Many industrial chemicals that are produced from fossil resources could be manufactured more sustainably through fermentation. Here we describe the development of a carbon-negative fermentation route to producing the industrially important chemicals acetone and isopropanol from abundant, low-cost waste gas feedstocks, such as industrial emissions and syngas. Using a combinatorial pathway library approach, we first mined a historical industrial strain collection for superior enzymes that we used to engineer the autotrophic acetogen Clostridium autoethanogenum. Next, we used omics analysis, kinetic modeling and cell-free prototyping to optimize flux. Finally, we scaled-up our optimized strains for continuous production at rates of up to ~3 g/L/h and ~90% selectivity. Life cycle analysis confirmed a negative carbon footprint for the products. Unlike traditional production processes, which result in release of greenhouse gases, our process fixes carbon. These results show that engineered acetogens enable sustainable, high-efficiency, high-selectivity chemicals production. We expect that our approach can be readily adapted to a wide range of commodity chemicals. Two industrial chemicals are sustainably produced at large scale by microbial gas fermentation. 

/pdf/carbon-negative-production-of-acetone-and-isopropanol-by-gas-1m4ydden.pdf

Carbon-negative production of acetone and isopropanol by gas fermentation at industrial pilot scale

Evolutionary warning system for COVID-19 severity: Colony predation algorithm enhanced extreme learning machine.

Valorization of C1 gases to value-added chemicals using acetogenic biocatalysts

Advanced strategies and tools to facilitate and streamline microbial adaptive laboratory evolution.

Synthesis gas, which is mainly produced from fossil fuels or biomass gasification, consists of C1 gases such as carbon monoxide, carbon dioxide, and methane as well as hydrogen. Acetogenic bacteria (acetogens) have emerged as an alternative solution to recycle C1 gases by converting them into value-added biochemicals using the Wood-Ljungdahl pathway. Despite the advantage of utilizing acetogens as biocatalysts, it is difficult to develop industrial-scale bioprocesses because of their slow growth rates and low productivities. To solve these problems, conventional approaches to metabolic engineering have been applied; however, there are several limitations owing to the lack of required genetic bioparts for regulating their metabolic pathways. Recently, synthetic biology based on genetic parts, modules, and circuit design has been actively exploited to overcome the limitations in acetogen engineering. This review covers synthetic biology applications to design and build industrial platform acetogens.

https://www.mdpi.com/1422-0067/21/20/7639/pdf

Synthetic Biology on Acetogenic Bacteria for Highly Efficient Conversion of C1 Gases to Biochemicals.

Acetogens are naturally capable of metabolizing carbon monoxide (CO), a component of synthesis gas (syngas), for autotrophic growth in order to produce biomass and metabolites such as acetyl-CoA via the Wood-Ljungdahl pathway. However, the autotrophic growth of acetogens is often inhibited by the presence of high CO concentrations because of CO toxicity, thus limiting their biosynthetic potential for industrial applications. Herein, we implemented adaptive laboratory evolution (ALE) for growth improvement of Eubacterium limosum ATCC 8486 under high CO conditions. The strain evolved under syngas conditions with 44% CO over 150 generations, resulting in a significant increased optical density (600 nm) and growth rate by 2.14 and 1.44 folds, respectively. In addition, the evolved populations were capable of proliferating under CO concentrations as high as 80%. These results suggest that cell growth is enhanced as beneficial mutations are selected and accumulated, and the metabolism is altered to facilitate the enhanced phenotype. To identify the causal mutations related to growth improvement under high CO concentrations, we performed whole genome resequencing of each population at 50-generation intervals. Interestingly, we found key mutations in CO dehydrogenase/acetyl-CoA synthase (CODH/ACS) complex coding genes, acsA and cooC. To characterize the mutational effects on growth under CO, we isolated single clones and confirmed that the growth rate and CO tolerance level of the single clone were comparable to those of the evolved populations and wild type strain under CO conditions. Furthermore, the evolved strain produced 1.34 folds target metabolite acetoin when compared to the parental strain while introducing the biosynthetic pathway coding genes to the strains. Consequently, this study demonstrates that the mutations in the CODH/ACS complex affect autotrophic growth enhancement in the presence of CO as well as the CO tolerance of E. limosum ATCC 8486.

/pdf/adaptive-laboratory-evolution-of-eubacterium-limosum-atcc-2bkpylpwbq.pdf

Adaptive Laboratory Evolution of Eubacterium limosum ATCC 8486 on Carbon Monoxide

Acetogenic bacteria use cellular redox energy to convert CO2 to acetate using the Wood-Ljungdahl (WL) pathway. Such redox energy can be derived from electrons generated from H2 as well as from inorganic materials, such as photoresponsive semiconductors. We have developed a nanoparticle-microbe hybrid system in which chemically synthesized cadmium sulfide nanoparticles (CdS-NPs) are displayed on the cell surface of the industrial acetogen Clostridium autoethanogenum The hybrid system converts CO2 into acetate without the need for additional energy sources, such as H2, and uses only light-induced electrons from CdS-NPs. To elucidate the underlying mechanism by which C. autoethanogenum uses electrons generated from external energy sources to reduce CO2, we performed transcriptional analysis. Our results indicate that genes encoding the metal ion or flavin-binding proteins were highly up-regulated under CdS-driven autotrophic conditions along with the activation of genes associated with the WL pathway and energy conservation system. Furthermore, the addition of these cofactors increased the CO2 fixation rate under light-exposure conditions. Our results demonstrate the potential to improve the efficiency of artificial photosynthesis systems based on acetogenic bacteria integrated with photoresponsive nanoparticles.

https://www.pnas.org/content/pnas/118/9/e2020552118.full.pdf

Acetogenic bacteria utilize light-driven electrons as an energy source for autotrophic growth.

Acetogens are anaerobic bacteria that utilise gaseous feedstocks such as carbon monoxide (CO) and carbon dioxide (CO2) to synthesise biomass and various metabolites via the energetically efficient Wood-Ljungdahl pathway. Because of this pathway, acetogens have been considered as a novel platform to produce biochemicals from gaseous feedstocks, potentially replacing the conventional thermochemical processes. Despite their advantages, a lack of systematic understanding of the transcriptional and translational regulation in acetogens during autotrophic growth limits the rational strain design to produce the desired products. To overcome this problem, we presented RNA sequencing and ribosome profiling data of four acetogens cultivated under heterotrophic and autotrophic conditions, providing data on genome-scale transcriptional and translational responses of acetogens during CO2 fixation. These data facilitate the discovery of regulatory elements embedded in their genomes, which could be utilised to engineer strains to achieve better growth and productivity. We anticipate that these data will expand our understanding of the processes of CO2 fixation and will help in the designing of strains for the desired biochemical production.

/pdf/transcriptome-and-translatome-of-co-2-fixing-acetogens-under-4e3abu62au.pdf

Jiyun Bae

Papers

Adaptive Laboratory Evolution of Eubacterium limosum ATCC 8486 on Carbon Monoxide

Acetogenic bacteria utilize light-driven electrons as an energy source for autotrophic growth.

Valorization of C1 gases to value-added chemicals using acetogenic biocatalysts

Synthetic Biology on Acetogenic Bacteria for Highly Efficient Conversion of C1 Gases to Biochemicals.

Transcriptome and translatome of CO 2 fixing acetogens under heterotrophic and autotrophic conditions