Control of D-lactic acid content in P(LA-3HB) copolymer in the yeast Saccharomyces cerevisiae using a synthetic gene expression system

IF 3.7 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
Anna Ylinen , Laura Salusjärvi , Mervi Toivari , Merja Penttilä
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引用次数: 0

Abstract

The fully biobased polyhydroxyalkanoate (PHA) polymers provide interesting alternatives for petrochemical derived plastic materials. The mechanical properties of some PHAs, including the common poly(3-hydroxybutyrate) (PHB), are limited, but tunable by addition of other monomers into the polymer chain. In this study we present a precise synthetic biology method to adjust lactate monomer fraction of a polymer by controlling the monomer formation in vivo at gene expression level, independent of cultivation conditions. We used the modified doxycycline-based Tet-On approach to adjust the expression of the stereospecific D-lactate dehydrogenase gene (ldhA) from Leuconostoc mesenteroides to control D-lactic acid formation in yeast Saccharomyces cerevisiae. The synthetic Tet-On transcription factor with a VP16 activation domain was continuously expressed and its binding to a synthetic promoter with eight transcription factor specific binding sites upstream of the ldhA gene was controlled with the doxycycline concentration in the media. The increase in doxycycline concentration correlated positively with ldhA expression, D-lactic acid production, poly(D-lactic acid) (PDLA) accumulation in vivo, and D-lactic acid content in the poly(D-lactate-co-3-hydroxybutyrate) P(LA-3HB) copolymer. We demonstrated that the D-lactic acid content of the P(LA-3HB) copolymer can be adjusted linearly from 6 mol% to 93 mol% in vivo in S. cerevisiae. These results highlight the power of controlling gene expression and monomer formation in the tuning of the polymer composition. In addition, we obtained 5.6% PDLA and 19% P(LA-3HB) of the cell dry weight (CDW), which are over two- and five-fold higher accumulation levels, respectively, than reported in the previous studies with yeast. We also compared two engineered PHA synthases and discovered that in S. cerevisiae the PHA synthase PhaC1437Ps6-19 produced P(LA-3HB) copolymers with lower D-lactic acid content, but with higher molecular weight, in comparison to the PHA synthase PhaC1Pre.

合成基因表达系统控制酿酒酵母P(LA-3HB)共聚物中d -乳酸含量
全生物基聚羟基烷酸酯(PHA)聚合物为石油化工衍生塑料材料提供了有趣的替代品。包括常见的聚(3-羟基丁酸酯)(PHB)在内的一些pha的机械性能是有限的,但可以通过在聚合物链中加入其他单体来调节。在这项研究中,我们提出了一种精确的合成生物学方法,通过在基因表达水平上控制单体的形成来调节聚合物的乳酸单体比例,而不依赖于培养条件。我们采用改良的基于多西环素的Tet-On方法调节mesenterostoc中立体特异性d -乳酸脱氢酶基因(ldhA)的表达,以控制酿酒酵母(Saccharomyces cerevisiae)中d -乳酸的形成。具有VP16活化结构域的合成Tet-On转录因子连续表达,其与ldhA基因上游具有8个转录因子特异性结合位点的合成启动子的结合受到培养基中强力霉素浓度的控制。多西环素浓度的升高与ldhA表达、d -乳酸生成、体内聚d -乳酸(PDLA)积累以及聚d -乳酸-co-3-羟基丁酸)P(LA-3HB)共聚物中d -乳酸含量呈正相关。我们证明了P(LA-3HB)共聚物的d -乳酸含量可以在酿酒酵母体内从6 mol%到93 mol%的范围内线性调节。这些结果突出了控制基因表达和单体形成在聚合物组成调节中的作用。此外,我们获得了细胞干重(CDW)的5.6%的PDLA和19%的P(LA-3HB),分别比以前用酵母研究报道的积累水平高出2倍和5倍。我们还比较了两种工程PHA合成酶,发现在酿酒酵母中,PHA合成酶PhaC1437Ps6-19产生的P(LA-3HB)共聚物与PHA合成酶PhaC1Pre相比,d -乳酸含量更低,但分子量更高。
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来源期刊
Metabolic Engineering Communications
Metabolic Engineering Communications Medicine-Endocrinology, Diabetes and Metabolism
CiteScore
13.30
自引率
1.90%
发文量
22
审稿时长
18 weeks
期刊介绍: Metabolic Engineering Communications, a companion title to Metabolic Engineering (MBE), is devoted to publishing original research in the areas of metabolic engineering, synthetic biology, computational biology and systems biology for problems related to metabolism and the engineering of metabolism for the production of fuels, chemicals, and pharmaceuticals. The journal will carry articles on the design, construction, and analysis of biological systems ranging from pathway components to biological complexes and genomes (including genomic, analytical and bioinformatics methods) in suitable host cells to allow them to produce novel compounds of industrial and medical interest. Demonstrations of regulatory designs and synthetic circuits that alter the performance of biochemical pathways and cellular processes will also be presented. Metabolic Engineering Communications complements MBE by publishing articles that are either shorter than those published in the full journal, or which describe key elements of larger metabolic engineering efforts.
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