Construction of engineered RuBisCO Kluyveromyces marxianus for a dual microbial bioethanol production system

Abstract

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) genes play important roles in CO2 fixation and redox balancing in photosynthetic bacteria. In the present study, the kefir yeast Kluyveromyces marxianus 4G5 was used as host for the transformation of form I and form II RubisCO genes derived from the nonsulfur purple bacterium Rhodopseudomonas palustris using the Promoter-based Gene Assembly and Simultaneous Overexpression (PGASO) method. Hungateiclostridium thermocellum ATCC 27405, a well-known bacterium for its efficient solubilization of recalcitrant lignocellulosic biomass, was used to degrade Napier grass and rice straw to generate soluble fermentable sugars. The resultant Napier grass and rice straw broths were used as growth media for the engineered K. marxianus. In the dual microbial system, H. thermocellum degraded the biomass feedstock to produce both C5 and C6 sugars. As the bacterium only used hexose sugars, the remaining pentose sugars could be metabolized by K. marxianus to produce ethanol. The transformant RubisCO K. marxianus strains grew well in hydrolyzed Napier grass and rice straw broths and produced bioethanol more efficiently than the wild type. Therefore, these engineered K. marxianus strains could be used with H. thermocellum in a bacterium-yeast coculture system for ethanol production directly from biomass feedstocks.

RNA polymerase II-driven CRISPR-Cas9 system for efficient non-growth-biased metabolic engineering of Kluyveromyces marxianus

“…Others have been successful at integrating more than one gene into a single site in K. marxianus , but the strategies required use of either complex PCR schemes to assemble multiple genes into a single donor fragment with very large homology sequences ( Li et al, 2021 ), or required simultaneous integration of a selection marker ( Chang et al, 2012 ; Ha-Tran et al, 2021 ; Lee et al, 2020 ; Lin et al, 2017 ). This study marks the first demonstration of dual markerless integrations at multiple sites in K. marxianus .…”

Standardization of Synthetic Biology Tools and Assembly Methods for Saccharomyces cerevisiae and Emerging Yeast Species

“… Yarrowia lipolytica is an oleaginous yeast species that is also widely used for metabolic engineering and biotechnological production especially for lipid-based, high-value products thanks to its lipid storage capabilities. − It has been also studied for the production of nonlipid chemicals such as sugar products, organic acids, and aromatic compounds. − For this reason, the availability of genetic tools plays an important role in the development of SynBio feasibilities for this important host Kluyveromyces marxianus is another nonconventional yeast with favorable characteristics, like utilizing a wide range of sugars, , hydrolyzing complex plant fructans, and growing at relatively high temperatures (>40 °C). , K. marxianus can also be used as an alternative platform for the production of valuable compounds such as biofuels, fragrances, and flavors. , Therefore, characterized parts and tools are needed to accelerate metabolic engineering in K. marxianus . …”

“…Kluyveromyces marxianus is another nonconventional yeast with favorable characteristics, like utilizing a wide range of sugars, , hydrolyzing complex plant fructans, and growing at relatively high temperatures (>40 °C). , K. marxianus can also be used as an alternative platform for the production of valuable compounds such as biofuels, fragrances, and flavors. , Therefore, characterized parts and tools are needed to accelerate metabolic engineering in K. marxianus .…”

“…122,123 K. marxianus can also be used as an alternative platform for the production of valuable compounds such as biofuels, fragrances, and flavors. 124,125 Therefore, characterized parts and tools are needed to accelerate metabolic engineering in K. marxianus. 126 which is also compatible with the hierarchical MoClo method (Figure 7).…”

Clostridium thermocellum as a Promising Source of Genetic Material for Designer Cellulosomes: An Overview

“…Generally, the incorporation of more catalytic enzymes onto the primary Scaf results in higher synergy for substrate decomposition [70]. The thermotolerant K. marxianus has several advantages (e.g., a wide range of sugars assimilation, thermo, salinity, and toxin tolerance, a wide range of pH, high ethanol yield at elevated temperature) for industrial applications [71,72], and therefore it could be used as an alternative to S. cerevisiae in specific conditions. Recently, the anchoring protein OlpB was successfully introduced and expressed in K. marxianus cells by Anandharaj and coworkers [73].…”