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1.
Proc Natl Acad Sci U S A ; 116(26): 12810-12815, 2019 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-31186357

RESUMO

The more than 50,000 isoprenoids found in nature are all derived from the 5-carbon diphosphates isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). Natively, IPP and DMAPP are generated by the mevalonate (MVA) and 2-C-methyl-d-erythritol-4-phosphate (MEP) pathways, which have been engineered to produce compounds with numerous applications. However, as these pathways are inherently constrained by carbon, energy inefficiencies, and their roles in native metabolism, engineering for isoprenoid biosynthesis at high flux, titer, and yield remains a challenge. To overcome these limitations, here we develop an alternative synthetic pathway termed the isoprenoid alcohol (IPA) pathway that centers around the synthesis and subsequent phosphorylation of IPAs. We first established a lower IPA pathway for the conversion of IPAs to isoprenoid pyrophosphate intermediates that enabled the production of greater than 2 g/L geraniol from prenol as well as limonene, farnesol, diaponeurosporene, and lycopene. We then designed upper IPA pathways for the generation of (iso)prenol from central carbon metabolites with the development of a route to prenol enabling its synthesis at more than 2 g/L. Using prenol as the linking intermediate further facilitated an integrated IPA pathway that resulted in the production of nearly 0.6 g/L total monoterpenoids from glycerol as the sole carbon source. The IPA pathway provides an alternative route to isoprenoids that is more energy efficient than native pathways and can serve as a platform for targeting a repertoire of isoprenoid compounds with application as high-value pharmaceuticals, commodity chemicals, and fuels.


Assuntos
Terpenos/síntese química , Monoterpenos Acíclicos/química , Biocatálise , Glicerol/química , Hemiterpenos/química , Pentanóis/química , Biologia Sintética
2.
Nat Chem Biol ; 15(9): 900-906, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31383974

RESUMO

Despite the potential of biotechnological processes for one-carbon (C1) bioconversion, efficient biocatalysts required for their implementation are yet to be developed. To address intrinsic limitations of native C1 biocatalysts, here we report that 2-hydroxyacyl CoA lyase (HACL), an enzyme involved in mammalian α-oxidation, catalyzes the ligation of carbonyl-containing molecules of different chain lengths with formyl-coenzyme A (CoA) to produce C1-elongated 2-hydroxyacyl-CoAs. We discovered and characterized a prokaryotic variant of HACL and identified critical residues for this newfound activity, including those supporting the hypothesized thiamine pyrophosphate-dependent acyloin condensation mechanism. The use of formyl-CoA as a C1 donor provides kinetic advantages and enables C1 bioconversion to multi-carbon products, demonstrated here by engineering an Escherichia coli whole-cell biotransformation system for the synthesis of glycolate and 2-hydroxyisobutyrate from formaldehyde and formaldehyde plus acetone, respectively. Our work establishes a new approach for C1 bioconversion and the potential for HACL-based pathways to support synthetic methylotrophy.


Assuntos
Enoil-CoA Hidratase/metabolismo , Álcoois Graxos/metabolismo , Rhodospirillales/enzimologia , Enoil-CoA Hidratase/classificação , Enoil-CoA Hidratase/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Álcoois Graxos/química , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Variação Genética , Humanos , Engenharia Metabólica , Modelos Moleculares , Mutagênese Sítio-Dirigida , Filogenia , Conformação Proteica
3.
Biotechnol Bioeng ; 116(5): 1116-1127, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30659582

RESUMO

Prenylated aromatics (PAs) are an important class of natural products with valuable pharmaceutical applications. To address current limitations of their sourcing from plants, here, we present a microbial platform for the in vivo synthesis of PAs based on the aromatic prenyltransferase NphB from Streptomyces sp. strain CL190. As proof of concept, we targeted the prenylation of phenolic/phenolcarboxylic acids, including orsellinic (OSA), divarinolic (DVA), and olivetolic (OLA) acids, whose prenylated products have important biopharmaceutical applications. Although the ability of wild-type NphB to catalyze the prenylation reaction with each acid was validated by in vitro characterization, improvement of product titers in vivo required protein modeling and rational design to engineer NphB variants with increased activity and product selectivity. When a designed NphB variant with eightfold improved catalytic efficiency toward OSA was expressed in an Escherichia coli host engineered to generate geranyl pyrophosphate at high flux through the mevalonate pathway, we observed up to 300 mg/L prenylated products by exogenously supplying OSA. The improved properties of engineered NphB were also utilized to demonstrate the diversification of this in vivo platform by using both different aromatic acceptors and different prenyl donors to generate various PA compounds, including medicinally important compounds such as cannabigerovarinic, cannabigerolic, and grifolic acids.


Assuntos
Proteínas de Bactérias , Benzoatos/metabolismo , Dimetilaliltranstransferase , Escherichia coli K12 , Engenharia Metabólica , Prenilação , Sesterterpenos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Dimetilaliltranstransferase/genética , Dimetilaliltranstransferase/metabolismo , Escherichia coli K12/genética , Escherichia coli K12/metabolismo , Streptomyces/enzimologia , Streptomyces/genética
4.
Metab Eng ; 48: 175-183, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29883803

RESUMO

Methane, the primary component of natural gas, is the second most abundant greenhouse gas (GHG) and contributes significantly to climate change. The conversion of methane to industrial platform chemicals provides an attractive opportunity to decrease GHG emissions and utilize this inexpensive and abundantly available gas as a carbon feedstock. While technologies exist for chemical conversion of methane to liquid fuels, the technical complexity of these processes mandate high capital expenditure, large-scale commercial facilities to leverage economies of scale that cannot be efficiently scaled down. Alternatively, bioconversion technologies capable of efficient small-scale operation with high carbon and energy efficiency can enable deployment at remote methane resources inaccessible to current chemical technologies. Aerobic obligate methanotrophs, specifically Methylomicrobium buryatense 5GB1, have recently garnered increased research interest for development of such bio-technologies. In this study, we demonstrate production of C-4 carboxylic acids non-native to the host, specifically crotonic and butyric acids, from methane in an engineered M. buryatense 5GB1C by diversion of carbon flux through the acetyl-CoA node of central 'sugar' linked metabolic pathways using reverse ß-oxidation pathway genes. The synthesis of short chain carboxylic acids through the acetyl-CoA node demonstrates the potential for engineering M. buryatense 5GB1 as a platform for bioconversion of methane to a number of value added industrial chemicals, and presents new opportunities for further diversifying the products obtainable from methane as the feedstock.


Assuntos
Acetilcoenzima A , Ácido Butírico/metabolismo , Crotonatos/metabolismo , Engenharia Metabólica , Metano/metabolismo , Methylococcaceae , Acetilcoenzima A/genética , Acetilcoenzima A/metabolismo , Methylococcaceae/genética , Methylococcaceae/metabolismo
5.
Metab Eng ; 45: 11-19, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29146470

RESUMO

An engineered reversal of the ß-oxidation cycle (r-BOX) and the fatty acid biosynthesis (FAB) pathway are promising biological platforms for advanced fuel and chemical production in part due to their iterative nature supporting the synthesis of various chain length products. While diverging in their carbon-carbon elongation reaction mechanism, iterative operation of each pathway relies on common chemical conversions (reduction, dehydration, and reduction) differing only in the attached moiety (acyl carrier protein (ACP) in FAB vs Coenzyme A in r-BOX). Given this similarity, we sought to determine whether FAB enzymes can be used in the context of r-BOX as a means of expanding available r-BOX components with a ubiquitous set of well characterized enzymes. Using enzymes from the type II FAB pathway (FabG, FabZ, and FabI) in conjunction with a thiolase catalyzing a non-decarboxylative condensation, we demonstrate that FAB enzymes support a functional r-BOX. Pathway operation with FAB enzymes was improved through computationally directed protein design to develop FabZ variants with amino acid substitutions designed to disrupt hydrogen bonding at the FabZ-ACP interface and introduce steric and electrostatic repulsion between the FabZ and ACP. FabZ with R126W and R121E substitutions resulted in improved carboxylic acid and alcohol production from one- and multiple-turn r-BOX compared to the wild-type enzyme. Furthermore, the ability for FAB enzymes to operate on functionalized intermediates was exploited to produce branched chain carboxylic acids through an r-BOX with functionalized priming. These results not only provide an expanded set of enzymes within the modular r-BOX pathway, but can also potentially expand the scope of products targeted through this pathway by operating with CoA intermediates containing various functional groups.


Assuntos
Oxirredutases do Álcool , Escherichia coli K12 , Ácidos Graxos , Complexos Multienzimáticos , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Escherichia coli K12/enzimologia , Escherichia coli K12/genética , Ácidos Graxos/biossíntese , Ácidos Graxos/genética , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo
6.
J Ind Microbiol Biotechnol ; 45(6): 379-391, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29675615

RESUMO

Convergence of market drivers such as abundant availability of inexpensive natural gas and increasing awareness of its global warming effects have created new opportunities for the development of small-scale gas-to-liquid (GTL) conversion technologies that can efficiently utilize methane, the primary component of natural gas. Leveraging the unique ability of methanotrophs that use methane as carbon and energy source, biological GTL platforms can be envisioned that are readily deployable at remote petroleum drilling sites where large chemical GTL infrastructure is uneconomical to set-up. Methylomicrobium buryatense, an obligate methanotroph, has gained traction as a potential industrial methanotrophic host because of availability of genetic tools and recent advances in its metabolic engineering. However, progress is impeded by low strain performance and lack of an industrial medium. In this study, we first established a small-scale cultivation platform using Hungate tubes for growth of M. buryatense at medium-to-high-throughput that also enabled 2X faster growth compared to that obtained in traditional glass serum bottles. Then, employing a synthetic biology approach we engineered M. buryatense with varying promoter (inducible and constitutive) and ribosome-binding site combinations, and obtained a strain capable of producing L-lactate from methane at a flux 14-fold higher than previously reported. Finally, we demonstrated L-lactate production in an industrial medium by replacing nitrate with less-expensive ammonium as the nitrogen source. Under these conditions, L-lactate was synthesized at a flux approximately 50-fold higher than that reported previously in a bioreactor system while achieving a titer of 0.6 g/L. These findings position M. buryatense closer to becoming an industrial host strain of choice, and pave new avenues for accelerating methane-to-chemical conversion using synthetic biology.


Assuntos
Reatores Biológicos , Ácido Láctico/biossíntese , Engenharia Metabólica , Metano/metabolismo , Methylococcaceae/metabolismo , Compostos de Amônio/química , Gases , Microbiologia Industrial , Nitratos/química , Nitrogênio/química , Petróleo , Ribossomos/química , Biologia Sintética
7.
J Ind Microbiol Biotechnol ; 45(7): 579-588, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29330665

RESUMO

Synthetic biology, encompassing the design and construction of novel artificial biological pathways and organisms and the redesign of existing natural biological systems, is rapidly expanding the number of applications for which biological systems can play an integral role. In the context of chemical production, the combination of synthetic biology and metabolic engineering approaches continues to unlock the ability to biologically produce novel and complex molecules from a variety of feedstocks. Here, we utilize a synthetic approach to design and build a pathway to produce 2-hydroxyisovaleric acid in Escherichia coli and demonstrate how pathway design can be supplemented with metabolic engineering approaches to improve pathway performance from various carbon sources. Drawing inspiration from the native pathway for the synthesis of the 5-carbon amino acid L-valine, we exploit the decarboxylative condensation of two molecules of pyruvate, with subsequent reduction and dehydration reactions enabling the synthesis of 2-hydroxyisovaleric acid. Key to our approach was the utilization of an acetolactate synthase which minimized kinetic and regulatory constraints to ensure sufficient flux entering the pathway. Critical host modifications enabling maximum product synthesis from either glycerol or glucose were then examined, with the varying degree of reduction of these carbons sources playing a major role in the required host background. Through these engineering efforts, the designed pathway produced 6.2 g/L 2-hydroxyisovaleric acid from glycerol at 58% of maximum theoretical yield and 7.8 g/L 2-hydroxyisovaleric acid from glucose at 73% of maximum theoretical yield. These results demonstrate how the combination of synthetic biology and metabolic engineering approaches can facilitate bio-based chemical production.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Engenharia Metabólica/métodos , Biologia Sintética , Valeratos/metabolismo , Escherichia coli/genética , Glicerol/metabolismo , Cinética , Ácido Pirúvico/metabolismo
8.
Nature ; 476(7360): 355-9, 2011 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-21832992

RESUMO

Advanced (long-chain) fuels and chemicals are generated from short-chain metabolic intermediates through pathways that require carbon-chain elongation. The condensation reactions mediating this carbon-carbon bond formation can be catalysed by enzymes from the thiolase superfamily, including ß-ketoacyl-acyl-carrier protein (ACP) synthases, polyketide synthases, 3-hydroxy-3-methylglutaryl-CoA synthases, and biosynthetic thiolases. Pathways involving these enzymes have been exploited for fuel and chemical production, with fatty-acid biosynthesis (ß-ketoacyl-ACP synthases) attracting the most attention in recent years. Degradative thiolases, which are part of the thiolase superfamily and naturally function in the ß-oxidation of fatty acids, can also operate in the synthetic direction and thus enable carbon-chain elongation. Here we demonstrate that a functional reversal of the ß-oxidation cycle can be used as a metabolic platform for the synthesis of alcohols and carboxylic acids with various chain lengths and functionalities. This pathway operates with coenzyme A (CoA) thioester intermediates and directly uses acetyl-CoA for acyl-chain elongation (rather than first requiring ATP-dependent activation to malonyl-CoA), characteristics that enable product synthesis at maximum carbon and energy efficiency. The reversal of the ß-oxidation cycle was engineered in Escherichia coli and used in combination with endogenous dehydrogenases and thioesterases to synthesize n-alcohols, fatty acids and 3-hydroxy-, 3-keto- and trans-Δ(2)-carboxylic acids. The superior nature of the engineered pathway was demonstrated by producing higher-chain linear n-alcohols (C ≥ 4) and extracellular long-chain fatty acids (C > 10) at higher efficiency than previously reported. The ubiquitous nature of ß-oxidation, aldehyde/alcohol dehydrogenase and thioesterase enzymes has the potential to enable the efficient synthesis of these products in other industrial organisms.


Assuntos
Biocombustíveis , Redes e Vias Metabólicas , Acetilcoenzima A/metabolismo , Acil Coenzima A/metabolismo , Álcool Desidrogenase/metabolismo , Álcoois/química , Álcoois/metabolismo , Aldeído Desidrogenase/metabolismo , Biocombustíveis/análise , Biocombustíveis/provisão & distribuição , Butanóis/metabolismo , Ácidos Carboxílicos/química , Ácidos Carboxílicos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Ácidos Graxos/biossíntese , Ácidos Graxos/química , Ácidos Graxos/metabolismo , Oxirredução , Tioléster Hidrolases/metabolismo
9.
Metab Eng ; 28: 202-212, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25638687

RESUMO

An engineered reversal of the ß-oxidation cycle was exploited to demonstrate its utility for the synthesis of medium chain (6-10-carbons) ω-hydroxyacids and dicarboxylic acids from glycerol as the only carbon source. A redesigned ß-oxidation reversal facilitated the production of medium chain carboxylic acids, which were converted to ω-hydroxyacids and dicarboxylic acids by the action of an engineered ω-oxidation pathway. The selection of a key thiolase (bktB) and thioesterase (ydiI) in combination with previously established core ß-oxidation reversal enzymes, as well as the development of chromosomal expression systems for the independent control of pathway enzymes, enabled the generation of C6-C10 carboxylic acids and provided a platform for vector based independent expression of ω-functionalization enzymes. Using this approach, the expression of the Pseudomonas putida alkane monooxygenase system, encoded by alkBGT, in combination with all ß-oxidation reversal enzymes resulted in the production of 6-hydroxyhexanoic acid, 8-hydroxyoctanoic acid, and 10-hydroxydecanoic acid. Following identification and characterization of potential alcohol and aldehyde dehydrogenases, chnD and chnE from Acinetobacter sp. strain SE19 were expressed in conjunction with alkBGT to demonstrate the synthesis of the C6-C10 dicarboxylic acids, adipic acid, suberic acid, and sebacic acid. The potential of a ß-oxidation cycle with ω-oxidation termination pathways was further demonstrated through the production of greater than 0.8 g/L C6-C10 ω-hydroxyacids or about 0.5 g/L dicarboxylic acids of the same chain lengths from glycerol (an unrelated carbon source) using minimal media.


Assuntos
Ácidos Carboxílicos/metabolismo , Engenharia Metabólica , Pseudomonas putida/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Oxirredução , Pseudomonas putida/genética , Tioléster Hidrolases/genética , Tioléster Hidrolases/metabolismo
10.
Appl Environ Microbiol ; 81(4): 1406-16, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25527535

RESUMO

We recently used a synthetic/bottom-up approach to establish the identity of the four enzymes composing an engineered functional reversal of the -oxidation cycle for fuel and chemical production in Escherichia coli (J. M. Clomburg, J. E. Vick, M. D. Blankschien, M. Rodriguez-Moya, and R. Gonzalez, ACS Synth Biol 1:541­554, 2012, http://dx.doi.org/10.1021/sb3000782).While native enzymes that catalyze the first three steps of the pathway were identified, the identity of the native enzyme(s) acting as the trans-enoyl coenzyme A (CoA) reductase(s) remained unknown, limiting the amount of product that could be synthesized (e.g., 0.34 g/liter butyrate) and requiring the overexpression of a foreign enzyme (the Euglena gracilis trans-enoyl-CoA reductase [EgTER]) to achieve high titers (e.g., 3.4 g/liter butyrate). Here, we examine several native E. coli enzymes hypothesized to catalyze the reduction of enoyl-CoAs to acyl-CoAs. Our results indicate that FabI, the native enoyl-acyl carrier protein (enoyl-ACP) reductase (ENR) from type II fatty acid biosynthesis, possesses sufficient NADH-dependent TER activity to support the efficient operation of a -oxidation reversal. Overexpression of FabI proved as effective as EgTER for the production of butyrate and longer-chain carboxylic acids. Given the essential nature of fabI, we investigated whether bacterial ENRs from other families were able to complement a fabI deletion without promiscuous reduction of crotonyl-CoA. These characteristics from Bacillus subtilis FabL enabled deltaffabI complementation experiments that conclusively established that FabI encodes a native enoyl-CoA reductase activity that supports the ß-oxidation reversal in E. coli.


Assuntos
Enoil-(Proteína de Transporte de Acila) Redutase (NADH)/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Acil Coenzima A/metabolismo , Catálise , Enoil-(Proteína de Transporte de Acila) Redutase (NADH)/química , Enoil-(Proteína de Transporte de Acila) Redutase (NADH)/genética , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Ácido Graxo Sintase Tipo II/química , Ácido Graxo Sintase Tipo II/genética , Ácido Graxo Sintase Tipo II/metabolismo , Ácidos Graxos/metabolismo , Cinética , NAD/metabolismo , Oxirredução
11.
J Ind Microbiol Biotechnol ; 42(3): 465-75, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25645093

RESUMO

The recently engineered reversal of the ß-oxidation cycle has been proposed as a potential platform for the efficient synthesis of longer chain (C ≥ 4) fuels and chemicals. Here, we demonstrate the utility of this platform for the synthesis of medium-chain length (C6-C10) products through the manipulation of key components of the pathway. Deletion of endogenous thioesterases provided a clean background in which the expression of various thiolase and termination components, along with required core enzymes, resulted in the ability to alter the chain length distribution and functionality of target products. This approach enabled the synthesis of medium-chain length carboxylic acids and primary alcohols from glycerol, a low-value feedstock. The use of BktB as the thiolase component with thioesterase TesA' as the termination enzyme enabled the synthesis of about 1.3 g/L C6-C10 saturated carboxylic acids. Tailoring of product formation to primary alcohol synthesis was achieved with the use of various acyl-CoA reductases. The combination of AtoB and FadA as the thiolase components with the alcohol-forming acyl-CoA reductase Maqu2507 from M. aquaeolei resulted in the synthesis of nearly 0.3 g/L C6-C10 alcohols. These results further demonstrate the versatile nature of a ß-oxidation reversal, and highlight several key aspects and control points that can be further manipulated to fine-tune the synthesis of various fuels and chemicals.


Assuntos
Álcoois/química , Álcoois/metabolismo , Ácidos Carboxílicos/química , Ácidos Carboxílicos/metabolismo , Escherichia coli/metabolismo , Aldeído Oxirredutases/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Engenharia Metabólica , Oxirredução , Biologia Sintética , Tioléster Hidrolases/deficiência
12.
Metab Eng ; 23: 100-15, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24569100

RESUMO

The modularity and versatility of an engineered functional reversal of the ß-oxidation cycle make it a promising platform for the synthesis of longer-chain (C≥4) products. While the pathway has recently been exploited for the production of n-alcohols and carboxylic acids, fully capitalizing on its potential for the synthesis of a diverse set of product families requires a system-level assessment of its biosynthetic capabilities. To this end, we utilized a genome scale model of Escherichia coli, in combination with Flux Balance Analysis and Flux Variability Analysis, to determine the key characteristics and constraints of this pathway for the production of a variety of product families under fermentative conditions. This analysis revealed that the production of n-alcohols, alkanes, and fatty acids of lengths C3-C18 could be coupled to cell growth in a strain lacking native fermentative pathways, a characteristic enabling product synthesis at maximum rates, titers, and yields. While energetic and redox constraints limit the production of target compounds from alternative platforms such as the fatty acid biosynthesis and α-ketoacid pathways, the metabolic efficiency of a ß-oxidation reversal allows the production of a wide range of products of varying length and functionality. The versatility of this platform was investigated through the simulation of various termination pathways for product synthesis along with the use of different priming molecules, demonstrating its potential for the efficient synthesis of a wide variety of functionalized compounds. Overall, specific metabolic manipulations suggested by this systems-level analysis include deletion of native fermentation pathways, the choice of priming molecules and specific routes for their synthesis, proper choice of termination enzymes, control of flux partitioning at the pyruvate node and the pentose phosphate pathway, and the use of an NADH-dependent trans-enoyl-CoA reductase instead of a ferredoxin-dependent enzyme.


Assuntos
Simulação por Computador , Escherichia coli/metabolismo , Ácidos Graxos/biossíntese , Metabolismo dos Lipídeos/fisiologia , Modelos Biológicos , Escherichia coli/genética , Ácidos Graxos/genética , Oxirredução
13.
Microb Cell Fact ; 12: 7, 2013 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-23347598

RESUMO

BACKGROUND: Due to its abundance and low-price, glycerol has become an attractive carbon source for the industrial production of value-added fuels and chemicals. This work reports the engineering of E. coli for the efficient conversion of glycerol into L-lactic acid (L-lactate). RESULTS: Escherichia coli strains have previously been metabolically engineered for the microaerobic production of D-lactic acid from glycerol in defined media by disrupting genes that minimize the synthesis of succinate, acetate, and ethanol, and also overexpressing the respiratory route of glycerol dissimilation (GlpK/GlpD). Here, further rounds of rationale design were performed on these strains for the homofermentative production of L-lactate, not normally produced in E. coli. Specifically, L-lactate production was enabled by: 1), replacing the native D-lactate specific dehydrogenase with Streptococcus bovis L-lactate dehydrogenase (L-LDH), 2) blocking the methylglyoxal bypass pathways to avoid the synthesis of a racemic mixture of D- and L-lactate and prevent the accumulation of toxic intermediate, methylglyoxal, and 3) the native aerobic L-lactate dehydrogenase was blocked to prevent the undesired utilization of L-lactate. The engineered strain produced 50 g/L of L-lactate from 56 g/L of crude glycerol at a yield 93% of the theoretical maximum and with high optical (99.9%) and chemical (97%) purity. CONCLUSIONS: This study demonstrates the efficient conversion of glycerol to L-lactate, a microbial process that had not been reported in the literature prior to our work. The engineered biocatalysts produced L-lactate from crude glycerol in defined minimal salts medium at high chemical and optical purity.


Assuntos
Escherichia coli/metabolismo , Glicerol/metabolismo , Ácido Láctico/biossíntese , Engenharia Metabólica , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Glicerol Quinase/genética , Glicerol Quinase/metabolismo , Glicerolfosfato Desidrogenase/genética , Glicerolfosfato Desidrogenase/metabolismo , Cinética , L-Lactato Desidrogenase/antagonistas & inibidores , L-Lactato Desidrogenase/genética , L-Lactato Desidrogenase/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Aldeído Pirúvico/metabolismo , Estereoisomerismo , Streptococcus bovis/enzimologia , Desidrogenase do Álcool de Açúcar/genética , Desidrogenase do Álcool de Açúcar/metabolismo
14.
Biotechnol Lett ; 35(6): 831-42, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23690047

RESUMO

Glycerol has attracted the attention of scientific and industrial communities due to its generation in bulk quantities as a byproduct of biofuel industries. With the rapid growth of these industries in recent years, glycerol is frequently treated as a very low-value byproduct or even a waste product with a disposal cost associated to it. Glycerol is not only abundant and inexpensive but also can generate more reducing equivalents than glucose or xylose. This unique characteristic of glycerol offers a tremendous opportunity for its biological conversion to valuable products at higher yield. This review focuses on research efforts to utilize glycerol as a carbon source for the production of a variety of fuels and chemicals by both native and metabolically engineered microorganisms.


Assuntos
Biocombustíveis , Biotecnologia/métodos , Glicerol/metabolismo , Carbono/metabolismo , Fermentação
15.
Biotechnol Bioeng ; 109(1): 187-98, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21858785

RESUMO

Availability, low price, and high degree of reduction have made glycerol a highly attractive and exploited carbon source for the production of fuels and reduced chemicals. Here we report the quantitative analysis of the fermentative metabolism of glycerol in Escherichia coli through the use of kinetic modeling and metabolic control analysis (MCA) to gain a better understanding of glycerol fermentation and identify key targets for genetic manipulation that could enhance product synthesis. The kinetics of glycerol fermentation in a batch culture was simulated using a dynamic model consisting of mass balances for glycerol, ethanol, biomass, and 11 intracellular metabolites, along with the corresponding kinetic expressions for the metabolism of each species. The model was then used to calculate metabolic control coefficients and elucidate the control structure of the pathways involved in glycerol utilization and ethanol synthesis. The calculated flux control coefficients indicate that the glycolytic flux during glycerol fermentation is almost exclusively controlled by the enzymes glycerol dehydrogenase (encoded by gldA) and dihydroxyacetone kinase (DHAK) (encoded by dhaKLM). In agreement with the MCA findings, overexpression of gldA and dhaKLM led to significant increase in glycerol utilization and ethanol synthesis fluxes. Moreover, overexpression of other enzymes involved in the pathways that mediate glycerol utilization and its conversion to ethanol had no significant impact on glycerol utilization and ethanol synthesis, further validating the MCA predictions. These findings were then applied as a means of increasing the production of ethanol: overexpression of glycerol dehyrdogenase and DHAK enabled the production of 20 g/L ethanol from crude glycerol, a by-product of biodiesel production, indicating the potential for industrial scale conversion of waste glycerol to ethanol under anaerobic conditions.


Assuntos
Escherichia coli/metabolismo , Etanol/metabolismo , Fermentação , Glicerol/metabolismo , Engenharia Metabólica , Biomassa , Reatores Biológicos , Expressão Gênica , Cinética , Redes e Vias Metabólicas/genética
16.
Microb Biotechnol ; 15(1): 289-304, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34699695

RESUMO

Most microorganisms can metabolize glycerol when external electron acceptors are available (i.e. under respiratory conditions). However, few can do so under fermentative conditions owing to the unique redox constraints imposed by the high degree of reduction of glycerol. Here, we utilize in silico analysis combined with in vivo genetic and biochemical approaches to investigate the fermentative metabolism of glycerol in Escherichia coli. We found that E. coli can achieve redox balance at alkaline pH by reducing protons to H2 , complementing the previously reported role of 1,2-propanediol synthesis under acidic conditions. In this new redox balancing mode, H2 evolution is coupled to a respiratory glycerol dissimilation pathway composed of glycerol kinase (GK) and glycerol-3-phosphate (G3P) dehydrogenase (G3PDH). GK activates glycerol to G3P, which is further oxidized by G3PDH to generate reduced quinones that drive hydrogenase-dependent H2 evolution. Despite the importance of the GK-G3PDH route under alkaline conditions, we found that the NADH-generating glycerol dissimilation pathway via glycerol dehydrogenase (GldA) and phosphoenolpyruvate (PEP)-dependent dihydroxyacetone kinase (DHAK) was essential under both alkaline and acidic conditions. We assessed system-wide metabolic impacts of the constraints imposed by the PEP dependency of the GldA-DHAK route. This included the identification of enzymes and pathways that were not previously known to be involved in glycerol metabolisms such as PEP carboxykinase, PEP synthetase, multiple fructose-1,6-bisphosphatases and the fructose phosphate bypass.


Assuntos
Proteínas de Escherichia coli , Escherichia coli , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fermentação , Glicerol , Redes e Vias Metabólicas/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética
17.
Nat Commun ; 13(1): 3058, 2022 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-35650184

RESUMO

Carbon-negative synthesis of biochemical products has the potential to mitigate global CO2 emissions. An attractive route to do this is the reverse ß-oxidation (r-BOX) pathway coupled to the Wood-Ljungdahl pathway. Here, we optimize and implement r-BOX for the synthesis of C4-C6 acids and alcohols. With a high-throughput in vitro prototyping workflow, we screen 762 unique pathway combinations using cell-free extracts tailored for r-BOX to identify enzyme sets for enhanced product selectivity. Implementation of these pathways into Escherichia coli generates designer strains for the selective production of butanoic acid (4.9 ± 0.1 gL-1), as well as hexanoic acid (3.06 ± 0.03 gL-1) and 1-hexanol (1.0 ± 0.1 gL-1) at the best performance reported to date in this bacterium. We also generate Clostridium autoethanogenum strains able to produce 1-hexanol from syngas, achieving a titer of 0.26 gL-1 in a 1.5 L continuous fermentation. Our strategy enables optimization of r-BOX derived products for biomanufacturing and industrial biotechnology.


Assuntos
Ciclo do Carbono , Escherichia coli , Processos Autotróficos , Escherichia coli/metabolismo , Fermentação , Oxirredução
18.
J Biol Chem ; 285(41): 31548-58, 2010 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-20667837

RESUMO

Pyruvate is located at a metabolic junction of assimilatory and dissimilatory pathways and represents a switch point between respiratory and fermentative metabolism. In Escherichia coli, the pyruvate dehydrogenase complex (PDHC) and pyruvate formate-lyase are considered the primary routes of pyruvate conversion to acetyl-CoA for aerobic respiration and anaerobic fermentation, respectively. During glucose fermentation, the in vivo activity of PDHC has been reported as either very low or undetectable, and the role of this enzyme remains unknown. In this study, a comprehensive characterization of wild-type E. coli MG1655 and a PDHC-deficient derivative (Pdh) led to the identification of the role of PDHC in the anaerobic fermentation of glucose. The metabolism of these strains was investigated by using a mixture of (13)C-labeled and -unlabeled glucose followed by the analysis of the labeling pattern in protein-bound amino acids via two-dimensional (13)C,(1)H NMR spectroscopy. Metabolite balancing, biosynthetic (13)C labeling of proteinogenic amino acids, and isotopomer balancing all indicated a large increase in the flux of the oxidative branch of the pentose phosphate pathway (ox-PPP) in response to the PDHC deficiency. Because both ox-PPP and PDHC generate CO(2) and the calculated CO(2) evolution rate was significantly reduced in Pdh, it was hypothesized that the role of PDHC is to provide CO(2) for cell growth. The similarly negative impact of either PDHC or ox-PPP deficiencies, and an even more pronounced impairment of cell growth in a strain lacking both ox-PPP and PDHC, provided further support for this hypothesis. The three strains exhibited similar phenotypes in the presence of an external source of CO(2), thus confirming the role of PDHC. Activation of formate hydrogen-lyase (which converts formate to CO(2) and H(2)) rendered the PDHC deficiency silent, but its negative impact reappeared in a strain lacking both PDHC and formate hydrogen-lyase. A stoichiometric analysis of CO(2) generation via PDHC and ox-PPP revealed that the PDHC route is more carbon- and energy-efficient, in agreement with its beneficial role in cell growth.


Assuntos
Dióxido de Carbono/metabolismo , Escherichia coli K12/enzimologia , Proteínas de Escherichia coli/metabolismo , Fermentação/fisiologia , Glucose/metabolismo , Via de Pentose Fosfato/fisiologia , Ativação Enzimática/fisiologia , Escherichia coli K12/genética , Escherichia coli K12/crescimento & desenvolvimento , Proteínas de Escherichia coli/genética , Formiato Desidrogenases , Glucose/genética , Hidrogenase , Liases/genética , Liases/metabolismo , Complexos Multienzimáticos , Oxirredução , Complexo Piruvato Desidrogenase
19.
Biotechnol Bioeng ; 108(4): 867-79, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21404260

RESUMO

Due to its availability, low-price, and high degree of reduction, glycerol has become an attractive carbon source for the production of fuels and reduced chemicals. Using the platform we have established from the identification of key pathways mediating fermentative metabolism of glycerol, this work reports the engineering of Escherichia coli for the conversion of glycerol into 1,2-propanediol (1,2-PDO). A functional 1,2-PDO pathway was engineered through a combination of overexpression of genes involved in its synthesis from the key intermediate dihydroxyacetone phosphate (DHAP) and the manipulation of the fermentative glycerol utilization pathway. The former included the overexpression of methylglyoxal synthase (mgsA), glycerol dehydrogenase (gldA), and aldehyde oxidoreductase (yqhD). Manipulation of the glycerol utilization pathway through the replacement of the native E. coli PEP-dependent dihydroxyacetone kinase (DHAK) with an ATP-dependent DHAK from C. freundii increased the availability of DHAP allowing for higher 1,2-PDO production. Analysis of the major fermentative pathways identified ethanol as a required co-product while increases in 1,2-PDO titer and yield were achieved through the disruption of the pathways for acetate and lactate production. Combination of these key metabolic manipulations resulted in an engineered E. coli strain capable of producing 5.6 g/L 1,2-PDO, at a yield of 21.3% (w/w). This strain also performed well when crude glycerol, a by-product of biodiesel production, was used as the substrate. The titer and yield achieved in this study were favorable to those obtained with the use of E. coli for the production of 1,2-PDO from common sugars.


Assuntos
Escherichia coli/enzimologia , Escherichia coli/metabolismo , Glicerol/metabolismo , Microbiologia Industrial/métodos , Propilenoglicol/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Engenharia Genética/métodos
20.
Nat Metab ; 3(10): 1385-1399, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34675440

RESUMO

Metabolic engineering often entails concurrent engineering of substrate utilization, central metabolism and product synthesis pathways, inevitably creating interdependency with native metabolism. Here we report an alternative approach using synthetic pathways for C1 bioconversion that generate multicarbon products directly from C1 units and hence are orthogonal to the host metabolic network. The engineered pathways are based on formyl-CoA elongation (FORCE) reactions catalysed by the enzyme 2-hydroxyacyl-CoA lyase. We use thermodynamic and stoichiometric analyses to evaluate FORCE pathway variants, including aldose elongation, α-reduction and aldehyde elongation. Promising variants were prototyped in vitro and in vivo using the non-methylotrophic bacterium Escherichia coli. We demonstrate the conversion of formate, formaldehyde and methanol into various products including glycolate, ethylene glycol, ethanol and glycerate. FORCE pathways also have the potential to be integrated with the host metabolism for synthetic methylotrophy by the production of native growth substrates as demonstrated in a two-strain co-culture system.


Assuntos
Carbono/metabolismo , Redes e Vias Metabólicas , Carbono-Carbono Liases/metabolismo , Catálise , Escherichia coli/metabolismo
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