Construct Whole-cell Biocatalyst and Produce (<i>S</i>)-Acetoin via Synthetic Biology Strategy

doi:10.13523/j.cb.20190408

China Biotechnology

2019, Vol. 39

Issue (4): 60-68 DOI: 10.13523/j.cb.20190408

Construct Whole-cell Biocatalyst and Produce (S)-Acetoin via Synthetic Biology Strategy

Jian-xiu LI^1,^2**,Xian-rui CHEN^1**,Xiao-ling CHEN¹,Yan-yan HUANG¹,Qi-wen MO¹,Neng-zhong XIE^1,^2***(

),Ri-bo HUANG^1,^2***(

)

1 National Engineering Research Center for Non-food Biorefinery, State Key Laboratory of Non-food Biomass Energy and Enzyme Technology, and Guangxi Key Laboratory of Biorefinery, Guangxi Academy of Sciences, Nanning 530007, China
2 State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, College of Life Science andTechnology, Guangxi University, Nanning 530004, China

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Abstract

Objective: The whole-cell biocatalyst, overexpressing diacetyl reductase (DAR) and introduced in situ-NADH regeneration systems was applied to improve (S)-acetoin production from prochiral diacetyl.Methods: The gene encoding DAR from Paenibacillus polymyxa was cloned and expressed in Escherichia coli. Recombine DAR was purified by HiTrap TALON affinity chromatography, then enzyme activities and molecular kinetic parameters of purified DAR were measured. NADH in situ regeneration system based on glucose dehydrogenase (GDH) from Bacillus subtilis was introduced. The whole-cell biocatalyst, overexpressing DAR and GDH was applied to (S)-acetoin produce and the reaction conditions were optimized.Results: DAR showed a high catalytic efficiency and enantioselective (enantiomeric purity 95.86%). The K_m, V_max and K_cat values of DAR for diacetyl were 2.59mmol/L, 1.64μmol/(L·min·mg) and 12.3/s, respectively. The whole-cell biocatalyst, introduced in situ-NADH regeneration systems resulted in higher (S)-acetoin concentration, productivity and yield form diacetyl. Under optimal conditions in fed-batch bioconversion, 51.26g/L (S)-acetoin was produced from 63g/L diacetyl with a productivity of 5.13g/(L·h).Conclusion: The compound of prochiral diacetyl was used as substrate for asymmetric synthesis of high value chiral (S)-acetoin. The results demonstrated that whole-cell biocatalyst, introduced in situ-NADH regeneration systems, can effectively improve the production of (S)-acetoin with good applicability and economic performance.

Key words： Whole-cell biocatalyst Cofactor regeneration Diacetyl reductase (S)-Acetoin

Received: 08 October 2018 Published: 08 May 2019

ZTFLH:

Q819

Corresponding Authors: Neng-zhong XIE,Ri-bo HUANG E-mail: xienengzhong@gxas.cn;guruace@gxas.cn

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	Articles by authors
	Jian-xiu LI
	Xian-rui CHEN
	Xiao-ling CHEN
	Yan-yan HUANG
	Qi-wen MO
	Neng-zhong XIE
	Ri-bo HUANG

Cite this article:

Jian-xiu LI,Xian-rui CHEN,Xiao-ling CHEN,Yan-yan HUANG,Qi-wen MO,Neng-zhong XIE,Ri-bo HUANG. Construct Whole-cell Biocatalyst and Produce (S)-Acetoin via Synthetic Biology Strategy. China Biotechnology, 2019, 39(4): 60-68.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20190408 OR https://manu60.magtech.com.cn/biotech/Y2019/V39/I4/60

Table1 Strains,plasmids and primers

Fig.1 Identification of the recombinant plasmid by enzyme digestion (a) Lane 1: pETDuet-dar digested by BamH I; Lane 2: pETDuet-dar digested by BamH I and Hind III; M1: Marker IV; M2: Marker DL2000 (b) M1: Marker DL2000; M2: Marker IV; Lane 1: pETDuet-dar/gdh digested by Nde I; Lane 2: pETDuet-dar/gdh digested by Nde I and Aat II

Fig.2 SDS-PAGE analysis of DAR and GDH expressed in E. coli BL21(DE3) and the purified recombinant DAR M: Marker; Lane 1: E. coli BL21(DE3)-pETDuet; Lane 2: E. coli BL21(DE3)-DAR; Lane 3: E. coli BL21(DE3)-DAR/GDH; Lane 4: Purified recombinant DAR

Table 2 The products of batch bioconversion with whole-cell biocatalyst

Fig.3 Optimization of biocatalysis conditions (a) Concentration of recombinant (b) pH (c) Temperature (d) Ratio of glucose to diacetyl (e) Shaking speeds (f) Concentration of diacetyl

Fig. 4 Fed-batch bioconversions under optimal condition ▲:Acetoin; ●:Diacetyl; ○:Glucose


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