Microbial production of 3-hydroxypropionic acid and poly(3-hydroxypropionate): Investigation of Lactobacillus reuteri propanediol utilization pathway enzymes

Concerns regarding environmental issues such as greenhouse gas emissions and climate change have led to a shift within the research community and chemical and energy industry sectors for finding sustainable routes for producing fuels and chemicals from renewable resources, thereby minimizing our dependence on petroleum. The C3-chemical 3-hydroxypropionic acid has been identifed as a top candidate for the biobased chemical industry. This platform chemical is a β-hydroxy acid containing two functional groups (hydroxyl and carboxyl) enabling its conversion into value-added chemicals such as 1,3-propanediol, acrolein, malonic acid, acrylamide and acrylic acid, which can be used in resins, coatings, paints, adhesives, lubricants, and in the textile industry as anti-static agent. Polymerized 3- HP, poly(3-hydroxypropionate) (poly(3-HP)), is a biodegradable and stable polymer which, besides its potential role as a biomaterial, can be degraded to 3-HP monomer. In recent years, a dramatic increase in the interest for microbial production of 3-HP and poly(3-HP) has been observed. Metabolic engineering and recombinant expression of various enzymatic pathways in a number of bacterial strains have been suggested and implemented, with mainly renewable glucose and glycerol as substrates. This thesis presents a novel pathway called the propanediol utilization pathway present in Lactobacillus reuteri that catalyzes dehydration of glycerol to 3- hydroxypropionaldehyde (3-HPA) and further to 3-HP by a series of reactions catalyzed by propionaldehyde dehydrogenase (PduP), phosphotransacylase (PduL) and propionate kinase (PduW). Through structural modeling and kinetic characterization of PduP, its 3-HPA consuming ability was confirmed and catalytic mechanism proposed. PduP, PduL and PduW-mediated conversion of 3-HPA to 3- HP was confirmed through their recombinant expression in Escherichia coli. 3-HPA produced from glycerol by L. reuteri was used as a substrate for conversion to 3-HP by the recombinant E. coli. A yield of 1 mol/mol was reached with a titer of 12 mM 3-HP. Depletion of the cofactor NAD+ required for the catalysis of 3-HP to 3-HPCoA, was deemed responsible for the low titer. Regeneration of NAD+, used up in PduP catalyzed reaction, was achieved by recombinant expression of NADH oxidase (Nox) from L. reuteri in E. coli expressing PduP, PduL and PduW. The final 3-HP titer by this recombinant strain was at least twice that of E. coli carrying solely PduP, PduL and PduW. For the production of poly(3-HP), PduL and PduW in the recombinant strain were replaced by polyhydroxyalkanoate synthase of Chromobacterium sp. that converts 3- HP-CoA to poly(3-HP). A poly(3-HP) content of up to 40% (w/w) cell dry weight was reached in an efficient and cheap process requring no additivies or expensive cofactors

Insight into Coenzyme A cofactor binding and the mechanism of acyl-transfer in an acylating aldehyde dehydrogenase from Clostridium phytofermentans

The breakdown of fucose and rhamnose released from plant cell walls by the cellulolytic soil bacterium Clostridium phytofermentans produces toxic aldehyde intermediates. To enable growth on these carbon sources, the pathway for the breakdown of fucose and rhamnose is encapsulated within a bacterial microcompartment (BMC). These proteinaceous organelles sequester the toxic aldehyde intermediates and allow the efficient action of acylating aldehyde dehydrogenase enzymes to produce an acyl-CoA that is ultimately used in substrate-level phosphorylation to produce ATP. Here we analyse the kinetics of the aldehyde dehydrogenase enzyme from the fucose/rhamnose utilisation BMC with different short-chain fatty aldehydes and show that it has activity against substrates with up to six carbon atoms, with optimal activity against propionaldehyde. We have also determined the X-ray crystal structure of this enzyme in complex with CoA and show that the adenine nucleotide of this cofactor is bound in a distinct pocket to the same group in NAD(+). This work is the first report of the structure of CoA bound to an aldehyde dehydrogenase enzyme and our crystallographic model provides important insight into the differences within the active site that distinguish the acylating from non-acylating aldehyde dehydrogenase enzymes

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

Structural and synthetic biology study of bacterial microcompartments

Bacterial microcompartments (BMCs) are proteinaceous metabolic compartments found in a wide range of bacteria, whose function it is to encapsulate pathways for the breakdown of various carbon sources, whilst retaining toxic and volatile intermediates formed from substrate breakdown. Examples of these metabolic processes are the 1,2- propanediol-breakdown pathway in Salmonella enterica (Pdu microcompartment), as well as the ethanolamine breakdown pathway in Clostridium difficile (Eut microcompartment). Some of the major challenges to exploiting BMCs as a tool in biotechnology are understanding how enzymes are targeted to microcompartments, as well as being able to engineer the protein shell of BMCs to make synthetic microcompartments that allow specific enzyme pathways to be targeted to their interior. Finally, the metabolic burden imposed by the production of large protein complexes requires a detailed knowledge of how the expression of these systems are controlled. This project explores the structure and biochemistry of an essential BMC pathway enzyme, the acylating propionaldehyde dehydrogenase. With crystal structures of the enzyme with the cofactors in the cofactor binding site and biochemical data presented to confirm the enzyme’s substrate. The project also focuses on the creation of synthetic biology tools to enable BMC engineering with a modular library of BMC shell protein parts; forward engineered ribosome binding sites (RBS) fused to BMC aldehyde dehydrogenase localisation sequences. The parts for this library were taken from the BMC loci found in Clostridium phytofermentans and Salmonella enterica. Using a synthetic biology toolkit will allow the rapid prototyping of BMC constructs for use in metabolic engineering. The shell protein parts were used to generate a number of transcriptional units, to assess the effect of overexpression of individual BMC shell components on the morphology of BMCs and the effect these had on their host chassis. Different strength forward engineered RBS and localisation constructs have been designed to assess the possibility of controlling the levels of heterologous proteins targeted to the interior of microcompartment shell to allow metabolic engineering of encapsulated pathways. Along with looking at overexpression of a single shell protein, to assess viability of BMCs as scaffold-like structures, recombinant BMCs can be explored for their utility in bioengineering and their potential role in generating biofuels

Utjecaj pojačane ekspresije gena biosintetskog puta za 3-hidroksipropionsku kiselinu na njezin prinos u bakteriji Lactobacillus reuteri

3-Hydroxypropionic acid (3-HP) is a novel antimicrobial agent against foodborne pathogens like Salmonella and Staphylococcus species. Lactobacillus reuteri converts glycerol into 3-HP using a coenzyme A-dependent pathway, which is encoded by propanediol utilization operon (pdu) subjected to catabolite repression. In a catabolite repression-deregulated L. reuteri RPRB3007, quantitative PCR revealed a 2.5-fold increase in the transcripts of the genes pduP, pduW and pduL during the mid-log phase of growth. The production of 3-HP was tested in resting cells in phosphate buff er and growing batch cultures in MRS broth of various glucose/glycerol ratios. Due to the upregulation of pathway genes, specific formation rate of 3-HP in the mutant strain was found to be enhanced from 0.167 to 0.257 g per g of cell dry mass per h. Furthermore, formation of 3-HP in resting cells was limited due to the substrate inhibition by reuterin at a concentration of (30±5) mM. In batch cultures, the formation of 3-HP was not observed during the logarithmic and stationary phases of growth of wild-type and mutant strains, which was confi rmed by NMR spectroscopy. However, the cells collected in these phases were found to produce 3-HP aft er washing and converting them to resting cells. Lactate and acetate, the primary end products of glucose catabolism, might be the inhibiting elements for 3-HP formation in batch cultures. This was confirmed when lactate (25±5 mM) or acetate (20±5 mM) were added to biotransformation medium, which prevented the 3-HP formation. Moreover, the removal of sodium acetate and glucose (carbon source for lactic acid production) was found to restore 3-HP formation in the MRS broth in a similar manner to that of the phosphate buff er. Even though the genetic repression was circumvented by the up-regulation of pathway genes using a mutant strain, 3-HP formation was further limited by the substrate and catabolite inhibition.3-Hidroksipropionska kiselina je novi antimikrobni agens koji se može upotrijebiti za suzbijanje patogenih bakterija u hrani, kao što su vrste iz rodova Salmonella i Staphylococcus. Bakterija Lactobacillus reuteri iz glicerola sintetizira 3-hidroksipropionsku kiselinu biosintetskim putem ovisnim o koenzimu A, kodiranim operonom za korištenje propandiola koji je reguliran kataboličkom represijom. U mutantu L. reuteri RPRB3007 u kojem nema kataboličke represije, ispitanom pomoću metode PCR, primijećeno je 2,5 puta više transkripata gena pduP, pduW i pduL, i to tijekom logaritamske faze rasta bakterije. Proizvodnja 3-hidroksipropionske kiseline određena je u stanicama koje se ne dijele, a bile su resuspendirane u fosfatnom puferu, te u šaržnim kulturama uzgojenim u podlozi MRS s različitim omjerima glukoze i glicerola. Utvrđeno je da se u mutantu zbog pojačane ekspresije gena biosintetskog puta povećala specifična brzina nastajanja 3-hidroksipropionske kiseline, i to s 0,167 na 0,257 g po gramu suhe biomase po satu. Osim toga, sinteza je 3-hidroksipropionske kiseline u stanicama koje se ne dijele bila usporena nakon dodatka reuterina u koncentraciji od (30±5) mM. U šaržnom uzgoju nije utvrđena prisutnost 3-hidroksipropionske kiseline tijekom logaritamske i stacionarne faze rasta divljeg soja i mutanta, što je potvrđeno i NMR spektroskopijom. Međutim, nakon ispiranja i povratka u stanje mirovanja ove su stanice ponovno proizvodile 3-hidroksipropionsku kiselinu. Zaključeno je da laktat i acetat, primarni produkti katabolizma glukoze, vjerojatno inhibiraju sintezu 3-hidroksipropionske kiseline u šaržnim kulturama, što je potvrđeno činjenicom da dodatak laktata u koncentraciji od (25±5) mM ili acetata u koncentraciji od (20±5) mM podlozi sprečava sintezu 3-hidroksipropionske kiseline. Osim toga, uklanjanjem je natrijevog acetata i glukoze (izvora ugljika za proizvodnju mliječne kiseline) potaknuta proizvodnja 3-hiroksipropionske kiseline u hranjivoj podlozi MRS na sličan način kao i uporabom fosfatnog pufera. Iako je genetička represija u mutantu izbjegnuta pojačanom ekspresijom gena biosintetskog puta, proizvodnja je 3-hidroksipropionske kiseline i dalje bila ograničena supstratom i kataboličkom inhibicijom

The Structural Basis of Coenzyme A Recycling in a Bacterial Organelle.

Bacterial Microcompartments (BMCs) are proteinaceous organelles that encapsulate critical segments of autotrophic and heterotrophic metabolic pathways; they are functionally diverse and are found across 23 different phyla. The majority of catabolic BMCs (metabolosomes) compartmentalize a common core of enzymes to metabolize compounds via a toxic and/or volatile aldehyde intermediate. The core enzyme phosphotransacylase (PTAC) recycles Coenzyme A and generates an acyl phosphate that can serve as an energy source. The PTAC predominantly associated with metabolosomes (PduL) has no sequence homology to the PTAC ubiquitous among fermentative bacteria (Pta). Here, we report two high-resolution PduL crystal structures with bound substrates. The PduL fold is unrelated to that of Pta; it contains a dimetal active site involved in a catalytic mechanism distinct from that of the housekeeping PTAC. Accordingly, PduL and Pta exemplify functional, but not structural, convergent evolution. The PduL structure, in the context of the catalytic core, completes our understanding of the structural basis of cofactor recycling in the metabolosome lumen

Functional analysis of the propanediol utilization microcompartment shell proteins PduB and PduB\u27 in Salmonella enterica

Bacterial microcompartments (MCPs) are proteinaceous sub-cellular organelles that are widely distributed among bacteria and that function in a variety of processes ranging from global carbon fixation to enteric pathogenesis. MCPs consist of metabolic enzymes encapsulated with a protein shell. The role of the MCP is to harbor a specific metabolic pathway that produces a toxic or volatile intermediate and confine that intermediate to minimize cellular toxicity and carbon loss. To date, the protein shells of MCPs have been shown to play a functional role in transport of small metabolites through selective pores and in the encapsulation of lumen enzymes through short N-or C-terminal peptide extensions. Interestingly, homologs of the propanediol utilization (Pdu) MCP shell protein PduB’ have been crystallized in two forms, one that is closed and another that forms a large channel. This suggested that these proteins undergo conformational changes that allow the transport of larger enzymatic cofactor that the MCP needs to properly function. However, no mutational work has been done to examine residues that are responsible for such a large conformational change and assess its physiological significance. Charged residues (R78, K81) and Ramachandran outlier (D79), which are located at the center of the PduB’, are key structural components that when substituted with alanine cause MCP instability. Interestingly, substitutions of a channel residue A53 appears to cause central pore opening. In addition, results indicate that there is a functional difference between PduB and PduB’ despite the fact that they are identical in sequence except for a 37 amino acid N-terminal extension on PduB. The smaller protein, PduB’, is dispensable for MCP formation but the PduB protein which contains a 37 amino acid N-terminal extension is integral to MCP assembly and formation

PduL is an evolutionary distinct phosphotransacylase involved in B12-dependent 1,2-propanediol degradation by Salmonella enterica serovar Typhimurium LT2 and is associated with the propanediol utilization microcompartments

Salmonella enterica degrades 1,2-propanediol (1,2-PD) in a coenzyme B12-dependent manner. Prior enzymatic assays of crude cell extracts indicated that a phosphotransacylase (PTAC) was needed for this process, but the enzyme involved was not identified. Here we show that the pduL gene encodes an evolutionarily distinct PTAC used for 1,2-PD degradation. Growth tests showed that pduL mutants were unable to ferment 1,2-PD and were also impaired for aerobic growth on this compound. Enzyme assays showed that cell extracts from a pduL mutant lacked measurable PTAC activity in a background that also carried a pta mutation (the pta gene was previously shown to encode a PTAC enzyme). Ectopic expression of pduL corrected the growth defects of pta mutant. PduL fused to 8 C-terminal histidine residues (PduL-his8) was purified and its kinetic constants determined: Vmax = 51.7y7.6 ymol min-1 mg-1; and Km for propionyl-PO42- and acetyl-PO42- = 0.61 and 0.97 mM, respectively. Sequence analyses showed that PduL is unrelated in amino acid sequence to known PTAC enzymes and that PduL homologues are distributed among at least 49 bacterial species, but are absent from the Archaea and Eukarya. Immunoblots showed that PduL was a component of propanediol utilization microcompartment

Genetic and nutrient modulation of acetyl-CoA levels in Synechocystis for <i>n</i>-butanol production

Background: There is a strong interest in using photosynthetic cyanobacteria as production hosts for biofuels and chemicals. Recent work has shown the benefit of pathway engineering, enzyme tolerance, and co-factor usage for improving yields of fermentation products. Results: An n-butanol pathway was inserted into a Synechocystis mutant deficient in polyhydroxybutyrate synthesis. We found that nitrogen starvation increased specific butanol productivity up to threefold, but cessation of cell growth limited total n-butanol titers. Metabolite profiling showed that acetyl-CoA increased twofold during nitrogen starvation. Introduction of a phosphoketolase increased acetyl-CoA levels sixfold at nitrogen replete conditions and increased butanol titers from 22 to 37 mg/L at day 8. Flux balance analysis of photoautotrophic metabolism showed that a Calvin-Benson-Bassham-Phosphoketolase pathway had higher theoretical butanol productivity than CBB-Embden-Meyerhof-Parnas and a reduced butanol ATP demand. Conclusion: These results demonstrate that phosphoketolase overexpression and modulation of nitrogen levels are two attractive routes toward increased production of acetyl-CoA derived products in cyanobacteria and could be implemented with complementary metabolic engineering strategies