Yeast cell factories have been increasingly employed for producing plant‐derived natural products. Unfortunately, the stability of plant natural product biosynthetic pathway genes, particularly when driven by the same sets of promoters and terminators, remains one of the biggest concerns for synthetic biology. Here we profile genomic loci flanked by essential genes as stable integration sites in a genome‐wide manner, for stable maintenance of multigene biosynthetic pathways in yeast. We demonstrate the application of our yeast integration platform in the construction of sanguinarine (24 expression cassettes) and ajmalicine (29 expression cassettes) de novo biosynthetic pathways for the first time. Moreover, we establish stable yeast cell factories that can produce 119.2 mg L−1 heteroyohimbine alkaloids (containing 61.4 mg L−1 ajmalicine) in shake flasks, representing the highest titer of monoterpene indole alkaloids (MIAs) ever reported and promising the complete biosynthesis of other high‐value MIAs (such as vinblastine) for biotechnological applications.
Purpose
Coronavirus disease 2019 (COVID-19) has become a topic of concern worldwide; however, the impacts of type 2 diabetes mellitus (T2DM) on disease severity, therapeutic effect, and mortality of patients with COVID-19 are unclear.
Methods
All consecutive patients with COVID-19 admitted to the Renmin Hospital of Wuhan University from January 11 to February 6, 2020, were included in this study.
Results
A total of 663 patients with COVID-19 were included, while 67 patients with T2DM accounted for 10.1% of the total. Compared with patients with COVID-19 without T2DM, those with T2DM were older (aged 66 years vs 57 years; P < 0.001) and had a male predominance (62.7% vs 37.3%; P = 0.019) and higher prevalence of cardiovascular diseases (61.2% vs 20.6%; P < 0.001) and urinary diseases (9% vs 2.5%; P = 0.014). Patients with T2DM were prone to developing severe (58.2% vs 46.3%; P = 0.002) and critical COVID-19 (20.9% vs 13.4%; P = 0.002) and having poor therapeutic effect (76.1% vs 60.4%; P = 0.017). But there was no obvious difference in the mortality between patients with COVID-19 with and without T2DM (4.5% vs 3.7%; P = 0.732). Multivariate logistic regression analysis identified that T2DM was associated with poor therapeutic effect in patients with COVID-19 (odd ratio [OR] 2.99; 95% confidence interval [CI], 1.07-8.66; P = 0.04). Moreover, having a severe and critical COVID-19 condition (OR 3.27; 95% CI, 1.02-9.00; P = 0.029) and decreased lymphocytes (OR 1.59; 95% CI, 1.10-2.34; P = 0.016) were independent risk factors associated with poor therapeutic effect in patients with COVID-19 with T2DM.
Conclusions
T2DM influenced the disease severity and therapeutic effect and was one of the independent risk factors for poor therapeutic effect in patients with COVID-19.
The Pyraloidea is one of the species-rich superfamilies of Lepidoptera and contains numerous economically important pest species that cause great loss in crop production. Here, we sequenced and annotated nine complete mitogenomes for Pyraloidea, and further performed various phylogenetic analyses, to improve our understanding of mitogenomic evolution and phylogeny of this superfamily. The nine mitogenomes were circular, double-stranded molecules, with the lengths ranging from 15,214 bp to 15,422 bp, which are comparable to other reported pyraloid mitogenomes in size. Gene content and arrangement were highly conserved and are typical of Lepidoptera. Based on the hitherto most extensive mitogenomic sampling, our various resulting trees showed generally congruent topologies among pyraloid subfamilies, which are almost in accordance with previous multilocus studies, indicating the suitability of mitogenomes in inferring high-level relationships of Pyraloidea. However, nodes linking subfamilies in the “non-PS clade” were not completely resolved in terms of unstable topologies or low supports, and future investigations are needed with increased taxon sampling and molecular data. Unexpectedly, Orybina Snellen, represented in a molecular phylogenetic investigation for the first time, was robustly placed as basal to the remaining Pyralidae taxa across our analyses, rather than nested in Pyralinae of Pyralidae as morphologically defined. This novel finding highlights the need to reevaluate Orybina monophyly and its phylogenetic position by incorporating additional molecular and morphological evidence.
The introduction of multi-gene metabolic pathways is generally the first step for the construction of microbial cell factories and plays an essential role in metabolic engineering and synthetic biology. Here, we developed a “PCR & Go” system for facile integration and assembly of multi-gene pathways into the chromosome of Saccharomyces cerevisiae. The core component of the “PCR & Go” system was an expression chassis, where eight promoter/terminator pairs were pre-installed into the yeast chromosome and PCR amplified gene fragments could be inserted directly for functional expression. In combination with the CRISPR/Cas9 system and a gRNA plasmid library, the β-carotene (three genes), zeaxanthin (four genes), and astaxanthin (five genes) biosynthetic pathways were integrated and assembled into the yeast genome with an efficiency of ~93, ~85, and 69%, respectively, using PCR amplified gene fragments with ~40 bp homology arms in a single step. Therefore, the “PCR & Go” system can be used for fast construction of yeast cell factories harboring multi-gene pathways with high efficiency and flexibility.
The complete mitochondrial genomes of three species of Odontiinae were newly sequenced: Dausara latiterminalis Yoshiyasu, Heortia vitessoides (Moore), and Pseudonoorda nigropunctalis (Hampson). These circular and double-stranded mitogenomes vary from 15,084 bp to 15,237 bp in size, including 13 protein-coding genes (PCGs), two ribosomal RNA genes (rRNAs), and 22 transfer RNA genes (tRNAs) and an A + T-rich region. The nucleotide composition indicated a strong A/T bias. Most PCGs are initiated with an ATN codon and terminated by a codon of TAR. All tRNAs could be folded into the clover-leaf structure with the exception of trnS1 (AGN), in which the dihydrouridine (DHU) arm formed a simple loop, and the motif ‘ATAG’ and ‘ATTTA’ in the A + T-rich region was also founded. The phylogenomic analyses covering Odontiinae + 11 subfamilies of Pyraloidea were conducted. Similar topologies were generated from both Bayesian inference (BI) and maximum likelihood (ML) analyses based on the nucleotide and amino acid sequence data. There was some discrepancy in the sister-group relationship of Odontiinae and Glaphyriinae, and the relationships among the subfamilies in the ‘CAMMSS clade’ of the Crambidae. The results of this study suggest that mitogenomic data are useful for resolving the deep-level relationships of Pyraloidea and the topologies generated from amino acid data might be more realistic and reliable. Moreover, more mitogenomic taxon sampling and larger scale analyses with more genes or a combination of mitogenomic and nuclear genes are needed to reconstruct a comprehensive framework of the pyraloid phylogeny.
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