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1. Leveraging synthetic biology for producing bioactive polyketides and non-ribosomal peptides in bacterial heterologous hosts

   https://doi.org/10.1039/C9MD00055K  

   Cook, Taylor B.; Pfleger, Brian F. (May 2019, MedChemComm)

   Bacteria have historically been a rich source of natural products ( e.g. polyketides and non-ribosomal peptides) that possess medically-relevant activities. Despite extensive discovery programs in both industry and academia, a plethora of biosynthetic pathways remain uncharacterized and the corresponding molecular products untested for potential bioactivities. This knowledge gap comes in part from the fact that many putative natural product producers have not been cultured in conventional laboratory settings in which the corresponding products are produced at detectable levels. Next-generation sequencing technologies are further increasing the knowledge gap by obtaining metagenomic sequence information from complex communities where production of the desired compound cannot be isolated in the laboratory. For these reasons, many groups are turning to synthetic biology to produce putative natural products in heterologous hosts. This strategy depends on the ability to heterologously express putative biosynthetic gene clusters and produce relevant quantities of the corresponding products. Actinobacteria remain the most abundant source of natural products and the most promising heterologous hosts for natural product discovery and production. However, researchers are discovering more natural products from other groups of bacteria, such as myxobacteria and cyanobacteria. Therefore, phylogenetically similar heterologous hosts have become promising candidates for synthesizing these novel molecules. The downside of working with these microbes is the lack of well-characterized genetic tools for optimizing expression of gene clusters and product titers. This review examines heterologous expression of natural product gene clusters in terms of the motivations for this research, the traits desired in an ideal host, tools available to the field, and a survey of recent progress. 

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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. Cellulosic biofuel production using emulsified simultaneous saccharification and fermentation (eSSF) with conventional and thermotolerant yeasts

   https://doi.org/10.1186/s13068-021-02008-7  

   Hoffman, Shannon M.; Alvarez, Maria; Alfassi, Gilad; Rein, Dmitry M.; Garcia-Echauri, Sergio; Cohen, Yachin; Avalos, José L. (July 2021, Biotechnology for Biofuels)

   Abstract BackgroundFuture expansion of corn-derived ethanol raises concerns of sustainability and competition with the food industry. Therefore, cellulosic biofuels derived from agricultural waste and dedicated energy crops are necessary. To date, slow and incomplete saccharification as well as high enzyme costs have hindered the economic viability of cellulosic biofuels, and while approaches like simultaneous saccharification and fermentation (SSF) and the use of thermotolerant microorganisms can enhance production, further improvements are needed. Cellulosic emulsions have been shown to enhance saccharification by increasing enzyme contact with cellulose fibers. In this study, we use these emulsions to develop an emulsified SSF (eSSF) process for rapid and efficient cellulosic biofuel production and make a direct three-way comparison of ethanol production betweenS. cerevisiae,O. polymorpha, andK. marxianusin glucose and cellulosic media at different temperatures. ResultsIn this work, we show that cellulosic emulsions hydrolyze rapidly at temperatures tolerable to yeast, reaching up to 40-fold higher conversion in the first hour compared to microcrystalline cellulose (MCC). To evaluate suitable conditions for the eSSF process, we explored the upper temperature limits for the thermotolerant yeastsKluyveromyces marxianusandOgataea polymorpha, as well asSaccharomyces cerevisiae, and observed robust fermentation at up to 46, 50, and 42 °C for each yeast, respectively. We show that the eSSF process reaches high ethanol titers in short processing times, and produces close to theoretical yields at temperatures as low as 30 °C. Finally, we demonstrate the transferability of the eSSF technology to other products by producing the advanced biofuel isobutanol in a light-controlled eSSF using optogenetic regulators, resulting in up to fourfold higher titers relative to MCC SSF. ConclusionsThe eSSF process addresses the main challenges of cellulosic biofuel production by increasing saccharification rate at temperatures tolerable to yeast. The rapid hydrolysis of these emulsions at low temperatures permits fermentation using non-thermotolerant yeasts, short processing times, low enzyme loads, and makes it possible to extend the process to chemicals other than ethanol, such as isobutanol. This transferability establishes the eSSF process as a platform for the sustainable production of biofuels and chemicals as a whole. 

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4. CRISPR-mediated multigene integration enables Shikimate pathway refactoring for enhanced 2-phenylethanol biosynthesis in Kluyveromyces marxianus

   https://doi.org/10.1186/s13068-020-01852-3  

   Li, Mengwan; Lang, Xuye; Moran Cabrera, Marcos; De Keyser, Sawyer; Sun, Xiyan; Da Silva, Nancy; Wheeldon, Ian (December 2021, Biotechnology for Biofuels)

   null (Ed.)

   Abstract Background 2-phenylethanol (2-PE) is a rose-scented flavor and fragrance compound that is used in food, beverages, and personal care products. Compatibility with gasoline also makes it a potential biofuel or fuel additive. A biochemical process converting glucose or other fermentable sugars to 2-PE can potentially provide a more sustainable and economical production route than current methods that use chemical synthesis and/or isolation from plant material. Results We work toward this goal by engineering the Shikimate and Ehrlich pathways in the stress-tolerant yeast Kluyveromyces marxianus . First, we develop a multigene integration tool that uses CRISPR-Cas9 induced breaks on the genome as a selection for the one-step integration of an insert that encodes one, two, or three gene expression cassettes. Integration of a 5-kbp insert containing three overexpression cassettes successfully occurs with an efficiency of 51 ± 9% at the ABZ1 locus and was used to create a library of K. marxianus CBS 6556 strains with refactored Shikimate pathway genes. The 3 3 -factorial library includes all combinations of KmARO4 , KmARO7 , and KmPHA2 , each driven by three different promoters that span a wide expression range. Analysis of the refactored pathway library reveals that high expression of the tyrosine-deregulated KmARO4 K221L and native KmPHA2 , with the medium expression of feedback insensitive KmARO7 G141S , results in the highest increase in 2-PE biosynthesis, producing 684 ± 73 mg/L. Ehrlich pathway engineering by overexpression of KmARO10 and disruption of KmEAT1 further increases 2-PE production to 766 ± 6 mg/L. The best strain achieves 1943 ± 63 mg/L 2-PE after 120 h fed-batch operation in shake flask cultures. Conclusions The CRISPR-mediated multigene integration system expands the genome-editing toolset for K. marxianus, a promising multi-stress tolerant host for the biosynthesis of 2-PE and other aromatic compounds derived from the Shikimate pathway. 

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5. Pyruvate Production by Escherichia coli by Use of Pyruvate Dehydrogenase Variants

   https://doi.org/10.1128/AEM.00487-21  

   Moxley, W. Chris; Eiteman, Mark A. (June 2021, Applied and Environmental Microbiology)

   Atomi, Haruyuki (Ed.)

   ABSTRACT Altering metabolic flux at a key branch point in metabolism has commonly been accomplished through gene knockouts or by modulating gene expression. An alternative approach to direct metabolic flux preferentially toward a product is decreasing the activity of a key enzyme through protein engineering. In Escherichia coli , pyruvate can accumulate from glucose when carbon flux through the pyruvate dehydrogenase complex is suppressed. Based on this principle, 16 chromosomally expressed AceE variants were constructed in E. coli C and compared for growth rate and pyruvate accumulation using glucose as the sole carbon source. To prevent conversion of pyruvate to other products, the strains also contained deletions in two nonessential pathways: lactate dehydrogenase ( ldhA ) and pyruvate oxidase ( poxB ). The effect of deleting phosphoenolpyruvate synthase ( ppsA ) on pyruvate assimilation was also examined. The best pyruvate-accumulating strains were examined in controlled batch and continuous processes. In a nitrogen-limited chemostat process at steady-state growth rates of 0.15 to 0.28 h −1 , an engineered strain expressing the AceE[H106V] variant accumulated pyruvate at a yield of 0.59 to 0.66 g pyruvate/g glucose with a specific productivity of 0.78 to 0.92 g pyruvate/g cells·h. These results provide proof of concept that pyruvate dehydrogenase complex variants can effectively shift carbon flux away from central carbon metabolism to allow pyruvate accumulation. This approach can potentially be applied to other key enzymes in metabolism to direct carbon toward a biochemical product. IMPORTANCE Microbial production of biochemicals from renewable resources has become an efficient and cost-effective alternative to traditional chemical synthesis methods. Metabolic engineering tools are important for optimizing a process to perform at an economically feasible level. This study describes an additional tool to modify central metabolism and direct metabolic flux to a product. We have shown that variants of the pyruvate dehydrogenase complex can direct metabolic flux away from cell growth to increase pyruvate production in Escherichia coli . This approach could be paired with existing strategies to optimize metabolism and create industrially relevant and economically feasible processes. 

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