The ethylene-forming enzyme (EFE) from Pseudomonas syringae catalyzes the synthesis of ethylene which can be easily detected in the headspace of closed cultures. A synthetic codon-optimized gene encoding N-terminal His-tagged EFE (EFEh) was expressed in Synechocystis sp. PCC 6803 (Synechocystis) and Escherichia coli (E. coli) under the control of diverse promoters in a self-replicating broad host-range plasmid. Ethylene synthesis was stably maintained in both organisms in contrast to earlier work in Synechococcus elongatus PCC 7942. The rate of ethylene accumulation was used as a reporter for protein expression in order to assess promoter strength and inducibility with the different expression systems. Several metal-inducible cyanobacterial promoters did not function in E. coli but were well-regulated in cyanobacteria, albeit at a low level of expression. The E. coli promoter Ptrc resulted in constitutive expression in cyanobacteria regardless of whether IPTG was added or not. In contrast, a Lac promoter variant, PA1lacO-1, induced EFE-expression in Synechocystis at a level of expression as high as the Trc promoter and allowed a fine level of IPTG-dependent regulation of protein-expression. The regulation was tight at low cell density and became more relaxed in more dense cultures. A synthetic quorum-sensing promoter system was also constructed and shown to function well in E. coli, however, only a very low level of EFE-activity was observed in Synechocystis, independent of cell density.

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.

Sphingomonas paucimobilis' P450SPα (CYP152B1) is a good candidate as industrial biocatalyst. This enzyme is able to use hydrogen peroxide as unique cofactor to catalyze the fatty acids conversion to α‐hydroxy fatty acids, thus avoiding the use of expensive electron‐donor(s) and redox partner(s). Nevertheless, the toxicity of exogenous H2O2 toward proteins and cells often results in the failure of the reaction scale‐up when it is directly added as co‐substrate. In order to bypass this problem, we designed a H2O2 self‐producing enzyme by fusing the P450SPα to the monomeric sarcosine oxidase (MSOX), as H2O2 donor system, in a unique polypeptide chain, obtaining the P450SPα‐polyG‐MSOX fusion protein. The purified P450SPα‐polyG‐MSOX protein displayed high purity (A417/A280 = 0.6) and H2O2‐tolerance (kdecay = 0.0021 ± 0.000055 min−1; ΔA417 = 0.018 ± 0.001) as well as good thermal stability (Tm: 59.3 ± 0.3°C and 63.2 ± 0.02°C for P450SPα and MSOX domains, respectively). The data show how the catalytic interplay between the two domains can be finely regulated by using 500 mM sarcosine as sacrificial substrate to generate H2O2. Indeed, the fusion protein resulted in a high conversion yield toward fat waste biomass‐representative fatty acids, that is, lauric acid (TON = 6,800 compared to the isolated P450SPα TON = 2,307); myristic acid (TON = 6,750); and palmitic acid (TON = 1,962).

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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.T o date, lactic acid has been produced with almost 100% conversion efficiency by several chemotrophic fermentative bacterial and yeast cell factories growing on various sugars (1, 2). Consequently, a large amount of effort is currently exerted to produce lactic acid with lactic acid bacteria (3), in combination with the use of a cheap substrate(s) such as lignocellulosic feedstock, though that feedstock requires an energy-intensive pretreatment of the biomass (1, 4). Lactic acid is used in food preservation, in the chemical and pharmaceutical industries, and as a building block for construction of polymers, the latter for use as an alternative to petroleum-derived plastics. It is compelling that the biodegradability and heat stability of this (bio)plastic depend on the blend of the two optically active, chiral isoforms of lactic acid (5).Employing a photosynthetic microorganism as the production host has the advantage of enabling the direct conversion of CO 2 into (poly)lactic acid (6-9). Such production, which is dependent on cyanobacterial cell factories, allows compound formation without the need to generate complex (plant) biomass first, only to break it down again later for its utilization by a chemotrophic fermentative microorganism (10).For living organisms, chirality plays an essential role. For example, amino acids are incorporated as L-enantiomers into proteins. Likewise, L-lactic acid seems to be the dominant enantiomeric form of this weak acid in nature. Thus, suitable and effective production hosts for D-lactic acid are more challenging to find and construct. Nonetheless, biosynthetic routes for both enantiomers, and for the corresponding products, exist in various (micro)organisms, facilitated by enantiomer-specific lactate dehydrogenases (LDH) (11), whereas chemical synthesis routinely results in a racemic mixture (12). Earlier, we constructed several L-lactic acid-producing Synechocystis sp. strain PCC6803 (here Synechocystis) variants (7, 13). In the framework of those experiments, we also tested the D-LDH of Escherichia coli (7), but we were not successful in producing D-lactic acid in the engineered Synechocystis strains at that time. However, in another cyanobacterium, Synechococcus elongatus PCC7942, synthesis of the latter enantiomer has been achieved through the expression of the same E. coli enzyme, although its extracell...