To bypass the thiolase in the pathway, we chose a new source for 3-ketovaleryl-CoA, namely betaoxidation of valeric acid. Like fatty acid oxidation, the new pathway first activates valeric acid, a biomass-derived chemical, into valeryl-CoA by a CoA ligase. An acyl-CoA dehydrogenase then oxidizes valeryl-CoA into 2-pentenyl-CoA, which is then transformed by an enoyl-CoA hydratase and a 3- hydroxyacyl-CoA dehydrogenase into 3-ketovaleryl-CoA . We believed this pathway would be more efficient than the PhaA-dependent pathway for bypassing the highly reversible and promiscuous thiolase-catalyzed reaction. We then tested the proposed beta-oxidation LMVA pathway, which accepts valeric acid into C6-isoprenol. Two plasmids were constructed to express the pathway : 1) pJL01, modified from pJL15, containing genes encoding NudB, Micrococcus luteus acyl-CoA dehydrogenase, and E. coli FadB, a bifunctional enzyme that catalyzes the hydration and dehydrogenation reactions ; 2) pJL02, modified from pJL10, containing the genes that encode the enzymes in the LMVA pathway and a Cannabis sativa acyl activating enzyme, CsAAE1, for valeryl-CoA production . E. coli BL21 transformed with pJL01 and pJL02 was grown in the presence of valeric acid and induced to express the pathway genes. GC-FID and GC-MS confirmed the production of C6-isoprenol at 27.3 mg/L . In the production broth, we also detected isoprenol at 5.8 mg/L . Compared to the thiolase LMVA pathway, C6- isoprenol production increased fourfold, and the ratio of C6-isoprenol with isoprenol increased to 4.7 from 1.3. Next, we screened CoA ligase and acyl-CoA dehydrogenase homologs, catalyzing the beta-oxidation pathway’s first two steps. We chose CoA ligase and acyl-CoA dehydrogenase candidates that have been overexpressed in E. coli and assayed in vitro in previous studies. Moreover, most of these candidates have known kinetic data for medium chain fatty acid substrates .
For the CoA ligase,plant bench indoor we selected the phenylacetate-CoA ligase from Streptomyces coelicolor A3 , ORF26 from Streptomyces aizumensis , the medium-chain fatty acyl-CoA ligase from Streptomyces sp. SN-593 , and the CoA ligase from Penicillium chrysogenum , in addition to CsAAE1 from Cannabis sativa. For the acyl-CoA dehydrogenase, we selected the short-chain acyl-CoA dehydrogenase from Pseudomonas putida KT2440 and the butyryl-CoA dehydrogenase from Megasphaera elsdenii , in addition to the acyl-CoA oxidase from Micrococcus luteus. The gene sequences were codon-optimized for E. coli and cloned into the pJL02 and pJL01 plasmid series . With the constructs in hand, we tested C6-isoprenol production in E. coli. First, we screened the CoA ligases, catalyzing the first step in beta-oxidation, with the well-characterized MeD as the acyl-CoA dehydrogenase. After production, we used GC-FID to quantify C6-isoprenol in the broths. The C6-isoprenol production varied substantially with different CoA ligases, suggesting this is a key step in the whole pathway. Among the genes we tested, PcCL gave rise to the highest production at 58.6 mg/L , a result consistent with the reported kinetics data . Also, we noticed that the combination of CsCL with MeD had a decreased production of 6.9 mg/L, down from the 27.3 mg/L produced by CsCL/MlD, suggesting that MlD is a better homolog for the second step . This hypothesis was supported by the screening results of the second step catalyzed by acyl-CoA dehydrogenase. The PcCL and MlD pair gave the highest C6-isoprenol production at 110.3 mg/L, so we used this pair of genes for the first two steps of the beta-oxidation pathway in the following experiments. Endpoint optical density analysis was performed to evaluate the impact of the expression of different beta-oxidation genes on cell growth. The results indicated the expression of the CoA ligases and acyl-CoA dehydrogenase barely impacts the growth. The slight growth inhibition effect in the high C6- isoprenol production, e.g., the PcCL/MlD, may result from the toxicity of C6-isoprenol.
After optimizing the pathway genes to increase the production efficiency, we turned to the host genes that may degrade the key intermediate, 3-ketovaleryl-CoA. As mentioned before, this intermediate can be degraded by a thiolase into acetyl-CoA and propionyl-CoA. Therefore we focused on two E. coli chromosomal thiolase genes, atoB and yqeF, which have substrate preference for short-chain betaketoacyl-CoA and were proposed to degrade 3-ketovalerylCoA. We conducted in-frame single-gene knockouts to delete these two genes in E. coli BL21 in sequence, and PCR confirmed the genotypes of the knockout mutants . The intermediate strain E. coli BL21 ΔatoBand the final double knockout strain E. coli BL21 ΔatoB ΔyqeF were then used for C6-isoprenol production using the plasmid combination of pJL01-MlD/pJL02-PcCL. The production of C6-isoprenol and the consumption of valeric acid were quantified. The results showed that while the knockout of atoB did not impact the C6-isoprenol titer, the double knockout strain, 6C02, increased the C6-isoprenol production to 390 mg/L, and the C6- isoprenol yield from valeric acid doubled to 44 mol% over the wild-type strain . We noticed even after knocking out the thiolase genes, only around half of the valeric acid is transformed into C6-isoprenol. The loss of valeric acid may come from the evaporation and the flux into side directions in the metabolic network, such as the degradation of 3-ketovaleryl-CoA by other thiolases in E. coli .In the C6-isoprenol runs, we noticed that the levels of isoprenol were generally negatively correlated to the levels of C6-isoprenol. For the sources of isoprenol, we reason that in addition to the E. coli native MEP pathway, the LMVA pathway may contribute the major portion via acetoacetylCoA, instead of 3-ketovaleryl-CoA, to C6-isoprenol. Through the LMVA pathway, the productions of C6-isoprenol and isoprenol use the same precursor, acetyl-CoA, resulting in the negatively correlated levels of C6-isoprenol and isoprenol. Also, the knockout of the short-chain acyl-CoA thiolase increased the supply of acetoacetyl-CoA and the production of IPP, resulting in an increased isoprenol titer of 14.4 mg/L compared to 2.1 mg/L for the wild-type strain.
Hence, we proposed that acetoacetyl-CoA is readily accepted by the LMVA pathway, and it’s betaoxidation precursor, butyric acid, might be a substate of the beta-oxidation LMVA pathway. To test this hypothesis, we fed 1 g/L butyric acid instead of valeric acid to strain 6C02 containing pJL01-MlD and pJL02-PcCL. After production, GC-FID and GC-MS confirmed the isoprenol production, quantified at 301.8 mg/L . This result validates butyric acid is a good substrate for the beta-oxidation LMVA pathway for IPP/isoprenol production. The successful transformation of butyric acid into C5 alcohols by C4A suggests that the beta-oxidation LMVA pathway has substrate promiscuity. To explore the substrate spaces of this pathway, we tested other fatty acids as substrates. Without supplementing hexanoic acid, E. coli 6C02 with the beta-oxidation LMVA pathway also produces a small amount of C7-isoprenol, confirmed by the synthetic standard using GC-FID and GC-MS . Supplementing hexanoic acid increased the production of C7-isoprenol significantly . Therefore, the betaoxidation LMVA pathway can activate hexanoic acid and transform it to C7-isoprenol, albeit with low efficiency. C7-isoprenol production without hexanoic acid supplementation is likely from endogenous hexanoyl-CoA in E. coli. We also tried fatty acid analogs with functional group substitutes, including 5- chloro-valeric acid,greenhouse rolling racks 4-pentenoic acid, 4-amino-butyric acid, 5,5,5-trifluorovaleric acid, and 4- bromobutyric acid. We conducted comparative GC-FID/GC-MS analysis and fragment search in GC-MS for lack of standards of the expected alcohol products. However, none of the expected substituted alcohols were detected, suggesting these fatty acid analogs are poor substrates for the beta-oxidation LMVA pathway . The substrate promiscuity assays suggest the beta-oxidation LMVA pathway can produce IPP and C7-IPP, expanding the product chemical space of the homoterpene biosynthesis platform. The isopentenols, including isoprenol and prenol, are drop-in alcohol biofuels and have versatile potential fuel applications: prenol is one of the top 10 Department of Energy Co-Optimization of Fuels & Engines gasoline blendstocks and has synergistic blending effects for research octane number , and isoprenol is the precursor of 1,4- dimethylcyclooctane , a high-performance jet fuel blend stock . Previous studies have shown that for alcohol biofuels, molecules with longer chain lengths have a better blend stability with conventional fuels, and are less hygroscopic than their shorter chain congeners . Our pathway to C6-isoprenol and C7-isoprenol from biomass-derived fatty acids makes it possible to produce these chain-extended isoprenol analogs sustainably. With the synthesized C6-isoprenol and C7-isoprenol, we were able to test some important fuel properties of these novel isoprenol analogs. We first estimated their water solubility based on their logP values. High water solubility contributes to the high hygroscopic nature of alcohol fuels, increasing the possibility of phase separation when blended with conventional hydrocarbon fuel. Also, for microbial bio-fuel production process, molecules with high logP and low water solubility will partition into the organic phase in a two-phase extractive fermentation, resulting in low product toxicity to the producing microbes . Increasing carbon chain length leads to decreasing polarity of alcohols, resulting in lower water solubilities. The logP of isoprenol analogs were determined using an HPLC method, with C4-C8 strain chain alcohols as references. The result revealed that the one-carbon increase of the chain length of isoprenol decreases the water solubility to 22.12 g/L from 65.36 g/L. Moreover, the addition of two carbons to isoprenol further decreases the water solubility to 6.6 g/L .
The decreasing trend of the water solubility was expected, and these data reflect the trend quantitatively. The energy density of the isoprenol analogs was determined by testing their gross heats of combustion using a standard method . The energy density tests revealed C6-isoprenol has a higher heating value of 35.524 MJ/kg, while C7-isoprenol had an HHV of 39.468 MJ/kg . These numbers are similar or higher to the HHVs of isopentenols tested in the same batch. We also determined the RONs of the 10% alcohol RBOB gasoline blends using the recently published AFIDA method . The results indicated that C6- and C7-isoprenols have comparable RON boosting effects to isopentenols, making these two chemicals potential blend stocks for gasoline blends. We previously constructed the homoterpene biosynthesis platform as a proof of concept that introduces terpene structural diversity at the precursor stage. Here we further optimized this platform towards practical application. The most significant change is the upstream pathway to the key intermediate, 3- ketovaleryl-CoA. Like the natural LMVA pathways, our previous pathway starts from propionyl-CoA, condensed by a thiolase into 3-ketovaleryl-CoA.First, thermodynamic analysis indicated the condensation reaction catalyzed by thiolase is endergonic with a positive Gibbs free energy change , suggesting the thiolase catalyzed condensation reaction is thermodynamically unfavorable. This calculation is consistent with the finding that PhaA homologs catalyze the degradation reaction better than the condensation reaction . Second, to our knowledge, almost all the reported thiolases that convert propionyl-CoA and acetyl-CoA into 3-ketovaleryl-CoA also convert two molecules of acetyl-CoA into acetoacetyl-CoA. Our later experiment using butyric acid as substrate in the beta-oxidation LMVA pathway suggests the LMVA pathway readily accepts acetoacetyl-CoA into IPP . Considering these two points and the relatively high concentration of acetyl-CoA in E. coli , we reasoned that it would be difficult for the thiolase LMVA pathway to make HIPP over IPP, complicating C16, C17 and C18 terpene biosynthesis. Instead, the beta-oxidation pathway is a more specific way to produce isopentenyl pyrophosphate analogs. Although background IPP production remains, either via the native MEP pathway or from endogenous acetoacetyl-CoA transformed by the LMVA pathway, the ratio of HIPP/IPP production is significantly improved, as the molar ratio of C6-isoprenol:isoprenol is over 60 in the production run using the E. coli BL21 ΔatoB host with 1 g/L valeric acid feeding. The high production of HIPP and its dominant content in the isopentenyl pyrophosphate analog pool will benefit future homoterpene biosynthesis efforts. Using isoprenol analogs as the final product, we successfully optimized the flux to HIPP in the homoterpene biosynthesis platform. The enzymes after the LMVA pathway leading to complex terpenes are more challenging to optimize because of their elusive enzymology and unusual substrates. Following HIPP production, an IDI is supposed to isomerize HIPP to HDMAPP. We could not detect the corresponding alcohol of HDMAPP, C6-prenol, in the E. coli production run with the expression of NudB and the thiolase LMVA pathway containing the IDI from Bombyx mori. While this IDI was confirmed in vitro to transform HIPP to HDMAPP specifically , the absence of C6-prenol in the production run suggests that this IDI does not work well in E. coli, or the hydrolyzed product of HDMAPP, HDMAP, is not well accepted by the E. coli endogenous phosphatases. Incorporating this IDI into the optimized beta-oxidation LMVA pathway may increase HDMAPP production by increasing substrate supply. Other Lepidopteran IDIs are also candidates for enzyme screening of this step.