Discovery of a Substrate Selectivity Switch in Tyrosine Ammonia-Lyase, a Member of the Aromatic Amino Acid Lyase Family

SummaryTyrosine ammonia-lyase (TAL) is a recently described member of the aromatic amino acid lyase family, which also includes phenylalanine (PAL) and histidine ammonia-lyases (HAL). TAL is highly selective for L-tyrosine, and synthesizes 4-coumaric acid as a protein cofactor or antibiotic precursor in microorganisms. In this report, we identify a single active site residue important for substrate selection in this enzyme family. Replacing the active site residue His89 with Phe in TAL completely switched its substrate selectivity from tyrosine to phenylalanine, thereby converting it into a highly active PAL. When a corresponding mutation was made in PAL, the enzyme lost PAL activity and gained TAL activity. The discovered substrate selectivity switch is a rare example of a complete alteration of substrate specificity by a single point mutation. We also show that the identity of the amino acid at the switch position can serve as a guide to predict substrate specificities of annotated aromatic amino acid lyases in genome sequences

Differences in the Fatty Acid Profile, Morphology, and Tetraacetylphytosphingosine-Forming Capability Between Wild-Type and Mutant Wickerhamomyces ciferrii

One tetraacetylphytosphingosine (TAPS)-producing Wickerhamomyces ciferrii mutant was obtained by exposing wild-type W. ciferrii to γ-ray irradiation. The mutant named 736 produced up to 9.1 g/L of TAPS (218.7 mg-TAPS/g-DCW) during batch fermentation in comparison with 1.7 g/L of TAPS (52.2 mg-TAPS/g-DCW) for the wild type. The highest production, 17.7 g/L of TAPS (259.6 mg-TAPS/g-DCW), was obtained during fed-batch fermentation by mutant 736. Fatty acid (FA) analysis revealed an altered cellular FA profile of mutant 736: decrease in C16:0 and C16:1 FA levels, and increase in C18:1 and C18:2 FA levels. Although a significant change in the cellular FA profile was observed, scanning electron micrographs showed that morphology of wild-type and mutant 736 cells was similar. Genetic alteration analysis of eight TAPS biosynthesis-related genes revealed that there are no mutations in these genes in mutant 736; however, mRNA expression analysis indicated 30% higher mRNA expression of TCS10 among the eight genes in mutant 736 than that in the wild-type. Collectively, these results imply that the enhancement of TAPS biosynthesis in mutant 736 may be a consequence of system-level genetic and physiological alterations of a complicated metabolic network. Reverse metabolic engineering based on system-level omics analysis of mutant 736 can make the mutant more suitable for commercial production of TAPS

Microbial Production of Bioactive Retinoic Acid Using Metabolically Engineered Escherichia coli

Microbial production of bioactive retinoids, including retinol and retinyl esters, has been successfully reported. Previously, there are no reports on the microbial biosynthesis of retinoic acid. Two genes (blhSR and raldhHS) encoding retinoic acid biosynthesis enzymes [β-carotene 15,15′-oxygenase (Blh) and retinaldehyde dehydrogenase2 (RALDH2)] were synthetically redesigned for modular expression. Co-expression of the blhSR and raldhHS genes on the plasmid system in an engineered β-carotene-producing Escherichia coli strain produced 0.59 ± 0.06 mg/L of retinoic acid after flask cultivation. Deletion of the ybbO gene encoding a promiscuous aldehyde reductase induced a 2.4-fold increase in retinoic acid production to 1.43 ± 0.06 mg/L. Engineering of the 5’-UTR sequence of the blhSR and raldhHS genes enhanced retinoic acid production to 3.46 ± 0.16 mg/L. A batch culture operated at 37 °C, pH 7.0, and 50% DO produced up to 8.20 ± 0.05 mg/L retinoic acid in a bioreactor. As the construction and culture of retinoic acid–producing bacterial strains are still at an early stage in the development, further optimization of the expression level of the retinoic acid pathway genes, protein engineering of Blh and RALDH2, and culture optimization should synergistically increase the current titer of retinoic acid in E. coli

Metagenomic Analysis of Antarctic Ocean near the King Sejong Station Reveals the Diversity of Carotenoid Biosynthetic Genes

Carotenoids, biotechnologically significant pigments, play crucial biological roles in marine microorganisms. While various environments have been explored to understand the diversity of carotenoids and their biosynthesis, the Antarctic Ocean remains relatively under-investigated. This study conducted a metagenomic analysis of seawater from two depths (16 and 25 m) near the King Sejong Station in the Antarctic Ocean. The analysis revealed a rich genetic diversity underlying C40 (astaxanthin, myxol, okenone, spheroidene, and spirilloxanthin), C30 (diaponeurosporene, diapolycopene, and staphyloxanthin), and C50 (C.p. 450) carotenoid biosynthesis in marine microorganisms, with notable differential gene abundances between depth locations. Exploring carotenoid pathway genes offers the potential for discovering diverse carotenoid structures of biotechnological value and better understanding their roles in individual microorganisms and broader ecosystems

Characterization of Carotenoid Biosynthesis in Newly Isolated Deinococcus sp. AJ005 and Investigation of the Effects of Environmental Conditions on Cell Growth and Carotenoid Biosynthesis

Our purpose was to characterize the structures of deinoxanthin from Deinococcus sp. AJ005. The latter is a novel reddish strain and was found to synthesize two main acyclic carotenoids: deinoxanthin and its derivative. The derivative (2-keto-deinoxanthin) contains a 2-keto functional group instead of a 2-hydroxyl group on a β-ionone ring. A deinoxanthin biosynthesis pathway of Deinococcus sp. AJ005 involving eight putative enzymes was proposed according to genome annotation analysis and chemical identification of deinoxanthin. Optimal culture pH and temperature for Deinococcus sp. AJ005 growth were pH 7.4 and 20 °C. Sucrose as a carbon source significantly enhanced the cell growth in comparison with glucose, glycerol, maltose, lactose, and galactose. When batch fermentation was performed in a bioreactor containing 40g/L sucrose, total carotenoid production was 650% higher than that in a medium without sucrose supplementation. The culture conditions found in this study should provide the basis for the development of fermentation strategies for the production of deinoxanthin and of its derivative by means of Deinococcus sp. AJ005

Engineering and application of synthetic nar promoter for fine-tuning the expression of metabolic pathway genes in Escherichia coli

Abstract Background Promoters regulate the expression of metabolic pathway genes to control the flux of metabolism. Therefore, fine-tuning of metabolic pathway gene expression requires an applicable promoter system. In this study, a dissolved oxygen-dependent nar promoter was engineered for fine-tuning the expression levels of biosynthetic pathway enzymes in Escherichia coli. To demonstrate the feasibility of using the synthetic nar promoters in production of biochemicals in E. coli, the d-lactate pathway consisting of one enzyme and the 2,3-butanediol (BDO) pathway consisting of three enzymes were investigated. Results The spacer sequence of 15 bp between the − 35 and − 10 elements of the upstream region of the wild-type nar promoter was randomized, fused to the GFP gene, transduced into E. coli, and screened by flow cytometry. The sorted synthetic nar promoters were divided into three groups according to fluorescence intensity levels: strong, intermediate, and weak. The selected three representative nar promoters of strong, intermediate, and weak intensities were used to control the expression level of the d-lactate and 2,3-BDO biosynthetic pathway enzymes in E. coli. When the ldhD gene encoding d-lactate dehydrogenase was expressed under the control of the strong synthetic nar promoter in fed-batch cultures of E. coli, the d-lactate titers were 105.6 g/L, 34% higher than those using the wild-type promoter (79.0 g/L). When the three 2,3-BDO pathway genes (ilvBN, aldB, and bdh1) were expressed under the control of combinational synthetic nar promoters (strong–weak–strong) in fed-batch cultures of E. coli, the titers of 2,3-BDO were 88.0 g/L, 72% higher than those using the wild-type promoter (51.1 g/L). Conclusions The synthetic nar promoters, which were engineered to have strong, intermediate, and weak intensities, were successfully applied to metabolic engineering of d-lactate and 2,3-BDO pathways in E. coli. By controlling expression levels of d-lactate and 2,3-BDO pathway enzymes using the synthetic nar promoters, the production of d-lactate and 2,3-BDO was increased over that using the wild-type promoter by 34 and 72%, respectively. Thus, this synthetic promoter module system will support the improved production of biochemicals and biofuels through fine-tuning of gene expression levels

Comparative Genomic Analysis of Agarolytic Flavobacterium faecale WV33T

Flavobacteria are widely dispersed in a variety of environments and produce various polysaccharide-degrading enzymes. Here, we report the complete genome of Flavobacterium faecale WV33T, an agar-degrading bacterium isolated from the stools of Antarctic penguins. The sequenced genome of F. faecale WV33T represents a single circular chromosome (4,621,116 bp, 35.2% G + C content), containing 3984 coding DNA sequences and 85 RNA-coding genes. The genome of F. faecale WV33T contains 154 genes that encode carbohydrate-active enzymes (CAZymes). Among the CAZymes, seven putative genes encoding agarases have been identified in the genome. Transcriptional analysis revealed that the expression of these putative agarases was significantly enhanced by the presence of agar in the culture medium, suggesting that these proteins are involved in agar hydrolysis. Pangenome analysis revealed that the genomes of the 27 Flavobacterium type strains, including F. faecale WV33T, tend to be very plastic, and Flavobacterium strains are unique species with a tiny core genome and a large non-core region. The average nucleotide identity and phylogenomic analysis of the 27 Flavobacterium-type strains showed that F. faecale WV33T was positioned in a unique clade in the evolutionary tree

Hot Spots of Phytoene Desaturase from Rhodobacter sphaeroides Influencing the Desaturation of Phytoene

Phytoene desaturase (CrtI, E.C. 1.3.99.31) shows variable desaturation activity, thereby introducing different numbers of conjugated double bonds (CDB) into the substrate phytoene. In particular, Rhodobacter sphaeroides CrtI is known to introduce additional 6 CDBs into the phytoene with 3 CDBs, generating neurosporene with 9 CDBs. Although in-depth studies have been conducted on the function and phylogenetic evolution of CrtI, little information exists on its range of CDB-introducing capabilities. We investigated the relationship between the structure and CDB-introducing capability of CrtI. CrtI of R. sphaeroides KCTC 12085 was randomly mutagenized to produce carotenoids of different CDBs (neurosporene for 9 CDBs, lycopene for 11 CDBs, and 3,4-didehydrolycopene for 13 CDBs). From six CrtI mutants producing different ratios of neurosporene/lycopene/3,4-didehydrolycopene, three amino acids (Leu163, Ala171, and Ile454) were identified that significantly determined carotenoid profiles. While the L163P mutation was responsible for producing neurosporene as a major carotenoid, A171P and I454T produced lycopene as the major product. Finally, according to the in silico model, the mutated amino acids are gathered in the membrane-binding domain of CrtI, which could distantly influence the FAD binding region and consequently the degree of desaturation in phytoene