Insights into 3-hydroxypropionic acid biosynthesis revealed by overexpressing native glycerol dehydrogenase in Klebsiella pneumoniae.

In Klebsiella pneumoniae, glycerol dissimilation involves parallel oxidation and reduction pathways. Oxidation pathway provides adenosine triphosphate (ATP) and cofactors to sustain cell growth, while reduction pathway presents 3-hydroxypropionic acid (3-HP) and 1,3-propanediol(1,3-PDO), which are commercially attractive platform chemicals. Previous metabolic engineering of K. pneumoniae focused on the intensification of reduction pathway; however, it failed to overproduce 3-HP or 1,3-PDO. Contrary to this strategy, here we show that overexpression of glycerol dehydrogenase (dhaD), the first functional enzyme in oxidation pathway, can efficiently stimulate cell growth and facilitate 3-HP accumulation. Under microaerobic conditions, although metabolic burden arising from plasmid replication, the recombinant K. pneumoniae overexpressing dhaD grew actively and showed 60% enhancement of 3-HP compared to the control. In particular, overexpression of dhaD increased the activity of glycerol dehydratase, indicating the concerted action of two enzymes and the interdependence between glycerol oxidation and reduction pathways. Moreover, the strain overexpressing dhaD produced more lactic acid yet less acetic acid than the control, implying the interplay between dhaD expression and the formation of byproducts. Together, not only showing that intensifying glycerol oxidation pathway is beneficial to 3-HP production, this study also reveals the structural rigidity of dha operon that mediates glycerol dissimilation in K. pneumoniae.

Engineered constitutive pathway in Klebsiella pneumoniae for 3-hydroxypropionic acid production and implications for decoupling glycerol dissimilation pathways.

3-Hydroxypropionic acid (3-HP) is a commercially important platform chemical from which a panel of chemicals can be generated. Klebsiella pneumoniae has been regarded as a promising host strain in glycerol-based 3-HP production for its exceptional ability to metabolize glycerol. Since the glycerol dissimilation mechanism governs the carbon flux distribution from glycerol, inducible strong promoters were usually employed to enhance the glycerol consumption and 3-HP production. Here, we report an alternative strategy that the native promoter of dhaB gene was applied to enhance 3-HP production in K. pneumoniae. The key enzyme genes (ald4 and dhaB) for 3-HP biosynthesis were co-expressed under this promoter. Metabolic analysis revealed that the 3-HP formation was partially coupled with cell metabolism. To optimize the production of 3-HP, the effects of glucose as energy source assistant were investigated based on the analysis of fermentation process kinetics. The highest 3-HP yield (3.77 g/L in flask) was observed upon optimized conditions. Since there were no additional inducers needed, the strategy of employing native promoter seems more feasible to industrial application. More importantly, the employment of constitutive promoter demonstrated an effective approach for decoupling the natural correlation between respiratory metabolism and glycerol dissimilation in K. pneumoniae.

Classifying DNA assembly protocols for devising cellular architectures.

DNA assembly is one of the most fundamental techniques in synthetic biology. Efficient methods can turn traditional DNA cloning into time-saving and higher efficiency practice, which is a foundation to accomplish the dreams of synthetic biologists for devising cellular architectures, reprogramming cellular behaviors, or creating synthetic cells. In this review, typical strategies of DNA assembly are discussed with special emphasis on the assembly of long and multiple DNA fragments into intact plasmids or assembled compositions. Constructively, all reported strategies were categorized into in vivo and in vitro types, and protocols are presented in a functional and practice-oriented way in order to portray the general nature of DNA assembly applications. Significantly, a five-step blueprint is proposed for devising cell architectures that produce valuable chemicals.

[Progress in metabolism and crucial enzymes of glycerol conversion to 1,3-propanediol].

1,3-propanediol production by microbial fermentation has become the research hot spot for its amiability with the environment. Here the molecular mechanism of glycerol bioconversion to 1,3-propanediol was outlined by elucidating the fermentation strains, metabolic pathways, regulon and key enzymes. Of enzymes, glycerol dehydrogenase, the velocity-limiting enzyme in glycerol reductive pathway, was emphatically discussed with regard to its molecular structure and reactivating factors. This paper aims to provide the basis for genetic modification of fermentation strains.

Progress in plant CACTA elements.

Transposable elements are DNA fragments that can insert new chromosomal locations. On the basis of the mechanism of transposition, transposable elements were divided into two classes. Class 1 elements were retroelements that used reverse transposase to transpose by an RNA intermediate. Class 2 elements or DNA transposons transposed directly from DNA to DNA. Of the Class 2 elements, CACTA superfamily, so far identified exclusively in plants and previously regarded as low-copy-transposon for the conserved mechanism of propagation, recently received considerable interest because of their increasing evidence reiterating their high copies in some plant genomes. This article aimed at outlining CACTA elements with regard to their structure, transposition, and utilization.

[The influence of soybean isoflavone on expression of low density lipoprotein receptor (LDLR) mRNA in ovariectomied rats].

OBJECTIVE: To investigate the effect of soybean isoflavone on blood lipid and the expression of LDLR mRNA in ovariectomied rats. METHODS: Forty eight Sprague-Dawley rats were divided randomly into four groups: Sham group (sham), OVX group (OVX), OVX + gemifibrozil (G) group (OVX + G) and OVX + isoflavone (ISO) group (OVX_ISO). in which the rats were treated with G or ISO for three months starting from two weeks after both sides of rat' s ovarietomy. Blood sample were taken out for determination of blood lipid. The liver tissue were taken out quickly, Isothiocyanate guanidine-phenol-chloroform was used to extract the total RNA from the liver and RT-PCR was used to detect the expression of LDLR mRNA. RESULTS: Compared with the OVX group, contents of TG, LDL-C and OX-LDL in serum in OVX + ISO group decreased remarkably, and the contents of HDL-C increased. LDLR mRNA in OVX + ISO group increased distinctively compared with OVX group and OVX + G group. CONCLUSION: The level of mRNA of LDLR in liver decreas after ovariotormy and the soybean isoflavone may increase it by regulating the expression level of LDLR through transcription.