Rational construction of genome-reduced Burkholderiales chassis facilitates efficient heterologous production of natural products from proteobacteria

Abstract

Heterologous expression of biosynthetic gene clusters (BGCs) avails yield improvements and mining of natural products, but it is limited by lacking of more efficient Gram-negative chassis. The proteobacterium Schlegelella brevitalea DSM 7029 exhibits potential for heterologous BGC expression, but its cells undergo early autolysis, hindering further applications. Herein, we rationally construct DC and DT series genome-reduced S. brevitalea mutants by sequential deletions of endogenous BGCs and the nonessential genomic regions, respectively. The DC5 to DC7 mutants affect growth, while the DT series mutants show improved growth characteristics with alleviated cell autolysis. The yield improvements of six proteobacterial natural products and successful identification of chitinimides from Chitinimonas koreensis via heterologous expression in DT mutants demonstrate their superiority to wild-type DSM 7029 and two commonly used Gram-negative chassis Escherichia coli and Pseudomonas putida. Our study expands the panel of Gram-negative chassis and facilitates the discovery of natural products by heterologous expression.

Random genome reduction coupled with polyhydroxybutyrate biosynthesis to facilitate its accumulation in Escherichia coli

“…Genome reduction based on rational analysis and design has gained great progress in construction of desirable chassis for different application purposes ( Sung et al, 2016 ; Chi et al, 2019 ). Owing to the inadequate understanding of genes still lacking annotation, as well as complex interactions of known genes and phenotypes, the effects of genomic deletion on cell physiology are difficult to predict and have to be tested experimentally, which is much laborious and may even cause the decay of cellular growth and performances ( Ara et al, 2007 ; Karcagi et al, 2016 ; Li et al, 2016 ; Tsuchiya et al, 2018 ; Kurokawa and Ying, 2020 ; Liu et al, 2021 ). In addition, rational design method only allows the sequential removal of a single DNA fragment from a strain, and obtains one or a few genome-reduced strains in final ( Posfai et al, 2006 ; Park et al, 2014 ; Liu et al, 2021 ).…”

“…Owing to the inadequate understanding of genes still lacking annotation, as well as complex interactions of known genes and phenotypes, the effects of genomic deletion on cell physiology are difficult to predict and have to be tested experimentally, which is much laborious and may even cause the decay of cellular growth and performances ( Ara et al, 2007 ; Karcagi et al, 2016 ; Li et al, 2016 ; Tsuchiya et al, 2018 ; Kurokawa and Ying, 2020 ; Liu et al, 2021 ). In addition, rational design method only allows the sequential removal of a single DNA fragment from a strain, and obtains one or a few genome-reduced strains in final ( Posfai et al, 2006 ; Park et al, 2014 ; Liu et al, 2021 ). In contrast, the TMRD can randomly and continuously reduce the E. coli genome for a given environment, and at the same times maintained a comparable deletion rate (about 25 Kb in size per cycle every 10 days) to rational design strategy.…”

“…The growth and surfactin production of Bacillus amyloliquefaciens was increased by deleting ∼4.18% of genome sequences ( Zhang et al, 2020 ). The DT (deletions of transposons) series genome-reduced Schlegelella brevitalea mutants were rationally constructed by sequential deletions 0.3%–4.9% of its genome, including forty-four transposases, two prophage-like regions, and seven genomic islands, and showed better growth characteristics with alleviated cell autolysis, and improved yields of six proteobacterial natural products ( Liu et al, 2021 ).…”

Remodeling Bacillus amyloliquefaciens Cell Wall Rigidity to Reduce Cell Lysis and Increase the Yield of Heterologous Proteins

“…SEM and TEM. Scanning electron microscopy (SEM) was performed to determine the cell morphology using a method described by Liu et al 25 Cells of B. amyloliquefaciens were collected by centrifugation (5000×g for 5 min) after 4, 6, 12, and 24 h of growth in a LB medium and washed with 0.1 M phosphate-buffered saline (PBS) (pH = 7.4) three times. Samples were fixed using 2.5% (v/v) glutaraldehyde in 0.1 M phosphate buffer (pH = 7.4) for 8 h at 4 °C.…”

Multiple Modular Engineering of Bacillus Amyloliquefaciens Cell Factories for Enhanced Production of Alkaline Proteases From B. Clausii

“…SEM was carried out based on reported method ( Liu et al, 2021 ) as follows: Cells of B. amyloliquefaciens were collected after 6, 10, 12 and 30 h of growth in LB medium were collected by centrifugation (5,000 g for 5 min) and washed in phosphate buffered saline (PBS) three times. Samples were fixed using 2.5% (v/v) glutaraldehyde in PBS overnight and washed three times with PBS to remove the remaining glutaraldehyde, then covered with platinum using a Q150R rotary-pumped sputter coater, and the shapes and appearances of cells were observed at 8,000 magnifications.…”