Recipient Organization
Massachusetts Institute of Technology
(N/A)
Cambridge,MA 02139
Performing Department
Chemical Engineering
Non Technical Summary
Increasing demand for renewable liquid transportation fuels has driven research into the production of next-generation alcohols from biomass-derived feedstock. This research has led to significant advancements in the microbial production of alcohols, such as n-butanol, that can could replace petroleum-based fuels. In fermentative systems n-butanol is produced by a bifunctional alcohol/aldehyde dehydrogenase (AdhE), which reduces the corresponding acyl-CoA in a two-step reaction. While this enzyme is an efficient producer of alcohols during fermentations, it lacks strict substrate selectivity and is capable of reducing substrates of varying chain-lengths. This results in the co-production of ethanol during fermentations, thereby decreasing the yield of n-butanol. This project aims to engineer the bifunctional alcohol/aldehyde dehydrogenase to alter the enzymes substrate specificity. Specifically, the primary goal is to engineer AdhE with increased substrate specificity towards butyryl-CoA/butyraldehyde, resulting in decreased ethanol production in fermentations. This project will utilize a recently described transcriptional regulator which can provide feedback to the presence of alcohols that are at least 4-carbons. Placing an essential gene under control of this regulator allows for selection of cells that are producing the alcohol. Importantly, this system can provide a dynamic response to the concentration of alcohol. Thus, cells that are producing more butanol are able to grow faster than those producing less butanol. A library of variant enzymes will be created and screened to identify enzymes that confer increased growth rate as a consequence of increased butanol production. Several iterations of mutant selection should identify altered enzymes that produce more butanol while also producing less ethanol, progressing towards homofermentative butanol production.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
1. Identify residues in the AdhE sequence that control substrate selectivity. 2. Engineer theE. coliAdhE to function with pentanoyl-CoA. In fermentation experiments where acetate and propionate are fed, the production of pentanol should be observed. 3. Engineer the C. acetobutyricum AdhE for increased activity with butyrl-CoA. After several iterations of mutagenesis variants should be identified which posess increased butanol production with decreased ethanol yields.
Project Methods
The primary goal of this project is to engineer an alcohol/aldehyde dehydrogenase with altered substrate specificity. This project will be performed using standard molecular biology and microbiological techniques. Attainment of the project goals will likely be determined by the success of a novel high-throughput screen to detect the fermentative production of butanol or pentanol. First, a strain of E. coli will be created that possesses the pSelect plasmid, which carries the genes and regulators that enable the high-throughput screen. This screen functions by allowing only those cells that are producing butanol or pentanol to grow. Furthermore, this screen has a dynamic response that allows the for growth rate to be coupled to the concentration of alcohol. Initial attempts to engineer AdhE will be made by using error-prone PCR to mutate the CoA-acetylating aldehyde dehydrogenase domain of AdhE. Next, the full-length gene will be expressed from a plasmid in E. coli. The same plasmid will also be used to express a CoA-transferase, which is needed to produce the acyl-CoA to be reduced by AdhE. Cells which show increased growth rates will have their aldehyde dehydrogenase domain sequenced by rolling circle amplification. Sequence analysis of the mutated genes should reveal several hotspots in the gene that encode for residues that are important for enzyme substrate specificity. Next, these hotspot locations will be further investigated using saturation mutagenesis. Degenerate primers will be designed that will allow for all possible residues to be encoded in these locations. These mutants will then be screened using the method described above. Strains will be further characterized using traditional fermentation experiments in which cells are grown anaerobically and the spent medium is analyzed for fermentation products using standard chromatographic separation. Results from these investigations will be described and disseminated to the scientific community through 1-2 journal publications. In addition, some of this work will be conducted by undergraduate students working in the lab for course credit. It is anticipated that 2-3 undergraduates will be trained in the lab each semester for the duration of the project. Project evaluation will be performed by measuring the fermentation profile ofE. colistrains harboring the engineered AdhE. The first iteration of mutagenesis should yield tens to hundreds of strains that have an increased growth as compared to a strain harboring the wild-type enzyme. These strains will then have their fermentation profiles characterized and success will be determined by observing increased butanol production relative to