Kinetic characterization of the C-terminal domain of Malonyl-CoA reductase.

Malonyl-CoA reductase utilizes two equivalents of NADPH to catalyze the reduction of malonyl-CoA to 3-hydroxypropionic acid (3HP). This reaction is part of the carbon fixation pathway in the phototrophic bacterium Chloroflexus aurantiacus. The enzyme is composed of two domains. The C-terminal domain catalyzes the reduction of malonyl-CoA to malonic semialdehyde, while the N-terminal domain catalyzes the reduction of the aldehyde to 3HP. The two domains can be produced independently and retain their enzymatic activity. This report focuses on the kinetic characterization of the C-terminal domain. Initial velocity patterns and inhibition studies showed the kinetic mechanism is ordered with NADPH binding first followed by malonyl-CoA. Malonic semialdehyde is released first, while CoA and NADP+ are released randomly. Analogs of malonyl-CoA showed that the thioester carbon is reduced, while the carboxyl group is needed for proper positioning. The enzyme transfers the pro-S hydrogen of NADPH to malonyl-CoA and pH rate profiles revealed that a residue with a pKa value of about 8.8 must be protonated for activity. Kinetic isotope effects indicated that NADPH is not sticky (that is, NADPH dissociates from the enzyme faster than the rate of product formation) and product release is partially rate-limiting. Moreover, the mechanism is stepwise with the pH dependent step occurring before or after hydride transfer. The findings from this study will aid in the development of an eco-friendly biosynthesis of 3HP which is an industrial chemical used in the production of plastics and adhesives.

Kinetic characterization of the N-terminal domain of Malonyl-CoA reductase.

Climate change is driving a search for environmentally safe methods to produce chemicals used in ordinary life. One such molecule is 3-hydroxypropionic acid, which is a platform industrial chemical used as a precursor for a variety of other chemical end products. The biosynthesis of 3-hydroxypropionic acid can be achieved in recombinant microorganisms via malonyl-CoA reductase in two separate reactions. The reduction of malonyl-CoA by NADPH to form malonic semialdehyde is catalyzed in the C-terminal domain of malonyl-CoA reductase, while the subsequent reduction of malonic semialdehyde to 3-hydroxypropionic acid is accomplished in the N-terminal domain of the enzyme. A new assay for the reverse reaction of the N-terminal domain of malonyl-CoA reductase from Chloroflexus aurantiacus activity has been developed. This assay was used to determine the kinetic mechanism and for isotope effect studies. Kinetic characterization using initial velocity patterns revealed random binding of the substrates NADP+ and 3-hydroxypropionic acid. Isotope effects showed substrates react to give products faster than they dissociate and that the products of the reverse reaction, NADPH and malonic semialdehyde, have a low affinity for the enzyme. Multiple isotope effects suggest proton and hydride transfer occur in a concerted fashion. This detailed kinetic characterization of the reaction catalyzed by the N-terminal domain of malonyl-CoA reductase could aid in engineering of the enzyme to make the biosynthesis of 3-hydroxypropionic acid commercially competitive with its production from fossil fuels.

Enhancing 3-hydroxypropionic acid production in Saccharomyces cerevisiae through enzyme localization within mitochondria.

Microbial 3-hydroxypropionic acid (3-HP) production can potentially replace petroleum-based production methods for acrylic acid. Here, we constructed a yeast strain that expressed enzymes related to 3-HP biosynthesis within the mitochondria. This approach aimed to enhance the 3-HP production by utilizing the mitochondrial acetyl-CoA, an important intermediate for synthesizing 3-HP. The strain that expressed 3-HP-producing enzymes in the mitochondria (YPH-mtA3HP) showed improved production of 3-HP compared to that shown by the strain expressing 3-HP-producing enzymes in the cytosol (YPH-cyA3HP). Additionally, cMCR was overexpressed, which regulates a rate-limiting reaction in synthesizing 3-HP. In this study, we aimed to further enhance 3-HP production by expressing multiple copies of cMCR in the mitochondria using the δ-integration strategy to optimize the expression level of cMCR (YPH-mtA3HPx*). The results of flask-scale cultivation showed that 3-HP production by cMCR δ-integration was significantly higher, exhibiting a yield of 160 mg/L in YPH-mtA3HP6* strain and 257 mg/L in YPH-mtA3HP22* strain. Notably, YPH-mtA3HP22*, exhibited the highest 3-HP titer, which was 3.2-fold higher than that of YPH-cyA3HP. Our results demonstrated the potential of utilizing the mitochondrial compartment within S. cerevisiae for enhancing 3-HP production.

Structural basis of a bi-functional malonyl-CoA reductase (MCR) from the photosynthetic green non-sulfur bacterium Roseiflexus castenholzii.

Malonyl-CoA reductase (MCR) is a NADPH-dependent bi-functional enzyme that performs alcohol dehydrogenase and aldehyde dehydrogenase (CoA-acylating) activities in the N- and C-terminal fragments, respectively. It catalyzes the two-step reduction of malonyl-CoA to 3-hydroxypropionate (3-HP), a key reaction in the autotrophic CO2 fixation cycles of Chloroflexaceae green non-sulfur bacteria and the archaea Crenarchaeota. However, the structural basis underlying substrate selection, coordination, and the subsequent catalytic reactions of full-length MCR is largely unknown. For the first time, we here determined the structure of full-length MCR from the photosynthetic green non-sulfur bacterium Roseiflexus castenholzii (RfxMCR) at 3.35 Å resolution. Furthermore, we determined the crystal structures of the N- and C-terminal fragments bound with reaction intermediates NADP+ and malonate semialdehyde (MSA) at 2.0 Å and 2.3 Å, respectively, and elucidated the catalytic mechanisms using a combination of molecular dynamics simulations and enzymatic analyses. Full-length RfxMCR was a homodimer of two cross-interlocked subunits, each containing four tandemly arranged short-chain dehydrogenase/reductase (SDR) domains. Only the catalytic domains SDR1 and SDR3 incorporated additional secondary structures that changed with NADP+-MSA binding. The substrate, malonyl-CoA, was immobilized in the substrate-binding pocket of SDR3 through coordination with Arg1164 and Arg799 of SDR4 and the extra domain, respectively. Malonyl-CoA was successively reduced through protonation by the Tyr743-Arg746 pair in SDR3 and the catalytic triad (Thr165-Tyr178-Lys182) in SDR1 after nucleophilic attack from NADPH hydrides. IMPORTANCE The bi-functional MCR catalyzes NADPH-dependent reduction of malonyl-CoA to 3-HP, an important metabolic intermediate and platform chemical, from biomass. The individual MCR-N and MCR-C fragments, which contain the alcohol dehydrogenase and aldehyde dehydrogenase (CoA-acylating) activities, respectively, have previously been structurally investigated and reconstructed into a malonyl-CoA pathway for the biosynthetic production of 3-HP. However, no structural information for full-length MCR has been available to illustrate the catalytic mechanism of this enzyme, which greatly limits our capacity to increase the 3-HP yield of recombinant strains. Here, we report the cryo-electron microscopy structure of full-length MCR for the first time and elucidate the mechanisms underlying substrate selection, coordination, and catalysis in the bi-functional MCR. These findings provide a structural and mechanistic basis for enzyme engineering and biosynthetic applications of the 3-HP carbon fixation pathways.

Engineering Rhodosporidium toruloides for production of 3-hydroxypropionic acid from lignocellulosic hydrolysate.

Microbial production of valuable bioproducts is a promising route towards green and sustainable manufacturing. The oleaginous yeast, Rhodosporidium toruloides, has emerged as an attractive host for the production of biofuels and bioproducts from lignocellulosic hydrolysates. 3-hydroxypropionic acid (3HP) is an attractive platform molecule that can be used to produce a wide range of commodity chemicals. This study focuses on establishing and optimizing the production of 3HP in R. toruloides. As R. toruloides naturally has a high metabolic flux towards malonyl-CoA, we exploited this pathway to produce 3HP. Upon finding the yeast capable of catabolizing 3HP, we then implemented functional genomics and metabolomic analysis to identify the catabolic pathways. Deletion of a putative malonate semialdehyde dehydrogenase gene encoding an oxidative 3HP pathway was found to significantly reduce 3HP degradation. We further explored monocarboxylate transporters to promote 3HP transport and identified a novel 3HP transporter in Aspergillus pseudoterreus by RNA-seq and proteomics. Combining these engineering efforts with media optimization in a fed-batch fermentation resulted in 45.4 g/L 3HP production. This represents one of the highest 3HP titers reported in yeast from lignocellulosic feedstocks. This work establishes R. toruloides as a host for 3HP production from lignocellulosic hydrolysate at high titers, and paves the way for further strain and process optimization towards enabling industrial production of 3HP in the future.

Cryo-EM structure of bifunctional malonyl-CoA reductase from Chloroflexus aurantiacus reveals a dynamic domain movement for high enzymatic activity.

The platform chemical 3-hydroxypropionic acid is used to synthesize various valuable materials, including bioplastics. Bifunctional malonyl-CoA reductase is a key enzyme in 3-hydroxypropionic acid biosynthesis as it catalyzes the two-step reduction of malonyl-CoA to malonate semialdehyde to 3-hydroxypropionic acid. Here, we report the cryo-EM structure of a full-length malonyl-CoA reductase protein from Chloroflexus aurantiacus (CaMCRFull). The EM model of CaMCRFull reveals a tandem helix architecture comprising an N-terminal (CaMCRND) and a C-terminal (CaMCRCD) domain. The CaMCRFull model also revealed that the enzyme undergoes a dynamic domain movement between CaMCRND and CaMCRCD due to the presence of a flexible linker between these two domains. Increasing the flexibility and extension of the linker resulted in a twofold increase in enzyme activity, indicating that for CaMCR, domain movement is crucial for high enzyme activity. We also describe the structural features of CaMCRND and CaMCRCD. This study reveals the protein structures underlying the molecular mechanism of CaMCRFull and thereby provides valuable information for future enzyme engineering to improve the productivity of 3-hydroxypropionic acid.

Engineering yeast mitochondrial metabolism for 3-hydroxypropionate production.

BACKGROUND: With unique physiochemical environments in subcellular organelles, there has been growing interest in harnessing yeast organelles for bioproduct synthesis. Among these organelles, the yeast mitochondrion has been found to be an attractive compartment for production of terpenoids and branched-chain alcohols, which could be credited to the abundant supply of acetyl-CoA, ATP and cofactors. In this study we explored the mitochondrial potential for production of 3-hydroxypropionate (3-HP) and performed the cofactor engineering and flux control at the acetyl-CoA node to maximize 3-HP synthesis. RESULTS: Metabolic modeling suggested that the mitochondrion serves as a more suitable compartment for 3-HP synthesis via the malonyl-CoA pathway than the cytosol, due to the opportunity to obtain a higher maximum yield and a lower oxygen consumption. With the malonyl-CoA reductase (MCR) targeted into the mitochondria, the 3-HP production increased to 0.27 g/L compared with 0.09 g/L with MCR expressed in the cytosol. With enhanced expression of dissected MCR enzymes, the titer reached to 4.42 g/L, comparable to the highest titer achieved in the cytosol so far. Then, the mitochondrial NADPH supply was optimized by overexpressing POS5 and IDP1, which resulted in an increase in the 3-HP titer to 5.11 g/L. Furthermore, with induced expression of an ACC1 mutant in the mitochondria, the final 3-HP production reached 6.16 g/L in shake flask fermentations. The constructed strain was then evaluated in fed-batch fermentations, and produced 71.09 g/L 3-HP with a productivity of 0.71 g/L/h and a yield on glucose of 0.23 g/g. CONCLUSIONS: In this study, the yeast mitochondrion is reported as an attractive compartment for 3-HP production. The final 3-HP titer of 71.09 g/L with a productivity of 0.71 g/L/h was achieved in fed-batch fermentations, representing the highest titer reported for Saccharomyces cerevisiae so far, that demonstrated the potential of recruiting the yeast mitochondria for further development of cell factories.

Overproduction of 3-hydroxypropionate in a super yeast chassis.

3-Hydroxypropionate (3-HP) is a platform chemical for production of acrylic acid, acrylamide and biodegradable polymers. Several microbial cell factories have been constructed for production of 3-HP from malonyl-CoA by using a malonyl-CoA reductase, which however suffer from inadequate supply of precursor and cofactor. Here 3-HP biosynthesis was optimized in a super yeast chassis with sufficient supply of precursor malonyl-CoA and cofactor NADPH, which had a 3-fold higher 3-HP (1.4 g/L) than that of wild-type background. The instability of the engineered strain was observed in fed-batch fermentation due to the plasmid loss, which may be caused by the toxic intermediate malonate semialdehyde. Genome integration of MCR-C encoding C-terminal of MCR enabled stable gene expression and much higher 3-HP production of 4.4 g/L under batch fermentation and 56.5 g/L under fed-batch fermentation with a yield of 0.31 g/g glucose. This was the highest 3-HP production reported from glucose in engineered microbes.

Production of 3-hydroxypropionic acid from acetate using metabolically-engineered and glucose-grown Escherichia coli.

Acetate can be used as carbon feedstock for the production of 3-hydroxypropionic acid (3-HP), but the production level was low due to inefficient cell growth on acetate. To better utilize acetate, a two-stage strategy, whereby glucose is used for cell growth and acetate for 3-HP formation, was attempted. Dissected malonyl-CoA reductase of Chloroflexus aurantiacus, alone or along with acetyl-CoA carboxylase and/or transhydrogenase, was overexpressed, and by-products formation pathway, glyoxylate shunt (GS) and gluconeogenesis were modified. When GS or gluconeogenesis was disrupted, cell growth on glucose was not hampered, while on acetate it was completely abolished. Consequently, 3-HP production, at production stage, were low, though 3-HP yield on acetate was increased, especially in the case of aceA deletion. In two-stage bioreactor, strain with upregulated GS produced 7.3 g/L 3-HP with yield of 0.26 mol/mol acetate. This study suggests that two-stage cultivation is a good strategy for 3-HP production from acetate.

Metabolic engineering of type II methanotroph, Methylosinus trichosporium OB3b, for production of 3-hydroxypropionic acid from methane via a malonyl-CoA reductase-dependent pathway.

We engineered a type II methanotroph, Methylosinus trichosporium OB3b, for 3-hydroxypropionic acid (3HP) production by reconstructing malonyl-CoA pathway through heterologous expression of Chloroflexus aurantiacus malonyl-CoA reductase (MCR), a bifunctional enzyme. Two strategies were designed and implemented to increase the malonyl-CoA pool and thus, increase in 3HP production. First, we engineered the supply of malonyl-CoA precursors by overexpressing endogenous acetyl-CoA carboxylase (ACC), substantially enhancing the production of 3HP. Overexpression of biotin protein ligase (BPL) and malic enzyme (NADP+-ME) led to a ∼22.7% and ∼34.5% increase, respectively, in 3HP titer in ACC-overexpressing cells. Also, the acetyl-CoA carboxylation bypass route was reconstructed to improve 3HP productivity. Co-expression of methylmalonyl-CoA carboxyltransferase (MMC) of Propionibacterium freudenreichii and phosphoenolpyruvate carboxylase (PEPC), which provides the MMC precursor, further improved the 3HP titer. The highest 3HP production of 49 mg/L in the OB3b-MCRMP strain overexpressing MCR, MMC and PEPC resulted in a 2.4-fold improvement of titer compared with that in the only MCR-overexpressing strain. Finally, we could obtain 60.59 mg/L of 3HP in 42 h using the OB3b-MCRMP strain through bioreactor operation, with a 6.36-fold increase of volumetric productivity compared than that in the flask cultures. This work demonstrates metabolic engineering of type II methanotrophs, opening the door for using type II methanotrophs as cell factories for biochemical production along with mitigation of greenhouse gases.

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 ß-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.

Production of 3-hydroxypropionic acid via the malonyl-CoA pathway using recombinant fission yeast strains.

3-Hydroxypropionic acid (3-HP) can be converted into derivatives such as acrylic acid, a source for producing super absorbent polymers. Although Escherichia coli has often been used for 3-HP production, it exhibits low tolerance to 3-HP. To circumvent this problem, we selected the fission yeast Schizosaccharomyces pombe as this microorganism has higher tolerance to 3-HP than E. coli. Therefore, we constructed S. pombe transformants overexpressing two genes, one encoding the S. pombe acetyl-CoA carboxylase (Cut6p) and the other encoding the malonyl-CoA reductase derived from Chloroflexus aurantiacus (CaMCR). To prevent the degradation of these expressed proteins, we employed an S. pombe protease-deficient strain. Moreover, to increase the cytosolic concentration of acetyl-CoA, we supplemented acetate to the medium, which improved 3-HP production. To further produce 3-HP by overexpressing Cut6p and CaMCR, we exploited the highly expressing S. pombe hsp9 promoter. Finally, culturing in high-density reached 3-HP production to 7.6 g/L at 31 h.

Malonyl-CoA pathway: a promising route for 3-hydroxypropionate biosynthesis.

3-Hydroxypropionate (3HP) is an attractive platform chemical, serving as a precursor to a variety of commodity chemicals like acrylate and acrylamide, as well as a monomer of a biodegradable plastic. To establish a sustainable way to produce these commercially important chemicals and materials, fermentative production of 3HP is widely investigated in recent years. It is reported that 3HP can be produced from several intermediates, such as glycerol, malonyl-CoA, and ß-alanine. Among all these biosynthetic routes, the malonyl-CoA pathway has some distinct advantages, including a broad feedstock spectrum, thermodynamic feasibility, and redox neutrality. To date, this pathway has been successfully constructed in various species including Escherichia coli, yeast and cyanobacteria, and optimized through carbon flux redirection, enzyme screening and engineering, and an increasing supply of energy and cofactors, resulting in significantly enhanced 3HP titer up to 40 g/L. These results show the feasibility of commercial manufacturing of 3HP and its derivatives in the future.

Functional balance between enzymes in malonyl-CoA pathway for 3-hydroxypropionate biosynthesis.

3-Hydroxypropionate (3HP) is an important platform chemical, and four 3HP biosynthetic routes were reported, in which the malonyl-CoA pathway has some expected advantages but presented the lowest 3HP yield. Here, we demonstrated that this low yield was caused by a serious functional imbalance between MCR-C and MCR-N proteins, responsible for the two-step reduction of malonyl-CoA to 3HP. Then we minimized the enzyme activity imbalance by directed evolution of rate-limiting enzyme MCR-C and fine tuning of MCR-N expression level. Combined with culture conditions optimization, our engineering approaches increased the 3HP titer 270-fold, from 0.15 g/L to 40.6 g/L, representing the highest 3HP production via malonyl-CoA pathway so far. This study not only significantly improved the 3HP productivity of recombinant Escherichia coli strain, but also proved the importance of metabolic balance in a multistep biosynthetic pathway, which should be always considered in any metabolic engineering study.

Enhanced production of 3-hydroxypropionic acid from glucose via malonyl-CoA pathway by engineered Escherichia coli.

In this study, production of 3-HP via malonyl-CoA was investigated by using metabolically engineered Escherichia coli carrying heterogeneous acetyl-CoA carboxylase (Acc) from Corynebacterium glutamicum and codon-optimized malonyl-CoA reductase (MCR) from Chloroflexus aurantiacus. Three engineered E. coli strains with different host-vector systems were constructed and investigated. The results indicated that the combination of E. coli BL21(DE3) and pET28a was the most efficient host-vector system for 3-HP production, and the highest concentration of 3-HP attained in shake flask cultivation reached 1.80g/L by the strain BE-MDA with induction at 0.25mM IPTG and 25°C, and supplementation of NaHCO3 and biotin. In fed-batch fermentation performed in a 5-L reactor, the concentration of 3-HP achieved 10.08g/L in 36h.

Production of 3-hydroxypropionic acid from glucose and xylose by metabolically engineered Saccharomyces cerevisiae.

Biomass, the most abundant carbon source on the planet, may in the future become the primary feedstock for production of fuels and chemicals, replacing fossil feedstocks. This will, however, require development of cell factories that can convert both C6 and C5 sugars present in lignocellulosic biomass into the products of interest. We engineered Saccharomyces cerevisiae for production of 3-hydroxypropionic acid (3HP), a potential building block for acrylates, from glucose and xylose. We introduced the 3HP biosynthetic pathways via malonyl-CoA or ß-alanine intermediates into a xylose-consuming yeast. Using controlled fed-batch cultivation, we obtained 7.37±0.17 g 3HP L-1 in 120 hours with an overall yield of 29±1% Cmol 3HP Cmol-1 xylose. This study is the first demonstration of the potential of using S. cerevisiae for production of 3HP from the biomass sugar xylose.