Abstract
BackgroundCo-production of polyhydroxyalkanoate (PHA) and amino acids makes bacteria effective microbial cell factories by secreting amino acids outside while accumulating PHA granules inside. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) is one of the PHAs with biocompatibility and fine mechanical properties, but its production is limited by the low level of intracellular propionyl-CoA.Resultsl-Isoleucine producing Corynebacterium glutamicum strain WM001 were analyzed by genome and transcriptome sequencing. The results showed that the most over-expressed genes in WM001 are relevant not only to l-isoleucine production but also to propionyl-CoA accumulation. Compared to the wild-type C. glutamicum ATCC13869, the transcriptional levels of the genes prpC2, prpD2, and prpB2, which are key genes relevant to propionyl-CoA accumulation, increased 26.7, 25.8, and 28.4-folds in WM001, respectively; and the intracellular level of propionyl-CoA increased 16.9-fold in WM001. When the gene cluster phaCAB for PHA biosynthesis was introduced into WM001, the recombinant strain WM001/pDXW-8-phaCAB produced 15.0 g/L PHBV with high percentage of 3-hydroxyvalerate as well as 29.8 g/L l-isoleucine after fed-batch fermentation. The maximum 3-hydroxyvalerate fraction in PHBV produced by WM001/pDXW-8-phaCAB using glucose as the sole carbon source could reach 72.5%, which is the highest reported so far.ConclusionsGenome and transcriptome analysis showed that C. glutamicum WM001 has potential to accumulate l-isoleucine and propionyl-CoA pool. This was experimentally confirmed by introducing the phaCAB gene cluster into WM001. The recombinant strain WM001/pDXW-8-phaCAB produced high levels of PHBV with high 3-hydroxyvalerate fraction as well as l-isoleucine. Because of its high level of intracellular propionyl-CoA pool, WM001 might be used for producing other propionyl-CoA derivatives.
Highlights
Co-production of polyhydroxyalkanoate (PHA) and amino acids makes bacteria effective microbial cell factories by secreting amino acids outside while accumulating PHA granules inside
Genome sequencing and transcriptome analysis of C. glutamicum WM001 Corynebacterium glutamicum has been remarkable platform bacteria for producing l-isoleucine [32]. l-Isoleucine-producing C. glutamicum strain WM001 was originally isolated from soil, and is closely related to C. glutamicum strain ATCC13869, based on their 16S rDNA sequences
The GC content of C. glutamicum WM001 is similar to the GC content of C. glutamicum ATCC13032 (53.8%), ATCC14067(54.1%), and ATCC13869 (54.2%) [37]
Summary
Co-production of polyhydroxyalkanoate (PHA) and amino acids makes bacteria effective microbial cell factories by secreting amino acids outside while accumulating PHA granules inside. Poly(3-hydroxybutyrate-co3-hydroxyvalerate) (PHBV) is one of the PHAs with biocompatibility and fine mechanical properties, but its production is limited by the low level of intracellular propionyl-CoA. Industrial producers of l-isoleucine were mostly Corynebacterium glutamicum [2]. Several l-isoleucine producers have been obtained by random mutation, site-directed mutagenesis, and rational metabolic engineering [1, 3,4,5,6,7,8,9,10]. Polyhydroxyalkanoates (PHAs) are natural biodegradable and biocompatible polyesters with piezoelectricity and flexible mechanical properties and are potential alternatives for petroleum-based plastics [11]. PHAs accumulated in various microorganisms and plants for storing carbon and energy under nutrient imbalance [12] and could be naturally degraded within 100 h in the soil [11]
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