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Studies on 3-Hydroxypropionate Metabolism in Rhodobacter sphaeroides

Carlson, Steven Joseph

Abstract Details

2018, Doctor of Philosophy, Ohio State University, Microbiology.
In this work, the involvement of multiple biochemical pathways used by the metabolically versatile Rhodobacter sphaeroides to assimilate 3-hydroxypropionate was investigated. In Chapter 2, evidence of a 3-hydroxypropionate oxidative path is presented. The mutant RsΔpdhAa2SJC was isolated which lacks pyruvate dehydrogenase activity and is unable to grow with pyruvate. Robust 3-hydropropionate growth with RsΔpdhAa2SJC indicated an alternative mechanism exists to maintain the acetyl-CoA pool. Further, RsΔdddCMA4, lacking the gene encoding a possible malonate semialdehyde dehydrogenase, was inhibited for growth with 3-hydroxypropionate providing support for a 3-hydroxypropionate oxidative pathway which involves conversion of malonate semialdehyde to acetyl-CoA. We propose that the 3-hydroxypropionate growth of RsΔpdhAa2SJC is due to the oxidative conversion of 3-hydroxypropionate to acetyl-CoA. In Chapter 3, the involvement of the ethylmalonyl-CoA pathway (EMCP) during growth with 3-hydroxypropionate was studied. Phenotypic analysis of mutants of the EMCP resulted in varying degrees of 3-hydroxypropionate growth. Specifically, a mutant lacking crotonyl-CoA carboxylase/reductase grew similar to wild type with 3-hydroxypropionate. However, mutants lacking subsequent enzymes in the EMCP exhibited 3-hydroxypropionate growth defects that became progressively more severe the later the enzyme participated in the EMCP. To resolve this finding, a late blockage EMCP strain has 3-hydroxypropionate growth restored by introducing an early blockage to the EMCP. Furthermore, the introduction of thioesterase YciA to inhibited mutant strains restored 3-hydroxypropionate growth with concomitant excretion of EMCP-derived metabolites showing a CoA-thioester intermediate accumulation most likely causes a decrease in free coenzyme A levels and the growth inhibition. The work confirms the EMCP is not essential for 3-hydroxypropionate growth. However, flux through the EMCP occurs. In Chapter 4, a novel way to alter flux through the EMCP was discovered. Late blockage EMCP mutants were inhibited for 3-hydroxypropionate growth, but spontaneously began growing after 100 hours. Whole genome sequencing of suppressor isolates identified a common mutation in the prkB gene, encoding phosphoribulokinase B of the Calvin-Benson-Bassham (CBB) cycle. The prkB mutation requirement for suppression was verified by introducing mutated alleles to the inhibited strains where 3-hydroxypropionate growth was restored. Finally, introduction of thioesterase YciA did not cause excretion of EMCP-derived metabolites during 3-hydroxypropionate growth in a suppressor strain indicating the prkB mutation decreases flux through the EMCP. In Chapter 5, the role of propionyl-CoA carboxylase during 3-hydroxypropionate, propionate, and acetate assimilation was investigated. Propionyl-CoA carboxylase (PccBA) catalyzes the conversion of propionyl-CoA to (2S)-methylmalonyl-CoA in the methylmalonyl-CoA pathway (MMCP) used for propionyl-CoA assimilation. The assimilation of acetyl-CoA and 3-hydroxypropionate also leads to formation of propionyl-CoA whereby the MMCP would be required. A pccB mutant strain could not grow with propionate/HCO3- confirming the requirement of the MMCP for propionyl-CoA assimilation. However, the same mutant could still grow with acetate and 3-hydroxypropionate. For acetate growth, metabolite analysis showed that propionate was excreted indicating a mechanism to prevent accumulation of propionyl-CoA formed during flux through the EMCP. For 3-hydroxypropionate growth, redirection of the carbon toward acetyl-CoA via the 3-hydroxypropionate oxidative pathway and entry into the EMCP was shown to allow growth when the 3-hydroxypropionate reductive pathway is blocked in R. sphaeroides.
Birgit E. Alber (Advisor)
F. Robert Tabita (Committee Member)
Venkat Gopalan (Committee Member)
Joseph A. Krzycki (Committee Member)
189 p.

Recommended Citations

Citations

  • Carlson, S. J. (2018). Studies on 3-Hydroxypropionate Metabolism in Rhodobacter sphaeroides [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1543496642308385

    APA Style (7th edition)

  • Carlson, Steven. Studies on 3-Hydroxypropionate Metabolism in Rhodobacter sphaeroides. 2018. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1543496642308385.

    MLA Style (8th edition)

  • Carlson, Steven. "Studies on 3-Hydroxypropionate Metabolism in Rhodobacter sphaeroides." Doctoral dissertation, Ohio State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=osu1543496642308385

    Chicago Manual of Style (17th edition)