Recipient Organization
DAST LLC dba Cyanosun Energy
1006 32nd Ave
Brookings,SD 57006
Performing Department
(N/A)
Non Technical Summary
The goal of the project is to genetically engineer photosynthetic cyanobacteria that will synthesize farnesene from carbon dioxide. The work will then involve shutting down pathways that compete for carbon in an attempt to maximize the amount of farnesene that can be produced. Then we will do tolerance testing by culturing the cyanobacteria in varying concentrations of the end product(s) in order to determine the optimal culturing conditions. This work will develop an engineered cyanobacterium capable of converting CO2 into farnesene (C15H24), a long-chain hydrocarbon with an energy density of 47 MJ/Kg, heat of vaporization of 0.24 MJ/Kg, and octane of 89.7. The cells would excrete farnesene into the recirculating culture fluid, where it would be recovered by low-cost phase separation for use as an energy-dense, infrastructure compatible biofuel. We have already established the validity of this "cyanofactory" platform by engineering Anabaena to produce the ten-carbon alcohol linalool. Farnesene has a much higher energy density and thus would be a more desirable 3rd generation biofuel.
Animal Health Component
100%
Research Effort Categories
Basic
(N/A)
Applied
100%
Developmental
(N/A)
Goals / Objectives
In Phase I, we will evaluate the feasibility of producing farnesene from an engineered cyanobacterium to achieve a titer of about 1 g/L. Low-cost phase separation will be used for direct recovery of farnesene, so that cells and culture fluid can be recycled. We anticipate that we will be able to achieve this level of farnesene production through a combination of approaches: Our Phase I objectives will include: 1) screening two cyanobacteria strains and three farnesene synthase (FaS) genes for farnesene production using a shuttle vector approach, 2) inserting the best FaS gene into the chromosome of the best cyanobacterium for stable expression, and 3) increasing carbon flow to farnesene by shutting off glycogen production.
Project Methods
Objective 1: Identify the best combination of cyanobacterial strains and farnesene synthase (FaS) genes for farnesene production by using the shuttle vector approach To quickly screen for the best combination of cyanobacterial strain and farnesene synthase (FaS) gene, we will use the shuttle vector method as it is both more efficient and faster than chromosomal integration. Since psbA promoter is highly conserved in cyanobacteria, we should be able to use these three constructs to screen the two cyanobacterial strains to identify the best farnesene-producing combination. The constructs will be individually introduced into the two cyanobacterial strains by conjugation. Transformants will be selected and grown to assess farnesene production. Objective 2: Integrate the best farnesene synthesis gene into the chromosome of the best cyanobacteria strain for stable expression. Using a shuttle vector will allow us to quickly screen for the best FaS gene - cyanobacteria combination, but the drawback is that an antibiotic selection pressure (extra cost) is needed to retain the plasmid in the cell during propagation. Once the ideal combination is identified, a stable farnesene-producing cyanobacterial strain will be created by the chromosomal integration approach. Objective 3: Block glycogen production to increase fixed carbon flow to farnesene, thus establishing the feasibility of using metabolic flux corrections to increase yield and productivity. To redirect carbon flow to the MEP pathway for farnesene production, we will block glycogen synthesis by inactivating glucose-1-phosphate adenylyltransferase, which catalyzes the first committed step in glycogen biosynthesis. Only one glucose-1-phosphate adenylyltransferase gene is found in the genome of cyanobacteria, thus we can block glycogen synthesis by inactivating the all4645 gene using double crossover knockout approach.Carbon flux-altered transgenic cultures will be evaluated for farnesene yield and productivity as described previously. Achieving this objective will demonstrate the feasibility of using additional metabolic flux corrections to further increase carbon flow to farnesene, hence boosting yield and productivity.