Evolution of protein molecules. I. Protein synthesis

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

Halophilic archaea, also referred to as haloarchaea, dominate hypersaline environments To survive under such extreme conditions, haloarchaea and their enzymes have evolved to function optimally in environments with high salt concentrations and, sometimes, with extreme pH and temperatures These features make haloarchaea attractive sources of a wide variety of biotechnological products, such as hydrolytic enzymes, with numerous potential applications in biotechnology The unique trait of haloarchaeal enzymes, haloenzymes, to sustain activity under hypersaline conditions has extended the range of already-available biocatalysts and industrial processes in which high salt concentrations inhibit the activity of regular enzymes In addition to their halostable properties, haloenzymes can also withstand other conditions such as extreme pH and temperature In spite of these benefits, the industrial potential of these natural catalysts remains largely unexplored, with only a few characterized extracellular hydrolases Because of the applied impact of haloarchaea and their specific ability to live in the presence of high salt concentrations, studies on their systematics have intensified in recent years, identifying many new genera and species This review summarizes the current status of the haloarchaeal genera and species, and discusses the properties of haloenzymes and their potential industrial applications

Systematics of haloarchaea and biotechnological potential of their hydrolytic 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

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.

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

Alcohol dehydrogenase (ADH; EC: 1111) is a key enzyme in production and utilization of ethanol In this study, the gene encoding for ADH of the haloalkaliphilic archaeon Natronomonas pharaonis (NpADH), which has a 1,068-bp open reading frame that encodes a protein of 355 amino acids, was cloned into the pET28b vector and was expressed in Escherichia coli Then, NpADH was purified by Ni-NTA affinity chromatography The recombinant enzyme showed a molecular mass of 413 kDa by SDS-PAGE The enzyme was haloalkaliphilic and thermophilic, being most active at 5 M NaCl or 4 M KCl and 70°C, respectively The optimal pH was 90 Zn2+ significantly inhibited activity The K
 m value for acetaldehyde was higher than that for ethanol It was concluded that the physiological role of this enzyme is likely the catalysis of the oxidation of ethanol to acetaldehyde

Characterization of alcohol dehydrogenase from the haloalkaliphilic archaeon Natronomonas pharaonis

Two strains, CN44(T) and CN47(T), isolated from marine sediment of the East China Sea, were characterized by using a polyphasic approach. The isolates were Gram-negative, strictly aerobic, non-spore-forming rods. The chemotaxonomic characteristics of these isolates included the presence of C(18 : 1)omega7c, C(16 : 0), iso-C(15 : 0) 2-OH and/or C(16 : 1)omega7c and C(10 : 0) 3-OH as the major cellular fatty acids and Q-8 as the predominant ubiquinone. The DNA G+C contents of strains CN44(T) and CN47(T) were 62.5 and 56.3 mol%, respectively. Phylogenetic analyses based on 16S rRNA gene sequences revealed that strain CN44(T) was related to members of the genus Marinobacterium. The most closely related described organism was the type strain of Marinobacterium rhizophilum (95.3 % sequence similarity). Strain CN47(T) showed the highest sequence similarity to the type strain of Marinobacterium stanieri (97.8 %) and <97 % similarity to other type strains of described Marinobacterium species. The level of DNA-DNA relatedness between strain CN47(T) and M. stanieri DSM 7027(T) was 46 %. On the basis of phenotypic and genotypic properties, strains CN44(T) and CN47(T) represent two novel species within the genus Marinobacterium, for which the names Marinobacterium nitratireducens sp. nov. (type strain, CN44(T) =CGMCC 1.7286(T) =JCM 15523(T)) and Marinobacterium sediminicola sp. nov. (type strain, CN47(T) =CGMCC 1.7287(T) =JCM 15524(T)) are proposed.

Marinobacterium nitratireducens sp. nov. and Marinobacterium sediminicola sp. nov., isolated from marine sediment.

Methylotrophic methanogenesis predominates at low temperatures in the cold Zoige wetland in Tibet. To elucidate the basis of cold-adapted methanogenesis in these habitats, Methanosarcina mazei zm-15 was isolated, and the molecular basis of its cold activity was studied. For this strain, aceticlastic methanogenesis was reduced 7.7-fold during growth at 15°C versus 30°C. Methanol-derived methanogenesis decreased only 3-fold under the same conditions, suggesting that it is more cold adaptive. Reverse transcription-quantitative PCR (RT-qPCR) detected <2-fold difference in the transcript abundances of mtaA1, mtaB1, and mtaC1, the methanol methyltransferase (Mta) genes, in 30°C versus 15°C culture, while ackA and pta mRNAs, encoding acetate kinase (Ack) and phosphotransacetylase (Pta) in aceticlastic methanogenesis, were 4.5- and 6.8-fold higher in 30°C culture than in 15°C culture. The in vivo half-lives of mtaA1 and mtaC1B1 mRNAs were similar in 30°C and 15°C cultures. However, the pta-ackA mRNA half-life was significantly reduced in 15°C culture compared to 30°C culture. Using circularized RNA RT-PCR, large 5' untranslated regions (UTRs) (270 nucleotides [nt] and 238 nt) were identified for mtaA1 and mtaC1B1 mRNAs, while only a 27-nt 5' UTR was present in the pta-ackA transcript. Removal of the 5' UTRs significantly reduced the in vitro half-lives of mtaA1 and mtaC1B1 mRNAs. Remarkably, fusion of the mtaA1 or mtaC1B1 5' UTRs to pta-ackA mRNA increased its in vitro half-life at both 30°C and 15°C. These results demonstrate that the large 5' UTRs significantly enhance the stability of the mRNAs involved in methanol-derived methanogenesis in the cold-adaptive M. mazei zm-15.

Mechanism for Stabilizing mRNAs Involved in Methanol-Dependent Methanogenesis of Cold-Adaptive Methanosarcina mazei zm-15

A sediment sample was collected from a deep-sea hydrothermal vent field located at a depth of 2 951 m on the Southwest Indian Ridge. Phylogenetic analyses were performed on the prokaryotic community using polymerase chain reaction (PCR) amplification of the 16S rRNA and nifH genes. Within the Archaea, the dominant clones were from marine benthic group E (MBGE) and marine group I (MGI) belonging to the phyla Euryarchaeota and Thaumarchaeota, respectively. More than half of the bacterial clones belonged to the Proteobacteria, and most fell within the Gammaproteobacteria. No epsilonproteobacterial sequence was observed. Additional phyla were detected including the Actinobacteria, Bacteroidetes, Planctomycetes, Acidobacteria, Nitrospirae, Chloroflexi, Chlorobi, Chlamydiae, Verrucomicrobia, and candidate divisions OD1, OP11, WS3 and TM6, confirming their existence in hydrothermal vent environments. The detection of nifH gene suggests that biological nitrogen fixation may occur in the hydrothermal vent field of the Southwest Indian Ridge. Phylogenetic analysis indicated that only Clusters I and III NifH were present. This is consistent with the phylogenetic analysis of the microbial 16S rRNA genes, indicating that Bacteria play the main role in nitrogen fixation in this hydrothermal vent environment.

Yi Cao

Papers

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

Characterization of alcohol dehydrogenase from the haloalkaliphilic archaeon Natronomonas pharaonis

Marinobacterium nitratireducens sp. nov. and Marinobacterium sediminicola sp. nov., isolated from marine sediment.

Mechanism for Stabilizing mRNAs Involved in Methanol-Dependent Methanogenesis of Cold-Adaptive Methanosarcina mazei zm-15

Microbial community structure and nitrogenase gene diversity of sediment from a deep-sea hydrothermal vent field on the Southwest Indian Ridge