Expanding the synthetic capabilities of chemoautotrophic metabolism.

The steep environmental costs of petrochemical-derived products have created a pressing need for sustainable approaches to chemical synthesis. To address this need, scientists and engineers have developed diverse methods for capturing and either storing or using greenhouse gases to make value-added chemicals. In this effort, many scientists have looked to Nature for inspiration. Nature has evolved many methods for capturing CO2 and using it as a carbon source for autotrophic growth. Of all the diverse forms of autotrophy, chemoautotropic metabolism is particularly remarkable for its versatile use of inorganic electron donors. If this versatility could be harnessed and coupled to sustainable sources of electrons and energy, chemoautotrophs may be used as a metabolic host for sustainable chemical synthesis./nThis thesis explores two approaches for the engineering of chemoautotrophic metabolism for the production of value-added chemicals. In the first approach, essential genes for the Wood-Ljungdahl carbon fixation pathway were investigated in Moorella thermoacetica, a model acetogenic bacteria. A panel of gene candidates were identified for the appropriate maturation of the acetyl-CoA synthase/CO dehydrogenase (ACS/CODH) complex, an important metalloenzyme complex in the Wood-Ljungdahl pathway. The most promising candidate, AcsF, was recombinantly expressed with ACS in Escherichia coli, showing an increase in ACS nickel content. Furthermore, methods were developed to produce knockouts in M. thermoacetica to assess the native role of our gene candidates and measure the in vitro and in vivo activity of a recombinant ACS/CODH complex from E.coli./nIn our second approach, we attempted to engineer Methanococcus maripaludis, a model methanogenic archaea, to produce 3-hydroxybutyrate (3HB) and polyhydroxybutyrate (PHB). NAD(H) pools in M. maripaludis were less than 5% of the pool in anaerobically-grown E.coli, suggesting that low NADH availability may be a metabolic bottleneck for biosynthesis. Using transcriptomic and proteomic studies, alanine dehydrogenase was identified as a critical native enzyme for in vivo NADH regeneration and improvements in product titers were observed in alanine-dependent growth. Multiple engineering strategies were used to precisely target and increase the cofactor pool, including heterologous expression of NAD+-dependent formate dehydrogenase from Candida boidinii and a synthetic nicotinamide salvage pathway. Engineered strains of M. maripaludis with improved NADH pools produced up to 150 ± 4 mg/L of PHB in McNA medium, a more than 2.5-fold improvement over the starting strain./nTo further characterize heterologous gene expression, native transcript structure in M. maripaludis was more carefully analyzed. Methanogenic archaea display characteristics of translation that are analogous to both eukaryotic and prokaryotic processes, but some features remain to be determined. To address this, we conducted a transcriptome assembly and determined the consensus Shine-Dalgarno (SD) sequence of GGAGGA from our 5’ UTR-containing sequences. Our predicted transcripts were verified using 3’ UTR sequencing. In addition, we found that a large fraction of our transcripts were leaderless, motivating us to investigate translation initiation for both leaderless and leadered transcripts. To probe this question, we developed a ribosome profiling methodology that could be used to precisely identify the ribosome’s position and interactions with mRNA. These developments help pave the way towards a robust heterologous gene pathway design strategy for this species. The metabolic engineering strategies presented in this study broaden the utility of M. maripaludis for sustainable chemical synthesis using CO2 and may be transferable to related archaeal species.

Advisor: Chang, Michelle C.Y.