Production of 3-Hydroxypropanoic Acid From Glycerol by Metabolically Engineered Bacteria

3-hydroxypropanoic acid (3-HP) is a valuable platform chemical with a high demand in the global market. 3-HP can be produced from various renewable resources. It is used as a precursor in industrial production of a number of chemicals, such as acrylic acid and its many derivatives. In its polymerized form, 3-HP can be used in bioplastic production. Several microbes naturally possess the biosynthetic pathways for production of 3-HP, and a number of these pathways have been introduced in some widely used cell factories, such as Escherichia coli and Saccharomyces cerevisiae. Latest advances in the field of metabolic engineering and synthetic biology have led to more efficient methods for bio-production of 3-HP. These include new approaches for introducing heterologous pathways, precise control of gene expression, rational enzyme engineering, redirecting the carbon flux based on in silico predictions using genome scale metabolic models, as well as optimizing fermentation conditions. Despite the fact that the production of 3-HP has been extensively explored in established industrially relevant cell factories, the current production processes have not yet reached the levels required for industrial exploitation. In this review, we explore the state of the art in 3-HP bio-production, comparing the yields and titers achieved in different microbial cell factories and we discuss possible methodologies that could make the final step toward industrially relevant cell factories

Recent Progress in the Understanding and Engineering of Coenzyme B-12-Dependent Glycerol Dehydratase

Coenzyme B-12-dependent glycerol dehydratase (GDHt) catalyzes the dehydration reaction of glycerol in the presence of adenosylcobalamin to yield 3-hydroxypropanal (3-HPA), which can be converted biologically to versatile platform chemicals such as 1,3-propanediol and 3-hydroxypropionic acid. Owing to the increased demand for biofuels, developing biological processes based on glycerol, which is a byproduct of biodiesel production, has attracted considerable attention recently. In this review, we will provide updates on the current understanding of the catalytic mechanism and structure of coenzyme B-12-dependent GDHt, and then summarize the results of engineering attempts, with perspectives on future directions in its engineering

2-Butanol and Butanone Production in Saccharomyces cerevisiae through Combination of a B-12 Dependent Dehydratase and a Secondary Alcohol Dehydrogenase Using a TEV-Based Expression System

2-Butanol and its chemical precursor butanone (methyl ethyl ketone -MEK) are chemicals with potential uses as biofuels and biocommodity chemicals. In order to produce 2-butanol, we have demonstrated the utility of using a TEV-protease based expression system to achieve equimolar expression of the individual subunits of the two protein complexes involved in the B-12-dependent dehydratase step (from the pdu-operon of Lactobacillus reuterii), which catalyze the conversion of meso-2,3-butanediol to butanone. We have furthermore identified a NADH dependent secondary alcohol dehydrogenase (Sadh from Gordonia sp.) able to catalyze the subsequent conversion of butanone to 2-butanol. A final concentration of 4 +/- 0.2 mg/L 2-butanol and 2 +/- 0.1 mg/L of butanone was found. A key factor for the production of 2-butanol was the availability of NADH, which was achieved by growing cells lacking the GPD1 and GPD2 isogenes under anaerobic conditions

Anaerobic radical enzymes for biotechnology

Enzymes that proceed through radical intermediates have a rich chemistry that includes functionalisation of otherwise unreactive carbon atoms, carbon-skeleton rearrangements, aromatic reductions, and unusual eliminations. Especially under anaerobic conditions, organisms have developed a wide range of approaches for managing these transformations that can be exploited to generate new biological routes towards both bulk and specialty chemicals. These routes are often either much more direct or allow access to molecules that are inaccessible through standard (bio)chemical approaches. This review gives an overview of some of the key enzymes in this area: benzoyl-CoA reductases (that effect the enzymatic Birch reduction), ketyl radical dehydratases, coenzyme B12-dependant enzymes, glycyl radical enzymes, and radical SAM (AdoMet radical) enzymes. These enzymes are discussed alongside biotechnological applications, highlighting the wide range of actual and potential uses. With the increased diversity in biotechnological approaches to obtaining these enzymes and information about them, even more of these amazing enzymes can be expected to find application in industrial processes

Bio-based C-3 Platform Chemical: Biotechnological Production and -Conversion of 3-Hydroxypropionaldehyde

Demands for efficient, greener, economical and sustainable production of chemicals, materials and energy have led to development of industrial biotechnology as a key technology area to provide such products from bio-based raw materials from agricultural-, forestry- and related industrial residues and by-products. For the bio-based industry, it is essential to develop a number of building blocks or platform chemicals for C2-C6 chemicals and even aromatic chemicals. 3-hydroxypropionaldehyde (3HPA) and 3-hydroxypropionic acid (3HP) are potential platform chemicals for C3 chemistry and even for producing polymers. This thesis presents investigations on the biotechnological routes for the production of a C3 platform chemical, 3HPA from glycerol and its conversion to 3HP. Glycerol, was used as the raw material for production of 3HPA using resting cells of the probiotic bacteria, Lactobacillus reuteri, as the biocatalyst. The antimicrobial effect of the bacteria is attributed to the secretion of “reuterin” that is an equilibrium mixture of 3HPA with its dimer and hydrate forms. Glycerol dehydratase, a Vitamin B12-dependent enzyme, presents in L. reuteri, catalyses the dehydration of glycerol to 3HPA. Production of 3HPA at high concentration results in strong inhibition of the enzyme activity and cell viability, which in turn limits the product yield and -productivity. Different means of in situ capture of 3HPA from the reaction were studied. Complexation of 3HPA with bisulfite in a fed-batch biotransformation of glycerol and subsequent removal through binding to an anion exchange resulted in increase in the production of 3HPA to 5.33 g/g biocatalyst from 0.45 g/g in a batch process. In another approach, in situ removal of 3HPA using semicarbazide-functionalized resin in a batch process, productivity was enhanced 2 fold than that without the resin. L. reuteri metabolizes 3HPA further to 1,3-propanediol (1,3PDO) and 3-hydroxypripionic acid (3HP) by reductive and oxidative pathways, respectively. The oxidative pathway, comprises 3 enzymes named propionaldehyde dehydrogenase (PduP), phosphotransacylase (PduL) and propionate kinase (PduW). Kinetic characterization and molecular modelling of the first enzyme, PduP, expressed in Escherichia coli was performed. The enzyme had a specific activity of 28.9 U/mg using propionaldehyde as substrate and 18 U/mg with 3HPA as substrate which is the highest specific activity reported up to date. All the Pdu enzymes were then expressed in E. coli in different combinations and used for bioconversion of 3HPA produced by native L. reuteri. Growing cells of the recombinant bacteria with all the three enzymes, E. coli pdu:P:L:W in a fed-batch mode gave 3HP yield of 0.5 mole/mole 3HPA with 1,3PDO as the co-product, while the resting cells gave 3HP yield of 1 mole /mole 3HPA. This showed the possibility of using of Pdu pathway of L. reuteri for production of 3HP

Microbial Production of Bio-Based Chemicals: A Biorefinery Perspective

A shift from fossil- to renewable biomass feedstock for the emerging bio-based economy requires the development and adoption of new sustainable technologies that are more suited for transformation of biomass components to chemicals, materials and energy. This thesis presents investigations on the development of processes based on industrial biotechnology as a key element for the production of chemicals from agro-/industrial by-products. The chemicals of interest are the ones that could potentially serve as building blocks, platforms, for other chemicals and polymers. Glycerol, a by-product of biodiesel production, was used as raw material for the production of propionic acid, 3-hydroxypropionaldehyde (3HPA) and 3-hydroxypropionic acid (3HP), while methacrylic acid (MA) was produced from 2-methyl-1,3-propanediol, a by-product of butanediol production. Different strategies to overcome the bottlenecks such as product inhibition existing in the bioprocesses for production of the chemicals were studied. Fermentation of glycerol to propionic acid was studied using Propionibacterium acidipropionici. High cell density cultivations were used to overcome the low production rate caused by slow microbial growth and product-mediated toxicity. Increasing the cell density by immobilization and sequential batch recycling improved the production rates by 2- and 6-fold, respectively, over that obtained using conventional batch fermentation. Potato juice, a by-product of potato starch processing, was shown to be a promising, inexpensive nitrogen/vitamin source for the growth of the organism and propionic acid production. Lactobacillus reuteri was employed as a whole cell biocatalyst for the conversion of glycerol to 3HPA and 3HP in aqueous solution. Production of 3HPA using glycerol dehydratase activity of the cells, limited by substrate inhibition and product toxicity, was performed in a fed-batch mode with in situ complexation of the hydroxyaldehyde with bisulfite, and subsequent removal through binding to an anion exchanger. This resulted in increase in production of 3HPA from 0.45 g/g biocatalyst in a batch process to 5.4 g/g. 3HP is formed as an oxidation product of 3HPA, however its accumulation as a product of glycerol metabolism in wild-type L. reuteri has not been reported earlier. The metabolic fluxes through the glycerol reductive and oxidative pathways were calculated using variable volume fed-batch operation. The glycerol feeding strategies were optimized to yield complete conversion of 3HPA into equimolar mixture of 3HP and 1,3PDO, the products that can be easily separated from each other. MA was quantitatively produced at high purity from 2-methyl-1,3-propanediol by a novel process involving integrated biological and chemical catalysis. Whole resting cells of Gluconobacter oxydans were used for selective oxidation of the substrate to the corresponding hydroxycarboxylic acid, which upon dehydration over TiO2 at 210 degree Celsius yielded MA. This process offers a potential, significantly greener alternative to the acetone-cyanohydrin process used for MA production, involving highly toxic substrates, large amounts of waste and greenhouse gas emissions

The cystobactamide pathway : enzymological investigation of unusual non-ribosomal peptide biosynthesis mechanisms

This thesis focused on the investigation of unusual biosynthetic reactions in the Cystobactamide pathway. Cystobactamides present an unusual hexapeptidic backbone mostly constituted from modified para-amino benzoic building blocks and a unique methoxy-isoasparaginyl moiety. Furthermore, the extensive in trans tailoring makes this pathway particularly interesting from an enzymological point of view. The present work details the in trans biosynthesis of the isopropoxyl decorations carried out on the two last para-amino benzoate units through in vitro reconstitution of these biochemical reactions including the generation of fully functional non-ribosomal peptide synthetase modules in vitro. Furthermore an in-depth biochemical investigation of the unprecedented bifunctional non-ribosomal peptide synthetase domain leading to the isomerization or dehydration of asparagine was performed in parallel with the characterization of the in trans hydroxylation and of the shuttling of this moiety. The self-resistance mechanisms of the producer strain Cystobacter velatus 34 were also investigated in comparison to the self-resistance mechanism for the related antibiotic Albicidin. Finally, attempts at complete in vitro reconstitution of the pathways were performed using the unique heterologous expression platform for non-ribosomal peptide synthetase modules developed during this thesis.Diese Dissertation beschäftigt sich mit der Untersuchung ungewöhnlicher biosynthetischer Reaktionen im Cystobactamid-Biosyntheseweg. Cystobactamide haben eine hexapeptidische Grundstruktur, die hauptsächlich aus modifizierten para-Aminobenzoesäure Bausteinen und einer einzigartigen Methoxyisoasparaginyleinheit besteht. Außerdem macht die extensive Verwendung von in trans enzymatischen Modifikationen diesen Biosyntheseweg aus enzymologischer Sichtweise besonders interessant. Die vorliegende Arbeit untersucht durch in vitro Rekonstitution dieser biochemischen Reaktionen, die in trans Biosynthese der Isopropoxyl Dekorationen, die auf den beiden letzten para-Aminobenzoateinheiten entsteht, einschließlich der Erzeugung voll funktionsfähiger nicht ribosomaler Peptidsynthetase-Module in vitro. Außerdem wurde eine umfassende biochemische Analyse der neuen bifunktionellen nicht ribosomalen Peptidsynthetase Domäne durchgeführt, die zur Isomerisierung oder Dehydratisierung von Asparagin führte, parallel zur Charakterisierung der in trans Hydroxylierung und des Transfer dieses Moleküls. Die Eigenresistenzmechanismen des Produzenten Cystobacter velatus 34 wurden im Vergleich zum Selbstresistenzmechanismus des verwandten Antibiotikums Albicidin untersucht. Letztendlich wurden Versuche einer kompletten in vitro Rekonstitution des Biosyntheseweges durchgeführt, mit Hilfe der in dieser Dissertation neuentwickelten heterologen Expressionsplattform für nicht ribosomale Peptidsynthetase-Module

Computational modeling of electronically excited states in cobalamin-dependent reactions.

The current understanding of the photolytic properties of Vitamin B12 derivatives or cobalamins are summarized from a computational point of view. The focus is on two non-alkylcobalamins, cyanocobalamin (CNCbl) and hydroxocobalamin (HOCbl), two alkylcobalamins, methylcobalamin (MeCbl) and adenosylcobalamin (AdoCbl), as well as the stable cob(II)alamin radical. Photolysis of alkylcobalamins involves low-lying singlet excited states where photo-dissociation of the Co-C bond forms singlet-born alkyl/cob(II)alamin radical pairs (RPs). Potential energy surfaces (PESs) of low-lying excited states as functions of both axial bonds provide the most reliable tool for analysis of photochemical and photophysical properties. Due to the size limitations associated with the cobalamins, the primary method for calculating ground state properties is density functional theory (DFT), with time-dependent DFT (TD-DFT) mainly used for electronically excited states. The energy pathways on the lowest singlet surfaces of the alkylcobalamins, connect metal-to-ligand charge transfer (MLCT) and ligand field (LF) minima associated with photo-homolysis of the Co-C bond observed experimentally. Additionally, energy pathways between minima and seams associated with crossing of S1/S0 surfaces are the most efficient for internal conversion (IC) to the ground state. Depending on the specific cobalamin, such IC may involve simultaneous elongation of both axial bonds (CNCbl), or detachment of axial base coupled with corrin ring distortion (MeCbl). The possible involvement of triplet RPs is also discussed, and a mechanism of intersystem crossing based on Landau-Zener theory is presented