Rearrangement of Coenzyme A-Acylated Carbon Chain Enables

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Letter

Rearrangement of Coenzyme A-acylated carbon chain enables synthesis of isobutanol via a novel pathway in Ralstonia eutropha William Black, Linyue Zhang, Cody Kamoku, James C Liao, and Han Li ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.7b00409 • Publication Date (Web): 11 Feb 2018 Downloaded from http://pubs.acs.org on February 12, 2018

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ACS Synthetic Biology

Rearrangement of Coenzyme A-acylated carbon chain enables synthesis of isobutanol via a novel pathway in Ralstonia eutropha William B. Black1, Linyue Zhang1, Cody Kamoku1, James C. Liao2, and Han Li1,* 1

Department of Chemical Engineering and Materials Science, University of California, Irvine, Irvine CA 90697, USA., 2Department of Chemical and Biomolecular Engineering, University of California, Los Angeles, Los Angeles, CA 90095, USA. *Corresponding author

Abstract Coenzyme A (CoA)-dependent pathways have been explored extensively for the biosynthesis of fuels and chemicals. While CoA-dependent mechanisms are widely used to elongate carbon chains in a linear fashion, branch-making chemistry has not been incorporated. In this study, we demonstrated the production of isobutanol, a branched-chain alcohol that can be used as a gasoline substitute, using a novel CoA-dependent pathway in recombinant Ralstonia eutropha H16. The designed pathway is constituted of three modules: chain elongation, rearrangement, and modification. We first integrated and optimized the chain elongation and modification modules, and we achieved the production of ~200mg/L n-butanol from fructose or ~30mg/L from formate by engineered R. eutropha. Subsequently, we incorporated the rearrangement module, which features a previously uncharacterized, native isobutyryl-CoA mutase in R. eutropha. The engineered strain produced ~30mg/L isobutanol from fructose. The carbon skeleton rearrangement chemistry demonstrated here may be used to expand the range of the chemicals accessible with CoA-dependent pathways.

Key words Ralstonia eutropha, isobutanol, CoA-dependent pathway, isobutyryl-CoA mutase, metabolic engineering

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Coenzyme A (CoA) is the universal carbon-carrying cofactor in metabolism. It activates carbon compounds in a broad range of biochemical reactions: forming, breaking, desaturating, functionalizing, and rearranging of carbon-carbon bonds. In metabolic engineering studies, CoAdependent synthetic pathways have been extensively explored for the bioproduction of fuels and chemicals1-8. A general scheme shared by diverse CoA-dependent biosynthetic pathways is the reverse βoxidation-like carbon chain elongation 2-4, which typically uses acetyl-CoA as the extender unit and adds two carbons in a linear fashion. The best understood example is the n-butanol production pathway from Clostridium species 1, 9-12. By reiterating the chain elongation cycle, nalcohols with lengths of up to C10 have become accessible 2-4. However, very few studies have employed the CoA-dependent pathways to produce branched-chain compounds 7, 13, despite their superior properties as fuels 7, 13 and wide applications in chemical industry14. In previous studies, branches of the carbon chains were only introduced at the initiation stage of chain elongation, which may restrict the positions where branches are created within the final products (see discussion below). In this study, we sought to explore alternative chemistry to rearrange linear CoA-acylated carbon chains and create branched carbon chains. To achieve this goal, we searched for biochemical tools in the bacterium Ralstonia eutropha H16, since this organism has been suggested to possess a versatile collection of CoA-dependent enzymes. For example, the R. eutropha genome has been predicted to encode over 50 acyl-CoA ligases, which enable the metabolism of various organic acids 15; R. eutropha also carries the genes of over 30 putative β-ketothiolases (PhaAs) and 18 acetoacetyl-CoA reductases (PhaBs), which support its capability to synthesize polyhydroxyalkanoates (PHAs) from different substrates 16-18. Recent studies continued to discover CoA-dependent enzymes with novel chemistry in R. eutropha 19-21. We identified three acyl-CoA mutases in R. eutropha H16 genome (encode by genes H16_B1842/B1841, sbm1, and sbm2) as potential catalysts for carbon chain rearrangement. Interestingly, although all three genes have been previously annotated as methylmalonyl-CoA mutases (MCMs), a recent study has suggested bioinformatically that sbm1 might encode a novel type of isobutyryl-CoA mutase (ICM) 21. ICMs are desirable catalysts for our purpose because it takes the linear butyryl-CoA, a reverse β-oxidation-like chain elongation product, and rearranges the carbon chain to yield the branched isobutyryl-CoA (Figure 1A). Protein sequence alignment showed that Sbm2 and H16_B1842 contain Y and R residues in the corresponding positions as shared by MCMs (Figure 1B) 21. On the other hand, Sbm1 contains the characteristic F and Q residues in the substrate binding site (Figure 1B), which are conserved in ICMs and are important for their preference for aliphatic substrates 21, 22. However, this alternative activity in Sbm1 has not been previously experimentally characterized. To confirm its ICM activity, we expressed Sbm1 in Escherichia coli as a His-tag fusion and purified it to apparent homogeneity with the size of ~120kD (Figure 1C). Using the purified protein, we demonstrated through enzyme assays that Sbm1 converted butyryl-CoA to isobutyryl-CoA with the specific activity of


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61 ± 11 nmol/min/mg. We next examined the activity of Sbm1 in its native host using crude cell lysate (Figure 1D, E, F). Slow conversion of butyryl-CoA to isobutyryl-CoA was detected using crude lysate of wildtype R. eutropha H16, indicating a basal level of Sbm1 expression (Figure 1D). When Sbm1 was overexpressed from a multiple-copy plasmid, significantly higher activity was observed (Figure 1E). The specific activity in the Sbm1-overexpressed crude lysate of R. eutropha was estimated to be ~21.4 nmole/min/mg as measured by the initial reaction rate (Figure 1F). These results represent the first biochemical characterization of R. eutropha Sbm1 as an ICM, and they demonstrated the feasibility to functionally overexpress this enzyme as a tool to convert linear to branched acyl-CoA. Using the newly characterized rearrangement enzyme, we designed a novel CoA-dependent pathway to produce isobutanol, a higher alcohol biofuel. The pathway is composed of three modules: chain elongation, rearrangement, and modification (Figure 2). Interestingly, although the idea of employing ICM-dependent rearrangement chemistry in isobutanol production has been postulated previously7, 23, it has not been experimentally realized possibly because of two reasons: First, heterologous expression of Coenzyme B12-dependent mutases has been shown challenging as 1) the enzymes often contain multiple subunits whose assembly is complex and relative concentrations need to be balanced 24, and 2) additional chaperon proteins from native hosts are required 25. To this end, our rearrangement module has advantages because Sbm1 is native to R. eutropha and it is a natural fusion of both small and large subunits as well as the Gprotein chaperon in a single polypeptide 21. Second, the concerted action of all three modules requires matching of the enzymes' kinetic properties which has not been experimentally explored before. As such, after demonstrating the function of the rearrangement module based on R. eutropha Sbm1, we next focused on establishing the other two modules and testing their function in vivo. Briefly, the chain elongation module contains the following steps (Figure 2): R. eutropha's native β-ketothiolase (encoded by phaA) and acetoacetyl-CoA reductase (encoded by phaB1) convert two acetyl-CoA into 3-hydroxybutyryl-CoA, which is also the intermediate in the host's highly active polyhydroxybutyrate (PHB) biosynthesis. Next, crotonyl-CoA is generated by an enoylCoA hydratase (encoded by PhaJ in Aeromonas caviae) 26. Subsequently, the trans-2-enoyl-CoA Reductase (TER) from Treponema denticola is used to reduce the double bond 9, 11. The final outcome of the chain elongation module is the C4 linear butyryl-CoA. The modification module contains a CoA-acylating aldehyde dehydrogenase (encoded by bldh from Clostridium saccharoperbutylacetonicum N1-4) and a broad-substrate range alcohol dehydrogenase (encoded by yqhD from E. coli) 12. Without the rearrangement module, the tandem action of chain elongation and modification modules is expected to produce n-butanol. To implement the pathway design mentioned above, we constructed a synthetic operon on the multiple-copy plasmid containing the four heterologous genes phaJ, ter, bldh, and yqhD, which was transformed into R. eutropha H16 to create strain LH201 (Figure 3A). LH201 produced ~30mg/L n-butanol in minimal medium with fructose as the sole carbon source (Figure 3B). To



improve the pathway flux, the first two steps of the pathway, which relied on the endogenous activity of the PHB biosynthesis pathway in LH201, were strengthened by overexpression of the R. eutropha phaA and phaB1 genes. In addition, a codon optimized ter (terOP) was used to replace the original ter gene in synthetic operon, since the TER activity was relatively low in LH201 (Figure 3E). These two changes resulted in strain LH202 (Figure 3A). Compared to LH201, LH202 has enhanced activities for phaA, phaB1, and TER (Figure 3C, D, E) based on enzyme assays using crude cell extract. LH202 also produced around ~2.5 fold higher n-butanol (~80ml/L) from fructose (Figure 3B). In summary, we have successfully established the chain elongation and modification modules and achieved the first production of n-butanol in R. eutropha. We further optimized the chain elongation and modification modules using n-butanol production as the readout. In strains LH201 and LH202, the synthetic operons are driven by a pre-existing promoter on the backbone of a multiple-copy plasmid, the chloramphenicol acetyl transferase (CAT) promoter. Although this promoter is widely used in R. eutropha, it may not provide the suitable strength of gene expression for biofuel production. To this end, we characterized promoters of several R. eutropha genes using β-galactosidase reporter assays (Figure 4A), including the promoters of pepck (encodes the phosphoenolpyruvate carboxykinase), pdh (encodes the pyruvate dehydrogenase), rrsC (produces the 16S ribosomal RNA), and phaC1 (encodes the Polyhydroxybutyrate polymerase). The results showed that the CAT promoter, albeit functional in a broad range of hosts, has low activity in R. eutropha compared with all of the native promoters tested. Two of the strongest promoters from the panel tested, the phd and phaC1 promoters, were used in place of Pcat to construct strains LH204 and LH205, respectively. These two strains exhibited a two-fold higher n-butanol production when compared to LH202 (Figure 4B). The best producer, LH205, generated ~200mg/L n-butanol in 3 days from fructose. In autotrophic conditions using formate as the sole carbon and energy source, LH205 produced ~30mg/L n-butanol in 3 days (Figure 4C). Next, we tested the compatibility of the modification module with the proposed rearrangement module, as the enzymes in the modification module need to be promiscuous to accommodate the rearranged carbon skeleton (Figure 2). The alcohol dehydrogenase yqhD from E.coli has been previously used to reduce isobutyraldehyde to isobutanol with high activity 14, 27. However, the CoA-acylating aldehyde dehydrogenase Bldh has not been tested for branched-chain substrates. We performed enzyme assays using crude cell extract of R. eutropha strain that overexpresses C. saccharoperbutylacetonicum N1-4 Bldh. The results showed that Bldh has comparable activity for the branched C4 substrate isobutyraldehyde as for the linear substrate butyraldehyde (Figure 5A). Finally, the rearrangement module was incorporated (Figure 2). We added sbm1 in the synthetic operon to construct strain LH206 (Figure 5B), which produced ~4mg/L isobutanol in minimal medium with fructose as the sole carbon source (Figure 5C). Noticeably, ~140mg/L n-butanol was also produced, indicating that the activity of the rearrangement module might need to be


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improved. Since the ICM family enzymes have been characterized previously to be vitamin B12 dependent 21, we hypothesized that supplementation of the vitamin B12 may enhance the enzyme activity. Consistently, we showed that the isobutanol production titer was positively correlated with the vitamin B12 levels in the medium (Figure 5C). R. eutropha strain LH206 produced ~32mg/L isobutanol under the optimized condition, a roughly 8-fold increase compared to no vitamin B12 supplementation. In conclusion, this work demonstrated the production of n-butanol and isobutanol using a CoAdependent pathway in R. eutropha H16 from renewable sources. In particular, the carbon chain rearrangement chemistry enabled the isobutanol production via a novel pathway. As mentioned above, branch-making chemistry in CoA-dependent pathways has previously been limited to the utilization of branched starter units derived from BCAA biosynthesis, namely isobutyryl-CoA, isovaleryl-CoA, and 2-methyl-butyryl-CoA7, 13. These 2- or 3-methyl acyl-CoAs only participate once in the CoA-dependent pathway at the beginning of carbon chain elongation, which generates only one branch at ω-2 or ω-3 position of the carbon chains. On the other hand, the carbon chain rearrangement chemistry demonstrated in this study does not utilized pre-formed branch structures, but it rather creates a branch de novo. Furthermore, the rearrangement module can potentially be incorporated after each cycle of chain elongation, offering flexibility on the number and positions that the branches could be generated. To realize this potential, acyl-CoA mutases that are active on longer chain aliphatic substrates than butyryl-CoA need to be discovered or engineered, which could be greatly accelerated by the recent understanding of the structural basis for substrate specificity in this enzyme class and by the advances in genome mining 22, 28. Paired with various modification modules, the proposed chemistry could also be applied to the biosynthesis of other branched compounds than alcohols, which may include fatty acids, aldehydes, and esters 3, 8, 29. R. eutropha H16 can utilize many different types of biomass-derived feedstocks including fructose, organic acids, glycerol, and plant oil. It can also fix CO2 using non-photosynthetic energy source such as H2 or formate. In this study, both n-butanol and isobutanol were produced from fructose. n-Butanol production was also achieved from formate, which can serve as a feedstock for “electrofuel” production with R. eutropha 27, 30. Compared to the previous studies 27, 30, 31 , the biofuel production levels reported here still need to be improved (the combined yield of n-butanol and isobutanol is roughly 10% of maximal theoretical yield from fructose or formate). One of the major limitations would be the oxygen-sensitivity of the CoA-acylating aldehyde dehydrogenase from Clostridium species. Replacement of this enzyme by oxygen-tolerant counterparts may improve the productivity 32. In addition, we observed back-consumption of accumulated n-butanol in the culture by R. eutropha at the rate of ~20mg/L per day, but not isobutanol (data not shown). Consistently, we detected high endogenous activity in wild type R. eutropha to oxidize butyraldehyde, but not isobutyraldehyde, in the presence of NADP+ (Supplementary Figure S1), suggesting that some native enzymes in R. eutropha may convert aldehydes to acids. These enzymes may need to be disrupted to preserve the aldehyde



intermediates and block the back-consumption of biofuel products. The high level of PHB accumulation up to 90% of cell dry weight33 in R. eutropha suggests that the metabolic network in this organism can supply a large amount of acetyl-CoA, which may make this pathway an attractive choice for biofuel production. Disruption of the phaC gene in PHB biosynthesis may represent another direction to further improve the biofuel productivity by directly eliminating competition for acetyl-CoA and draining of 3-hydroxybutyryl-CoA. Isobutanol has been shown to be toxicity to R. eutropha at concentrations above 0.5% (v v-1) 34. While the titer reported here is still well below the toxicity limit, future work might benefit from utilizing the alcohol-tolerant R. eutropha strains 34.

Methods Bacterium strain, medium, and production condition Ralstonia eutropha H16 strain was purchased from American Type Culture Collection (ATCC). R. eutropha strains were regularly cultured in rich medium (16g/L nutrient broth, 10g/L Yeast extract, 5g/L (NH4)SO4) at 30°C. If the strains contained plasmids, 200mg/L kanamycin was added. For biofuel production from fructose, strains were cultured in German minimal medium 35 with 10g/L fructose in rubber-capped test tubes at 30°C. Culture samples were taken daily to assay for biofuel levels by gas chromatography. For biofuel production from formate, German minimal medium 35 was used with 20mM sodium formate initially. 20% formic acid was continuously added to the production medium according to the pH changes caused by formate consumption. Air was bubbled through the medium for aeration and constant removal of alcohol product. Evaporated alcohols in vented gas were condensed with a Graham condenser and collected. Daily samples of culture broth and condensation liquid were taken, and alcohols were quantified using gas chromatography according to previously reported methods11. Introduction of the plasmid into R. eutropha cells was performed using previously reported electroporation method 27. Plasmid construction All cloning and plasmid preparation was performed using E. coli XL1-blue cells (Stratagene, La Jolla, CA). LB medium was used to culture E. coli. Detailed information about plasmids and primers used in this study can be found in supplementary material Table S1 and Table S2. Enzyme assays with crude cell extract


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R. eutropha crude cell extract for phaA, phaB, and TER assays was prepared by Qiagen Tissuelyser using lysis buffer (50mM Tris-HCl pH=7.5, DTT 1mM). The enzyme activities were measured using a previously reported method 11. Briefly, phaA activity was monitored by the decrease in OD303nm, corresponding to the disappearance of the substrate acetoacetyl-CoA. PhaB activity was monitored by decrease in OD340nm, corresponding to the disappearance of the substrate NADPH. TER activity was monitored by decrease in OD340nm, corresponding to the disappearance of the substrate NADH. The β-galactosidase reporter assays were performed using previously reported method 27. The R. eutropha crude cell extract for Bldh assay was prepared anaerobically in sealed vials by Qiagen Tissuelyser II. The lysis buffer contained 50mM potassium phosphate buffer pH=7.5, 10mM 2-Mercaptoethanol, and 1mM MgSO4. The cell extract was kept on ice in anaerobic environment at all times. The activity was measured in the oxidation direction. The assay system contains 50mM Tris-HCl pH=9.0, 10mM DTT, 2mM NADP+, 0.2mM CoA, and 10mM butyraldehyde or isobutyraldehyde. Activity was monitored by the increase of OD340nm. CoA was omitted to assay the endogenous non-CoA-acylating aldehyde dehydrogenase activity, which convert aldehyde to acid, independent of CoA. CoA-acylating activity of Bldh was calculated by subtracting the activity level without CoA addition from the activity level with CoA. The R. eutropha crude cell extract for Sbm1 assay was prepared by Qiagen Tissuelyser II with the buffer A containing 50mM potassium phosphate pH=7.4, 50mM KCl, 10mM MgSO4, and 50µM Vitamin B12. The reaction was performed in 200µl volume with buffer A, appropriate amount of crude cell extract, and 1mM of butyryl-CoA. The reaction was incubated at 30°C. 100µl 2N KOH was used to stop the reaction followed by addition of 100ul 15% H2SO4. Next, 500mg of NaCl was added to saturate the solution. 250µl of ethyl acetate was used to extract the product. The samples were assayed by gas chromatography. Sbm1 Protein Purification E. coli strain BW25113 overexpressing 6xHis-Sbm1 was cultured in 2XYT rich media with antibiotics in shake flasks at 14°C. 24 hours after expression the cells were pelleted and resuspended in buffer A containing 50mM potassium phosphate buffer pH=7.4, 50mM KCl, 10mM MgSO4, 0.05mM VitaminB12, and 2.75mM GTP. Cells were homogenized with glass beads (Benchmark Scientific, Edison, NJ). Cell lysate was centrifuged for 20 minutes at 4°C at 15,000rpm, and the supernatant was used for protein purification. The his-tagged Sbm1 was purified using His-Spin Protein MiniPrep (Zymo Research, Irvine, CA). Enzyme Assay with Purified protein The enzyme assay method of ICM was adapted from previous studies 21. The assay system contained 50mM HEPES pH=7.4, 50mM NaCl, 10mM MgSO4 0.1mM Vitamin B12, and 4mM GTP. After Sbm1 was allowed to equilibrate for 10 minutes, 2mM of butyryl-CoA was added to



begin the reaction. The reaction was incubated at 30°C. 50µl of 2N KOH was used to stop the 100uL reaction at different time points followed by the addition of 50µl of 15% H2SO4. 250mg of NaCl was added to saturate the solution, and 125µl ethyl acetate was used to extract the products. The samples were assayed by gas chromatography using previous reported methods 36. Supporting Information Supporting information contains details on plasmids, primers, and strains used in this study and measurement of CoA-independent aldehyde dehydrogenase activity in R. eutropha. Author information Corresponding author: Han Li, Department of Chemical Engineering and Materials Science, University of California, Irvine, Irvine CA 90697, USA. Email: [email protected] Author contributions: H.L. and J.C.L. conceived the study. W.B.B., L.Z., C.K., and H.L. performed the experiments. All authors reviewed the data. W.B.B., L.Z., and H.L. wrote the manuscript. Conflict of Interest: The authors declare no conflict of interest. Acknowledgement: The work is supported by start-up fund of UC Irvine.

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Fukui, T., Shiomi, N., and Doi, Y. (1998) Expression and characterization of (R)-specific enoyl coenzyme A hydratase involved in polyhydroxyalkanoate biosynthesis by Aeromonas caviae, J Bacteriol 180, 667-673. Li, H., Opgenorth, P. H., Wernick, D. G., Rogers, S., Wu, T. Y., Higashide, W., Malati, P., Huo, Y. X., Cho, K. M., and Liao, J. C. (2012) Integrated electromicrobial conversion of CO2 to higher alcohols, Science 335, 1596. Kitanishi, K., Cracan, V., and Banerjee, R. (2015) Engineered and Native Coenzyme B12dependent Isovaleryl-CoA/Pivalyl-CoA Mutase, J Biol Chem 290, 20466-20476. Rodriguez, G. M., Tashiro, Y., and Atsumi, S. (2014) Expanding ester biosynthesis in Escherichia coli, Nat Chem Biol 10, 259-265. Liu, C., Colon, B. C., Ziesack, M., Silver, P. A., and Nocera, D. G. (2016) Water splittingbiosynthetic system with CO(2) reduction efficiencies exceeding photosynthesis, Science 352, 1210-1213. Lu, J., Brigham, C. J., Gai, C. S., and Sinskey, A. J. (2012) Studies on the production of branchedchain alcohols in engineered Ralstonia eutropha, Appl Microbiol Biotechnol 96, 283-297. Lan, E. I., Ro, S., and Liao, J. C. (2013) Oxygen-tolerant Coenzyme A-acylating aldehyde dehydrogenase facilitates efficient photosynthetic n-butanol biosynthesis in cyanobacteria, Energy & Environmental Science. Spiekermann, P., Rehm, B. H., Kalscheuer, R., Baumeister, D., and Steinbuchel, A. (1999) A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds, Arch Microbiol 171, 73-80. Bernardi, A. C., Gai, C. S., Lu, J., Sinskey, A. J., and Brigham, C. J. (2016) Experimental evolution and gene knockout studies reveal AcrA-mediated isobutanol tolerance in Ralstonia eutropha, Journal of bioscience and bioengineering 122, 64-69. Schlegel, H. G., Kaltwasser, H., and Gottschalk, G. (1961) Ein Submersverfahren zur Kultur wasserstoffoxydierender Bakterien: Wachstumsphysiologische Untersuchungen, Archives of Microbiology 38, 209-222. Birch, A., Leiser, A., and Robinson, J. A. (1993) Cloning, sequencing, and expression of the gene encoding methylmalonyl-coenzyme A mutase from Streptomyces cinnamonensis, J Bacteriol 175, 3511-3519.


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Figure legend Figure 1 Characterization of Ralstonia eutropha Sbm1. A) Reaction catalyzed by isobutyryl-CoA mutase (ICM). B) Alignment of protein sequences at active sites of R. eutropha Sbm1, Sbm2, H16_B1842, and a characterized ICM from Streptomyces cinnamonensis (Sc) and a characterized methylmalonyl-CoA mutase (MCM) from Propionibacterium shermanii (Ps). C) SDS-PAGE of purified R. eutropha Sbm1. D) and E) Typical gas chromatography trace of isobutyryl-CoA mutase enzyme assay end point samples using crude cell extract of wildtype R. eutropha cells or cells with Sbm1 overexpression, respectively. Butyryl-CoA served as substrate. F) Time course of isobutyryl-CoA mutase enzyme assay using crude extract of R. eutropha cells with Sbm1 overexpression, with 1mM butyryl-CoA as substrate. The reaction reached equilibrium in approximately 60min. The initial reaction rate was calculated using the slope of the first five minutes. Error bars represent standard deviation of 3 replicate experiments (n=3). Figure 2 Pathway design for n-butanol and isobutanol biosynthesis using CoA-dependent pathway in Ralstonia eutropha. The pathway for n-butanol biosynthesis is composed of a chain elongation module and a modification module. If an acyl-CoA rearrangement module is added, the pathway can be adapted to produce isobutanol. Figure 3 Establishing the chain elongation and modification modules in Ralstonia eutropha. A) Synthetic operons for n-butanol production in stain LH201 and LH202. Ac, Aeromonas caviae , Td, Treponema denticola, Cs, Clostridium saccharoperbutylacetonicum N1-4 , Ec, Escherichi coli , Re, Ralstonia eutropha H16. terOP, ter gene codon optimized for R. eutropha. B) n-butanol production by LH201 and LH202 in minimal medium with fructose as the sole carbon source. C), D), E) β-ketothiolase (PhaA), NADPH-dependent acetoacetyl-CoA reductase (PhaB1), and NADH-dependent trans-2-Enoyl-CoA Reductase (TER) activities , repectively, in wild type, LH201, and LH202 strains as measured using enzyme assays with crude cell extract. Error bars represent standard deviation of 3 replicate experiments (n=3). Figure 4 Improvement of n-butanol production by altering promoter strength of the synthetic operons and autotrophic n-butanol production from formate. A) Expression strength of the broad host-range promoter Pcat and several native promoters of Ralstonia eutropha H16 as measured by the β-galactosidase reporter assays. pepck encodes the phosphoenolpyruvate carboxykinase, pdh encodes the pyruvate dehydrogenase, rrsC produces the 16S ribosomal RNA, and phaC1 encodes the Polyhydroxybutyrate polymerase. B) Replacement of the Pcat in LH202 with Ppdh in LH204 and PphaC1 in LH205, repectively, increased the n-butanol production from fructose. C) The strain LH205 produced n-butanol using formate as the sole carbon and energy source. Error bars represent standard deviation of 3 replicate experiments (n=3). Figure 5 Rearrangement of CoA-acylated carbon chain enabled isobutanol production. A) CoA-acylating aldehyde dehydrogenase activity for isobutyraldehyde and butyraldehyde assayed using crude lysate of wild type Ralstonia eutropha cells or cells with CoA-acylating aldehyde dehydrogenase (encoded by bldh from Clostridium saccharoperbutylacetonicum N1-4) overexpressed. B) Synthetic operon for n-butanol and isobutanol co-production in stain LH206. C) Production of isobutanol by LH206 from fructose and its dependence on vitamin B12 levels. Error bars represent standard deviation of 3 replicate experiments (n=3).



Figures Figure 1

Figure 2


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Figure 3

Figure 4



Figure 5


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78x43mm (300 x 300 DPI)


ACS Synthetic Biology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

A

B

O O

ICM

CoA

CoA

Butyryl-CoA

Isobutyryl-CoA

Ladder 200 kD →

sbm1 Re 596 DPTRMFAGEG sbm2 Re 52 WTIRQYGGYG H16_B1842 Re 63 WTIRQYSGFG MCM Ps 84 WTIRQYAGFS ICM Sc 75 WTIRQFAGFG

605 61 72 73 84

740 170 181 202 193

KEDQGQNTCI KEFMVRNTWI KEYVARGTWI KEFMVRNTYI KEYIAQKEWL

749 179 190 211 202

O

D

C

Page 16 of 20

CoA

Sbm1 wildtype O

116.2 kD →

F

Concentration (mM) 1.0

isobutyryl-CoA Butyryl-CoA

0.8 CoA

0.6

E

0.4

Sbm1 overexpression CoA

CoA

Initial reaction rate =21.4 nmole/min/mg

0.2

O

O

0.0

0 7.6

8.1

8.6

Retention Time (min)


10

20 30 40 Time (min)

50

60

Page 17 of 20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41


formate

fructose

CO2

Chain Elongation

Rearrangement NADH

O CoA

CoA

Acetyl-CoA

Crotonyl-CoA

PhaA

TER

H2 O

PhaJ

O

O

OH CoA

Acetoacetyl-CoA

PhaB1

Bldh CoA

O

YqhD

Butyraldehyde

Butyryl-CoA

n-butanol

NADPH

NAD(P)H Sbm1 O

NADPH

OH

O

O

O

Modification

OH

O CoA

Bldh

YqhD

CoA

3-hydroxybutyryl-CoA

Isobutyryl-CoA

Polyhydroxybutyrate (PHB)


Isobutyraldehyde

Isobutanol


A

PCAT

B

LH201 phaJ

ter

bldh

yqhD

(Ac)

(Td)

(Cs)

(Ec)

PCAT

LH202

n-butanol (mg/L)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 60000 21 22 50000 23 24 40000 25 26 30000 27 28 20000 29 30 10000 31 0 32 33 34 35 36 37 38 39 40 41

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phaJ phaA phaB1 terOP bldh (Ac)

(Re)

(Re)

(Td)

C

(Cs)

yqhD

80 70 60 50 40 30 20 10 0

(Ec)

LH201 LH202

0

1

phaA

WT H16

LH201 LH202

1200 1000 800 600 400 200 0

3

Time (Day)

D Activity (nmole/min/mg)

2

E Activity (nmole/min/mg)

phaB1

200 150 100

Activity (nmole/min/mg)

TER

50 0

WT H16

LH201 LH202


H16 WT

LH201

LH202

Page 19 of 20

A

B Activity (miller unit)

C

200

LH202 LH204 LH205

150

n-butanol (mg/L)

8000 7000 6000 5000 4000 3000 2000 1000 0

n-butanol (mg/L)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47


100 50 0

CAT pepck rrsC phaC1 pdh

0

1 2 Time (Day)


3

30 25 20 15 10 5 0

LH205

0

1 2 Time (Day)

3

ACS Synthetic Biology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

A

C

Activity (nmole/min/mg)

35 25

WT H16

30

20

LH206 +Bldh

25

Page 20 of 20

Isobutanol (mg/L)

20

15

15

10

10

5

5 0

0 Isobutyraldehyde

0nM

Butyraldehyde

200nM 500nM 1000nM Vitamin B12

PphaC1

B LH206

phaJ

Sbm1

(Ac)

(Re)

phaA phaB1 terOP bldh (Re)

(Re)

(Td)


(Cs)

yqhD (Ec)

Rearrangement of Coenzyme A-Acylated Carbon Chain Enables

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