Creation and evaluation of a pyruvate decarboxylase dependent ethanol fermentation pathway in Geobacillus thermoglucosidasius

Abstract

Bioethanol, produced from organic waste as a second-generation biofuel, is an important renewable energy source. Here, recalcitrant carbohydrate sources, such as municipal and agricultural waste, and plants grown on land not suitable for food crops, are exploited. The thermophilic, Gram-positive bacterium Geobacillus thermoglucosidasius is naturally very flexible in its growth substrates and produces a variety of fermentation products, including lactate, formate, acetate and ethanol. TMO Renewables Ltd. used metabolic engineering to enhance ethanol production, creating the production strain TM242 (NCIMB 11955 ∆ldh, ∆pfl, pdhup). Ethanol yield has been increased to 82% of the theoretical maximum on glucose and up to 92% of the theoretical maximum on cellobiose. However, this strain still produces acetate, presumably derived from the overproduction of acetyl-CoA through the upregulated pdh gene encoding the pyruvate dehydrogenase complex. An alternative to the mixed fermentation pathway found in G. thermoglucosidasius is to introduce a homoethanologenic pathway. Yeast and a very limited range of mesophilic bacteria use the homoethanol fermentation pathway, which employs pyruvate decarboxylase (PDC) in conjunction with alcohol dehydrogenase (ADH), to convert pyruvate to ethanol. Despite extensive screening, no PDC has yet been identified in a thermophilic organism. Using the thermophile G. thermoglucosidasius as a host platform, we endeavoured to develop a thermophilic version of the homoethanol pathway for use in Geobacillus spp. This Thesis reports the in vitro characterization and crystal structure of one of the most thermostable bacterial PDCs from the mesophile Zymobacter palmae (ZpPDC) and describes strategies to improve expression of active PDC at high growth temperatures. This includes codon harmonization and the successful development of a PET (producer of ethanol) operon. Furthermore, ancestral sequence reconstruction was explored as an alternative engineering approach, but did not yield a PDC more thermostable than ZpPDC. In vitro ZpPDC is most active at 65°C with a denaturation temperature of 70°C, when sourced from a recombinant mesophilic host. Codon harmonization improved detectable PDC activity in G. thermoglucosidasius cultures grown up to 65°C by up to 42%. Pairing this PDC with G. thermoglucosidasius ADH6 produced a PET functional up to 65°C with ethanol yields of 87% of the theoretical maximum on glucose. This increase in yield at temperatures of up to 15°C higher than previously reported for any PDC expressed 

Date of Award	1 Mar 2017
Original language	English
Awarding Institution	University of Bath
Sponsors	Biotechnology and Biological Sciences Research Council & TMO Renewables Ltd
Supervisor	Michael Danson (Supervisor) & David Leak (Supervisor)