RESUMO
Bioproduction of chiral amines is limited by low transaminase (TA) activity on nonnatural substrates, leading to the need for protein engineering. To address the challenge of quickly and precisely identifying the engineering targets, a strategy was proposed based on analyzing the mode changes in the high-energy intermediate state (H-state) of the substrate-enzyme complex during catalysis. By substituting the residues with minimal structural changes in catalytically active mode (A-mode) and distance-free mode (F-mode) of the H-state complex with more conserved ones to stabilize it, a TA mutant M5(T295C/L387A/V436A) with 121.9-fold higher activity for synthesizing the (S)-Rivastigmine precursor (S)-1-(3-methoxyphenyl)ethylamine was created. The applicability of this strategy was also validated by engineering another TA for 1.52-fold higher activity and >99% selectivity toward (R)-3-amino-1-butanol biopreparation. The much higher stereoselectivity of the mutant compared with the wild type (28.3%) demonstrated that this strategy is not only advantageous in engineering enzyme activity but also applicable for modulating stereoselectivity.
Assuntos
Engenharia de Proteínas , Transaminases , Transaminases/genética , Transaminases/metabolismo , Aminas/química , Especificidade por SubstratoRESUMO
Lutein, as a carotenoid with strong antioxidant capacity and an important component of macular pigment in the retina, has wide applications in pharmaceutical, food, feed, and cosmetics industries. Besides extraction from plant and algae, microbial fermentation using engineered cell factories to produce lutein has emerged as a promising route. However, intra-pathway competition between the lycopene cyclases and the conflict between cell growth and production are two major challenges. In our previous study, de novo synthesis of lutein had been achieved in Saccharomyces cerevisiae by dividing the pathway into two stages (δ-carotene formation and conversion) using temperature as the input signal to realize sequential cyclation of lycopene. However, lutein production was limited to microgram level, which is still too low to meet industrial demand. In this study, a dual-signal hierarchical dynamic regulation system was developed and applied to divide lutein biosynthesis into three stages in response to glucose concentration and culture temperature. By placing the genes involved in δ-carotene formation under the glucose-responsive ADH2 promoter and genes involved in the conversion of δ-carotene to lutein under temperature-responsive GAL promoters, the growth-production conflict and intra-pathway competition were simultaneously resolved. Meanwhile, the rate-limiting lycopene ε-cyclation and carotene hydroxylation reactions were improved by screening for lycopene ε-cyclase with higher activity and fine tuning of the P450 enzymes and their redox partners. Finally, a lutein titer of 19.92 mg/L (4.53 mg/g DCW) was obtained in shake-flask cultures using the engineered yeast strain YLutein-3S-6, which is the highest lutein titer ever reported in heterologous production systems.
Assuntos
Luteína , Saccharomyces cerevisiae , Luteína/metabolismo , Licopeno/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Carotenoides/metabolismo , Regiões Promotoras GenéticasRESUMO
The nylon 12 (PA12) monomer ω-aminododecanoic acid (ω-AmDDA) could be synthesized from lauric acid (DDA) through multi-enzyme cascade transformation using engineered E. coli, with the P450 catalyzing terminal hydroxylation of DDA as a rate-limiting enzyme. Its activity is jointly determined by the heme domain and the reductase domain. To obtain a P450 mutant with higher activity, directed evolution was conducted using a colorimetric high-throughput screening (HTS) system with DDA as the real substrate. After two rounds of directed evolution, a positive double-site mutant (R14R/D629G) with 90.3% higher activity was obtained. Molecular docking analysis, kinetic parameter determination and protein electrophoresis suggested the improved soluble expression of P450 resulting from the synonymous mutation near the N-terminus and the shortened distance of the electron transfer between FMN and FAD caused by D629G mutation as the major reasons for activity improvement. The significantly increased kcat and unchanged Km provided further evidence for the increase in electron transfer efficiency. Considering the important role of heme in P450, its supply was strengthened by the metabolic engineering of the heme synthesis pathway. By combining P450-directed evolution and enhancing heme synthesis, 2.02 ± 0.03 g/L of ω-AmDDA was produced from 10 mM DDA, with a yield of 93.6%.
Assuntos
Sistema Enzimático do Citocromo P-450 , Escherichia coli , Sistema Enzimático do Citocromo P-450/metabolismo , Simulação de Acoplamento Molecular , Escherichia coli/metabolismo , Hidroxilação , Heme/químicaRESUMO
Retinoic acid (RA), a vitamin A (retinol)-derived lipophilic compound, is involved in various physiological functions. The demand for RA is growing in the pharmaceutical industry, but RA biosynthesis is still in its infancy compared to other forms of retinoids such as retinol and retinal, largely due to the lack of efficient retinal dehydrogenases. To achieve effective biosynthesis of RA, the catalytic activities of exogenous retinal dehydrogenases were comparatively analyzed in a previously constructed retinoids-producing Saccharomyces cerevisiae strain, followed by mining of endogenous enzymes with higher retinal dehydrogenase activities using homology-based search. After confirming the retinal oxidation activity of the endogenous aldehyde dehydrogenase Hfd1 using in vivo and in vitro experiments, it was overexpressed in multiple copies, and the resulting strain produced 99.71 mg/L of RA in shake-flask cultures. Finally, 545.28 mg/L of RA was produced in fed-batch fermentation. This study suggests the yeast endogenous Hfd1 as a potent catalyst for RA biosynthesis, and demonstrates the potential of yeast as a platform for RA production.
Assuntos
Aldeído Desidrogenase , Tretinoína , Retinal Desidrogenase/genética , Retinoides , Saccharomyces cerevisiae/genética , Vitamina ARESUMO
The past decade has witnessed the rapid progress in development of synthetic biology, and advances in construction of yeast cell factories open vast opportunities for green and sustainable production of chemicals. Focusing on the progress in yeast engineering for production of plant natural products in the last 5 years, this review introduces different yeast chassis used for cell factory construction, including Saccharomyces cerevisiae, Yarrowia lipolytica and Komagataella phaffii, together with the emerging genome editing tools. The metabolic regulation strategies developed for yeast engineering are highlighted, such as subcellular pathway localization dynamic regulation, and transporter engineering. C1-based chemical bioproduction by engineered yeast is also covered. Finally, the existing challenges and future prospects in creating efficient yeast cell factories are summarized.
Assuntos
Saccharomyces cerevisiae , Yarrowia , Edição de Genes , Engenharia Metabólica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Biologia Sintética , Yarrowia/genética , Yarrowia/metabolismoRESUMO
The market-expanding lutein is currently mainly supplied by plant extraction, with microbial fermentation using engineered cell factory emerging as a promising substitution. During construction of lutein-producing yeast, α-carotene formation through asymmetric ε- and ß-cyclization of lycopene was found as the main limiting step, attributed to intra-pathway competition of the cyclases for lycopene, forming ß-carotene instead. To solve this problem, temperature-responsive expression of ß-cyclase was coupled to constitutive expression of ε-cyclase for flux redirection to α-carotene by allowing ε-cyclization to occur first. Meanwhile, the ε-cyclase was engineered and re-localized to the plasma membrane for further flux reinforcement towards α-carotene. Finally, pathway extension with proper combination of carotenoid hydroxylases enabled lutein (438 µg/g dry cells) biosynthesis in S. cerevisiae. The success of heterologous lutein biosynthesis in yeast suggested temporospatial pathway control as a potential strategy in solving intra-pathway competitions, and may also be applicable for promoting the biosynthesis of other natural products.
Assuntos
Liases Intramoleculares , Luteína , Licopeno , Saccharomyces cerevisiae/genética , beta CarotenoRESUMO
As a natural phenolic acid product of plant source, caffeic acid displays diverse biological activities and acts as an important precursor for the synthesis of other valuable compounds. Limitations in chemical synthesis or plant extraction of caffeic acid trigger interest in its microbial biosynthesis. Recently, Saccharomyces cerevisiae has been reported for the biosynthesis of caffeic acid via episomal plasmid-mediated expression of pathway genes. However, the production was far from satisfactory and even relied on the addition of precursor. In this study, we first established a controllable and stable caffeic acid pathway by employing a modified GAL regulatory system to control the genome-integrated pathway genes in S. cerevisiae and realized biosynthesis of 222.7 mg/L caffeic acid. Combinatorial engineering strategies including eliminating the tyrosine-induced feedback inhibition, deleting genes involved in competing pathways, and overexpressing rate-limiting enzymes led to about 2.6-fold improvement in the caffeic acid production, reaching up to 569.0 mg/L in shake-flask cultures. To our knowledge, this is the highest ever reported titer of caffeic acid synthesized by engineered yeast. This work showed the prospect for microbial biosynthesis of caffeic acid and laid the foundation for constructing biosynthetic pathways of its derived metabolites. KEY POINTS: Genomic integration of ORgTAL, OHpaB, and HpaC for caffeic acid production in yeast. Feedback inhibition elimination and Aro10 deletion improved caffeic acid production. The highest ever reported titer (569.0 mg/L) of caffeic acid synthesized by yeast.
Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Ácidos Cafeicos , Engenharia Metabólica , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Biosynthesis of Nylon 12 monomer using dodecanoic acid (DDA) or its esters as the renewable feedstock typically involves ω-hydroxylation, oxidation and ω-amination. The dependence of hydroxylation and oxidation-catalyzing enzymes on redox cofactors, and the requirement of L-alanine as the co-substrate and pyridoxal 5'-phosphate (PLP) as the coenzyme for transamination, raise the issue of redox imbalance and cofactor shortage, challenging the development of efficient biocatalysts. Simultaneous regeneration of the redox equivalents, PLP and L-alanine required in the artificial pathway was enabled by its interfacing with the native metabolism of the host using glucose dehydrogenase (GDH), L-alanine dehydrogenase (AlaDH) and an exogenous ribose 5-phosphate (R5P)-dependent PLP synthesis pathway as bridges. Further engineering of the host by blocking ß-oxidation and enhancing substrate uptake improved the ω-aminododecanoic acid (ω-AmDDA) yield to 96.5%. This study offers a strategy to resolve the cofactor imbalance issue commonly encountered in whole-cell biocatalysis and meanwhile lays a solid foundation for Nylon 12 bioproduction.
Assuntos
Coenzimas , Nylons , Biocatálise , Vias Biossintéticas , Coenzimas/metabolismoRESUMO
(R)-3-Chloro-1-phenyl-1-propanol ((R)-CPPO) is an important chiral intermediate for antidepressants. For its efficient biosynthesis, the carbonyl reductase EbSDR8 was engineered to asymmetrically reduce the unnatural substrate 3-chloro-1-phenyl-1-propanone (3-CPP) at high concentrations. Molecular docking and molecular dynamics simulations of the resulting mutants suggested enlarged substrate binding pocket and more reasonable interactions between the enzyme and the substrate or cofactor as the reasons for the enhanced catalytic activity and thus the remarkably improved conversion of high-concentration 3-CPP. Using the best mutant EbSDR8G94A/L153I/Y188A/Y202M as the whole-cell biocatalyst, reduction of 3-CPP (1.0 M) was conducted using 100% isopropanol as both the solvent and co-substrate for NADH regeneration, delivering (R)-CPPO with Ë 99% eep and 95.5% conversion. This result suggests EbSDR8G94A/L153I/Y188A/Y202M as a potential biocatalyst for green production of (R)-CPPO at the industrial scale. KEY POINTS: ⢠Rational design of EbSDR8 by modulating steric hindrance and molecular interactions; ⢠Non-aqueous biocatalysis using isopropanol as both the solvent and co-substrate; ⢠Whole-cell catalyzed production of 161 g/L enantiopure (R)-CPPO from 1.0 M of 3-CPP. Graphical Abstract.
Assuntos
1-Propanol , Oxirredutases do Álcool , Álcoois Benzílicos , Simulação de Acoplamento MolecularRESUMO
As a chiral precursor for the important anticoagulant Edoxaban, enantioselective synthesis of (S)-3-cyclohexene-1-carboxylic acid is of great significance. The complicated procedures and generation of massive solid waste discourage its chemical synthesis, and the alternative biocatalysis route calls for an enzyme capable of asymmetric hydrolysis of racemic methyl-3-cyclohexene-1-carboxylate. To this end, we engineered the E. coli esterase BioH for improved S-enantioselectivity via rational design. By combinatorial modulation of steric and aromatic interactions, a positive mutant Mu3 (L24A/W81A/L209A) with relatively high S-selectivity in hydrolyzing racemic methyl-3-cyclohexene-1-carboxylate was obtained, improving the enantiomeric excess from 32.3% (the wild type) to 70.9%. Molecular dynamics simulation was conducted for both (R)- or (S)- complexes of the wild type and Mu3 to provide hints for the mechanism behind the increased S-selectivity. Moreover, the reaction conditions of Mu3 in methyl-3-cyclohexene-1-carboxylate hydrolysis was optimized to improve the conversion rate to 2 folds.
Assuntos
Ácidos Carboxílicos/química , Cicloexenos/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Cinética , Simulação de Dinâmica Molecular , Mutação , EstereoisomerismoRESUMO
Pyricularia oryzae is a plant pathogen causing rice blast, a serious disease spreading in cultivated rice globally. Transcription factors play important regulatory roles in fungal development and pathogenicity. Here, we characterized the biological functions of Crf1, a basic helix-loop-helix (bHLH) transcription factor, in the development and pathogenicity of P. oryzae with functional genetics, molecular and biochemical approaches. We found that CRF1 is necessary for virulence and plays an indispensable role in the regulation of carbohydrate and lipid metabolism in P. oryzae. Deletion of CRF1 led to defects in utilization of lipids, ethanol, glycerol and L-arabinose, and down-regulation of many important genes in lipolysis, ß-oxidation, gluconeogenesis, as well as glycerol and arabinose metabolism. CRF1 is also essential for peroxisome and vacuole function, and conidial cell death during appressorium formation. The appressorium turgor, penetration ability and virulence in Δcrf1 were restored by supplementation of exogenous glucose. The virulence of Crf1 mutant was also recovered by adding exogenous D-xylose, but not by addition of ethanol, pyruvate, leucine or L-arabinose. These data showed that Crf1 plays an important role in the complex regulatory network of carbohydrate and lipid metabolism that governs fungal development and pathogenicity.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Metabolismo dos Carboidratos , Proteínas Fúngicas/metabolismo , Metabolismo dos Lipídeos , Magnaporthe/metabolismo , Magnaporthe/patogenicidade , Oryza/microbiologia , Doenças das Plantas/microbiologia , Fatores de Transcrição/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Magnaporthe/genética , Magnaporthe/crescimento & desenvolvimento , Deleção de Sequência , Esporos Fúngicos/genética , Esporos Fúngicos/crescimento & desenvolvimento , Esporos Fúngicos/metabolismo , Esporos Fúngicos/patogenicidade , Fatores de Transcrição/genética , VirulênciaRESUMO
Conflict between cell growth and product accumulation is frequently encountered in biosynthesis of secondary metabolites. Herein, a temperature-dependent dynamic control strategy was developed by modifying the GAL regulation system to facilitate two-stage fermentation in yeast. A temperature-sensitive Gal4 mutant Gal4M9 was created by directed evolution, and used as a protein switch in ΔGAL80 yeast. After EGFP-reported validation of its temperature-responsive induction capability, the sensitivity and stringency of this system in multi-gene pathway regulation was tested, using lycopene as an example product. When Gal4M9 was used to control the expression of PGAL -driven pathway genes, growth and production was successfully decoupled upon temperature shift during fermentation, accumulating 44% higher biomass and 177% more lycopene than the control strain with wild-type Gal4. This is the first example of adopting temperature as an input signal for metabolic pathway regulation in yeast cell factories.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação Fúngica da Expressão Gênica/efeitos da radiação , Engenharia Metabólica/métodos , Metabolismo/efeitos da radiação , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Anti-Inflamatórios/metabolismo , Proteínas de Ligação a DNA/genética , Licopeno/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Temperatura , Fatores de Transcrição/genéticaRESUMO
With increasing concerns in sustainable development, biocatalysis has been recognized as a competitive alternative to traditional chemical routes in the past decades. As nature's biocatalysts, enzymes are able to catalyze a broad range of chemical transformations, not only with mild reaction conditions but also with high activity and selectivity. However, the insufficient activity or enantioselectivity of natural enzymes toward non-natural substrates limits their industrial application, while directed evolution provides a potent solution to this problem, thanks to its independence on detailed knowledge about the relationship between sequence, structure, and mechanism/function of the enzymes. A proper high-throughput screening (HTS) method is the key to successful and efficient directed evolution. In recent years, huge varieties of HTS methods have been developed for rapid evaluation of mutant libraries, ranging from in vitro screening to in vivo selection, from indicator addition to multi-enzyme system construction, and from plate screening to computation- or machine-assisted screening. Recently, there is a tendency to integrate directed evolution with metabolic engineering in biosynthesis, using metabolites as HTS indicators, which implies that directed evolution has transformed from molecular engineering to process engineering. This paper aims to provide an overview of HTS methods categorized based on the reaction principles or types by summarizing related studies published in recent years including the work from our group, to discuss assay design strategies and typical examples of HTS methods, and to share our understanding on HTS method development for directed evolution of enzymes involved in specific catalytic reactions or metabolic pathways.
Assuntos
Evolução Molecular Direcionada , Enzimas , Ensaios de Triagem em Larga Escala/métodos , Biocatálise , Vias Biossintéticas , Engenharia Metabólica , Engenharia de ProteínasRESUMO
Current studies on microbial isoprene biosynthesis have mostly focused on regulation of the upstream mevalonic acid (MVA) or methyl-erythritol-4-phosphate (MEP) pathway. However, the downstream bottleneck restricting isoprene biosynthesis capacity caused by the weak expression and low activity of plant isoprene synthase (ISPS) under microbial fermentation conditions remains to be alleviated. Here, based on a previously constructed Saccharomyces cerevisiae strain with enhanced precursor supply, we strengthened the downstream pathway through increasing both the expression and activity of ISPS to further improve isoprene production. Firstly, a two-level expression enhancement system was developed for the PGAL1-controlled ISPS by overexpression of GAL 4. Meanwhile, the native GAL1/7/10 promoters were deleted to avoid competition for the transcriptional activator Gal4p, and GAL80 was disrupted to eliminate the dependency of gene expression on galactose induction. The IspS expression was obviously elevated upon enhanced Gal4p supply, and the isoprene production was improved from 6.0mg/L to 23.6mg/L in sealed-vial cultures with sucrose as carbon source. Subsequently, a novel high-throughput screening method was developed based on precursor toxicity and used for ISPS directed evolution towards enhanced catalytic activity. Combinatorial mutagenesis of the resulting ISPS mutants generated the best mutant ISPSM4, introduction of which into the GAL4-overexpressing strain YXM29 achieved 50.2mg/L of isoprene in sealed vials, and the isoprene production reached 640mg/L and 3.7g/L in aerobic batch and fed-batch fermentations, respectively. These results demonstrated the effectiveness of the proposed combinatorial engineering strategy in isoprene biosynthesis, which might also be feasible and instructive for biotechnological production of other valuable chemicals.
Assuntos
Alquil e Aril Transferases/metabolismo , Butadienos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Evolução Molecular Direcionada/métodos , Hemiterpenos/metabolismo , Engenharia Metabólica/métodos , Pentanos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Fatores de Transcrição/metabolismo , Vias Biossintéticas/genética , Butadienos/isolamento & purificação , Proteínas de Ligação a DNA/genética , Regulação Bacteriana da Expressão Gênica/genética , Regulação Enzimológica da Expressão Gênica/genética , Melhoramento Genético/métodos , Hemiterpenos/isolamento & purificação , Redes e Vias Metabólicas/genética , Pentanos/isolamento & purificação , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Regulação para Cima/genéticaRESUMO
Optically pure methyl (R)-o-chloromandelate and (R)-acetyl-o-mandelic acid are key intermediates for the synthesis of (S)-clopidogrel, which could be prepared with 100 % theoretical yield by sequential hydrolysis and racemization. At the moment, efficient sequential hydrolysis and racemization are hindered by the low catalytic activity of mandelate racemase (MR) toward (S)-o-chloromandelic acid ((S)-2-CMA). In the present work, we proposed to improve the catalytic performance of MR toward (S)-2-CMA by directed evolution and developed an enantioselective oxidation system for high-throughput screening (HTS) of MR libraries. Based on this HTS method, a triple mutant V22I/V29I/Y54F (MRDE1) with 3.5-fold greater relative activity as compared to the native MR was obtained. Kinetic analysis indicated that the enhanced catalytic efficiency mainly arose from the elevated k cat. Further insight into the source of improved catalytic activity was gained by molecular simulations, finding that substrate binding and product release were possibly made easier by decreased steric bulk and increased hydrophobicity of substrate binding sites. In addition, the substrate (S)-2-CMA in the enzyme-substrate complex of MRDE1 seemed to have a lower binding free energy comparing with the complex of wild-type MR. The HTS method developed in this work and the successful directed evolution of MR based on this method provide an example for racemase engineering and may inspire directed evolution of other racemases toward enhanced catalytic performance on non-natural substrates.
Assuntos
Evolução Molecular Direcionada/métodos , Ensaios de Triagem em Larga Escala/métodos , Racemases e Epimerases/genética , Catálise , Simulação por Computador , Hidrólise , Cinética , Mutagênese Sítio-Dirigida , Pseudomonas putida/genética , Racemases e Epimerases/metabolismo , Especificidade por SubstratoRESUMO
Isoprene is facing a growing global market due to its wide industrial applications. Current industrial production of isoprene is almost entirely petroleum-based, which is influenced by the shrinking C5 supply, while the natural emission of isoprene is predominantly contributed by plants. To bridge the need gap, a highly efficient fermentation-based process for isoprene production might be a suitable and sustainable solution, and extensive research works have been performed to achieve this goal. Here we review the accomplishments in this field by summarizing the history and prospects of microbial isoprene production. The natural producers and biosynthesis pathways of isoprene, the key enzyme isoprene synthase and the metabolic engineering strategies adopted for developing isoprene-producing microorganisms are introduced. In particular, strategies employed for achieving engineered strains with improved performance indices are discussed based on the published papers and patents. The perspectives on further performance improvements and potential future strategies are presented as well.
Assuntos
Alquil e Aril Transferases/metabolismo , Fenômenos Fisiológicos Bacterianos/genética , Fermentação/fisiologia , Engenharia Metabólica/métodos , Redes e Vias Metabólicas/fisiologia , Saccharomyces cerevisiae/fisiologia , Terpenos/metabolismo , Alquil e Aril Transferases/genética , Vias Biossintéticas/fisiologia , Melhoramento Genético/métodos , Análise do Fluxo Metabólico/métodos , Terpenos/isolamento & purificaçãoRESUMO
Metabolic engineering of microorganisms for heterologous biosynthesis is a promising route to sustainable chemical production which attracts increasing research and industrial interest. However, the efficiency of microbial biosynthesis is often restricted by insufficient activity of pathway enzymes and unbalanced utilization of metabolic intermediates. This work presents a combinatorial strategy integrating modification of multiple rate-limiting enzymes and modular pathway engineering to simultaneously improve intra- and inter-pathway balance, which might be applicable for a range of products, using isoprene as an example product. For intra-module engineering within the methylerythritol-phosphate (MEP) pathway, directed co-evolution of DXS/DXR/IDI was performed adopting a lycopene-indicated high-throughput screening method developed herein, leading to 60% improvement of isoprene production. In addition, inter-module engineering between the upstream MEP pathway and the downstream isoprene-forming pathway was conducted via promoter manipulation, which further increased isoprene production by 2.94-fold compared to the recombinant strain with solely protein engineering and 4.7-fold compared to the control strain containing wild-type enzymes. These results demonstrated the potential of pathway optimization in isoprene overproduction as well as the effectiveness of combining metabolic regulation and protein engineering in improvement of microbial biosynthesis. Biotechnol. Bioeng. 2016;113: 2661-2669. © 2016 Wiley Periodicals, Inc.
Assuntos
Vias Biossintéticas/genética , Evolução Molecular Direcionada/métodos , Escherichia coli/fisiologia , Melhoramento Genético/métodos , Hemiterpenos/biossíntese , Engenharia Metabólica/métodos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Butadienos , Técnicas de Química Combinatória/métodos , Hemiterpenos/genética , Taxa de Depuração Metabólica , Pentanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Phospholipase C (PLC) catalyzes the hydrolysis of phospholipids to produce phosphate monoesters and diacylglycerol. It has many applications in the enzymatic degumming of plant oils. PLC Bc , a bacterial PLC from Bacillus cereus, is an optimal choice for this activity in terms of its wide substrate spectrum, high activity, and approved safety. Unfortunately, its large-scale production and reliable high-throughput screening of PLC Bc remain challenging. Herein, we summarize the research progress regarding PLC Bc with emphasis on the screening methods, expression systems, catalytic mechanisms and inhibitor of PLC Bc . This review hopefully will inspire new achievements in related areas, to promote the sustainable development of PLC Bc and its application.
Assuntos
Bacillus cereus/enzimologia , Inibidores Enzimáticos/farmacologia , Fosfolipases Tipo C/biossíntese , Bacillus cereus/química , Bacillus cereus/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Especificidade por Substrato , Fosfolipases Tipo C/química , Fosfolipases Tipo C/genética , Fosfolipases Tipo C/isolamento & purificaçãoRESUMO
Improved supply of farnesyl diphosphate (FPP) is often considered as a typical strategy for engineering Saccharomyces cerevisiae towards efficient terpenoid production. However, in the engineered strains with enhanced precursor supply, the production of the target metabolite is often impeded by insufficient capacity of the heterologous terpenoid pathways, which limits further conversion of FPP. Here, we tried to assemble an unimpeded biosynthesis pathway by combining directed evolution and metabolic engineering in S. cerevisiae for lycopene-overproduction. First, the catalytic ability of phytoene syntheses from different sources was investigated based on lycopene accumulation. Particularly, the lycopene cyclase function of the bifunctional enzyme CrtYB from Xanthophyllomyces dendrorhous was inactivated by deletion of functional domain and directed evolution to obtain mutants with solely phytoene synthase function. Coexpression of the resulting CrtYB11M mutant along with the CrtE and CrtI genes from X. dendrorhous, and the tHMG1 gene from S. cerevisiae led to production of 4.47 mg/g DCW (Dry cell weight) of lycopene and 25.66 mg/g DCW of the by-product squalene. To further increase the FPP competitiveness of the lycopene synthesis pathway, we tried to enhance the catalytic performance of CrtE by directed evolution and created a series of pathway variants by varying the copy number of Crt genes. Finally, fed-batch fermentation was conducted for the diploid strain YXWPD-14 resulting in accumulation of 1.61 g/L (24.41 mg/g DCW) of lycopene, meanwhile, the by-production of squalene was reduced to below 1 mg/g DCW.
Assuntos
Carotenoides , Evolução Molecular Direcionada/métodos , Engenharia Metabólica/métodos , Saccharomyces cerevisiae , Carotenoides/biossíntese , Carotenoides/genética , Licopeno , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
Balanced utilization of metabolic intermediates and controllable expression of genes in biosynthetic pathways are key issues for the effective production of value-added chemicals in microbes. An inducer/repressor-free sequential control strategy regulated by glucose concentration in the growth environment was proposed to address these issues, and its efficiency was validated using heterologous beta-carotenoid biosynthesis in Saccharomyces cerevisiae as an example. Through sequential control of the downstream, upstream, and competitive pathways of farnesyl diphosphate (FPP), the crucial metabolic node in the biosynthesis of terpenoids, in a predetermined order, a carotenoid production of 1156 mg/L (20.79 mg/g DCW) was achieved by high-cell density fermentation. Quantitative PCR analysis of the regulated genes demonstrated that the transcription patterns were controlled in a sequential manner as expected. The inducer/repressor-free nature of this strategy offers a both practical and economically efficient approach to improved biosynthetic production of value-added chemicals.