Safety Evaluation of a Novel Algal Feed Additive for Poultry Production.

Feed additives are critical components for poultry health and the economic viability of antibiotic-free poultry production. The aim of the present study is to evaluate the safety of a novel algal-derived feed additive, a dried biomass powder produced from Chlamydomonas reinhardtii strain crAL082, modified to express an N-acetylmuramoyl-L-alanine amidase (EC 3.5.1.28) and a lysozyme-type enzyme (EC 3.2.1.17). A 42-day oral toxicity study showed that the crAL082 dried biomass powder was fully tolerated by broiler chicken based on the lack of detrimental effects found in performance, mortality, hematology, blood clinical chemistry, and histopathologic results compared with those of a nontreated control group, resulting in a "No Observed Adverse Effect Level" of 5000 ppm, the highest dose tested. The study demonstrates the first-ever safety result of a C. reinhardtii microalgae dried biomass powder used as a feed additive in broiler chickens. Furthermore, safety is shown for the two additional enzymes expressed within the C. reinhardtii crAL082 strain and ingested by the birds.

Evaluación de seguridad de un nuevo aditivo alimentario con base en algas para la producción avícola. Los aditivos alimentarios son componentes críticos para la salud avícola y la viabilidad económica de la producción avícola libre de antibióticos. El objetivo del presente estudio es evaluar la seguridad de un nuevo aditivo alimentario derivado de algas, un polvo de biomasa seca producido a partir de la cepa crAL082 de Chlamydomonas reinhardtii, que fue modificado para expresar una N-acetilmuramoil-Lalanina amidasa (EC 3.5.1.28) y una enzima tipo lisozima (EC 3.2.1.17). Un estudio de toxicidad oral de 42 días mostró que el polvo de biomasa seca crAL082 fue totalmente tolerado por los pollos de engorde con base a la ausencia de efectos perjudiciales encontrados en el rendimiento, la mortalidad, la hematología, la química clínica sanguínea y los resultados histopatológicos en comparación con los del grupo de control no tratado, lo que resultó en un "Nivel sin efecto adverso observado" ("No Observed Adverse Effect Level" = de 5000 ppm, la dosis más alta probada. El estudio demuestra el primer resultado de seguridad de un polvo de biomasa seca de microalgas C. reinhardtii utilizado como aditivo alimentario en pollos de engorde. Además, se muestra la seguridad de las dos enzimas adicionales expresadas dentro de la cepa crAL082 de C. reinhardtii e ingeridas por las aves.

Directed evolution for soluble and active periplasmic expression of bovine enterokinase in Escherichia coli.

Bovine enterokinase light chain (EKL) is an industrially useful protease for accurate removal of affinity-purification tags from high-value biopharmaceuticals. However, recombinant expression in Escherichia coli produces insoluble inclusion bodies, requiring solubilisation, refolding, and autocatalytic activation to recover functional enzyme. Error-prone PCR and DNA shuffling of the EKL gene, T7 promoter, lac operon, ribosome binding site, and pelB leader sequence, yielded 321 unique variants after screening ~ 6500 colonies. The best variants had > 11,000-fold increased total activity in lysates, producing soluble enzyme that no longer needed refolding. Further characterisation identified the factors that improved total activity from an inactive and insoluble starting point. Stability was a major factor, whereby melting temperatures > 48.4 °C enabled good expression at 37 °C. Variants generally did not alter catalytic efficiency as measured by kcat/Km, which improved for only one variant. Codon optimisation improved the total activity in lysates produced at 37 °C. However, non-optimised codons and expression at 30 °C gave the highest activity through improved protein quality, with increased kcat and Tm values. The 321 variants were statistically analysed and mapped to protein structure. Mutations detrimental to total activity and stability clustered around the active site. By contrast, variants with increased total activity tended to combine stabilising mutations that did not disrupt the active site.