Dataset Information

Chirality Matters: Synthesis and Consumption of the d-Enantiomer of Lactic Acid by Synechocystis sp. Strain PCC6803.

ABSTRACT: Both enantiomers of lactic acid, l-lactic acid and d-lactic acid, can be produced in a sustainable way by a photosynthetic microbial cell factory and thus from CO2, sunlight, and water. Several properties of polylactic acid (a polyester of polymerized lactic acid) depend on the controlled blend of these two enantiomers. Recently, cyanobacterium Synechocystis sp. strain PCC6803 was genetically modified to allow formation of either of these two enantiomers. This report elaborates on the d-lactic acid production achieved by the introduction of a d-specific lactate dehydrogenase from the lactic acid bacterium Leuconostoc mesenteroides into Synechocystis. A typical batch culture of this recombinant strain initially shows lactic acid production, followed by a phase of lactic acid consumption, until production "outcompetes" consumption at later growth stages. We show that Synechocystis is able to use d-lactic acid, but not l-lactic acid, as a carbon source for growth. Deletion of the organism's putative d-lactate dehydrogenase (encoded by slr1556), however, does not eliminate this ability with respect to d-lactic acid consumption. In contrast, d-lactic acid consumption does depend on the presence of glycolate dehydrogenase GlcD1 (encoded by sll0404). Accordingly, this report highlights the need to match a product of interest of a cyanobacterial cell factory with the metabolic network present in the host used for its synthesis and emphasizes the need to understand the physiology of the production host in detail.

SUBMITTER: Angermayr SA

PROVIDER: S-EPMC4751861 | biostudies-literature | 2016 Feb

REPOSITORIES: biostudies-literature

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Chirality Matters: Synthesis and Consumption of the d-Enantiomer of Lactic Acid by Synechocystis sp. Strain PCC6803.

Angermayr S Andreas SA van der Woude Aniek D AD Correddu Danilo D Kern Ramona R Hagemann Martin M Hellingwerf Klaas J KJ

Applied and environmental microbiology 20151218 4

Both enantiomers of lactic acid, l-lactic acid and d-lactic acid, can be produced in a sustainable way by a photosynthetic microbial cell factory and thus from CO2, sunlight, and water. Several properties of polylactic acid (a polyester of polymerized lactic acid) depend on the controlled blend of these two enantiomers. Recently, cyanobacterium Synechocystis sp. strain PCC6803 was genetically modified to allow formation of either of these two enantiomers. This report elaborates on the d-lactic a ...[more]

PMID: 26682849

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Project description:BackgroundMolecular engineering of the intermediary physiology of cyanobacteria has become important for the sustainable production of biofuels and commodity compounds from CO2 and sunlight by "designer microbes." The chemical commodity product L-lactic acid can be synthesized in one step from a key intermediary metabolite of these organisms, pyruvate, catalyzed by a lactate dehydrogenase. Synthetic biology engineering to make "designer microbes" includes the introduction and overexpression of the product-forming biochemical pathway. For further optimization of product formation, modifications in the surrounding biochemical network of intermediary metabolism have to be made.ResultsTo improve light-driven L-lactic acid production from CO2, we explored several metabolic engineering design principles, using a previously engineered L-lactic acid producing mutant strain of Synechocystis sp. PCC6803 as the benchmark. These strategies included: (i) increasing the expression level of the relevant product-forming enzyme, lactate dehydrogenase (LDH), for example, via expression from a replicative plasmid; (ii) co-expression of a heterologous pyruvate kinase to increase the flux towards pyruvate; and (iii) knockdown of phosphoenolpyruvate carboxylase to decrease the flux through a competing pathway (from phosphoenolpyruvate to oxaloacetate). In addition, we tested selected lactate dehydrogenases, some of which were further optimized through site-directed mutagenesis to improve the enzyme's affinity for the co-factor nicotinamide adenine dinucleotide phosphate (NADPH). The carbon partitioning between biomass and lactic acid was increased from about 5% to over 50% by strain optimization.ConclusionAn efficient photosynthetic microbial cell factory will display a high rate and extent of conversion of substrate (CO2) into product (here: L-lactic acid). In the existing CO2-based cyanobacterial cell factories that have been described in the literature, by far most of the control over product formation resides in the genetically introduced fermentative pathway. Here we show that a strong promoter, in combination with increased gene expression, can take away a significant part of the control of this step in lactic acid production from CO2. Under these premises, modulation of the intracellular precursor, pyruvate, can significantly increase productivity. Additionally, production enhancement is achieved by protein engineering to increase co-factor specificity of the heterologously expressed LDH.

Project description:BackgroundThis study aimed at exploring the molecular physiological consequences of a major redirection of carbon flow in so-called cyanobacterial cell factories: quantitative whole-cell proteomics analyses were carried out on two (14)N-labelled Synechocystis mutant strains, relative to their (15)N-labelled wild-type counterpart. Each mutant strain overproduced one specific commodity product, i.e. ethanol or lactic acid, to such an extent that the majority of the incoming CO2 in the organism was directly converted into the product.ResultsIn total, 267 proteins have been identified with a significantly up- or down-regulated expression level. In the ethanol-producing mutant, which had the highest relative direct flux of carbon-to-product (>65%), significant up-regulation of several components involved in the initial stages of CO2 fixation for cellular metabolism was detected. Also a general decrease in abundance of the protein synthesizing machinery of the cells and a specific induction of an oxidative stress response were observed in this mutant. In the lactic acid overproducing mutant, that expresses part of the heterologous l-lactate dehydrogenase from a self-replicating plasmid, specific activation of two CRISPR associated proteins, encoded on the endogenous pSYSA plasmid, was observed. RT-qPCR was used to measure, of nine of the genes identified in the proteomics studies, also the adjustment of the corresponding mRNA level.ConclusionThe most striking adjustments detected in the proteome of the engineered cells were dependent on the specific product formed, with, e.g. more stress caused by lactic acid- than by ethanol production. Up-regulation of the total capacity for CO2 fixation in the ethanol-producing strain was due to hierarchical- rather than metabolic regulation. Furthermore, plasmid-based expression of heterologous gene(s) may induce genetic instability. For selected, limited, number of genes a striking correlation between the respective mRNA- and the corresponding protein expression level was observed, suggesting that for the expression of these genes regulation takes place primarily at the level of gene transcription.

Dataset Information

Chirality Matters: Synthesis and Consumption of the d-Enantiomer of Lactic Acid by Synechocystis sp. Strain PCC6803.

Publications

Chirality Matters: Synthesis and Consumption of the d-Enantiomer of Lactic Acid by Synechocystis sp. Strain PCC6803.

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