A major challenge in metabolic engineering and synthetic biology is to balance the flux of an engineered heterologous metabolic pathway to achieve high yield and productivity in a target organism. Here, we report a simple, efficient and programmable approach named ‘customized optimization of metabolic pathways by combinatorial transcriptional engineering (COMPACTER)’ for rapid tuning of gene expression in a heterologous pathway under distinct metabolic backgrounds. Specifically, a library of mutant pathways is created by de novo assembly of promoter mutants of varying strengths for each pathway gene in a target organism followed by high-throughput screening/selection. To demonstrate this approach, a single round of COMPACTER was used to generate both a xylose utilizing pathway with near-highest efficiency and a cellobiose utilizing pathway with highest efficiency that were ever reported in literature for both laboratory and industrial yeast strains. Interestingly, these engineered xylose and cellobiose utilizing pathways were all host-specific. Therefore, COMPACTER represents a powerful approach to tailor-make metabolic pathways for different strain backgrounds, which is difficult if not impossible to achieve by existing pathway engineering methods.

Customized optimization of metabolic pathways by combinatorial transcriptional engineering.

Agar is a mixture of heterogeneous galactans, mainly composed of 3,6-anhydro-l-galactoses (or l-galactose-6-sulfates) d-galactoses and l-galactoses (routinely in the forms of 3,6-anhydro-l-galactoses or l-galactose-6-sulfates) alternately linked by β-(1,4) and α-(1,3) linkages. It is a major component of the cell walls of red algae and has been used in a variety of laboratory and industrial applications, owing to its jellifying properties. Many microorganisms that can hydrolyze and metabolize agar as a carbon and energy source have been identified in seawater and marine sediments. Agarolytic microorganisms commonly produce agarases, which catalyze the hydrolysis of agar. Numerous agarases have been identified in microorganisms of various genera. They are classified according to their cleavage pattern into three types—α-agarase, β-agarase, and β-porphyranase. Although, in a broad sense, many other agarases are involved in complete hydrolysis of agar, most of those identified are β-agarases. In this article we review agarolytic microorganisms and their agar-hydrolyzing systems, covering β-agarases as well as α-agarases, α-neoagarobiose hydrolases, and β-porphyranases, with emphasis on the recent discoveries. We also present an overview of the biochemical and structural characteristics of the various types of agarases. Further, we summarize and compare the agar-hydrolyzing systems of two specific microorganisms: Gram-negative Saccharophagus degradans 2–40 and Gram-positive Streptomyces coelicolor A3(2). We conclude with a brief discussion of the importance of agarases and their possible future application in producing oligosaccharides with various nutraceutical activities and in sustainably generating stock chemicals for biorefinement and bioenergy.

Agar degradation by microorganisms and agar-degrading enzymes

The genus Vibrio , belonging to Gammaproteobacteria of the phylum Proteobacteria , is a genetically and ecologically diverse group of heterotrophic bacteria, that are ubiquitous in marine environments, especially in coastal areas. In particular, vibrios dominate, i.e. up to 10% of the readily culturable marine bacteria in these habitats. The distribution of Vibrio spp. is shaped by various environmental parameters, notably temperature, salinity and dissolved organic carbon. Vibrio spp. may utilize a wide range of organic carbon compounds, including chitin (this may be metabolized by most Vibrio spp.), alginic acid and agar. Many Vibrio spp. have very short replication times (as short as ~ 10 min), which could facilitate them developing into high biomass content albeit for relatively short durations. Although Vibrio spp. usually comprise a minor portion (typically ~1% of the total bacterioplankton in coastal waters) of the total microbial population, they have been shown to proliferate explosively in response to various nutrient pulses, e.g., organic nutrients from algae blooms and iron from Saharan dust. Thus, Vibrio spp. may exert large impacts on marine organic carbon cycling especially in marginal seas. Genomics and related areas of investigation will reveal more about the molecular components and mechanisms involved in Vibrio -mediated biotransformation and remineralization processes.

Significance of Vibrio species in the marine organic carbon cycle—A review

Streptomyces coelicolor can degrade agar, the main cell wall component of red macroalgae, for growth. To constitute a crucial carbon source for bacterial growth, the alternating α-(1,3) and β-(1,4) linkages between the 3,6-anhydro-L-galactoses and D-galactoses of agar must be hydrolyzed by α/β-agarases. In S. coelicolor, DagA was confirmed to be an endo-type β-agarase that degrades agar into neoagarotetraose and neoagarohexaose. Genomic sequencing data of S. coelicolor revealed that Sco3487, annotated as a putative hydrolase, has high similarity to the glycoside hydrolase (GH) GH50 β-agarases. Sco3487 encodes a primary translation product (88.5 kDa) of 798 amino acids, including a 45-amino-acid signal peptide. The sco3487 gene was cloned and expressed under the control of the ermE promoter in Streptomyces lividans TK24. β-Agarase activity was detected in transformant culture broth using the artificial chromogenic substrate p-nitrophenyl-β-D-galactopyranoside. Mature Sco3487 (83.9 kDa) was purified 52-fold with a yield of 66% from the culture broth. The optimum pH and temperature for Sco3487 activity were 7.0 and 40°C, respectively. The K(m) and V(max) for agarose were 4.87 mg/ml (4 × 10(-5) M) and 10.75 U/mg, respectively. Sco3487 did not require metal ions for its activity, but severe inhibition by Mn(2+) and Cu(2+) was observed. Thin-layer chromatography analysis, matrix-assisted laser desorption ionization-time of flight mass spectrometry, and Fourier transform-nuclear magnetic resonance spectrometry of the Sco3487 hydrolysis products revealed that Sco3487 is both an exo- and endo-type β-agarase that degrades agarose, neoagarotetraose, and neoagarohexaose into neoagarobiose.

Identification and Biochemical Characterization of Sco3487 from Streptomyces coelicolor A3(2), an Exo- and Endo-Type β-Agarase-Producing Neoagarobiose

Traditional use of enzymes for food processing and production of food ingredients resulted in fast-growing enzyme industries world over The advances in technologies gave rise to exploring newer enzymes and/or modified enzymes for specific application Search for novel enzymes that can augment catalytic efficiency and advances in molecular biology techniques including sequencing has targeted microbial diversity through metagenomic approaches for sourcing enzymes from difficult to culture organisms Such mining studies have received more attention in characterizing hydrolases, their prevalence, broad substrate specificities, stability, and independence of cofactors The focus on glycosyl hydrolases from metagenome for their application in food sector is reviewed

https://onlinelibrary.wiley.com/doi/pdf/10.1111/1750-3841.12677

Diversity of glycosyl hydrolase enzymes from metagenome and their application in food industry.

A new agarase, AgaACN41, cloned from Vibrio sp. strain CN41, consists of 990 amino acids, with only 49% amino acid sequence identity with known β-agarases. AgaACN41 belongs to the GH50 (glycoside hydrolase 50) family but yields neoagarotetraose as the end product. AgaACN41 was expressed and characterized.

Na Yi

Papers

Cloning, Expression, and Characterization of a New β-Agarase from Vibrio sp. Strain CN41