Heterologous production of acrylic acid: current challenges and perspectives

Acrylic acid (AA) is a chemical with high market value used in industry to produce diapers, paints, adhesives and coatings, among others. AA available worldwide is chemically produced mostly from petroleum derivatives. Due to its economic relevance, there is presently a need for innovative and sustainable ways to synthesize AA. In the past decade, several semi-biological methods have been developed and consist in the bio-based synthesis of 3-hydroxypropionic acid (3-HP) and its chemical conversion to AA. However, more recently, engineered Escherichia coli was demonstrated to be able to convert glucose or glycerol to AA. Several pathways have been developed that use as precursors glycerol, malonyl-CoA or β-alanine. Some of these pathways produce 3-HP as an intermediate. Nevertheless, the heterologous production of AA is still in its early stages compared, for example, to 3-HP production. So far, only up to 237 mg/L of AA have been produced from glucose using β-alanine as a precursor in fed-batch fermentation. In this review, the advances in the production of AA by engineered microbes, as well as the hurdles hindering high-level production, are discussed. In addition, synthetic biology and metabolic engineering approaches to improving the production of AA in industrial settings are presented.This study was funded by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of the UIDB/04469/2020 unit.info:eu-repo/semantics/publishedVersio

Production of 3-hydroxypropionate from biomass

Biorenewable technology is a developing field of science that researches alternative sources for petroleum products like fuels, plastics, paints, etc. The DoE biomass program has identified the 12 top chemicals that can be produced from biomass and further processed to replace many petroleum products; 3-hydroxypropionate (3HP) is a top 12 chemical that can be used to produce plastics, paints, tires, and other consumer products. This thesis describes the attempts to produce 3HP from glucose in E. coli and from syngas in R. rubrum. Malonyl CoA reductase from C. aurantiacus and R. castenholzii was cloned and expressed in E. coli and R. rubrum respectively. This enzyme converts malonyl-CoA (a central metabolic intermediate) to 3-HP. Gas chromatography-mass spectrometry showed recombinant E. coli and R. rubrum produced 3HP in low levels. The quantity of 3-HP produced is not economical for commercial production so further studies were conducted to increase 3HP production

Enzymic synthesis and reduction of malonyl semialdehyde-coenzyme A

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/32442/1/0000524.pd

Metabolic Engineering of Yeast for the Production of 3-Hydroxypropionic Acid

The beta-hydroxy acid 3-hydroxypropionic acid (3-HP) is an attractive platform compound that can be used as a precursor for many commercially interesting compounds. In order to reduce the dependence on petroleum and follow sustainable development, 3-HP has been produced biologically from glucose or glycerol. It is reported that 3-HP synthesis pathways can be constructed in microbes such as Escherichia coli, Klebsiella pneumoniae and the yeast Saccharomyces cerevisiae. Among these host strains, yeast is prominent because of its strong acid tolerance which can simplify the fermentation process. Currently, the malonyl-CoA reductase pathway and the β-alanine pathway have been successfully constructed in yeast. This review presents the current developments in 3-HP production using yeast as an industrial host. By combining genome-scale engineering tools, malonyl-CoA biosensors and optimization of downstream fermentation, the production of 3-HP in yeast has the potential to reach or even exceed the yield of chemical production in the future

A kinetic model of the central carbon metabolism for acrylic acid production in Escherichia coli

Acrylic acid is an economically important chemical compound due to its high market value. Nevertheless, the majority of acrylic acid consumed worldwide its produced from petroleum derivatives by a purely chemical process, which is not only expensive, but it also contributes towards environment deterioration. Hence, justifying the current need for sustainable novel production methods that allow higher profit margins. Ideally, to minimise production cost, the pathway should consist in the direct bio-based production from microbial feedstocks, such as Escherichia coli, but the current yields achieved are still too low to compete with conventional method. In this work, even though the glycerol pathway presented higher yields, we identified the malonyl-CoA route, when using glucose as carbon source, as having the most potential for industrial-scale production, since it is cheaper to implement. Furthermore, we also identified potential optimisation targets for all the tested pathways, that can help the bio-based method to compete with the conventional process.This study was supported by the Portuguese Foundation for Science and Technology(FCT) under the scope of the strategic funding of UIDB/04469/2020 unit. This article is also a result of the project 22231/01/SAICT/2016: “Biodata.pt – Infraestrutura Portuguesa de Dados Biolo´gicos”, by Lisboa Portugal Regional Operational Programme (Lisboa2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). Alexandre Oliveira holds a doctoral fellowship (2020.10205.BD) provided by the FCT. Oscar Dias acknowledge FCT for the Assistant Research contract obtained under CEEC Individual 2018. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.info:eu-repo/semantics/publishedVersio

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

Studies on the microbial production of acrylic acid

Thesis (M.S.)--Massachusetts Institute of Technology, Dept. of Nutrition and Food Science, 1980.MICROFICHE COPY AVAILABLE IN ARCHIVES AND ENGINEERING.Bibliography: leaves 201-212.Rajen Kantilal Dalal.M.S

Functional compartmentalization and metabolic separation in a prokaryotic cell

The prokaryotic cell is traditionally seen as a “bag of enzymes,” yet its organization is much more complex than in this simplified view. By now, various microcompartments encapsulating metabolic enzymes or pathways are known for Bacteria. These microcompartments are usually small, encapsulating and concentrating only a few enzymes, thus protecting the cell from toxic intermediates or preventing unwanted side reactions. The hyperthermophilic, strictly anaerobic Crenarchaeon Ignicoccus hospitalis is an extraordinary organism possessing two membranes, an inner and an energized outer membrane. The outer membrane (termed here outer cytoplasmic membrane) harbors enzymes involved in proton gradient generation and ATP synthesis. These two membranes are separated by an intermembrane compartment, whose function is unknown. Major information processes like DNA replication, RNA synthesis, and protein biosynthesis are located inside the “cytoplasm” or central cytoplasmic compartment. Here, we show by immunogold labeling of ultrathin sections that enzymes involved in autotrophic CO2 assimilation are located in the intermembrane compartment that we name (now) a peripheric cytoplasmic compartment. This separation may protect DNA and RNA from reactive aldehydes arising in the I. hospitalis carbon metabolism. This compartmentalization of metabolic pathways and information processes is unprecedented in the prokaryotic world, representing a unique example of spatiofunctional compartmentalization in the second domain of life

Realization of a New-to-Nature Carboxylation Pathway

Most inorganic carbon enters the biosphere via the Calvin-Benson-Bassham (CBB) cycle by its key enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). An unproductive side reaction of RuBisCO with oxygen leads to the formation of 2-phosphoglycolate (2-PG), which is recycled via complex pathways into 3-phosphoglycerate (3-PGA), releasing carbon dioxide in the process. The tartronyl-CoA pathway represents a synthetic pathway that was designed to recycle 2-PG more efficiently, avoiding the release of carbon dioxide, and fixing carbon dioxide instead. It consists of four main reactions steps, which are not known to take part in any natural metabolic pathway. These steps are the activation of glycolate to glycolyl-CoA, the carboxylation of glycolyl-CoA to tartronyl-CoA as its key reaction, and the subsequent two reductions giving rise to glycerate. In this work, all required enzymes were identified or established by engineering and the tartronyl-CoA pathway was realized in vitro. Promiscuous enzyme candidates performing analogous reactions with similar substrates were screened and further improved to perform their desired functions. These include engineered glycolyl-CoA synthetase and glycolyl-CoA carboxylase (GCC), as well as a tartronyl-CoA reductase. For the engineering of GCC, rational design as well as high-throughput directed evolution was applied resulting in a new-to-nature carboxylase that matches the kinetic properties of natural carboxylases. Moreover, a 1.96 Å resolution cryogenic electron microscopy (cryo-EM) structure of GCC was obtained, highlighting and corroborating the effects of the introduced mutations. The concerted function of all tartronyl-CoA pathway enzymes was confirmed in the context of photorespiration in vitro. The in vitro reconstitution also included the optimization of reaction parameters as well as efficient cofactor recycling. Besides its function as photorespiratory bypass, the tartronyl-CoA pathway was shown to be functional as an additional carbon fixing module, able to connect a synthetic carbon dioxide fixation cycle to central carbon metabolism. Furthermore, the tartronyl-CoA pathway was successfully employed for the in vitro conversion of the plastic waste component ethylene glycol into the central carbon metabolite glycerate. In an initial attempt of an in vivo implementation of the tartronyl-CoA pathway for ethylene glycol assimilation, it was shown that GCC, the key enzyme of the tartronyl-CoA pathway, can be functionally produced in Pseudomonas putida