Construction of Recombinant <i>Bacillus subtilis</i> as Catalyst for Preparing D- p-Hydroxyphenylglycine

doi:10.13523/j.cb.20190310

China Biotechnology

2019, Vol. 39

Issue (3): 75-86 DOI: 10.13523/j.cb.20190310

Construction of Recombinant Bacillus subtilis as Catalyst for Preparing D- p-Hydroxyphenylglycine

Fa-bin LI,Lu LIU,Yan DU,Rui Ban(

)

1 School of Chemical Engineering and Technology, Key Laboratory of Systems Biotechnology of Ministry of Education,Tianjin University, Tianjin 300350, China

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Abstract

Objective: To construct the recombinant Bacillus subtilis with D-hydantoinase(DHase, hyd ene) and D-carbamoylase(DCase, adc gene) activity as catalyst to produce D-p-hydroxyphenylglycine(D-HPG) by hydantoinase process. Methods: The hyd gene expression plasmids were constructed. The effects of divalent metal ions in medium on the DHase activity was investigated. The acoR gene was over-expressed to investigate the correlation between the activator protein AcoR and P_acoA-hyd gene expression. The optimal promoter used to express adc gene was screened from the P_AE, P_spoVG, P_cdd and P_lytR. The hyd and adc gene co-expression plasmid was constructed and its catalytic properties was characterized. Results: The hyd gene expression plasmid pHPS and pUBS were successfully constructed. Mn ²⁺ has a strong activation effect on DHase and the activity of the 168N/pUBS reached 956U/gDCW when 0.8mmol/L MnCl₂·4H₂O was added to the medium. Adding a copy of the P_cdd-acoR gene, DHase activity of the LSL02/pUBS reached 1 470 U/gDCW. The LN04 strain integrated with the P_AE-adc had the highest DCase activity. The co-expression plasmid pUBSC was constructed, and under the optimum conditions of pH 8.0 and 40℃, with initial substrate concentration of 20g/L, the catalytic activity of LSL02/pUBSC could last for 12h to generate 14.32g/L of D-HPG with the conversion rate of 95%, yield of 82.4%. Conclusion: The recombinant strain with higher dual enzyme activity can be obtained when heterologous hyd and adc were expressed in Bacillus subtilis and it is technically feasible and has the application prospect for preparing D-HPG by hydantoinase process.

Key words： Bacillus subtilis Heterologous expression D-hydantoinase D-carbamoylase D-p-Hydroxyphenylglycine

Received: 20 September 2018 Published: 12 April 2019

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Corresponding Authors: Rui Ban E-mail: rbprofessor@163.com

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Cite this article:

Fa-bin LI,Lu LIU,Yan DU,Rui Ban. Construction of Recombinant Bacillus subtilis as Catalyst for Preparing D- p-Hydroxyphenylglycine. China Biotechnology, 2019, 39(3): 75-86.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20190310 OR https://manu60.magtech.com.cn/biotech/Y2019/V39/I3/75

Fig.1 The double-enzyme catalyzed reaction process of D-HPG produced by DL-HPH

Table 1 Strains and plasmids used in this study

Primer name	Primer sequence (5'-3')	Size(bp)
ASU1	ATCTCGTTTCGGGAAATTAC	20
ASU2	TCAGTCTTCTCATCTTCACT	20
Sd1	AGTGAAGATGAGAAGACTGA	20
Sd2	GCGACATCCTCACCGATCAACAATCACAATGGAGGACAAT	40
ASD1	TTGATCGGTGAGGATGTCGC	20
ASD2	ATGGGTGCTTTAGTTGAAGATTATCTGGTGTCGGCAAAT	39
CR1/ CR2	TCTTCAACTAAAGCACCCAT/TTATTCATTCAGTTTTCGTG	20/20
ASG1	CACGAAAACTGAATGAATAACACATTGCGGATCTTGATAA	40
ASG2	CCATCTCTTACATTCCTCCTT	21
PHSD1	CCCAAGCTTAACGGCACGATAGACTGTATG	30
PHSD2	CGGGATCCCAATCACAATGGAGGACAATATG	31
PURS1	AAAACTGCAGCAATCACAATGGAGGACAATATG	33
PURS2	AACGGCACGATAGACTGTATG	21
PUB1	CATACAGTCTATCGTGCCGTTAGTGCCGACCAAAACCATAAAAC	44
PUB2	AAAACTGCAGCAGCACAATTCCAAGAAAAACA	32
sacBU1	TCCTCATAGCCAAGAATCC	19
sacBU2	GCAATGTTTCCGATAAAGTCTGGTAGCCGTGATAGTT	37
PacoR1/ PacoR2	GACTTTATCGGAAACATTGC/AATAGGAACGCCGTTATATG	20/20
AP1/AP2	GACAAACATCACGCTCTTG/GTGTAAATTCCTCCCTTACCT	19/21
sacBUAE	GATCAGTATATCACAGCGTTCTTGGTAGCCGTGATAGTT	39
sacBPAE1	AGAACGCTGTGATATACTGATC	22
sacBpAE2	CGTTTGGGACCGAGTTCATTCTTTACCCTCTCCTTTT	37
acoR1	ATGAACTCGGTCCCAAACG	19
sacBD1	CATATAACGGCGTTCCTATTTTGATCCTAACGATGTAACC	40
sacBG2	GGAGTCAGTGAACAGGTAC	19
pelU1/ pelU2	CGTTGTTATTCTGGCTTGAT/TGTTCCGCTATCCTATTGC	40
AP1s	GCAATAGGATAGCGGAACAGACAAACATCACGCTCTTG	38
AP2s	AAGTATCATCTGACGTGTCATGTGTAAATTCCTCCCTTACCT
spoVG1	GCAATAGGATAGCGGAACATGCGGAAGTAAACGAAGTG	38
spoVG2	GTGTACATTTCACCTCCTTTCTATATAAAAGCATTAGTGT	40
peladcA /peladcB	ATGACACGTCAGATGATACTT/GCATCGTTTGACTGAATAGC	42
pelD1	TACCAAGGAGGAGTTATAGCGGATCAAGTGACAGCAA	37
pelG2	AGTTAGCACCGTTGGAAG	18
lytRU1/ lytRU2	CTACACTATCACTGACGCTAA/AAATTACTTTCATTATGAG	21/19
AEC1/ AEC2	AGAACGCTGTGATATACTGATC/GCATCGTTTGACTGAATAGC	22/20
PUBR1	CGGGATCCCAGCACAATTCCAAGAAAAACA	30
PUBC2	GATCAGTATATCACAGCGTTCTAGTGCCGACCAAAACCATAAAAC	45
adc1	AGAACGCTGTGATATACTGATC	22
cdh2	CATACAGTCTATCGTGCCGTTCGCTATAACTCCTCCTTGGTA	42
acoA1	AACGGCACGATAGACTGTATG	21
hyd2	CGGGATCCCAATCACAATGGAGGACAATATG	31

Table 2 Primers used in this study

Fig.2 The schematic of D-hydantoinase expression plasmids pHPS(a) and pUBS(b)

Fig.3 Chromatogram of solution before and after reaction (a) and SDS-PAGE analysis of intracellular proteins of recombinant strains(b) The blue and red line refer to the solution before and after reaction, respectively

Fig.4 The effects of divalent metal ions on D-hydantoinase activity The concentration gradient (mmol/L) of metal ions in medium is as follows: Fe²⁺ and Mn²⁺ , 0.2、0.4、0.6、0.8、1.0; Mg²⁺and EDTA , 2、4、6、8、10; Zn²⁺, 0.1、0.2、0.3、0.4、0.5

Table 3 The transcription analysis of acoR using qRT-PCR

Fig.5 The relationship between the level of intracellular AcoR and the expression level of different copy number of P_acoA-hyd

Table 4 The transcription analysis of adc using qRT-PCR

Fig.6 The comparison of D-carbamoylase activity of different recombinant strains. LN01, LN02, LN03 and LN04 refer to the strain using promoter P_cdd, P_spoVG, P_lytR and P_AE respectively

Fig.7 The schematic of recombinant plasmid pUBSC

Fig.8 The effects of components in medium on whole-cell catalytic activity. The SPM medium served as a control. The yeast powder and soy powder were replaced by yeast extract and tryptone, respectively, and glucose/glycerol was added into SPM medium

Fig.9 Time profiles of D-HPG concentrations

Fig.10 Chromatograms of solution at different reaction time at 40℃

Fig.11 The Catalytic reaction process of LSL02/pUBSC strain at different concentration of D, L-HPH


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