The Calvin–Benson–Bassham (CBB) cycle is presumably evolved for optimal synthesis of C3 sugars, but not for the production of C2 metabolite acetyl-CoA. The carbon loss in producing acetyl-CoA from decarboxylation of C3 sugar limits the maximum carbon yield of photosynthesis. Here we design a synthetic malyl-CoA-glycerate (MCG) pathway to augment the CBB cycle for efficient acetyl-CoA synthesis. This pathway converts a C3 metabolite to two acetyl-CoA by fixation of one additional CO2 equivalent, or assimilates glyoxylate, a photorespiration intermediate, to produce acetyl-CoA without net carbon loss. We first functionally demonstrate the design of the MCG pathway in vitro and in Escherichia coli. We then implement the pathway in a photosynthetic organism Synechococcus elongates PCC7942, and show that it increases the intracellular acetyl-CoA pool and enhances bicarbonate assimilation by roughly 2-fold. This work provides a strategy to improve carbon fixation efficiency in photosynthetic organisms.

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e20511 Background: Mutations in KRAS and NRAS have been found in a number of malignancies, particularly metastatic colorectal cancer, lung adenocarcinoma, and thyroid cancer. Several studies have shown that specific mutations in KRAS and NRAS are less likely to respond to anti-EGFR therapies. Several diagnostics have been developed to identify such mutations. However, in a large number of cancer patients, tumor biopsy poses a significant health risk thereby limiting the tissue available for current molecular diagnostics. Non-invasive liquid biopsy diagnostics are revolutionizing cancer treatment selection, patient prognosis, and monitoring. Low abundance circulating tumor DNA (ctDNA) is purified from patient plasma and used as an alternative source of genomic material for detecting tumor mutations. Currently available qPCR kits for detecting RAS mutations have a limited sensitivity and coverage since they are not optimized for liquid biopsy purposes. Methods: We have developed a two-step PCR to qPCR method to identify down to single mutant copies of KRAS or NRAS in ctDNA isolated from plasma. The mutant copies are first enriched by a novel multiplexed formula of primers and wildtype (WT) allele blockers. The mutations are then identified by EntroGen’s RAS Mutation Screening Panel or next generation sequencing (NGS). Synthetic mutant DNA was spiked in a background of WT DNA purified from plasma to yield a range of mutant allelic burden over WT background. The dilutions were analyzed to determine the method’s sensitivity. We subsequently used the method to screen 53 non-small cell lung cancer (NSCLC) patient samples. Results: Our two-step method detects single digit mutant copies of 35 clinically significant KRAS and NRAS mutations. Furthermore, NGS experiments demonstrated the two-step method can enrich mutations more than 52-fold. KRAS 12/13 mutations were identified in 6 out of 53 (11%) NSCLC patients. Conclusions: ctDNA-based liquid biopsy tests have been employed by few laboratories but are generally available only as a service. EntroGen’s platform agnostic liquid biopsy RAS assay provides clinicians the tools necessary to determine cancer treatment and monitor patients using most PCR and qPCR instruments.