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1. A Perspective in Future Biomanufacturing: Challenges in Industrial Fermentation—Understanding and Controlling Microbial Lifespan and Aging

   https://doi.org/10.35534/sbe.2023.10019  

   Yang, Shang-Tian; Wang, Geng; Qin, Zhen (December 2023, Synthetic Biology and Engineering)

   Zeng, A; Yang, ST (Ed.)

   Biomanufacturing with broad applications in various industries is projected to reach a market value of ~30 trillion USD by 2030, accounting for more than one third of the global manufacturing output. Future biomanufacturing of industrial products will use novel synthetic biology tools and advanced bioprocesses to convert abundant biomass and waste resources into value-added products with comparable or superior properties to replace current petroleum-based products, thus enabling circular bioeconomy with affordable energy, economic growth, and innovation in renewable energy and chemicals production. However, biomanufacturing faces many challenges in its development that requires fundamental research in synthetic biology and novel bioprocesses involving multidisciplinary teams and academic-industry partnerships. In particular, aging and lifespan of microbial cells have been largely overlooked in industrial fermentation. Only recently have microbiologists realized that many microorganisms including yeasts (e.g., Saccharomyces cerevisiae) and bacteria (e.g., Escherichia coli) have chronological and replicative life spans which dramatically impact cell viability and longevity. In this article, we will give our perspective on how synthetic biology may contribute to overcoming some challenges facing industrial biotechnology for fuels and chemicals production from renewable sources, highlighting the importance of understanding and regulating microorganism’s lifespan and aging. 

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2. Hydrolysis of lignocellulose by anaerobic fungi produces free sugars and organic acids for two‐stage fine chemical production with Kluyveromyces marxianus

   https://doi.org/10.1002/btpr.3172  

   Hillman, Ethan_T; Li, Mengwan; Hooker, Casey_A; Englaender, Jacob_A; Wheeldon, Ian; Solomon, Kevin_V (May 2021, Biotechnology Progress)

   Abstract Development of the bioeconomy is driven by our ability to access the energy‐rich carbon trapped in recalcitrant plant materials. Current strategies to release this carbon rely on expensive enzyme cocktails and physicochemical pretreatment, producing inhibitory compounds that hinder subsequent microbial bioproduction. Anaerobic fungi are an appealing solution as they hydrolyze crude, untreated biomass at ambient conditions into sugars that can be converted into value‐added products by partner organisms. However, some carbon is lost to anaerobic fungal fermentation products. To improve efficiency and recapture this lost carbon, we built a two‐stage bioprocessing system pairing the anaerobic fungusPiromyces indianaewith the yeastKluyveromyces marxianus, which grows on a wide range of sugars and fermentation products. In doing so we produce fine and commodity chemicals directly from untreated lignocellulose.P.indianaeefficiently hydrolyzed substrates such as corn stover and poplar to generate sugars, fermentation acids, and ethanol, whichK.marxianusconsumed while producing 2.4 g/L ethyl acetate. An engineered strain ofK.marxianuswas also able to produce 550 mg/L 2‐phenylethanol and 150 mg/L isoamyl alcohol fromP.indianaehydrolyzed lignocellulosic biomass. Despite the use of crude untreated plant material, production yields were comparable to optimized rich yeast media due to the use of all available carbon including organic acids, which formed up to 97% of free carbon in the fungal hydrolysate. This work demonstrates that anaerobic fungal pretreatment of lignocellulose can sustain the production of fine chemicals at high efficiency by partnering organisms with broad substrate versatility. 

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3. Reverse β-oxidation pathways for efficient chemical production

   https://doi.org/10.1093/jimb/kuac003  

   Tarasava, Katia; Lee, Seung Hwan; Chen, Jing; Köpke, Michael; Jewett, Michael C; Gonzalez, Ramon (February 2022, Journal of Industrial Microbiology and Biotechnology)

   Abstract Microbial production of fuels, chemicals, and materials has the potential to reduce greenhouse gas emissions and contribute to a sustainable bioeconomy. While synthetic biology allows readjusting of native metabolic pathways for the synthesis of desired products, often these native pathways do not support maximum efficiency and are affected by complex regulatory mechanisms. A synthetic or engineered pathway that allows modular synthesis of versatile bioproducts with minimal enzyme requirement and regulation while achieving high carbon and energy efficiency could be an alternative solution to address these issues. The reverse β-oxidation (rBOX) pathways enable iterative non-decarboxylative elongation of carbon molecules of varying chain lengths and functional groups with only four core enzymes and no ATP requirement. Here, we describe recent developments in rBOX pathway engineering to produce alcohols and carboxylic acids with diverse functional groups, along with other commercially important molecules such as polyketides. We discuss the application of rBOX beyond the pathway itself by its interfacing with various carbon-utilization pathways and deployment in different organisms, which allows feedstock diversification from sugars to glycerol, carbon dioxide, methane, and other substrates. 

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4. Comparison of metagenomes from fermentation of various agroindustrial residues suggests a common model of community organization

   https://doi.org/10.3389/fbioe.2023.1197175  

   Myers, Kevin S.; Ingle, Abel T.; Walters, Kevin A.; Fortney, Nathaniel W.; Scarborough, Matthew J.; Donohue, Timothy J.; Noguera, Daniel R. (May 2023, Frontiers in Bioengineering and Biotechnology)

   Strik, David (Ed.)

   The liquid residue resulting from various agroindustrial processes is both rich in organic material and an attractive source to produce a variety of chemicals. Using microbial communities to produce chemicals from these liquid residues is an active area of research, but it is unclear how to deploy microbial communities to produce specific products from the different agroindustrial residues. To address this, we fed anaerobic bioreactors one of several agroindustrial residues (carbohydrate-rich lignocellulosic fermentation conversion residue, xylose, dairy manure hydrolysate, ultra-filtered milk permeate, and thin stillage from a starch bioethanol plant) and inoculated them with a microbial community from an acid-phase digester operated at the wastewater treatment plant in Madison, WI, United States. The bioreactors were monitored over a period of months and sampled to assess microbial community composition and extracellular fermentation products. We obtained metagenome assembled genomes (MAGs) from the microbial communities in each bioreactor and performed comparative genomic analyses to identify common microorganisms, as well as any community members that were unique to each reactor. Collectively, we obtained a dataset of 217 non-redundant MAGs from these bioreactors. This metagenome assembled genome dataset was used to evaluate whether a specific microbial ecology model in which medium chain fatty acids (MCFAs) are simultaneously produced from intermediate products (e.g., lactic acid) and carbohydrates could be applicable to all fermentation systems, regardless of the feedstock. MAGs were classified using a multiclass classification machine learning algorithm into three groups, organisms fermenting the carbohydrates to intermediate products, organisms utilizing the intermediate products to produce MCFAs, and organisms producing MCFAs directly from carbohydrates. This analysis revealed common biological functions among the microbial communities in different bioreactors, and although different microorganisms were enriched depending on the agroindustrial residue tested, the results supported the conclusion that the microbial ecology model tested was appropriate to explain the MCFA production potential from all agricultural residues. 

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5. Engineering of a Highly Efficient Escherichia coli Strain for Mevalonate Fermentation through Chromosomal Integration

   https://doi.org/10.1128/AEM.02178-16  

   Wang, Jilong; Niyompanich, Suthamat; Tai, Yi-Shu; Wang, Jingyu; Bai, Wenqin; Mahida, Prithviraj; Gao, Tuo; Zhang, Kechun; Vieille, C. (November 2016, Applied and Environmental Microbiology)

   ABSTRACT Chromosomal integration of heterologous metabolic pathways is optimal for industrially relevant fermentation, as plasmid-based fermentation causes extra metabolic burden and genetic instabilities. In this work, chromosomal integration was adapted for the production of mevalonate, which can be readily converted into β-methyl-δ-valerolactone, a monomer for the production of mechanically tunable polyesters. The mevalonate pathway, driven by a constitutive promoter, was integrated into the chromosome of Escherichia coli to replace the native fermentation gene adhE or ldhA . The engineered strains (CMEV-1 and CMEV-2) did not require inducer or antibiotic and showed slightly higher maximal productivities (0.38 to ∼0.43 g/liter/h) and yields (67.8 to ∼71.4% of the maximum theoretical yield) than those of the plasmid-based fermentation. Since the glycolysis pathway is the first module for mevalonate synthesis, atpFH deletion was employed to improve the glycolytic rate and the production rate of mevalonate. Shake flask fermentation results showed that the deletion of atpFH in CMEV-1 resulted in a 2.1-fold increase in the maximum productivity. Furthermore, enhancement of the downstream pathway by integrating two copies of the mevalonate pathway genes into the chromosome further improved the mevalonate yield. Finally, our fed-batch fermentation showed that, with deletion of the atpFH and sucA genes and integration of two copies of the mevalonate pathway genes into the chromosome, the engineered strain CMEV-7 exhibited both high maximal productivity (∼1.01 g/liter/h) and high yield (86.1% of the maximum theoretical yield, 30 g/liter mevalonate from 61 g/liter glucose after 48 h in a shake flask). IMPORTANCE Metabolic engineering has succeeded in producing various chemicals. However, few of these chemicals are commercially competitive with the conventional petroleum-derived materials. In this work, chromosomal integration of the heterologous pathway and subsequent optimization strategies ensure stable and efficient (i.e., high-titer, high-yield, and high-productivity) production of mevalonate, which demonstrates the potential for scale-up fermentation. Among the optimization strategies, we demonstrated that enhancement of the glycolytic flux significantly improved the productivity. This result provides an example of how to tune the carbon flux for the optimal production of exogenous chemicals. 

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