A cotransformation system of the unicellular red alga Cyanidioschyzon merolae with blasticidin S deaminase and chloramphenicol acetyltransferase selectable markers

Background The unicellular red alga Cyanidioschyzon merolae exhibits a very simple cellular and genomic architecture. In addition, procedures for genetic modifications, such as gene targeting by homologous recombination and inducible/repressible gene expression, have been developed. However, only two markers for selecting transformants, uracil synthase (URA) and chloramphenicol acetyltransferase (CAT), are available in this alga. Therefore, manipulation of two or more different chromosomal loci in the same strain in C. merolae is limited. Results This study developed a nuclear targeting and transformant selection system using an antibiotics blasticidin S (BS) and the BS deaminase (BSD) selectable marker by homologous recombination in C. merolae. In addition, this study has succeeded in simultaneously modifying two different chromosomal loci by a single-step cotransformation based on the combination of BSD and CAT selectable markers. A C. merolae strain that expresses mitochondrion-targeted mSCARLET (with the BSD marker) and mVENUS (with the CAT marker) from different chromosomal loci was generated with this procedure. Conclusions The newly developed BSD selectable marker enables an additional genetic modification to the already generated C. merolae transformants based on the URA or CAT system. Furthermore, the cotransformation system facilitates multiple genetic modifications. These methods and the simple nature of the C. merolae cellular and genomic architecture will facilitate studies on several phenomena common to photosynthetic eukaryotes.

Structural characterization of a Type B chloramphenicol acetyltransferase from the emerging pathogen Elizabethkingia anophelis NUHP1

Elizabethkingia anophelis is an emerging multidrug resistant pathogen that has caused several global outbreaks. E. anophelis belongs to the large family of Flavobacteriaceae, which contains many bacteria that are plant, bird, fish, and human pathogens. Several antibiotic resistance genes are found within the E. anophelis genome, including a chloramphenicol acetyltransferase (CAT). CATs play important roles in antibiotic resistance and can be transferred in genetic mobile elements. They catalyse the acetylation of the antibiotic chloramphenicol, thereby reducing its effectiveness as a viable drug for therapy. Here, we determined the high-resolution crystal structure of a CAT protein from the E. anophelis NUHP1 strain that caused a Singaporean outbreak. Its structure does not resemble that of the classical Type A CATs but rather exhibits significant similarity to other previously characterized Type B (CatB) proteins from Pseudomonas aeruginosa, Vibrio cholerae and Vibrio vulnificus, which adopt a hexapeptide repeat fold. Moreover, the CAT protein from E. anophelis displayed high sequence similarity to other clinically validated chloramphenicol resistance genes, indicating it may also play a role in resistance to this antibiotic. Our work expands the very limited structural and functional coverage of proteins from Flavobacteriaceae pathogens which are becoming increasingly more problematic.

Single mutation at a highly conserved region of chloramphenicol acetyltransferase enables isobutyl acetate production directly from cellulose by Clostridium thermocellum at elevated temperatures

Background Esters are versatile chemicals and potential drop-in biofuels. To develop a sustainable production platform, microbial ester biosynthesis using alcohol acetyltransferases (AATs) has been studied for decades. Volatility of esters endows high-temperature fermentation with advantageous downstream product separation. However, due to the limited thermostability of AATs known, the ester biosynthesis has largely relied on use of mesophilic microbes. Therefore, developing thermostable AATs is important for ester production directly from lignocellulosic biomass by the thermophilic consolidated bioprocessing (CBP) microbes, e.g., Clostridium thermocellum. Results In this study, we engineered a thermostable chloramphenicol acetyltransferase from Staphylococcus aureus (CATSa) for enhanced isobutyl acetate production at elevated temperatures. We first analyzed the broad alcohol substrate range of CATSa. Then, we targeted a highly conserved region in the binding pocket of CATSa for mutagenesis. The mutagenesis revealed that F97W significantly increased conversion of isobutanol to isobutyl acetate. Using CATSa F97W, we demonstrated direct conversion of cellulose into isobutyl acetate by an engineered C. thermocellum at elevated temperatures. Conclusions This study highlights that CAT is a potential thermostable AAT that can be harnessed to develop the thermophilic CBP microbial platform for biosynthesis of designer bioesters directly from lignocellulosic biomass.

Single mutation at a highly conserved region of chloramphenicol acetyltransferase enables thermophilic isobutyl acetate production directly from cellulose by Clostridium thermocellum

ABSTRACTBackgroundEsters are versatile chemicals and potential drop-in biofuels. To develop a sustainable production platform, microbial ester biosynthesis using alcohol acetyltransferases (AATs) has been studied for decades. Volatility of esters endows thermophilic production with advantageous downstream product separation. However, due to the limited thermal stability of AATs known, the ester biosynthesis has largely relied on use of mesophilic microbes. Therefore, developing thermostable AATs is important for thermophilic ester production directly from lignocellulosic biomass by the thermophilic consolidated bioprocessing (CBP) microbes, e.g., Clostridium thermocellum.ResultsIn this study, we engineered a thermostable chloramphenicol acetyltransferase from Staphylococcus aureus (CATSa) for enhanced isobutyl acetate production at elevated temperature. We first analyzed the broad alcohol substrate range of CATSa. Then, we targeted a highly conserved region in the binding pocket of CATSa for mutagenesis. The mutagenesis revealed that F97W significantly increased conversion of isobutanol to isobutyl acetate. Using CATSa F97W, we demonstrated the engineered C. thermocellum could produce isobutyl acetate directly from cellulose.ConclusionsThis study highlights that CAT is a potential thermostable AAT that can be harnessed to develop the thermophilic CBP microbial platform for biosynthesis of designer bioesters directly from lignocellulosic biomass.