Effect of ldhL Gene Knock out Mutant on Lactobacillus delbrueckii subsp. blgaricus Producing L-lactic Acid

doi:10.13523/j.cb.20190809

China Biotechnology

2019, Vol. 39

Issue (8): 66-73 DOI: 10.13523/j.cb.20190809

Orginal Article

Effect of ldhL Gene Knock out Mutant on Lactobacillus delbrueckii subsp. blgaricus Producing L-lactic Acid

WANG Gang^1,²,XIAO Yu¹,LI Yi²,LIU Zhi-gang²,PEI Cheng-li²,WU Li-da²,LI Yan-li¹,WANG Xi-qing¹,ZHANG Ming-lei¹,CHEN Guang^1,^3,^**(

),TONG Yi^2,^**(

)

1 College of Life Science, Jilin Agricultural University,Changchun 130118,China
2 Cofco Biochemical Co, LTD of Jilin, Changchun 130118,China
3 Key Laboratory for Straw Biology and Utilization of the Ministry of Education, Jilin Agricultural University,Changchun 130118,China

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Abstract

To construct ldhL gene deletion strain in Lactobacillus delbrueckii subsp. bulgaricus.Bulgaria lactobacillus CICC21101 was employed as the original strain, amplified the upstream sequence ldhL1 and downstream sequence ldhL2 by PCR, obtained successfully ldhL gene deletion fragment which includes both upstream and downstream sequence, then connected to knockout plasmid pGhost4, electricity to Bulgaria lactobacillus CICC21101, screening at low temperature. The results indicated that the ldhL gene knocked out deficient mutant was obtained.By gene knockout, the yield of D-lactic acid decreased from 30.5g/L to 4.8g/L, the yield of L-lactic acid increased from 25.4g/L to 58.3g/L, and the optical purity increased from 54.56% to 90%. It was a foundation to study the detail functions of ldh in Lactobacillus delbrueckii subsp. bulgaricus,it will lay the foundation of constuction on D-lactic acid engineering bacteria.

Key words： Lactobacillus delbrueckii subsp.bulgaricus L-lactate dehydrogenase Homologous recombination Knock out pGhost4

Received: 22 January 2019 Published: 18 September 2019

ZTFLH:

Q93

Corresponding Authors: Guang CHEN,Yi TONG E-mail: chg61@163.com;tongyi@cofco.com

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	Articles by authors
	Gang WANG
	Yu XIAO
	Yi LI
	Zhi-gang LIU
	Cheng-li PEI
	Li-da WU
	Yan-li LI
	Xi-qing WANG
	Ming-lei ZHANG
	Guang CHEN
	Yi TONG

Cite this article:

WANG Gang,XIAO Yu,LI Yi,LIU Zhi-gang,PEI Cheng-li,WU Li-da,LI Yan-li,WANG Xi-qing,ZHANG Ming-lei,CHEN Guang,TONG Yi. Effect of ldhL Gene Knock out Mutant on Lactobacillus delbrueckii subsp. blgaricus Producing L-lactic Acid. China Biotechnology, 2019, 39(8): 66-73.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20190809 OR https://manu60.magtech.com.cn/biotech/Y2019/V39/I8/66

Fig.1 The Pyruvate metabolic pathway of Lactobacillus delbrueckii subsp. bulgaricus

Fig.2 Strategy employed in this study for the deletation of ldh gene based on red-recombinat technology

Fig.3 PCR confirmation of recombinant clone M:Marker DL2000;1-2:The PCR results of recombinant clone plasmid

Fig.4 The result of restriction endonuclease digestion M:Marker DL5000;1:Recombinant clone plasmid by restriction enzyme digestion

Fig.5 PCR confirmation of recombinant plasmid M: Marker DL5000;1-3: Recombinant expression plasmid

Fig.6 PCR confirmation of mutant strain M:Marker DL5000;1-2: Wide type strain; 3-4: Mutant strain

Fig.7 Growth curve of wild type and mutant strain

Table 1 The effect of knock out ldb0094 on lactic acid production

Fig.8 Engineering bacteria determination of the gene expression results


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