Improved sorbitol production and growth in cyanobacteria using promiscuous haloacid dehalogenase-like hydrolase.

Sorbitol can be produced photosynthetically in the model cyanobacterium Synechocystis sp. PCC 6803 harboring sorbitol-6-phosphate (S6P) dehydrogenase (S6PDH) from apple, representing a promising metabolic engineering strategy for environmentally friendly carbon-based compound production. However, no gene for S6P phosphatase has been reported to date. We previously found that members of the Escherichia coli haloacid dehalogenase-like hydrolase (HAD) superfamily, a group of promiscuous hydrolases, exert phosphatase activity to S6P in vitro. Here, we examined the effects of HADs on sorbitol production in cyanobacterial cells. Overexpression of E. coli HAD1 improved sorbitol production induced by toxic S6PDH, whereas it suppressed growth even without induction. Moreover, overexpression of HAD1 in combination with engineering of other pathways successfully allowed for the constitutive expression of toxic S6PDH. Consequently, the sorbitol production was highly improved, which in turn suppressed the growth suppression effect of promiscuous HAD1. These results provide a good example of a novel metabolic engineering strategy using a combination of a promiscuous enzyme with an abundant supply of one of its substrates.

Detection of active sorbitol-6-phosphate phosphatase in the haloacid dehalogenase-like hydrolase superfamily.

Sorbitol-6-phosphatase (EC 3.1.3.50) catalyzes sorbitol production from sorbitol-6-phosphate in certain organisms, but has not been identified unequivocally. We screened the activity of the haloacid dehalogenase-like hydrolases (HAD) superfamily and identified four HAD proteins from Escherichia coli as sorbitol-6-phosphatase. Of these proteins, HAD2 (YfbT) exhibited catalytic activity (kcat/Km) that was better than that of the previously reported "preferred" substrate. HAD1 (YniC) and HAD2 exhibited higher sorbitol-6-phosphatase activity than that of HAD12 (YbiV) and HAD13 (YidA). Therefore, genes of HAD may be useful for metabolic engineering of effective sorbitol production.

Sorbitol production and optimization of photosynthetic supply in the cyanobacterium Synechocystis PCC 6803.

Biochemicals production is a major theme in the application of photosynthesis to address global warming and organic-resource problems. Among biochemicals, sugar alcohols have attracted research attention because they are directly derived from two photosynthetic products, sugars and reductants. Here, we produced sorbitol photosynthetically by using cyanobacteria and modified the supply of its substrates through genetic engineering. Expression of an NADPH-dependent enzyme that generates sorbitol-6-phosphate, S6PDH, was highly toxic to cyanobacteria likely due to the sorbitol production, whereas expression of an NADH-dependent enzyme, SrlD2, yielded no sorbitol. The toxicity was partly overcome by introducing a theophylline-inducible riboswitch for S6PDH expression and optimizing induction, but sorbitol production was still low and severely inhibited growth. Co-expression of fructose-1,6-bisphosphatase drastically alleviated the growth inhibition, but did not increase short-term sorbitol production. The NADPH/NADP+ ratio decreased during sorbitol production. Overexpression of a membrane-bound transhydrogenase for NADPH generation from NADH elevated the short-term sorbitol production, but only partly alleviated the growth inhibition. Notably, a strain overexpressing all three enzymes exhibited sustainable sorbitol production at 312 mg/L, which was nearly 27-fold higher than the yield of the initial S6PDH-overexpressing strain. We discuss these results in relation to the optimization of photosynthetic supply for sorbitol production in cyanobacteria.

Photosynthetic production of itaconic acid in Synechocystis sp. PCC6803.

Here, we report the photosynthetic production of itaconic acid (IA), a promising building block, from carbon dioxide (CO2) by Synechocystis sp. PCC6803. The engineered PCC6803 strain expressing cis-aconitate decarboxylase, the key enzyme in IA biosynthesis, produced 0.9 mg/L and 14.5 mg/L of IA at production rates of 42.8 µgL(-1)day(-1) and 919.0 µgL(-1)day(-1), under conditions of constant bubbling with air and 5% CO2, respectively. This is the first report on the possibility of IA production from CO2 via the photosynthetic process in cyanobacteria.

Production of itaconic acid in Escherichia coli expressing recombinant α-amylase using starch as substrate.

Several studies on fermentative production of a vinyl monomer itaconic acid from hydrolyzed starch using Aspergillus terreus have been reported. Herein, we report itaconic acid production by Escherichia coli expressing recombinant α-amylase, using soluble starch as its sole carbon source. To express α-amylase in E. coli, we first constructed recombinant plasmids expressing α-amylases by using cell surface display technology derived from two amylolytic bacteria, Bacillus amyloliquefaciens NBRC 15535(T) and Streptococcus bovis NRIC 1535. The recombinant α-amylase from S. bovis (SBA) showed activity at 28°C, which is the optimal temperature for production of itaconic acid, while α-amylase from B. amyloliquefaciens displayed no noticeable activity. E. coli cells expressing SBA produced 0.15 g/L itaconic acid after 69 h cultivation under pH-stat conditions, using 1% starch as the sole carbon source. In fact, E. coli cells expressing SBA had similar growth rates when grown in the presence of 1% glucose or starch, thereby highlighting the expression of an active α-amylase that enabled utilization of starch to produce itaconic acid in E. coli.

Production of itaconic acid using metabolically engineered Escherichia coli.

An Escherichia coli system was engineered for the heterologous production of itaconic acid via the expression of cis-aconitate decarboxylase gene (cad), and then maximal itaconic acid levels produced by engineered E. coli were evaluated. Expression of cad in E. coli grown in Luria-Bertani (LB) medium without glucose in a test tube resulted in 0.07 g/L itaconic acid production after 78 h at 20°C. To increase itaconic acid production, E. coli recombinants were constructed by inactivating the isocitrate dehydrogenase gene (icd) and/or the isocitrate lyase gene (aceA). Expression of cad and inactivation of icd resulted in 0.35 g/L itaconic acid production after 78 h, whereas aceA inactivation had no effect on itaconic acid production. The intracellular itaconate concentration in the Δicd strain was higher than that in the cad-expressing strain without icd inactivation, which suggests that the extracellular secretion of itaconate in E. coli is the rate-determining step during itaconic acid production. pH-stat cultivation using the cad-expressing Δicd strain in LB medium with 3% glucose in a jar fermenter resulted in 1.71 g/L itaconic acid production after 97 h at 28°C. To further increase itaconic acid production, the aconitase B gene (acnB) was overexpressed in the cad-expressing Δicd strain. Simultaneous overexpression of acnB with the expression of cad in the Δicd strain led to 4.34 g/L itaconic acid production after 105 h. Our findings indicate that icd inactivation and acnB overexpression considerably enhance itaconic acid production in cad-expressing E. coli.