Rational modification of tricarboxylic acid cycle for improving l-lysine production in Corynebacterium glutamicum

Abstract

BackgroundOxaloacetate (OAA) and l-glutamate are essential precursors for the biosynthesis of l-lysine. Reasonable control of all potentially rate-limiting steps, including the precursors supply rate, is of vital importance to maximize the efficiency of l-lysine fermentation process.ResultsIn this paper, we have rationally engineered the tricarboxylic acid (TCA) cycle that increased the carbon yield (from 36.18 to 59.65%), final titer (from 14.47 ± 0.41 to 23.86 ± 2.16 g L−1) and productivity (from 0.30 to 0.50 g L−1 h−1) of l-lysine by Corynebacterium glutamicum in shake-flask fermentation because of improving the OAA and l-glutamate availability. To do this, the phosphoenolpyruvate–pyruvate–oxaloacetate (PEP–pyruvate–OAA) node’s genes ppc and pyc were inserted in the genes pck and odx loci, the P1 promoter of the TCA cycle’s gene gltA was deleted, and the nature promoter of glutamate dehydrogenase-coding gene gdh was replaced by Ptac-M promoter that resulted in the final engineered strain C. glutamicum JL-69Ptac-M gdh. Furthermore, the suitable addition of biotin accelerates the l-lysine production in strain JL-69Ptac-M gdh because it elastically adjusts the carbon flux for cell growth and precursor supply. The final strain JL-69Ptac-M gdh could produce 181.5 ± 11.74 g L−1 of l-lysine with a productivity of 3.78 g L−1 h−1 and maximal specific production rate (qLys, max.) of 0.73 ± 0.16 g g−1 h−1 in fed-batch culture during adding 2.4 mg L−1 biotin with four times.ConclusionsOur results reveal that sufficient biomass, OAA and l-glutamate are equally important in the development of l-lysine high-yielding strain, and it is the first time to verify that fed-batch biotin plays a positive role in improving l-lysine production.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-0958-z) contains supplementary material, which is available to authorized users.

Metabolic engineering of carbohydrate metabolism systems in Corynebacterium glutamicum for improving the efficiency of l-lysine production from mixed sugar

“…A sample was taken from shake flasks or fermenter every 2 or 4 h. A half of sample was used to measure biomass using a spectrophotometer at 600 nm after an appropriate dilution. According to the previous description [57], JL-69P tac-M gdh Fed-batch 181.5 3.78 0.65 [3] JOV2-C7 a Batch 13.2 0.33 0.31 [62] LYS-12 Fed-batch 120 4.0 0.55 [10] MH20-22B∆leuA a,b Batch 21.6 0.3 0.22 [63] the correlation factor between dry cell weight (DCW) and OD 600 was determined as 0.318 (1 OD 600 = 0.318 g DCW). The other half of sample was diluted 100-fold, and then used to determine glucose and l-lysine concentration using an SBA-40E immobilized enzyme biosensor (Shandong, China).…”

“…Inoculum was obtained from a seed culture at ∆OD 562 = 0.45 − 0.50 (at a dilution of 25-fold). The seed medium was prepared according to the description reported by Xu et al [3]. The fermentation medium contained (per liter): 80 g glucose, 40 g beet molasses, 30 g corn steep liquor, 50 g (NH 4 ) 2 SO 4 , 1.5 g KH 2 SO 4 , 1.0 g MgSO 4 •7H 2 O, 0.02 g FeSO 4 , 0.02 g MnSO 4 , 0.5 g l-methionine, 0.05 g glycine betaine, 2.4 mg biotin (add in 4 portions), 400 μg thiamine-HCl and 2 mL antifoam.…”

“…Corynebacterium glutamicum K-1 (i.e., C. glutamicum JL-6 ∆pck::ppc ∆odx::pyc ∆P1gltA/P tac-M gdh) is a l-lysine high-yielding strain with l-lysine production of 181.5 ± 7.65 g/L after cultivating 48 h in fed-batch culture [3]. However, the productivity of C. glutamicum K-1 is lower as compared with the previous results (3.78 g/L/h v.s.…”

Efficient bioproduction of 5-aminolevulinic acid, a promising biostimulant and nutrient, from renewable bioresources by engineered Corynebacterium glutamicum

“…Overexpression of phosphoenolpyruvate carboxylase (PPC) has been proven to be effective to improve biosynthesis of oxaloacetate derivatives such as l-lysine, l-glutamate, and succinate [40][41][42]. Therefore, the native PPC of C. glutamicum was overexpressed under the control of the same P trc promoter with ALAS in C. glutamicum strain CA1.…”

Improving phytosterol biotransformation at low nitrogen levels by enhancing the methylcitrate cycle with transcriptional regulators PrpR and GlnR of Mycobacterium neoaurum

“…The primers for qRT-PCR were listed in Table 1. The messenger RNA (mRNA) level of the 16S rRNA gene was used as the housekeeping gene (internal control) to normalize the sampling errors [38]. Relative gene expression levels were calculated by the comparative Ct method (2 −ΔΔCt method) [39].…”