Effect of Polyhydroxybutyrate (PHB) storage on l-arginine production in recombinant Corynebacterium crenatum using coenzyme regulation

Abstract

BackgroundCorynebacterium crenatum SYPA 5 is the industrial strain for l-arginine production. Poly-β-hydroxybutyrate (PHB) is a kind of biopolymer stored as bacterial reserve materials for carbon and energy. The introduction of the PHB synthesis pathway into several strains can regulate the global metabolic pathway. In addition, both the pathways of PHB and l-arginine biosynthesis in the cells are NADPH-dependent. NAD kinase could upregulate the NADPH concentration in the bacteria. Thus, it is interesting to investigate how both PHB and NAD kinase affect the l-arginine biosynthesis in C. crenatum SYPA 5.ResultsC. crenatum P1 containing PHB synthesis pathway was constructed and cultivated in batch fermentation for 96 h. The enzyme activities of the key enzymes were enhanced comparing to the control strain C. crenatum SYPA 5. More PHB was found in C. crenatum P1, up to 12.7 % of the dry cell weight. Higher growth level and enhanced glucose consumptions were also observed in C. crenatum P1. With respect to the yield of l-arginine, it was 38.54 ± 0.81 g/L, increasing by 20.6 %, comparing to the control under the influence of PHB accumulation. For more NADPH supply, C. crenatum P2 was constructed with overexpression of NAD kinase based on C. crenatum P1. The NADPH concentration was increased in C. crenatum P2 comparing to the control. PHB content reached 15.7 % and 41.11 ± 1.21 g/L l-arginine was obtained in C. crenatum P2, increased by 28.6 %. The transcription levels of key l-arginine synthesis genes, argB, argC, argD and argJ in recombinant C. crenatum increased 1.9–3.0 times compared with the parent strain.ConclusionsAccumulation of PHB by introducing PHB synthesis pathway, together with up-regulation of coenzyme level by overexpressing NAD kinase, enables the recombinant C. crenatum to serve as high-efficiency cell factories in the long-time l-arginine fermentation. Furthermore, batch cultivation of the engineered C. crenatum revealed that it could accumulate both extracellular l-arginine and intracellular PHB simultaneously. All of these have a potential biotechnological application as a strategy for high-yield l-arginine.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0414-x) contains supplementary material, which is available to authorized users.

Increasing Ascomycin Yield in Streptomyces Hygroscopicus var. Ascomyceticus by Using Polyhydroxybutyrate as an Intracellular Carbon Supply Station

“…Different from the defective growth of knockout strains mentioned above, the co-overexpressed strain accumulated more biomass and produced more FK520 due to the time difference between the accumulation and degradation of PHB. Previous study showed that the introduction of PHB gene cluster in Corynebacterium crenatum could improve the growth level of strains by promoting the consumption of glucose [20]. Here, the comparative transcriptomic analysis re ected that when carbon sources were abundant in the environment, polyhydroxybutyrate stored in the form of polymer and stimulated strain growth by promoting the utilization of carbon sources.…”

“…In fact, it had been reported that the introduction of PHB synthesis pathway could promote the production of some primary metabolites, such as L-threonine, succinate and L-arginine, by driving the carbon ux of related primary metabolic pathways [18][19][20]. Even so, PHB metabolism was also once thought to be an ineffective cycle in the biosynthesis of dicarboxylic acids in the EMCP.…”

“…For example, the introduction of the PHB biosynthesis pathway in Escherichia coli QZ1111 improved the output of succinate by increasing the ux of pyruvate into citrate cycle (TCA) [19]. And the introduction of the PHB synthesis pathway into Corynebacterium crenatum increased the L-arginine yield by regulating the corresponding carbon metabolic ows [20]. However, the effect of PHB on antibiotic production has not been reported to date.…”

Recent Advances of L-ornithine Biosynthesis in Metabolically Engineered Corynebacterium glutamicum

“…NADPH, an important cofactor, is widely used in the biosynthesis of various amino acids, such as lysine (Wu et al, 2019), valine (Zhang H. et al, 2018), methionine (Li et al, 2016), ornithine (Hwang and Cho, 2012), and arginine (Chen M. et al, 2015). Intracellular supplementation of NADPH can be increased by various strategies, such as improving the metabolic flux of the pentose phosphate pathway (Siedler et al, 2013), overexpression of NAD kinase (Lindner et al, 2010;Xu et al, 2016), and developing a NADP-dependent glyceraldehyde 3-phosphate dehydrogenase (Takeno et al, 2010(Takeno et al, , 2016Hoffmann et al, 2018). NADPH is a rate-limiting factor in the biosynthesis of metabolites in C. glutamicum.…”

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) co-produced with l-isoleucine in Corynebacterium glutamicum WM001

“…Nocardia and Rhodococcus could naturally accumulate PHBV but are commercially non-viable [ 20 ], Bacillus could produce PHBV [ 19 ] but requires addition of extra threonine and cyanocobalamin, Corynebacterium could produce PHBV [ 22 ] but requires the addition of propionate in the medium. PHA could be co-produced with amino acids, such as l -glutamate [ 23 ], l -tryptophan [ 24 ], l -arginine [ 25 ], and succinate [ 26 ]. The co-productions positively affected the transcription of key enzymes, increased the product yield, rearranged the cofactor flux, and improved the cell growth.…”