Green Biologics (United Kingdom)

The objective of this project was to engineer novel Clostridium strains to produce n-butanol from low-cost lignocellulosic biomass and gases (CO2 and H2). Butanol is an important industrial solvent and potentially a superior fuel that can fit the existing fuel infrastructure and replace gasoline in auto engines without modification. This project focused on metabolic engineering of cellulolytic Clostridium cellulovorans for directly converting cellulose to n-butanol and carboxydotrophic Clostridium carboxidivorans to convert CO2 and H2 to butanol and ethanol. These clostridial strains were engineered using synthetic biology, gene overexpression and gene knockout strategies to produce n-butanol as the main product from lignocellulose and gaseous substrates. With these metabolically engineered strains, high-titer and high-yield n-butanol production from lignocellulosic biomass were achieved with minimal CO2 released to the environment in a sustainable and co-cultured consolidated bioprocess (CBP) integrated with in situ butanol separation by gas stripping. A well-to-pump life cycle analysis showed that the integrated biobutanol production process could reduce CO2 and greenhouse gas (GHG) emissions by ~75% compared to traditional chemical process. Moreover, the production of cellulosic butanol from CBP would not require enzymatic hydrolysis, an expensive step in current biorefinery using lignocellulosic biomass. The successful development of CBP using engineered clostridia for n-butanol production from lignocellulosic biomass and CO2 can meet the BETO 2022 cost target of $\$ $3/gallon gasoline equivalent (gge) for advanced biofuels. With the higher energy content, butanol will be cheaper than ethanol and similar to gasoline at $\$ $2.50/gal in terms of energy cost ($\$ $ per MJ). With the reduced production cost and product value, biobutanol from lignocellulose and CO2 can one day become a major liquid transportation biofuel, replacing bioethanol and gasoline. Strain development via metabolic engineering and fermentation process optimization, assisted by omic analysis of the mutation and process effects, helped us to achieve our goal of producing n-butanol and ethanol (biofuel) from lignocellulosic biomass at a cost of $3/gge (gallon gasoline equivalent) or less. The co-fermentation technology using both cellulose and CO2/H2 for biofuel production can greatly increase product yield from the biomass feedstock while also reduce GHG emissions, and is novel and advantageous compared to existing ethanol and acetone-butanol-ethanol fermentation technologies. The same metabolic engineering and process approaches can also be applied to other microorganisms to produce bio-based chemicals from renewable resources. Overall, the CBP technology using the novel engineered clostridia is unique and innovative, and complements well with the Biochemical Conversion technologies in the BETO Multi-Year Program Plan (MYPP), which largely focuses on the hydrolysis of cellulosic biomass and utilization of the hydrolysates for ethanol production.

ABSTRACTThe solventogenic clostridia have long been known for their ability to convert sugars from complex feedstocks into commercially important solvents. Although the acetone-butanol-ethanol process fell out of favour decades ago, renewed interest in sustainability and ‘green’ chemistry has re-established our appetite for reviving technologies such as these, albeit with 21st century improvements. As CRISPR-Cas genome editing tools are being developed and applied to the solventogenic clostridia, their industrial potential is growing. Through integration of new pathways, the beneficial traits and historical track record of clostridial fermentation can be exploited to generate a much wider range of industrially relevant products. Here we show the application of genome editing using the endogenous CRISPR-Cas mechanism of Clostridium saccharoperbutylacetonicum N1-4(HMT), to generate a deletion, SNP and to integrate new DNA into the genome. These technological advancements pave the way for application of clostridial species to the production of an array of products.

<p>The ButaNexT project aims to demonstrate the techno-economic feasibility of the conversion of two</p> <p>sustainable lignocellulosic feedstocks (wheat straw and miscanthus) available at EU level, into biobutanol through the</p> <p>integration of different technology developments. The project has ended recently and the main achievements of the</p> <p>consortium have been the following: (1) design, construction and operation of a micronizing pilot prototype for</p> <p>controlled biomass particle size production up to 0.5 mm with a more reduced energy consumption and able to be</p> <p>scalable to industrial size; (2) development of a flexible and easy to adapt two-step fractionation process based on the</p> <p>combination of the micronizing prototype with a continuous thermochemical pretreatment; (3) development of</p> <p>tailored enzyme cocktails (MetZyme® SUNO™) that address harsh process-specific conditions and facilitate</p> <p>improved hydrolysis of pretreated biomass to monosaccharides; (4) development of a Clostridial strain able to</p> <p>ferment cellulosic sugars effectively (target butanol yield > 0.25 g/g pure sugar) and having 20% higher butanol</p> <p>tolerance than baseline strain; (5) development of a patent-pending “In situ product recovery process” based on</p> <p>improved pervaporation fluxes obtained during fermentation that allows continuous operation; (6) validation of the</p> <p>technical performance of the individual stages cited above in a pilot facility using wheat straw as feedstock from</p> <p>handling to product recovery in a 100 L bioreactor; (7) testing of properties and performance of butanol as a blend</p> <p>component in both diesel and gasoline fuels and (8) calculation of GHG emissions of biobutanol in a number of</p> <p>different scenarios based on the use of wheat straw and miscanthus as feedstocks and for the provision of heat,</p> <p>electricity and waste water treatment.</p>

The acetone–butanol–ethanol (ABE) fermentation was one of the largest biotechnological operations ever performed, being beaten in size only by the ethanolic fermentation. ABE had its peak time between 1915 and 1950, but several plants were even operating until 2004. Meanwhile, the process might be revived, especially in China. Workhorses of the fermentation are solventogenic clostridia, namely, Clostridium acetobutylicum, C. beijerinckii, C. saccharobutylicum, and C. saccharoperbutylacetonicum. Their features in product formation, enzymes, regulation, genes and genomes, threats by phage infection, and newly developed sophisticated genetic tools for targeted strain improvement are summarized in this chapter.

In this study, we demonstrate the sensitivity of two-dimensional perturbation-correlation moving windows (PCMW) to characterize conformational transitions in antibodies. An understanding of how physiochemical properties affect protein stability and instigate aggregation is essential for the engineering of antibodies. In order to establish the potential of PCMW as a technique for early identification of aggregation mechanisms during antibody development, five antibodies with varying propensity to aggregate were compared. Raman spectra were acquired, using a 532 nm excitation wavelength as the protein samples were heated from 56 to 78 °C and analyzed with PCMW. Initial principal component analysis confirmed a trend between the observed spectral variations and increasing temperature for all five samples. Analysis using PCMW revealed that when spectral variations were directly related to temperature, distinct differences in conformational changes could be determined between samples related to protein stability, providing a greater understanding of the aggregation mechanisms of problematic antibody variants.