bAlginate lyases are enzymes that degrade alginate through ␤-elimination of the glycosidic bond into smaller oligomers. We investigated the alginate lyases from Vibrio splendidus 12B01, a marine bacterioplankton species that can grow on alginate as its sole carbon source. We identified, purified, and characterized four polysaccharide lyase family 7 alginates lyases, AlyA, AlyB, AlyD, and AlyE, from V. splendidus 12B01. The four lyases were found to have optimal activity between pH 7.5 and 8.5 and at 20 to 25°C, consistent with their use in a marine environment. AlyA, AlyB, AlyD, and AlyE were found to exhibit a turnover number (k cat ) for alginate of 0.60 ؎ 0.02 s ؊1 , 3.7 ؎ 0.3 s ؊1 , 4.5 ؎ 0.5 s ؊1 , and 7.1 ؎ 0.2 s ؊1 , respectively. The K m values of AlyA, AlyB, AlyD, and AlyE toward alginate were 36 ؎ 7 M, 22 ؎ 5 M, 60 ؎ 2 M, and 123 ؎ 6 M, respectively. AlyA and AlyB were found principally to cleave the ␤-1,4 bonds between ␤-D-mannuronate and ␣-L-guluronate and subunits; AlyD and AlyE were found to principally cleave the ␣-1,4 bonds involving ␣-L-guluronate subunits. The four alginate lyases degrade alginate into longer chains of oligomers. Alginate is an abundant polysaccharide found within the cell wall of brown seaweeds (1) and comprises approximately 40% of the dry weight of the plant (2). Alginate is a copolymer consisting of the 1,4-linked epimers ␣-L-guluronate (G) and ␤-Dmannuronate (M). The individual monomeric subunits are organized in short stretches of polyguluronate (polyG), polymannuronate (polyM), or alternating sequences of mannuronate and guluronate (polyMG) (3). Alginate recently has been considered as a source for bioenergy, as it offers several potential advantages over terrestrial biomass. In particular, the farming of algae does not impinge on arable land; hence, it avoids the conflict between food and energy (4, 5). Algae also display higher growth rates than terrestrial plants (6). Finally, algae lack crystalline cellulose and lignin (7,8), bypassing a key obstacle to biofuel production.Alginate lyases are enzymes that degrade alginate through the ␤-elimination of the glycosidic bond into smaller oligomers. These enzymes can be classified based on the specific dyad G-G (EC 4.2.2.11) (9, 10), M-M (EC 4.2.2.3) (11), and M-G/G-M (12) bonds that they cleave. Additionally, alginate lyases can have either endocleaving or exocleaving specificity, with the majority of alginate lyases having an endocleaving preference (3). Along with the substrate specificity, alginate lyases also are characterized by their structure, termed polysaccharide lyase (PL) families. A total of seven PL families have been identified: PL5, PL6, PL7, PL14, PL15, PL17, and PL18 (13, 14). The most prevalent of these families is PL7 (Carbohydrate Active Enzymes database; http://www .cazy.org). The PL7 domain contains a ␤-jelly roll that consists of ␤-sheets in an antiparallel, adjacent barrel forming a cleft (12). This cleft contains three adjacent ␤-sheets that contain the catalytic residues (15). PL7 contains enzymes...

Polysaccharide degradation by marine microbes represents one of the largest and most rapid heterotrophic transformations of organic matter in the environment. Microbes employ systems of complementary carbohydrate-specific enzymes to deconstruct algal or plant polysaccharides (glycans) into monosaccharides. Because of the high diversity of glycan substrates, the functions of these enzymes are often difficult to establish. One solution to this problem may lie within naturally occurring microdiversity; varying numbers of enzymes, due to gene loss, duplication, or transfer, among closely related environmental microbes create metabolic differences akin to those generated by knock-out strains engineered in the laboratory used to establish the functions of unknown genes. Inspired by this natural fine-scale microbial diversity, we show here that it can be used to develop hypotheses guiding biochemical experiments for establishing the role of these enzymes in nature. In this work, we investigated alginate degradation among closely related strains of the marine bacterium One strain, 13B01, exhibited high extracellular alginate lyase activity compared with other strains. To identify the enzymes responsible for this high extracellular activity, we compared 13B01 with the previously characterized 12B01, which has low extracellular activity and lacks two alginate lyase genes present in 13B01. Using a combination of genomics, proteomics, biochemical, and functional screening, we identified a polysaccharide lyase family 7 enzyme that is unique to 13B01, secreted, and responsible for the rapid digestion of extracellular alginate. These results demonstrate the value of querying the enzymatic repertoires of closely related microbes to rapidly pinpoint keyproteins with beneficial functions.

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Background: HER2-targeted therapies have substantially improved outcomes for patients with HER2-positive breast and gastric cancers. Several other cancers exhibit HER2 expression and/or amplification of its gene (ERBB2), suggesting that HER2-targeted agents may have broader therapeutic utility. Zanidatamab is a humanized, novel bispecific antibody directed against two non-overlapping domains of HER2. The aim of this Phase 1 dose-escalation and expansion study (NCT02892123) was to evaluate the safety and efficacy of zanidatamab across a range of solid tumors. Parallel to drug development, there has been rapid advancements in NGS technologies including the Guardant360 assay that can specifically sequence ctDNA and detect amplifications of the ERBB2 gene, which can lead to overexpression of HER2. FISH, the current gold standard for HER2 amplification detection, is a tissue-based assay that assesses the raw ERBB2 copy number as well as ratio of ERBB2 to a centromeric protein of chromosome 17 where the ERBB2 gene resides. We evaluated concordance of the FISH and Guardant360 assays to detect ERBB2 amplification in plasma samples. Unlike gene copy number in tissue analysis, the observed plasma copy number (pCN) is also a function of the tumor burden and rate of tumor shedding of ctDNA into the bloodstream. Methods: HER2 status was determined from a fresh tumor biopsy or in archival FFPE tissue samples by IHC and FISH according to ASCO-CAP guidelines from the Phase 1 study with zanidatamab in multiple cancer types (cholangiocarcinoma [21], colorectal carcinoma [27], all other [87]). Plasma samples were collected prior to the first cycle of zanidatamab and on-treatment for testing with Guardant360, 74 gene ctDNA NGS-based assays. Results: A concordance of 82% was observed in ERBB2/HER2 amplifications between the Guardant360 and FISH assays. An exploratory adjustment method based on tumor DNA shedding was developed by Guardant using the maximum mutant allele fraction (maxVAF) as a surrogate for tumor content. Majority of patients experienced a decrease in HER2 pCN post treatment, with 9 PD patients having the least and 21 PR patients the largest changes in ctDNA fraction (maxVAF). Conclusion: These results indicate that ERBB2 amplification detected by the Guardant360 assay could be used as a surrogate for FISH analysis in lieu of invasive surgical procedures. Citation Format: Diana Shpektor, Daryanaz Dargahi, Antonios Samiotakis, Sara Wienke, Ali Livernois, Arielle Yablonovitch, Geethika Yalamanchili, Elaina Gartner, Funda Meric-Bernstam. ERBB2 amplification detected in ctDNA as a surrogate for tumor tissue FISH analysis of HER2 status in a phase 1 study with zanidatamab for the treatment of locally advanced or metastatic HER2 expressing cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT278.