APseudomonas putidadouble mutant deficient in butanol assimilation: a promising step for engineering a biological biofuel production platform

Abstract

Biological production in heterologous hosts is of interest for the production of the C4 alcohol (butanol) and other chemicals. However, some hurdles need to be overcome in order to achieve an economically viable process; these include avoiding the consumption of butanol and maintaining tolerance to this solvent during production. Pseudomonas putida is a potential host for solvent production; in order to further adapt P. putida to this role, we generated mini-Tn5 mutant libraries in strain BIRD-1 that do not consume butanol. We analyzed the insertion site of the mini-Tn5 in a mutant that was deficient in assimilation of butanol using arbitrary PCR followed by Sanger sequencing and found that the transposon was inserted in the malate synthase B gene. Here, we show that in a second round of mutagenesis a double mutant unable to take up butanol had an insertion in a gene coding for a multisensor hybrid histidine kinase. The genetic context of the histidine kinase sensor revealed the presence of a set of genes potentially involved in butanol assimilation; qRT-PCR analysis showed induction of this set of genes in the wild type and the malate synthase mutant but not in the double mutant.

Metabolic and stress responses of Acinetobacter oleivorans DR1 during long‐chain alkane degradation

“…The aceA gene encoding an isocitrate lyase in glyoxylate bypass was proven to be important for acetate, ethanol, poly‐3‐hydroxybutyrate, alkane metabolisms (Sabirova et al ., , ; Jung et al ., ; Zhang and Bryant, ; Ahn et al ., ; Dunn et al ., ). Interestingly, butanol metabolism appeared to occur through the glyoxylate bypass in Pseudomonas putida BIRD‐1 (Cuenca Mdel et al ., ,b) and loss of ethanol and acetate metabolisms was also observed in Candida albicans lacking the isocitrate lyase (Lorenz and Fink, ). Alkane degradation generates the TCA cycle intermediates from acetyl‐CoA via the glyoxylate bypass.…”

Twenty‐first‐century chemical odyssey: fuels versus commodities and cell factories versus chemical plants

“…However, the process was restricted by solvent‐sensitivity and the limited activity of the Lactococcus lactis keto‐acid decarboxylase used. The same pathway was explored in solvent‐tolerant aerobic Pseudomonas strains; however, the strain metabolizes butanol, and different metabolic blocks were needed to achieve significant butanol productivity (Cuenca et al ., ).…”

“…However, the process was restricted by solvent-sensitivity and the limited activity of the Lactococcus lactis keto-acid decarboxylase used. The same pathway was explored in solvent-tolerant aerobic Pseudomonas strains; however, the strain metabolizes butanol, and different metabolic blocks were needed to achieve significant butanol productivity (Cuenca et al, 2016). Atsumi et al (2008) reported titres of 22 g l À1 of isobutanol with engineered Escherichia coli strains that produced this alcohol using diversion of central metabolites in the valine biosynthetic pathway, and Baez et al (2011) reported that the engineered E. coli (JCL260) strain produced more than 50 g l À1 in 72 h if isobutanol was removed from the bioreactor using gas stripping.…”

Specific Integration of Temperate Phage Decreases the Pathogenicity of Host Bacteria

“…In addition to the lytic cycle, temperate phages also have a lysogenic cycle, in which phages incorporate their genomes into a host chromosome (or maintain their genome extrachromosomally) and replicate with it without lysing their host cells (Howard-Varona et al, 2017). Therefore, temperate phages are considered as natural vectors for gene transmission among bacteria and play important roles in virulence of bacterial pathogens (Boyd, 2012;Cuenca Mdel et al, 2016).…”

“…Many effector proteins, which are bacterial virulent factors and are injected into host cells by bacterial type III secretion system to help bacterial invasion and survival, were identified in Salmonella typhimurium phages (Figueroa-Bossi et al, 2001). Infection with such temperate phages might lead to the transmission of the virulent genes, thus increases the virulence of host bacteria (Cuenca Mdel et al, 2016). Additionally, it was reported that temperate staphylococcal phage 80α could efficiently encapsidate and transfer bacterial pathogenicity island, SaPI1, to a recipient strain (Ruzin et al, 2001).…”