3-Hydroxypropionic acid (3-HP) is an important platform chemical proposed by the United States Department of Energy. 3-HP can be converted to a series of bulk chemicals. Biological production of 3-HP has made great progress in recent years. However, low yield of 3-HP restricts its commercialization. In this study, systematic optimization was conducted towards high-yield production of 3-HP in Klebsiella pneumoniae. We first investigated appropriate promoters for the key enzyme (aldehyde dehydrogenase, ALDH) in 3-HP biosynthesis, and found that IPTG-inducible tac promoter enabled overexpression of an endogenous ALDH (PuuC) in K. pneumoniae. We optimized the metabolic flux and found that blocking the synthesis of lactic acid and acetic acid significantly increased the production of 3-HP. Additionally, fermentation conditions were optimized and scaled-up cultivation were investigated. The highest 3-HP titer was observed at 83.8 g/L with a high conversion ratio of 54% on substrate glycerol. Furthermore, a flux distribution model of glycerol metabolism in K. pneumoniae was proposed based on in silico analysis. To our knowledge, this is the highest 3-HP production in K. pneumoniae. This work has significantly advanced biological production of 3-HP from renewable carbon sources.

In Klebsiella pneumoniae, 3-hydroxypropaldehyde is converted to 3-hydroxypropionic acid (3-HP) by aldehyde dehydrogenase (ALDH) with NAD(+) as a cofactor. Although ALDH overexpression stimulates the formation of 3-HP, it ceases to accumulate when NAD(+) is exhausted. Here we show that NAD(+) regeneration, together with ALDH overexpression, facilitates 3-HP production and benefits cell growth. Three distinct NAD(+)-regenerating enzymes: NADH oxidase and NADH dehydrogenase from K. pneumoniae, and glycerol-3-phosphate dehydrogenase (GPD1) from Saccharomyces cerevisiae, were individually expressed in K. pneumoniae. In vitro assay showed their higher activities than that of the control, indicating their capacities to regenerate NAD(+). When they were respectively co-expressed with ALD4, an ALDH from S. cerevisiae, the activities of ALD4 were significantly elevated compared with that expressing ALD4 alone, suggesting that the regenerated NAD(+) enhanced the activity of ALD4. More interestingly, the growth rates of all NAD(+)-regenerating strains were prolonged in comparison with the control, indicating that NAD(+) regeneration stimulated cell proliferation. This study not only reveals the reliance of ALD4 activity on NAD(+) availability but also provides a method for regulating the dha regulon.

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Berberine (BBR) is a traditional Chinese medicine which recently was applied as a biological pesticide. Here, we studied the antimicrobial mode of BBR and its impact on soil bacterial diversity. BBR was more effective against fungi than bacteria due to the specific interaction between BBR and glucan. Also, BBR was degraded rapidly in soil, leading to the limited effect on soil bacterial diversity. Collectively, BBR is an environment-friendly pesticide and it is promising in dealing with fungal plant diseases.

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In Klebsiella pneumoniae, aldehyde dehydrogenases (ALDH) convert 3-hydroxypropionaldehyde (3-HPA) into 3-hydroxypropionic acid (3-HP). Although ALDHs can increase the production of 3-HP in K. pneumoniae, the substrate specificity of ALDH homologues from other microorganisms toward 3-HPA is less documented. Here we report that DhaS, a putative ALDH from Bacillus subtilis, shows high specificity toward 3-HPA when heterologously expressed in K. pneumoniae. Using NAD(+) as a cofactor, DhaS exhibited higher catalytic activity (2.3 U mg(-1)) and lower K m value (0.4 mmol l(-1)) toward 3-HPA than that toward other aldehydes. Under shake-flask conditions, the recombinant strain produced 2.1 g 3-HP l(-1) in 24 h, which is 3.9-fold of that in a control harboring a blank vector. Under non-optimized bioreactor conditions, the recombinant strain produced 18 g 3-HP l(-1) and 1,3-propanediol (1,3-PDO) at 27 g l(-1) in 24 h. The overall conversion rate from glycerol to 3-HP and 1,3-PDO reached 59.4 mol mol(-1). Homology modeling of DhaS illustrates substrate specificity and NAD(+)-binding site. DhaS is thus a 3-HPA-specific enzyme useful for production of 3-HP.

Berberine (BBR) is an effective drug for human intestinal inflammation by preventing intestinal adhesion of bacterial pathogens, while its antibacterial activity is ineffective. Although the antimicrobial mechanisms of BBR are intensively studied at high concentrations, the response of pathogens to its low concentrations remains poorly understood. Here we demonstrated that low concentrations of BBR (3 and 6 μg/mL) conferred by hormesis accelerated cell growth of an important Gram-negative pathogen, Klebsiella pneumoniae, in vitro, while higher concentrations (25 and 50 μg/mL) resulted in the opposite. Transcriptome analysis of K. pneumoniae revealed the up-regulated expression of the KmrA efflux pump and further confirmed it was hypersensitive to BBR stress. Strikingly, when cultivated in tetracycline, the growth-promoting effect of BBR became more significant, while this effect was reversed in the presence of the efflux pump inhibitor cyanide-m-chlorophenylhydrazone. The hormesis was also found in Enterobacter cloacae and Acinetobacter baumannii. More importantly, the presence of BBR at low concentrations resulted in higher minimal inhibitory concentrations of efflux-related antibiotics such as rifampicin and azithromycin. Overall, our data demonstrated the hormesis of BBR and revealed the potential risk of its applications against Gram-negative pathogens at low concentrations.