1,3-Propanediol is a type of bulk chemical widely used as a solvent in the cosmetics industry, and particularly in polymer industries as a building block for the synthesis of polytrimethylene terephthalate (PTT) [1]. PTT has many advantages over its polyester cousin polyethylene terephthalate (PET) and polybutylene terephthalate (PBT). PTT has high resiliency, superior elastic recovery, high bulk, and soft handle, which makes it attractive for making carpets, textile fibers, and nonwovens [2]. PTT can replace PET in all applications, but the high price of 1,3-propanediol limits the competitiveness of PTT in the market. Therefore, producing 1,3-propanediol with high efficiency is a hot topic worldwide. 1,3-propanediol is a metabolite of many microorganisms cultured with glycerol as the feedstock, such as Citrobacter freundii [3], Clostridium butyricum [4], Lactobacillus reuteri [5], and Klebsiella pneumoniae [6]. Among them, K. pneumoniae is the best one, having the highest conversion ratios and highest titers. Since glycerol is a by-product of biodiesel production, it is available at low prices. In general, during biodiesel production 10% (w/w) of glycerol is produced [7]. The technology of 1,3-propanediol production using glycerol as the feedstock and K. pneumoniae as the producer has been already industrialized in China.

The pathway of 1,3-propanediol synthesis from glycerol is a dismutation process, which contains two branches [8]. In the oxidization branch, glycerol is converted to dihydroxyacetone which is catalyzed by glycerol dehydrogenase. Dihydroxyacetone is phosphorylated to phosphate dihydroxyacetone and flows into the glycolysis pathway. This reaction is catalyzed by two kinases [9]. In the reduction branch, glycerol is converted to 3-hydroxypropionaldehyde which is catalyzed by glycerol dehydratase. This enzyme contains three subunits and is a vitamin B12-dependent enzyme. 3-Hydroxypropionaldehyde is further reduced to 1,3-propanedioland catalyzed by 1,3-propanediol dehydrogenase [10]. During the conversion of glycerol to 1,3-propanediol, one molecule of NADH is consumed. NADH, on the other hand, is produced in the oxidation branch and in other catabolic pathways to maintain the balance of NAD+ and NADH [6]. Glycerol dehydratases, 1,3-propanediol dehydrogenase, glycerol dehydrogenase, dihydroxyacetone kinase I and II are encoded by dhaBCE, dhaT, dhaD, dhaK, and dhaK123, respectively. These genes and a regulatory protein DhaR form a dha operon. This operon is induced by dihydroxyacetone (Fig. 1) [8]. Besides the dha operon, the pdu regulon which is responsible for 1,2-propanediol catabolism was also involved in 1,3-propanediol synthesis from glycerol in K. pneumoniae. pduCDE encodes a diol dehydratase, which is an isoenzyme of the dhaBCE encoded glycerol dehydratase. Diol dehydratase and other enzymes of the pdu regulon are located in the Pdu microcompartment, a bacteria organelle, rather than in the cytoplasm of the cell [11].

Many studies in metabolic engineering have focused on the production of 1,3-propanediol by K. pneumoniae. Lactic acid is a by-product of 1,3-propanediol production and is synthesized from pyruvate. ldhA encoded lactate dehydrogenase catalyzes this reaction. It has been proven that ldhA disrupted K. pneumoniae strain can lead to the increase of 1,3-propanediol concentration [12] 2,3-butanediol is also a by-product in the synthesis of 1,3-propanediol from glycerol, and the key enzyme in this process is decarboxylase encoded by budA. However, a budA disrupted K. pneumoniae was unable to synthesize 2,3-butanediol, and at the same time the concentration of 1,3-propanediol was also reduced [13].

2,3-butanediol is a main catabolite of K. pneumoniae during cultivation on glucose as the substrate. 2,3-butanediol is synthesized from pyruvate, in a way that two molecules of pyruvate form one molecule of acetolactate which is catalyzed by a budB encoded acetolactate synthetase. Acetolactate is further converted to acetoin, catalyzed by a budA encoded decarboxylase, while the acetoin is converted to 2,3-butanediol, catalyzed by butanediol dehydrogenase (Fig. 1).

The conversion of pyruvate to acetyl-CoA is a central reaction of living organisms’ metabolic pathways. AcoABCD concedes a pyruvate dehydrogenase. This enzyme catalyzes the conversion of pyruvate to acetyl-CoA and CO2, with one molecule of NAD+ converted to NADH. At the same time, a pflB encoded pyruvate formatelyase catalyzes pyruvate conversion to acetyl-CoA and formic acid, but in this reaction NADH is not generated. The two enzymic reactions are both active in the wild type strain of K. pneumonia (Fig. 1). In our previous research, a pflB knock out K. pneumonia strain was constructed, and the formation of acetyl-CoA from pyruvate must undergo the reaction catalyzed by pyruvate dehydrogenase. Thus, more NADH was generated in this strain than that of the wild type strain, and the 1,3-propanediol production of this strain was improved. However, small amounts of formic acid were still produced in the broth of this strain [14].

Hydrogen is an anaerobic metabolic product of many Enterobacteria. The mechanism of hydrogen production by bacteria is triggered by anaerobic conditions. Under aerobic conditions, the carbon sources are completely oxidized to CO2. Electrons formed in the process are finally transferred to O2 through the respiratory chain to form H2O. Under anaerobic conditions, some catabolites accept electrons to form incomplete oxidative compounds, such as lactic acid, acetic acid, formic acid, ethanol, etc. However, the production of organic acids leads to a decrease in the pH of the environment, and too low a pH is lethal to bacteria. H2 production is a mechanism used by some bacteria to reduce the drop in pH of the environment under anaerobic conditions. Normally, formic acid is produced by bacteria and released into the environment. When the pH of the environment decreases, the formic acid is reabsorbed by the cell and converted to H2 and CO2 by catalysis of the formate hydrogenlyase complex [15]. There are four hydrogenases encoding genes in the genome of E. coli, and formate hydrogenlyase complex (hydrogenase-3) is responsible for nearly all H2 production. The structure of this complex has been well-resolved. It is membrane-bounded and contains a formate dehydrogenase and a [NiFe] hydrogenase. Formate dehydrogenase is encoded by fdhF. Hydrogenase contains 6 subunits and they are encoded by hycBCDEFG, respectively. Formate dehydrogenase catalyzes the oxidation of formic acid to form CO2 and proton. Two electrons are transferred to hycE and driving proton reduction to H2 [16].

As a kind of clean energy, H2 production by microorganisms especially by Enterobacteria has been a hotspot in recent years [17]. As the Thauer limit, scientists generally assume that a maximum of 4 moles of H2 can be produced from one molecule of glucose. However, there are some reports that H2 production has exceeded the Thauer limit [18]. These results are dependent on external electro-power input. The reaction of H2 production from formic acid is reversible, and this reaction has been used for CO2 fixation [19]. Although the efficiency of CO2 fixation is low, this has been investigated a lot in recent years.

Although there are some reports on H2 production by K. pneumonia [20], [21], [22], [23], there are no reports on K. pneumonia losing its ability to synthesize H2. In this work, the encoding genes of hydrogenase-3 in K. pneumoniae were knocked out, and their effects on the production of 1,3-propanediol and 2,3-butanediol, have been studied in detail. Results show that the inactivation of hydrogenase-3 has a distinct enhancement on 1,3-propanediol and 2,3-butanediol production by K. pneumoniae, even at microaerobic cultivation conditions.