ABSTRACT
The maize glossy2 and glossy2-like genes are homologs, which encode proteins that belong to the BAHD family of acyltransferases. In planta genetic studies have demonstrated that these genes may be involved in the elongation of very long chain fatty acids (VLCFAs) that are precursors of the cuticular wax fraction of the plant cuticle. VLCFAs are synthesized by a fatty acyl-CoA elongase complex (FAE) that consists of four component enzymes. Previously, we functionally identified the maize FAE component enzymes by their ability to complement haploid Saccharomyces cerevisiae strains that carry lethal deletion alleles for each FAE component enzyme. In this study we used these complemented haploid strains and wild-type diploid strains to evaluate whether the co-expression of either GLOSSY2 or GLOSSY2-LIKE with individual maize FAE component enzymes affects the VLCFA product-profile of the FAE system. Wild-type diploid strains produced VLCFAs of up to 28-carbon chain length. Co-expression of GLOSSY2 or GLOSSY2-LIKE with a combination of maize 3-ketoacyl-CoA synthases stimulated the synthesis of longer VLCFAs, up to 30-carbon chain lengths. However, such results could not be recapitulated when these co-expression experiments were conducted in the yeast haploid mutant strains that lacked individual components of the endogenous FAE system. Specifically, lethal yeast mutant strains that are genetically complemented by the expression of maize FAE-component enzymes produce VLCFAs that range between 20- and 26-carbon chain lengths. However, expressing either GLOSSY2 or GLOSSY2-LIKE in these complemented strains does not enable the synthesis of longer chain VLCFAs. These results indicate that the apparent stimulatory role of GLOSSY2 or GLOSSY2-LIKE to enable the synthesis of longer chain VLCFAs in diploid yeast cells may be associated with mixing plant enzyme components with the endogenous FAE complex.
ABSTRACT
Consolidated bioprocessing (CBP) of lignocellulosic biomass holds promise to realize economic production of second-generation biofuels/chemicals, and Clostridium thermocellum is a leading candidate for CBP due to it being one of the fastest degraders of crystalline cellulose and lignocellulosic biomass. However, CBP by C. thermocellum is approached with co-cultures, because C. thermocellum does not utilize hemicellulose. When compared with a single-species fermentation, the co-culture system introduces unnecessary process complexity that may compromise process robustness. In this study, we engineered C. thermocellum to co-utilize hemicellulose without the need for co-culture. By evolving our previously engineered xylose-utilizing strain in xylose, an evolved clonal isolate (KJC19-9) was obtained and showed improved specific growth rate on xylose by â¼3-fold and displayed comparable growth to a minimally engineered strain grown on the bacteria's naturally preferred substrate, cellobiose. To enable full xylan deconstruction to xylose, we recombinantly expressed three different ß-xylosidase enzymes originating from Thermoanaerobacterium saccharolyticum into KJC19-9 and demonstrated growth on xylan with one of the enzymes. This recombinant strain was capable of co-utilizing cellulose and xylan simultaneously, and we integrated the ß-xylosidase gene into the KJC19-9 genome, creating the KJCBXint strain. The strain, KJC19-9, consumed monomeric xylose but accumulated xylobiose when grown on pretreated corn stover, whereas the final KJCBXint strain showed significantly greater deconstruction of xylan and xylobiose. This is the first reported C. thermocellum strain capable of degrading and assimilating hemicellulose polysaccharide while retaining its cellulolytic capabilities, unlocking significant potential for CBP in advancing the bioeconomy.
Subject(s)
Clostridium thermocellum , Metabolic Engineering , Polysaccharides , Clostridium thermocellum/metabolism , Clostridium thermocellum/genetics , Polysaccharides/metabolism , Polysaccharides/genetics , Xylose/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Cellulose/metabolism , Xylosidases/metabolism , Xylosidases/geneticsABSTRACT
Acetyl-CoA synthetase (ACS) is one of several enzymes that generate the key metabolic intermediate, acetyl-CoA. In microbes and mammals ACS activity is regulated by the post-translational acetylation of a key lysine residue. ACS in plant cells is part of a two-enzyme system that maintains acetate homeostasis, but its post-translational regulation is unknown. This study demonstrates that the plant ACS activity can be regulated by the acetylation of a specific lysine residue that is positioned in a homologous position as the microbial and mammalian ACS sequences that regulates ACS activity, occurring in the middle of a conserved motif, near the carboxyl-end of the protein. The inhibitory effect of the acetylation of residue Lys-622 of the Arabidopsis ACS was demonstrated by site-directed mutagenesis of this residue, including its genetic substitution with the non-canonical N-ε-acetyl-lysine residue. This latter modification lowered the catalytic efficiency of the enzyme by a factor of more than 500-fold. Michaelis-Menten kinetic analysis of the mutant enzyme indicates that this acetylation affects the first half-reaction of the ACS catalyzed reaction, namely, the formation of the acetyl adenylate enzyme intermediate. The post-translational acetylation of the plant ACS could affect acetate flux in the plastids and overall acetate homeostasis.
ABSTRACT
The aerobic, thermophilic Actinobacterium, Thermobifida fusca has been proposed as an organism to be used for the efficient conversion of plant biomass to fatty acid-derived precursors of biofuels or biorenewable chemicals. Despite the potential of T. fusca to catabolize plant biomass, there is remarkably little data available concerning the natural ability of this organism to produce fatty acids. Therefore, we determined the fatty acids that T. fusca produces when it is grown on different carbon sources (i.e., glucose, cellobiose, cellulose and avicel) and at two different growth temperatures, namely at the optimal growth temperature of 50°C and at a suboptimal temperature of 37°C. These analyses establish that T. fusca produces a combination of linear and branched chain fatty acids (BCFAs), including iso-, anteiso-, and 10-methyl BCFAs that range between 14- and 18-carbons in length. Although different carbon sources and growth temperatures both quantitatively and qualitatively affect the fatty acid profiles produced by T. fusca, growth temperature is the greater modifier of these traits. Additionally, genome scanning enabled the identification of many of the fatty acid biosynthetic genes encoded by T. fusca.