BioChem - DOS

Systematically redesigning and optimizing the expression of D-lactate dehydrogenase efficiently produces high-optical-purity D-lactic acid in Saccharomyces cerevisiae

Biochemical engineering journal

Zhong, Wei; Yang, Maohua; Mu, Tingzhen; Wu, Fan; Hao, Xuemi; Chen, Ruonan; Sharshar, Moustafa Mohamed; Thygesen, Anders; Wang, Qinhong; Xing, Jianmin

<P><B>Abstract</B></P> <P> <SUB>D</SUB>-lactic acid (<SUB>D</SUB>-LA) is gaining increased attention as it can improve the thermostability of poly lactic acid. Acid-tolerant <I>Saccharomyces cerevisiae</I> is a good host for <SUB>D</SUB>-LA fermentation. High catalytic efficiency of <SUB>D</SUB>-lactate dehydrogenase (<SUB>D</SUB>-LDH, EC 1.1.1.28) is crucial for the production of <SUB>D</SUB>-LA in yeast. Here, a synthetic biology approach was used to construct high-producing <SUB>D</SUB>-LA strains by redesigning and optimization of <SUB>D</SUB>-LDH expression by a combination of different promoters, terminators and <SUB>D</SUB>-LDHs. The pyruvate decarboxylase-deficient mutant strain TAMH was used as host strain for optimizing the 40 <SUB>D</SUB>-LDH expression cassettes. The TCSt strain harboring the pTCSt plasmid with the <I>TEF1</I> promoter, <I>E. coli</I> <SUB>D</SUB>-LDH and <I>Synth25</I> synthetic short terminator produced 5.8 g/L <SUB>D</SUB>-LA with an optical purity of 99.9%. The production of <SUB>D</SUB>-LA was further improved by integrating this high expression cassette into the <I>Ty1</I> transposable element of the YIP-01 strain with deleted <I>Pdc1</I>and <I>Pdc6</I>. The resulting strain YIP-pTCSt-301(CGMCC2.5726) was screened by a double enzyme-coupled system. Genomes sequencing of the strain revealed three copies of the <SUB>D</SUB>-LDH expression cassette. This strain was further improved by deleting the <I>Jen1</I>, <I>Cyb2</I>, <I>Dld1</I>, and <I>Adh1</I> genes and the resulting strain YIP-J-C-D-A1 (CGMCC2.5783) produced 80.0 g/L <SUB>D</SUB>-LA with a yield of 0.6 g/g glucose and a volumetric productivity of 1.1 g/L/h in fed-batch fermentation under non-neutralization conditions.</P> <P><B>Highlights</B></P> <P> <UL> <LI> The expression of <SUB>D</SUB>-lactate dehydrogenase was systematically optimized. </LI> <LI> The repetitive Ty1 site was chosen as the integration site. </LI> <LI> Double enzyme-coupled system was developed to screen efficient strains. </LI> <LI> Three copies of <I>E. coli</I> <SUB>D</SUB>-LDH were inserted at one time. </LI> <LI> The resulting strain produced 80 g/L <SUB>D</SUB>-LA with a yield of 0.6 g/g glucose. </LI> </UL> </P>

2019

Elsevier

1369-703x

144

pp.217-226

10.1016/j.bej.2018.09.013

http://click.ndsl.kr/servlet/OpenAPIDetailView?keyValue=05787966&target=NART&cn=NART95412464

Synthetic biology; D-lactic acid; Double enzyme-coupled system; Chromosomal integration; Saccharomyces cerevisiae