Yarrowia lipolytica is categorized as a generally recognized as safe (GRAS) organism and is a heavily documented, unconventional yeast that has been widely incorporated into multiple industrial fields to produce valuable biochemicals. This study describes the construction of a CRISPR-Cas9 system for genome editing in Y. lipolytica using a single plasmid (pCAS1yl or pCAS2yl) to transport Cas9 and relevant guide RNA expression cassettes, with or without donor DNA, to target genes. Two Cas9 target genes, TRP1 and PEX10, were repaired by non-homologous end-joining (NHEJ) or homologous recombination, with maximal efficiencies in Y. lipolytica of 85.6 % for the wild-type strain and 94.1 % for the ku70/ku80 double-deficient strain, within 4 days. Simultaneous double and triple multigene editing was achieved with pCAS1yl by NHEJ, with efficiencies of 36.7 or 19.3 %, respectively, and the pCASyl system was successfully expanded to different Y. lipolytica breeding strains. This timesaving method will enable and improve synthetic biology, metabolic engineering and functional genomic studies of Y. lipolytica.

Bifidobacteria are considered one of the most important intestinal probiotics because of their significant health impact. However, this ability is usually limited by gastrointestinal fluid and temperature sensitivity. Emulsification and internal gelation is an encapsulation technique with great potential for probiotic protection during storage and the gastrointestinal transit process. This study prepared microcapsules using an emulsification and internal gelation encapsulation method with sodium alginate, chitosan, and Bifidobacterium longum as wall material, coating material, and experimental strain, respectively. Optical, scanning electron, and focal microscopes were used to observe the microcapsule surface morphology and internal viable cell distribution, and a laser particle size analyzer and zeta potentiometer were used to evaluate the chitosan-coating characteristics. In addition, microcapsule probiotic viability after storage, heat treatment, and simulated gastrointestinal fluid treatment were examined. Alginate microcapsules and chitosan-coated alginate microcapsules both had balling properties and uniform bacterial distribution. The latter kept its balling properties after freeze-drying, verified by scanning electronic microscopy (SEM), and had a clear external coating, observed by an optical microscope. The particle size of chitosan-coated alginate microcapsules was slightly larger than the uncoated microcapsules. The zeta potential of alginate and chitosan-coated alginate microcapsules was negative and positive, respectively. Heat, acid and bile salt tolerance, and stability tests revealed that the decrease of viable cells in the chitosan-coated alginate microcapsule group was significantly lower than that in uncoated microcapsules. These experimental results indicate that the chitosan-coated alginate microcapsules protect B. longum from gastrointestinal fluid and high-temperature conditions.

Amycolatopsis orientalis is the type species of the genus and its industrial strain HCCB10007, derived from ATCC 43491, has been used for large-scale production of the vital antibiotic vancomycin. However, to date, neither the complete genomic sequence of this species nor a systemic characterization of the vancomycin biosynthesis cluster (vcm) has been reported. With only the whole genome sequence of Amycolatopsis mediterranei available, additional complete genomes of other species may facilitate intra-generic comparative analysis of the genus. The complete genome of A. orientalis HCCB10007 comprises an 8,948,591-bp circular chromosome and a 33,499-bp dissociated plasmid. In total, 8,121 protein-coding sequences were predicted, and the species-specific genomic features of A. orientalis were analyzed in comparison with that of A. mediterranei. The common characteristics of Amycolatopsis genomes were revealed via intra- and inter-generic comparative genomic analyses within the domain of actinomycetes, and led directly to the development of sequence-based Amycolatopsis molecular chemotaxonomic characteristics (MCCs). The chromosomal core/quasi-core and non-core configurations of the A. orientalis and the A. mediterranei genome were analyzed reciprocally, with respect to further understanding both the discriminable criteria and the evolutionary implementation. In addition, 26 gene clusters related to secondary metabolism, including the 64-kb vcm cluster, were identified in the genome. Employing a customized PCR-targeting-based mutagenesis system along with the biochemical identification of vancomycin variants produced by the mutants, we were able to experimentally characterize a halogenase, a methyltransferase and two glycosyltransferases encoded in the vcm cluster. The broad substrate spectra characteristics of these modification enzymes were inferred. This study not only extended the genetic knowledge of the genus Amycolatopsis and the biochemical knowledge of vcm-related post-assembly tailoring enzymes, but also developed methodology useful for in vivo studies in A. orientalis, which has been widely considered as a barrier in this field.