Effect of fed-batch and chemostat cultivation processes of C. glutamicum CP for L-leucine production

Figures & data

Table 1. Composition of chemostat culture feed media with different C/N ratios

C/N ratio (mol/mol)	(NH4)2SO4 (g L^−1)	(NH4)2C2O4 (g L^−1)	CH3COONH4 (g L^−1)	NH4Cl (g L^−1)
99.2	0	0	0	0
72.9	0.5	0.5	0.6	0.4
57.6	1.0	1.0	1.2	0.8
40.6	2.0	1.9	2.5	1.6
25.5	4.0	3.9	5.1	3.2

Table 2. Primers used for quantitative real-time PCR

Primer No.	Target gene		Sequence (5ʹ-3ʹ)	Amplicon size (bp)
1	leuA	Forward	AGGTTGAGTTCTCCACCGTTGTC	124
2		Reverse	ATCTGCTCAACTGGTGCGGT
3	leuB	Forward	TTGCTGTTATTGGTGGAGATGGTAT	147
4		Reverse	AAGGTCATAATCGGTGGTCTCG
5	leuC	Forward	ATGACCAGCCCCGTGGAGAAC	105
6		Reverse	GTCGGGCTCGCCGTTTTCT
7	leuD	Forward	ATCCCAGCCGTCTACCTCAAG	141
8		Reverse	CGAATCGTGAGGAGAAGACAGC
9	brnE	Forward	TTGCGATTACAGTGGTGGCG	119
10		Reverse	CCTGCTGGCATCCACATCG
11	brnF	Forward	CGGCGTGGCGACTTATCTCA	159
12		Reverse	GGCAGGAATCCAAAGTCAGCGT
13	alaT	Forward	GTCTATCCAAGGCATACCGCG	134
14		Reverse	TGGGACATTTGGGCAGAGTC
15	avtA	Forward	CAGCCCAGGAAACCCAACG	136
16		Reverse	TGCCAGCGGACGACCAAAGC
17	16S rRNA	Forward	CGATACGGGCATAACTTGA	181
18		Reverse	GTTTACGGCATGGACTACC

Figure 1. The growth, product/byproduct generation, and glucose consumption profiles of C. glutamicum CP in fed-batch culture

Figure 2. Effect of dilution rate on the chemostat culture of C. glutamicum CP. (a) Biomass, (b) L-leucine, (c) L-alanine, (d) Glucose. Data are presented as means ± standard deviations from three independent experiments. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001

Figure 3. The effect of nitrogen source and C/N ratio on the chemostat culture of C. glutamicum CP in the steady state. (a) Biomass, (b) L-leucine, (c) L-alanine, (d) Glucose

Figure 4. Comparison of gene expression levels and activities of enzymes related to the synthesis of L-leucine and L-alanine. The expression levels of the genes were measured using fluorescence quantitative RT-PCR from RNA extracted in the stationary phases during the fed-batch process and the steady state of the chemostat process. Specific activity of IPMS, IPMD, AlaT, and AvtA in crude extracts of C. glutamicum CP were determined, respectively. Data are presented as means ± standard deviations from three independent experiments. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001

Table 3. The representative strains for production of L-leucine

Strain	Relevant characteristics	Media	Culture mode	L-leucine titer g L^−1		L-alanine titer g L^−1	yield mol mol^−1	Productivity g·L^−1·h^−1	Reference
C. glutamicum CP	Mutation of ilvBN and leuA	semi-defined culture medium	Fed-batch, glucose 80%, 32 °C, 44 h		53.0 ± 1.2	8.9 ± 0.3	0.30	1.2	This work
C. glutamicum CP	Mutation of ilvBN and leuA	semi-defined culture medium	Chemostat, glucose 10%, 32 °C, 100 h		24.8 ± 0.2	0.8 ± 0.1	0.33	1.9	This work
C. glutamicum WL-14	Deletion of ltbR, ppc, avtA, insertion of terminator T3 in front of alaT, replacement of ilvBNC and leuA promoter by Ptuf, pECXK99E-leuA	semi-defined culture medium	shake-flasks, glucose 13%, 30 °C, 72 h		28.5 ± 0.4	-	-	-	[45]
C. glutamicum XQ-9	L-leucine producing C. glutamicum strain created by random mutagenesis	semi-defined culture medium	shake-flasks, glucose 13%, 30 °C, 72 h		23.3 ± 0.2	-	0.26	0.3	[47]
C. glutamicum FA-1	pECXK99E-aspB, deletion of ilvE	semi-defined culture medium	shake-flasks, glucose 13%, 30 °C, 72 h		20.8 ± 0.3	2.38	-	0.289	[44]
C. glutamicum MV-LeuF2	Deletion of avtA; deletion of iolR; deletion of ltbR; reduction of gltA; introduction of ilvBN^r; integration of leuA-B018; replacement of leuA-B018 promoter by Ptuf	chemically defined medium	Fed-batch, glucose 50%, 30 °C, 50 h		24	1.8	0.26	0.4	[27]
E.coli K-12 No.103	Resistance of L-leucine	chemically defined medium	shake-flasks, glucose 6%, 32 °C, 48 h		1.7	-	-	0.035	[48]
E.coli K-12 No.55	Mutation of leuA^r	chemically defined medium	shake-flasks, glucose 6%, 32 °C, 48 h		5.2	-	-	0.108	[49]
E.coli K-12 505	Mutation of ilvE (double L-valine and L-isoleucine auxotrophy); overexpression of tyrB, pACYC-tyrB	semi-defined culture medium	shake-flasks, glucose 6%, 32 °C, 48 h		2.7	-	-	0.038	[50]
E.coli 57	Feedback-resistant leuA*; inactivation of ilvE; replacement of sucAB promoter by Ptac−21	defined culture medium	shake-flasks, glucose 6%, 34 °C, 72 h		11.4	-	-	0.158	[51]

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