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.
Highlights
The thermotolerant yeast Kluyveromyces marxianus is a promising candidate for fuel ethanol production as it possesses several advantageous characteristics for biotechnological applications such as the faster growth rate relative to Kluyveromyces lactis [1] or Saccharomyces cerevisiae [2], the ability to assimilate a wide range of sugars [3], thermotolerance [4,5,6], secretion of lytic enzymes and the ability to produce ethanol at elevated temperatures [7]
Gene cassettes for Promoter-based Gene Assembly and Simultaneous Overexpression (PGASO) technique were amplified with KOD plus DNA polymerase kit (TOYOBO Biotech) with specific primers listed in the Table 1
The selection gene neomycin phosphotransferase G418 (1955 bp) essential for kanamycin resistance was linked with promoter Glyceraldehyde 3phosphate dehydrogenase (GapDH) in the gene cassette 4 KlPGapDH
Summary
The thermotolerant yeast Kluyveromyces marxianus is a promising candidate for fuel ethanol production as it possesses several advantageous characteristics for biotechnological applications such as the faster growth rate relative to Kluyveromyces lactis [1] or Saccharomyces cerevisiae [2], the ability to assimilate a wide range of sugars [3], thermotolerance [4,5,6], secretion of lytic enzymes and the ability to produce ethanol at elevated temperatures [7]. By the Ministry of Science and Technology (https:// www.most.gov.tw/) [104-2621-M-005-003-MY3, 107-2621-M-005-007- MY3, 107-2321-B-005004, and 108-2321-B-005-009 to CCH and 1072621-M-005-001 to SCL
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