace synthetic colorants of health concerns. Escherichia coli has been employed to produce natural colorants including carotenoids, indigo, anthocyanins, and violacein. However, production of natural green and navy colorants has not been reported. Many natural products are hydrophobic, which are accumulated inside or on the cell membrane. This causes cell growth limitation and consequently reduces production of target chemicals. Here, integrated membrane engineering strategies are reported for the enhanced production of rainbow colorants-three carotenoids and four violacein derivatives-as representative hydrophobic natural products in E. coli. By integration of systems metabolic engineering, cell morphology engineering, inner- and outer-membrane vesicle formation, and fermentation optimization, production of rainbow colorants are significantly enhanced to 322 mg L-1 of astaxanthin (red), 343 mg L-1 of β-carotene (orange), 218 mg L-1 of zeaxanthin (yellow), 1.42 g L-1 of proviolacein (green), 0.844 g L-1 of prodeoxyviolacein (blue), 6.19 g L-1 of violacein (navy), and 11.26 g L-1 of deoxyviolacein (purple). The membrane engineering strategies reported here are generally applicable to microbial production of a broader range of hydrophobic natural products, contributing to food, cosmetic, chemical, and pharmaceutical industries.
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Project description:BackgroundRational engineering studies for deoxycytidine production were initiated due to low intracellular levels and tight regulation. To achieve high-level production of deoxycytidine, a useful precursor of decitabine, genes related to feed-back inhibition as well as the biosynthetic pathway were engineered. Additionally, we predicted the impact of individual gene expression levels on a complex metabolic network by microarray analysis. Based on these findings, we demonstrated rational metabolic engineering strategies capable of producing deoxycytidine.ResultsTo prepare the deoxycytidine producing strain, we first deleted 3 degradation enzymes in the salvage pathway (deoA, udp, and deoD) and 4 enzymes involved in the branching pathway (dcd, cdd, codA and thyA) to completely eliminate degradation of deoxycytidine. Second, purR, pepA and argR were knocked out to prevent feedback inhibition of CarAB. Third, to enhance influx to deoxycytidine, we investigated combinatorial expression of pyrG, T4 nrdCAB and yfbR. The best strain carried pETGY (pyrG-yfbR) from the possible combinatorial plasmids. The resulting strain showed high deoxycytidine yield (650 mg/L) but co-produced byproducts. To further improve deoxycytidine yield and reduce byproduct formation, pgi was disrupted to generate a sufficient supply of NADPH and ribose. Overall, in shake-flask cultures, the resulting strain produced 967 mg/L of dCyd with decreased byproducts.ConclusionsWe demonstrated that deoxycytidine could be readily achieved by recombineering with biosynthetic genes and regulatory genes, which appeared to enhance the supply of precursors for synthesis of carbamoyl phosphate, based on transcriptome analysis. In addition, we showed that carbon flux rerouting, by disrupting pgi, efficiently improved deoxycytidine yield and decreased byproduct content.
| S-EPMC4491880 | biostudies-literature
Project description:Significant achievements in polyketide gene expression have made Escherichia coli one of the most promising hosts for the heterologous production of pharmacologically important polyketides. However, attempts to produce glycosylated polyketides, by the expression of heterologous sugar pathways, have been hampered until now by the low levels of glycosylated compounds produced by the recombinant hosts. By carrying out metabolic engineering of three endogenous pathways that lead to the synthesis of TDP sugars in E. coli, we have greatly improved the intracellular levels of the common deoxysugar intermediate TDP-4-keto-6-deoxyglucose resulting in increased production of the heterologous sugars TDP-L-mycarose and TDP-D-desosamine, both components of medically important polyketides. Bioconversion experiments carried out by feeding 6-deoxyerythronolide B (6-dEB) or 3-?-mycarosylerythronolide B (MEB) demonstrated that the genetically modified E. coli B strain was able to produce 60- and 25-fold more erythromycin D (EryD) than the original strain K207-3, respectively. Moreover, the additional knockout of the multidrug efflux pump AcrAB further improved the ability of the engineered strain to produce these glycosylated compounds. These results open the possibility of using E. coli as a generic host for the industrial scale production of glycosylated polyketides, and to combine the polyketide and deoxysugar combinatorial approaches with suitable glycosyltransferases to yield massive libraries of novel compounds with variations in both the aglycone and the tailoring sugars.
| S-EPMC3815289 | biostudies-literature
Project description:BackgroundMonounsaturated fatty acids (MUFAs) are the best components for biodiesel when considering the low temperature fluidity and oxidative stability. However, biodiesel derived from vegetable oils or microbial lipids always consists of significant amounts of polyunsaturated and saturated fatty acids (SFAs) alkyl esters, which hampers its practical applications. Therefore, the fatty acid composition should be modified to increase MUFA contents as well as enhancing oil and lipid production.ResultsThe model microorganism Escherichia coli was engineered to produce free MUFAs. The fatty acyl-ACP thioesterase (AtFatA) and fatty acid desaturase (SSI2) from Arabidopsis thaliana were heterologously expressed in E. coli BL21 star(DE3) to specifically release free unsaturated fatty acids (UFAs) and convert SFAs to UFAs. In addition, the endogenous fadD gene (encoding acyl-CoA synthetase) was disrupted to block fatty acid catabolism while the native acetyl-CoA carboxylase (ACCase) was overexpressed to increase the malonyl coenzyme A (malonyl-CoA) pool and boost fatty acid biosynthesis. The finally engineered strain BL21?fadD/pE-AtFatAssi2&pA-acc produced 82.6 mg/L free fatty acids (FFAs) under shake-flask conditions and FFAs yield on glucose reached about 3.3% of the theoretical yield. Two types of MUFAs, palmitoleate (16:1?9) and cis-vaccenate (18:1?11) made up more than 75% of the FFA profiles. Fed-batch fermentation of this strain further enhanced FFAs production to a titer of 1.27 g/L without affecting fatty acid compositions.ConclusionsThis study demonstrated the possibility to regulate fatty acid composition by using metabolic engineering approaches. FFAs produced by the recombinant E. coli strain consisted of high-level MUFAs and biodiesel manufactured from these fatty acids would be more suitable for current diesel engines.
| S-EPMC4021618 | biostudies-literature
Project description:Microbial production of bioactive retinoids, including retinol and retinyl esters, has been successfully reported. Previously, there are no reports on the microbial biosynthesis of retinoic acid. Two genes (blhSR and raldhHS) encoding retinoic acid biosynthesis enzymes [β-carotene 15,15'-oxygenase (Blh) and retinaldehyde dehydrogenase2 (RALDH2)] were synthetically redesigned for modular expression. Co-expression of the blhSR and raldhHS genes on the plasmid system in an engineered β-carotene-producing Escherichia coli strain produced 0.59 ± 0.06 mg/L of retinoic acid after flask cultivation. Deletion of the ybbO gene encoding a promiscuous aldehyde reductase induced a 2.4-fold increase in retinoic acid production to 1.43 ± 0.06 mg/L. Engineering of the 5'-UTR sequence of the blhSR and raldhHS genes enhanced retinoic acid production to 3.46 ± 0.16 mg/L. A batch culture operated at 37 °C, pH 7.0, and 50% DO produced up to 8.20 ± 0.05 mg/L retinoic acid in a bioreactor. As the construction and culture of retinoic acid-producing bacterial strains are still at an early stage in the development, further optimization of the expression level of the retinoic acid pathway genes, protein engineering of Blh and RALDH2, and culture optimization should synergistically increase the current titer of retinoic acid in E. coli.
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Project description:BackgroundVanillin is one of the most important aromatic flavour compounds used in the food and cosmetic industries. Natural vanillin is extracted from vanilla beans and is relatively expensive. Moreover, the consumer demand for natural vanillin highly exceeds the amount of vanillin extracted by plant sources. This has led to the investigation of other routes to obtain this flavour such as the biotechnological production from ferulic acid. Studies concerning the use of engineered recombinant Escherichia coli cells as biocatalysts for vanillin production are described in the literature, but yield optimization and biotransformation conditions have not been investigated in details.ResultsEffect of plasmid copy number in metabolic engineering of E. coli for the synthesis of vanillin has been evaluated by the use of genes encoding feruloyl-CoA synthetase and feruloyl hydratase/aldolase from Pseudomonas fluorescens BF13. The higher vanillin production yield was obtained using resting cells of E. coli strain JM109 harbouring a low-copy number vector and a promoter exhibiting a low activity to drive the expression of the catabolic genes. Optimization of the bioconversion of ferulic acid to vanillin was accomplished by a response surface methodology. The experimental conditions that allowed us to obtain high values for response functions were 3.3 mM ferulic acid and 4.5 g/L of biomass, with a yield of 70.6% and specific productivity of 5.9 micromoles/g x min after 3 hours of incubation. The final concentration of vanillin in the medium was increased up to 3.5 mM after a 6-hour incubation by sequential spiking of 1.1 mM ferulic acid. The resting cells could be reused up to four times maintaining the production yield levels over 50%, thus increasing three times the vanillin obtained per gram of biomass.ConclusionFerulic acid can be efficiently converted to vanillin, without accumulation of undesirable vanillin reduction/oxidation products, using E. coli JM109 cells expressing genes from the ferulic acid-degrader Pseudomonas fluorescens BF13. Optimization of culture conditions and bioconversion parameters, together with the reuse of the biomass, leaded to a final production of 2.52 g of vanillin per liter of culture, which is the highest found in the literature for recombinant strains and the highest achieved so far applying such strains under resting cells conditions.
| S-EPMC1857700 | biostudies-literature
Project description:cis-Abienol, a natural diterpene-diol isolated from balsam fir (Abies balsamea), can be employed as precursors for the semi-synthesis of amber compounds, which are sustainable replacement for ambergris and widely used in the fragmented industry. This study combinatorially co-expressed geranyl diphosphate synthase, geranylgeranyl diphosphate synthase, Labda-13-en-8-ol diphosphate synthase and diterpene synthase, with the best combination achieving ~ 0.3 mg/L of cis-abienol. An additional enhancement of cis-abienol production (up to 8.6 mg/L) was achieved by introducing an exogenous mevalonate pathway which was divided into the upper pathway containing acetyl-CoA acetyltransferase/HMG-CoA reductase and HMG-CoA synthase and the lower pathway containing mevalonate kinase, phosphomevalonate kinase, pyrophosphate mevalonate decarboxylase and isopentenyl pyrophosphate isomerase. The genetically modified strain carrying chromosomal copy of low genes of the mevalonate with the trc promoter accumulated cis-abienol up to 9.2 mg/L in shake flask. Finally, cis-abienol titers of ~ 220 mg/L could be achieved directly from glucose using this de novo cis-abienol-producing E. coli in high-cell-density fermentation. This study demonstrates a microbial process to apply the E. coli cell factory in the biosynthesis of cis-abienol.
| S-EPMC7545193 | biostudies-literature
Project description:Background(-)-α-Bisabolol, also known as levomenol, is an unsaturated sesquiterpene alcohol that has mainly been used in pharmaceutical and cosmetic products due to its anti-inflammatory and skin-soothing properties. (-)-α-Bisabolol is currently manufactured mainly by steam-distillation of the essential oils extracted from the Brazilian candeia tree that is under threat because its natural habitat is constantly shrinking. Therefore, microbial production of (-)-α-bisabolol plays a key role in the development of its sustainable production from renewable feedstock.ResultsHere, we created an Escherichia coli strain producing (-)-α-bisabolol at high titer and developed an in situ extraction method of (-)-α-bisabolol, using natural vegetable oils. We expressed a recently identified (-)-α-bisabolol synthase isolated from German chamomile (Matricaria recutita) (titer: 3 mg/L), converted the acetyl-CoA to mevalonate, using the biosynthetic mevalonate pathway (12.8 mg/L), and overexpressed farnesyl diphosphate synthase to efficiently supply the (-)-α-bisabolol precursor farnesyl diphosphate. Combinatorial expression of the exogenous mevalonate pathway and farnesyl diphosphate synthase enabled a dramatic increase in (-)-α-bisabolol production in the shake flask culture (80 mg/L) and 5 L bioreactor culture (342 mg/L) of engineered E. coli harboring (-)-α-bisabolol synthase. Fed-batch fermentation using a 50 L fermenter was conducted after optimizing culture conditions, resulting in efficient (-)-α-bisabolol production with a titer of 9.1 g/L. Moreover, a green, downstream extraction process using vegetable oils was developed for in situ extraction of (-)-α-bisabolol during fermentation and showed high yield recovery (>98%).ConclusionsThe engineered E. coli strains and economically viable extraction process developed in this study will serve as promising platforms for further development of microbial production of (-)-α-bisabolol at large scale.
| S-EPMC5101696 | biostudies-literature