Hao Xie: Pathway Engineering for Butanol Production in Cyanobacteria

Abstract

Cyanobacteria, photosynthetic microorganisms, are emerging as promising platforms for sustainable biofuel production, leveraging their capabilities to utilize solar energy, carbon dioxide, and water in a direct process. Among various biofuels, isobutanol (IB) and 3-methyl-1-butanol (3M1B) are superior cadidates as gasoline replacements. The unicellular cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis) has been successfully engineered for IB and 3M1B production via heterologous expression of α-ketoisovalerate decarboxylase (KivdS286T). However, the production levels remain low. This thesis focuses on further enhancing photosynthetic IB and 3M1B bioproduction via the 2-keto acid pathway in Synechocystis through multifaceted metabolic engineering strategies.

For the first time, a functional 2-keto acid pathway, consisting of acetolactate synthase (AlsS), acetohydroxy-acid isomeroreductase (IlvC), dihydroxy-acid dehydratase (IlvD), KivdS286T, and alcohol dehydrogenase (Adh), was successfully introduced in Synechocystis. By modulating kivdS286T copy number, a stepwise increase of IB and 3M1B production was observed. Building on these successes, additional genetic engineering strategies were employed. First, by overexpressing selected genes involved in central carbon metabolism, substantial production improvements were achieved. Second, a dCas12a-mediated CRISPR interference (CRISPRi-dCas12a) system was developed and integrated into an IB/3M1B producer strain to allow for elimination of potential competing pathways. Repression of ten out of fifteen target genes resulted in improved IB and 3M1B production. A strain with dual repression of ppc and gltA demonstrated 2.6-fold and 14.8-fold increases in IB and 3M1B production per cell, respectively. In addition to genetic engineering, protein engineering is another powerful tool in metabolic engineering. Manipulating kivdS286Tcopy number effectively increased overall cellular KivdS286T expression level, while KivdS286T directed evolution offered an opportunity to improve catalytic activity. After screening 1,600 variants, one KivdS286T variant (1B12), featuring dual T186S and K419E substitutions, exhibited a significant increase of 55% in IB and 50% in 3M1B production per cell. This is the first demonstration of using directed evolution to enhance bioproduction in Synechocystis. Long-term cultivation of two selected strains in plug-sealed tissue culture flasks resulted in maximal cumulative IB and 3M1B titers of 1.2 g L-1 and 0.4 g L-1, respectively, representing new records of photosynthetic IB and 3M1B production levels in cyanobacteria.

In summary, these advancements highlight the feasibility of applying various metabolic engineering strategies to develop Synechocystis as a biofuel producer. Furthermore, the engineered biofuel-producing strains establish a strong foundation for further research into industrial-scale biofuel production process, aiming to replace fossil fuels with sustainable, carbon-neutral alternatives.