Abstract:Microbial production of fuels, chemicals and materials has the potential to reduce greenhouse gas emissions and contribute to a sustainable bioeconomy. While synthetic biology allows readjusting of native metabolic pathways for the synthesis of desired products, often these native pathways do not support maximum efficiency and are affected by complex regulatory mechanisms. A synthetic or engineered pathway that allows modular synthesis of versatile bioproducts with minimal enzyme requirement and regulation whi… Show more
“…Cyclic and iterative pathways offer the unique advantage of providing access to hundreds of molecules with different chemistries and chain lengths from one core pathway. Reverse β-oxidation (r-BOX) is one such cyclic pathway that is highly modular and has been demonstrated as a promising route to many sustainable molecules with different chemistries and carbon chain length 5 , 6 . For example, r-BOX has been successfully used in Escherichia coli to produce high titers of C4-C10 saturated 7 and α,β-unsaturated carboxylic acids 8 (Fig.…”
Carbon-negative synthesis of biochemical products has the potential to mitigate global CO2 emissions. An attractive route to do this is the reverse β-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.
“…Cyclic and iterative pathways offer the unique advantage of providing access to hundreds of molecules with different chemistries and chain lengths from one core pathway. Reverse β-oxidation (r-BOX) is one such cyclic pathway that is highly modular and has been demonstrated as a promising route to many sustainable molecules with different chemistries and carbon chain length 5 , 6 . For example, r-BOX has been successfully used in Escherichia coli to produce high titers of C4-C10 saturated 7 and α,β-unsaturated carboxylic acids 8 (Fig.…”
Carbon-negative synthesis of biochemical products has the potential to mitigate global CO2 emissions. An attractive route to do this is the reverse β-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.
“…In particular, the choice of a tri-functional enzyme complex from β-oxidation in place of separate thiolase, hydroxybutyryl-CoA dehydrogenase (Hbd), and short-chain enoyl-CoA hydratase (Crt) enzymes from Clostridium solventogenic or PHA biosynthetic pathways enables the synthesis of longer chain-length products. In R-βox studies to date, products from assembled pathways using enzymes from these other sources have been limited to a maximum C 6 and C 10 chain length, respectively, although it is unclear if this is due to the limit of these enzymes or if those pathways were limited by the paired thiolase. The choice to use an enzyme complex from β-oxidation, however, also comes with the consequence that the vf FadBA complex evolved in the context of oxidative flux instead of the reductive.…”
Section: Discussionmentioning
confidence: 99%
“…These advantages make R-βox attractive for oleochemical production in microorganisms. Significant progress has been made in demonstrating the potential to expand product classes and incorporate additional functionalization of the carbon chain through α- and ω-substitutions . The main remaining challenge that must now be overcome in the development of the R-βox pathway is that of achieving highly specific product chain lengths at high titers.…”
The β-oxidation pathway, normally involved in the catabolism of fatty acids, can be functionally made to act as a fermentative, iterative, elongation pathway when driven by the activity of a trans-enoyl-CoA reductase (TER). The terminal acyl-CoA reduction to alcohol can occur on substrates with varied chain lengths, leading to a broad distribution of fermentation products in vivo. Tight control of the average chain length and product profile is desirable, as the chain length greatly influences the molecular properties and the commercial value. Lacking a termination enzyme with a narrow chain length preference, we sought alternative factors that could influence the product profile and pathway flux in the iterative pathway. In this study, we reconstituted the reversed β-oxidation (R-βox) pathway in vitro with a purified tri-functional complex (FadBA) responsible for the thiolase, enoyl-CoA hydratase, and hydroxyacyl-CoA dehydrogenase activities, a TER, and an acyl-CoA reductase. Using this system, we determined the rate-limiting step of the elongation cycle and demonstrated that by controlling the ratio of these three enzymes and the ratio of NADH and NADPH, we can influence the average chain length of the alcohol product profile.
“…Finally, the double bond at the α-carbon of the enoyl-CoA is reduced at the expense of one NADH molecule by a trans-enoyl-CoA reductase, generating an acyl-CoA molecule. This acyl-CoA molecule is available for a new elongation cycle or can be used by a termination enzyme or a different pathway to produce a wide variety of products like alcohols, acids, polyketides, or volatile esters among others [ 56 ]. Fig.…”
Background
Medium-chain fatty acids are molecules with applications in different industries and with growing demand. However, the current methods for their extraction are not environmentally sustainable. The reverse β-oxidation pathway is an energy-efficient pathway that produces medium-chain fatty acids in microorganisms, and its use in Saccharomyces cerevisiae, a broadly used industrial microorganism, is desired. However, the application of this pathway in this organism has so far either led to low titers or to the predominant production of short-chain fatty acids.
Results
We genetically engineered Saccharomyces cerevisiae to produce the medium-chain fatty acids hexanoic and octanoic acid using novel variants of the reverse β-oxidation pathway. We first knocked out glycerolphosphate dehydrogenase GPD2 in an alcohol dehydrogenases knock-out strain (△adh1-5) to increase the NADH availability for the pathway, which significantly increased the production of butyric acid (78 mg/L) and hexanoic acid (2 mg/L) when the pathway was expressed from a plasmid with BktB as thiolase. Then, we tested different enzymes for the subsequent pathway reactions: the 3-hydroxyacyl-CoA dehydrogenase PaaH1 increased hexanoic acid production to 33 mg/L, and the expression of enoyl-CoA hydratases Crt2 or Ech was critical to producing octanoic acid, reaching titers of 40 mg/L in both cases. In all cases, Ter from Treponema denticola was the preferred trans-enoyl-CoA reductase. The titers of hexanoic acid and octanoic acid were further increased to almost 75 mg/L and 60 mg/L, respectively, when the pathway expression cassette was integrated into the genome and the fermentation was performed in a highly buffered YPD medium. We also co-expressed a butyryl-CoA pathway variant to increase the butyryl-CoA pool and support the chain extension. However, this mainly increased the titers of butyric acid and only slightly increased that of hexanoic acid. Finally, we also tested the deletion of two potential medium-chain acyl-CoA depleting reactions catalyzed by the thioesterase Tes1 and the medium-chain fatty acyl CoA synthase Faa2. However, their deletion did not affect the production titers.
Conclusions
By engineering the NADH metabolism and testing different reverse β-oxidation pathway variants, we extended the product spectrum and obtained the highest titers of octanoic acid and hexanoic acid reported in S. cerevisiae. Product toxicity and enzyme specificity must be addressed for the industrial application of the pathway in this organism.
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