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Karst tiankengs in China are globally significant locations for studying ecological environments and plant diversity. However, there are few reports on how the unique geographical environment of tiankengs affects plant genetic diversity and genetic structure. This study used Hyper-seq gene sequencing technology to develop large-scale genomic SNPs of Manglietia aromatica, both within and outside the tiankengs. Its aim was to investigate the impact of tiankengs on the genetic diversity and genetic structure of the M. aromatica population. The analysis results indicate that the genetic diversity of the populations within the tiankeng (π = 0.2044) is higher than that of the populations outside of it (π = 0.1671), indicating that the tiankengs have a positive impact on species diversity. The genetic differentiation coefficient (FST) between the populations inside and outside the tiankeng was 0.0534 and the FST values of populations within the tiankeng were 0.077, 0.082, and 0.141, meaning that the genetic variation in the tiankengs is very high. The genetic similarity outside the tiankengs is also very high, indicating that the tiankengs are effectively preserving the genetic diversity of M. aromatica. Furthermore, the gene introgression analysis results gave no proof of gene flow between the three tiankeng populations. This suggests that the tiankengs not only protect species diversity, but also hinder gene flow between populations to some extent. However, this hindrance may gradually subside with the evolution of the tiankengs. The genetic structure analysis revealed that the M. aromatica population in Guangxi, China, can be classified into three subpopulations. The first is the tiankeng subpopulation, including all the populations in tiankengs. The second subpopulation consists of populations surrounding the tiankengs. These two subpopulations are distributed in Leye County in northwestern Guangxi, China, and are very close to each other. The third is the Huanjiang subpopulation, which is located far away from the tiankengs. Considering the direction of gene flow and genetic structure, it is speculated that the populations in the tiankengs evolved from the populations near the pit mouth. This study confirms that the tiankengs are shelters and provide a suitable habitat for the endangered plant M. aromatica, because its genetic diversity is well conserved and the species is well adapted to the habitat within the tiankengs.

The incidence of cancer is high worldwide, and biological factors such as viruses and bacteria play an important role in the occurrence of cancer. Helicobacter pylori, human papillomavirus, hepatitis B viruses and other organisms have been identified as carcinogens. Cancer is a disease driven by the accumulation of genome changes. Viruses can directly cause cancer by changing the genetic composition of the human body, such as cervical cancer caused by human papillomavirus DNA integration and liver cancer caused by hepatitis B virus DNA integration. Recently, bacterial DNA has been found around cancers such as pancreatic cancer, breast cancer and colorectal cancer, and the idea that bacterial genes can also be integrated into the human genome has become a hot topic. In the present paper, we reviewed the latest phenomenon and specific integration mechanism of bacterial DNA into the human genome. Based on these findings, we also suggest three sources of bacterial DNA in cancers: bacterial DNA around human tissues, free bacterial DNA in bacteremia or sepsis, and endogenous bacterial DNA in the human genome. Clarifying the theory that bacterial DNA integrates into the human genome can provide a new perspective for cancer prevention and treatment.

Pseudonocardia species are emerging as important microorganisms of global concern with unique and increasingly significant ecological roles and represent a prominent source of bioactive natural products, but genetic engineering of these organisms for biotechnological applications is greatly hindered due to the limitation of efficient genetic manipulation tools. In this regard, we report here the establishment of an efficient genetic manipulation system for a newly isolated strain, Pseudonocardia alni Shahu, based on plasmid conjugal transfer from Escherichia coli to Pseudonocardia. Conjugants were yielded upon determining the optimal ratio between the donor and recipient cells, and designed genome modifications were efficiently accomplished, including exogenous gene integration based on an integrative plasmid and chromosomal stretch removal by homologous recombination using a suicidal non-replicating vector. Collectively, this work has made the P. alni Shahu accessible for genetic engineering, and provided an important reference for developing genetic manipulation methods in other rare actinomycetes.

Given the rapid development of genome mining in this decade, the substrate channel of paclitaxel might be identified in the near future. A robust microbial cell factory with gene dbat, encoding a key rate-limiting enzyme 10-deacetylbaccatin III-10-O-transferase (DBAT) in paclitaxel biosynthesis to synthesize the precursor baccatin III, will lay out a promising foundation for paclitaxel de novo synthesis. Here, we integrated gene dbat into the wild-type Escherichia coli BW25113 to construct strain BWD01. Yet, it was relatively unstable in baccatin III synthesis. Mutant gene dbatS189V with improved thermostability was screened out from a semi-rational mutation library of DBAT. When it was over-expressed in an engineered strain N05 with improved acetyl-CoA generation, combined with carbon source optimization of fermentation engineering, the production level of baccatin III was significantly increased. Using this combination, integrated strain N05S01 with mutant dbatS189V achieved a 10.50-fold increase in baccatin III production compared with original strain BWD01. Our findings suggest that the combination of protein engineering and metabolic engineering will become a promising strategy for paclitaxel production.

Abstract Most DNA assembly methods require bacterial amplification steps, which restrict its application to genes that can be cloned in the bacterial host without significant toxic effects. However, genes that cannot be cloned in bacteria do not necessarily exert toxic effects on the final host. In order to tackle this issue, we adapted two DNA assembly workflows for rapid, cloning‐free construction and genomic integration of expression cassettes in Saccharomyces cerevisiae. One method is based on a modified Gibson assembly, while the other relies on a direct assembly and integration of linear PCR products by yeast homologous recombination. The methods require few simple experimental steps, and their performance was evaluated for the assembly and integration of unclonable zeaxanthin epoxidase expression cassettes in yeast. Results showed that up to 95% integration efficiency can be reached with minimal experimental effort. The presented workflows can be employed as rapid gene integration tools for yeast, especially tailored for integrating unclonable genes.