Ameliorating end-product inhibition to improve cadaverine production in engineered Escherichia coli and its application in the synthesis of bio-based diisocyanates.
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ABSTRACT:
SUBMITTER: Wang X
PROVIDER: S-EPMC8446744 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Bio-production of cadaverine from cheap carbon sources for synthesizing bio-based polyamides is becoming more common. Here, a novel fermentation process for cadaverine bio-production from glucose was implemented by using a microbial consortium of two engineered Escherichia coli strains to relieve the toxic effect of cadaverine on fermentation efficiency. To achieve controllable growth of strains in the microbial consortium, two engineered E. coli strains grown separately on different carbon sources were first constructed. The strains were, an L-lysine-producing E. coli NT1004 with glucose as carbon source, and a cadaverine-producing E. coli CAD03 with glucose metabolism deficiency generated by modifying the PTSGlc system with CRISPR-Cas9 technology and inactivating cadaverine degradation pathways. Co-culturing these two engineered E. coli strains with a mixture of glucose and glycerol led to successful production of cadaverine. After optimizing cultivation conditions, a cadaverine titer of 28.5 g/L was achieved with a multi-stage constant-speed feeding strategy.
| S-EPMC6018084 | biostudies-literature
Project description:Citramalic acid is a central intermediate in a combined biocatalytic and chemocatalytic route to produce bio-based methylmethacrylate, the monomer used to manufacture Perspex and other high performance materials. We developed an engineered E. coli strain and a fed-batch bioprocess to produce citramalate at concentrations in excess of 80 g l-1 in only 65 h. This exceptional efficiency was achieved by designing the production strain and the fermentation system to operate synergistically. Thus, a single gene encoding a mesophilic variant of citramalate synthase from Methanococcus jannaschii, CimA3.7, was expressed in E. coli to convert acetyl-CoA and pyruvate to citramalate, and the ldhA and pflB genes were deleted. By using a bioprocess with a continuous, growth-limiting feed of glucose, these simple interventions diverted substrate flux directly from central metabolism towards formation of citramalate, without problematic accumulation of acetate. Furthermore, the nutritional requirements of the production strain could be satisfied through the use of a mineral salts medium supplemented only with glucose (172 g l-1 in total) and 1.4 g l-1 yeast extract. Using this system, citramalate accumulated to 82±1.5 g l-1, with a productivity of 1.85 g l-1 h-1 and a conversion efficiency of 0.48 gcitramalate g-1glucose. The new bioprocess forms a practical first step for integrated bio- and chemocatalytic production of methylmethacrylate.
| S-EPMC5882075 | biostudies-literature
Project description:BackgroundGeraniol, an acyclic monoterpene alcohol, is found as a primary constituent in the essential oils of plants such as geranium, lemongrass and rose. The floral-like scent of geraniol has made it a popular constituent of flavour and fragrance products. Over recent decades biotechnology has made significant progress towards the development of industrial platforms for the production of commercially valuable monoterpenoids, such as geraniol, through expression of recombinant terpene biosynthetic pathways in microbial hosts. Titres, however, have been hindered due to the inherent toxicity of these compounds-which are often utilised for anti-microbial and anti-fungal functions in their host plant.ResultsIn this study we modified an Escherichia coli strain, engineered to express a heterologous mevalonate pathway, by replacement of the terpene synthase with a geraniol synthase from Ocimum basilicum for the production of geraniol, and co-expressed an alcohol acyltransferase (AAT) from Rosa hybrida for the specific acetylation of geraniol. The low water solubility of geranyl acetate facilitated its partition into the organic phase of a two-phase system, relieving the cellular toxicity attributed to the build-up of geraniol in the aqueous phase. In a partially optimised system this strain produced 4.8 g/L geranyl acetate (based on the aqueous volume) which, on a molar equivalent basis, represents the highest monoterpene titre achieved from microbial culture to date. It was also found that esterification of geraniol prevented bioconversion into other monoterpenoids, leading to a significant improvement in product specificity, with geranyl acetate being the sole product observed.ConclusionIn this study we have shown that it is possible to both overcome the toxicity limit impeding the production of the monoterpene alcohol geraniol and mitigate product loss in culture through endogenous metabolism by using an in vivo esterification strategy. This strategy has resulted in the highest geraniol (equivalent) titres achieved from a microbial host, and presents esterification as a viable approach to increasing the titres obtained in microbial monoterpenoid production.
| S-EPMC6556219 | biostudies-literature
Project description:BackgroundStyrene is a large-volume commodity petrochemical, which has been used in a wide range of polymer industry as the main building block for the construction of various functional polymers. Despite many efforts to produce styrene in microbial hosts, the production titers are still low and are not enough to meet the commercial production of styrene.ResultsPreviously, we developed a high L-phenylalanine producer (E. coli YHP05), and it was used as a main host for de novo synthesis of styrene. First, we introduced the co-expression system of phenylalanine-ammonia lyase (PAL) and ferulic acid decarboxylase (FDC) genes for the synthesis of styrene from L-phenylalanine. Then, to minimize cell toxicity and enhance the recovery of styrene, in situ product recovery (ISPR) with n-dodecane was employed, and culture medium with supplementation of complex sources was also optimized. As a result, 1.7 ± 0.1 g/L of styrene was produced in the flask cultures. Finally, fed-batch cultivations were performed in lab-scale bioreactor, and to minimize the loss of volatile styrene during the cultivation, three consecutive bottles containing n-dodecane were connected to the air outlet of bioreactor for gas-stripping. To conclude, the total titer of styrene was as high as 5.3 ± 0.2 g/L, which could be obtained at 60 h.ConclusionWe successfully engineered E. coli strain for the de novo production of styrene in both flask and fed-batch cultivation, and could achieve the highest titer for styrene in bacterial hosts reported till date. We believe that our efforts in strain engineering and ISPR strategy with organic solvent will provide a new insight for economic and industrial production of styrene in a biological platform.
| S-EPMC6498506 | biostudies-literature
Project description:BackgroundHydroxycinnamoyl anthranilates, also known as avenanthramides (avns), are a group of phenolic alkaloids with anti-inflammatory, antioxidant, anti-itch, anti-irritant, and antiatherogenic activities. Some avenanthramides (avn A-H and avn K) are conjugates of hydroxycinnamic acids (HC), including p-coumaric acid, caffeic acid, and ferulic acid, and anthranilate derivatives, including anthranilate, 4-hydroxyanthranilate, and 5-hydroxyanthranilate. Avns are primarily found in oat grain, in which they were originally designated as phytoalexins. Knowledge of the avns biosynthesis pathway has now made it possible to synthesize avns through a genetic engineering strategy, which would help to further elucidate their properties and exploit their beneficial biological activities. The aim of the present study was to synthesize natural avns in Escherichia coli to serve as a valuable resource.ResultsWe synthesized nine avns in E. coli. We first synthesized avn D from glucose in E. coli harboring tyrosine ammonia lyase (TAL), 4-coumarate:coenzyme A ligase (4CL), anthranilate N-hydroxycinnamoyl/benzoyltransferase (HCBT), and anthranilate synthase (trpEG). A trpD deletion mutant was used to increase the amount of anthranilate in E. coli. After optimizing the incubation temperature and cell density, approximately 317.2 mg/L of avn D was synthesized. Avn E and avn F were then synthesized from avn D, using either E. coli harboring HpaBC and SOMT9 or E. coli harboring HapBC alone, respectively. Avn A and avn G were synthesized by feeding 5-hydroxyanthranilate or 4-hydroxyanthranilate to E. coli harboring TAL, 4CL, and HCBT. Avn B, avn C, avn H, and avn K were synthesized from avn A or avn G, using the same approach employed for the synthesis of avn E and avn F from avn D.ConclusionsUsing different HCs, nine avns were synthesized, three of which (avn D, avn E, and avn F) were synthesized from glucose in E. coli. These diverse avns provide a strategy to synthesize both natural and unnatural avns, setting a foundation for exploring the biological activities of diverse avns.
| S-EPMC5863376 | biostudies-literature
Project description:Although the routes of de novo pyridoxal 5'-phosphate (PLP) biosynthesis have been well described, studies of the engineering of an intracellular PLP supply are limited, and the effects of cellular PLP levels on PLP-dependent enzyme-based whole-cell biocatalyst activity have not been described. To investigate the effects of PLP cofactor availability on whole-cell biocatalysis, the ribose 5-phosphate (R5P)-dependent pathway genes pdxS and pdxT of Bacillus subtilis were introduced into the lysine decarboxylase (CadA)-overexpressing Escherichia coli strain BL-CadA. This strain was then used as a whole-cell biocatalyst for cadaverine production from L-lysine. Co-expression strategies were evaluated, and the culture medium was optimised to improve the biocatalyst performance. As a result, the intracellular PLP concentration reached 1144 nmol/gDCW, and a specific cadaverine productivity of 25 g/gDCW/h was achieved; these values were 2.4-fold and 2.9-fold higher than those of unmodified BL-CadA, respectively. Additionally, the resulting strain AST3 showed a cadaverine titre (p = 0.143, α = 0.05) similar to that of the BL-CadA strain with the addition of 0.1 mM PLP. These approaches for improving intracellular PLP levels to enhance whole-cell lysine bioconversion activity show great promise for the engineering of a PLP cofactor to optimise whole-cell biocatalysis.
| S-EPMC4614675 | biostudies-literature
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.
| S-EPMC3020529 | biostudies-literature
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.
| S-EPMC4200411 | biostudies-literature
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:Bridging broken DNA ends via nonhomologous end-joining (NHEJ) contributes to the evolution and stability of eukaryote genomes. Although some bacteria possess a simplified NHEJ mechanism, the human commensal Escherichia coli is thought to rely exclusively on homology-directed mechanisms to repair DNA double-strand breaks (DSBs). We show here that laboratory and pathogenic E. coli strains possess a distinct end-joining activity that repairs DSBs and generates genome rearrangements. This mechanism, named alternative end-joining (A-EJ), does not rely on the key NHEJ proteins Ku and Ligase-D which are absent in E. coli. Differently from classical NHEJ, A-EJ is characterized by extensive end-resection largely due to RecBCD, by overwhelming usage of microhomology and extremely rare DNA synthesis. We also show that A-EJ is dependent on the essential Ligase-A and independent on Ligase-B. Importantly, mutagenic repair requires a functional Ligase-A. Although generally mutagenic, accurate A-EJ also occurs and is frequent in some pathogenic bacteria. Furthermore, we show the acquisition of an antibiotic-resistance gene via A-EJ, refuting the notion that bacteria gain exogenous sequences only by recombination-dependent mechanisms. This finding demonstrates that E. coli can integrate unrelated, nonhomologous exogenous sequences by end-joining and it provides an alternative strategy for horizontal gene transfer in the bacterial genome. Thus, A-EJ contributes to bacterial genome evolution and adaptation to environmental challenges. Interestingly, the key features of A-EJ also appear in A-NHEJ, an alternative end-joining mechanism implicated in chromosomal translocations associated with human malignancies, and we propose that this mutagenic repair might have originated in bacteria.
| S-EPMC2836643 | biostudies-literature