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  • 1. Cheon, Seungwoo
    et al.
    Kim, Hye Mi
    Gustavsson, Martin
    KTH, School of Biotechnology (BIO), Bioprocess Technology (closed 20130101).
    Lee, Sang Yup
    Recent trends in metabolic engineering of microorganisms for the production of advanced biofuels2016In: Current opinion in chemical biology, ISSN 1367-5931, E-ISSN 1879-0402, Vol. 35, 10-21 p.Article in journal (Refereed)
    Abstract [en]

    As climate change has become one of the major global risks, our heavy dependence on petroleum-derived fuels has received much public attention. To solve such problems, production of sustainable fuels has been intensively studied over the past years. Thanks to recent advances in synthetic biology and metabolic engineering technologies, bio-based platforms for advanced biofuels production have been developed using various microorganisms. The strategies for production of advanced biofuels have converged upon four major metabolic routes: the 2-ketoacid pathway, the fatty acid synthesis (FAS) pathway, the isoprenoid pathway, and the reverse β-oxidation pathway. Additionally, the polyketide synthesis pathway has recently been attracting interest as a promising alternative biofuel production route. In this article, recent trends in advanced biofuels production are reviewed by categorizing them into three types of advanced biofuels: alcohols, biodiesel and jet fuel, and gasoline. Focus is given on the strategies of employing synthetic biology and metabolic engineering for the development of microbial strains producing advanced fuels. Finally, the prospects for future advances needed to achieve much more efficient bio-based production of advanced biofuels are discussed, focusing on designing advanced biofuel production pathways coupled with screening, modifying, and creating novel enzymes.

  • 2.
    Chung, Hannah
    et al.
    Korean Adanced Institute of Science and Technology (KAIST).
    Yang, Jung Eun
    Korean Adanced Institute of Science and Technology (KAIST).
    Ha, Ji Yeon
    Korean Adanced Institute of Science and Technology (KAIST).
    Chae, Tong Un
    Korean Adanced Institute of Science and Technology (KAIST).
    Shin, Jae Ho
    Korean Adanced Institute of Science and Technology (KAIST).
    Gustavsson, Martin
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Lee, Sang Yup
    Korean Adanced Institute of Science and Technology (KAIST).
    Bio-based production of monomers and polymers by metabolically engineered microorganisms2015In: Current Opinion in Biotechnology, ISSN 0958-1669, E-ISSN 1879-0429, Vol. 36, 73-84 p.Article, review/survey (Refereed)
    Abstract [en]

    Recent metabolic engineering strategies for bio-based production of monomers and polymers are reviewed. In the case of monomers, we describe strategies for producing polyamide precursors, namely diamines (putrescine, cadaverine, 1,6-diaminohexane), dicarboxylic acids (succinic, glutaric, adipic, and sebacic acids), and ω-amino acids (γ-aminobutyric, 5-aminovaleric, and 6-aminocaproic acids). Also, strategies for producing diols (monoethylene glycol, 1,3-propanediol, and 1,4-butanediol) and hydroxy acids (3-hydroxypropionic and 4-hydroxybutyric acids) used for polyesters are reviewed. Furthermore, we review strategies for producing aromatic monomers, including styrene, p-hydroxystyrene, p-hydroxybenzoic acid, and phenol, and propose pathways to aromatic polyurethane precursors. Finally, in vivo production of polyhydroxyalkanoates and recombinant structural proteins having interesting applications are showcased.

  • 3.
    Gustavsson, Martin
    KTH, School of Biotechnology (BIO), Bioprocess Technology (closed 20130101).
    Influence of recombinant passenger properties and process conditions on surface expression using the AIDA-I autotransporter2013Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Surface expression has attracted much recent interest, and it has been suggested for a variety of applications. Two such applications are whole-cell biocatalysis and the creation of live vaccines. For successful implementation of these applications there is a need for flexible surface expression systems that can yield a high level of expression with a variety of recombinant fusion proteins. The aim of this work was thus to create a surface expression system that would fulfil these requirements.

     

    A novel surface expression system based on the AIDA-I autotransporter was created with the key qualities being are good, protein-independent detection of the expression through the presence of two epitope tags flanking the recombinant protein, and full modularity of the different components of the expression cassette. To evaluate the flexibility of this construct, 8 different model proteins with potential use as live-vaccines or biocatalysts were expressed and their surface expression levels were analysed.

     

    Positive signals were detected for all of the studied proteins using antibody labelling followed by flow cytometric analysis, showing the functionality of the expression system. The ratio of the signal from the two epitope tags indicated that several of the studied proteins were present mainly in proteolytically degraded forms, which was confirmed by Western blot analysis of the outer membrane protein fraction. This proteolysis was suggested to be due to protein-dependent stalling of translocation intermediates in the periplasm, with indications that larger size and higher cysteine content had a negative impact on expression levels. Process design with reduced cultivation pH and temperature was used to increase total surface expression yield of one of the model proteins by 400 %, with a simultaneous reduction of proteolysis by a third. While not sufficient to completely remove proteolysis, this shows that process design can be used to greatly increase surface expression. Thus, it is recommended that future work combine this with engineering of the bacterial strain or the expression system in order to overcome the observed proteolysis and maximise the yield of surface expressed protein.

  • 4.
    Gustavsson, Martin
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Surface expression using the AIDA autotransporter:  Towards live vaccines and whole-cell biocatalysis2011Licentiate thesis, comprehensive summary (Other academic)
    Abstract [en]

    The area of surface expression has gathered a lot of interest from research groups all over the world and much work is performed in the area. Autotransporters have been used for surface expression in Gram-negative bacteria. One of the more commonly used autotransporters is the Adhesin Involved in Diffuse Adherence (AIDA) of pathogenic Escherichia coli. The surface expression of enzymes and vaccine epitopes offer several advantages. Surface expressed enzymes gain similar properties to immobilised enzymes, mainly simplified handling and separation using centrifugation. Surface expressed vaccine epitopes can have longer half-lives inside the animal that is to be immunized and surface groups on the host cell can act as adjuvants, increasing the immune response and leading to a better immunisation.

      

    However, while much basic research is directed towards mechanisms of surface expression using autotransporters there are few reports regarding production of surface expressed protein. Thus the aim of this work was the optimisation of the yield and productivity of surface expressed protein. Protein Z, an IgG-binding domain of Staphylococcal protein A, was used as a model protein for the investigation of which cultivation parameters influenced surface expression. The choice of cultivation medium gave the largest impact on expression, which was attributed to effects based on the induction of the native promoter of AIDA. The AIDA system was then used for the expression of two Salmonella surface proteins, SefA and H:gm, with potential for use as vaccine epitopes. SefA was verified located on the cell surface, and H:gm was found in the outer membrane of the host cell, though only in proteolytically truncated forms lacking the His6-tag used for detection. This proteolysis persisted in E. coli strains deficient for the outer membrane protease OmpT and was concluded to be dependent on other proteases. The removal of proteolysis and further optimisation of the yield of surface-expressed protein are important goals of further work.

  • 5.
    Gustavsson, Martin
    et al.
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Bäcklund, Emma
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Larsson, Gen
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Optimisation of surface expression using the AIDA autotransporter2011In: Microbial Cell Factories, ISSN 1475-2859, Vol. 10Article in journal (Refereed)
    Abstract [en]

    Background: Bacterial surface display is of interest in many applications, including live vaccine development, screening of protein libraries and the development of whole cell biocatalysts. The goal of this work was to understand which parameters result in production of large quantities of cells that at the same time express desired levels of the chosen protein on the cell surface. For this purpose, staphylococcal protein Z was expressed using the AIDA autotransporter in Escherichia coli.

    Results: The use of an OmpT-negative E. coli mutant resulted in successful expression of the protein on the surface, while a clear degradation pattern was found in the wild type. The expression in the mutant resulted also in a more narrow distribution of the surface anchored protein within the population. Medium optimisation showed that minimal medium with glucose gave more than four times as high expression as LB-medium. Glucose limited fed-batch was used to increase the cell productivity and the highest protein levels were found at the highest feed rates. A maintained high surface expression up to cell dry weights of 18 g l(-1) could also be achieved by repeated glucose additions in batch cultivation where production was eventually reduced by low oxygen levels. In spite of this, the distribution in the bacterial population of the surface protein was narrower using the batch technique.

    Conclusions: A number of parameters in recombinant protein production were seen to influence the surface expression of the model protein with respect both to the productivity and to the display on the individual cell. The choice of medium and the cell design to remove proteolytic cleavage were however the most important. Both fed-batch and batch processing can be successfully used, but prolonged batch processing is probably only possible if the chosen strain has a low acetic acid production.

  • 6.
    Gustavsson, Martin
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Do, T. -H
    Lüthje, P.
    Tran, N. T.
    Brauner, A.
    Samuelson, Patrik
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Truong, N. H.
    Larsson, Gen
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Improved cell surface display of Salmonella enterica serovar Enteritidis antigens in Escherichia coli2015In: Microbial Cell Factories, ISSN 1475-2859, Vol. 14, no 1, 47Article in journal (Refereed)
    Abstract [en]

    Background: Salmonella enterica serovar Enteritidis (SE) is one of the most potent pathogenic Salmonella serotypes causing food-borne diseases in humans. We have previously reported the use of the β-autotransporter AIDA-I to express the Salmonella flagellar protein H:gm and the SE serotype-specific fimbrial protein SefA at the surface of E. coli as live bacterial vaccine vehicles. While SefA was successfully displayed at the cell surface, virtually no full-length H:gm was exposed to the medium due to extensive proteolytic cleavage of the N-terminal region. In the present study, we addressed this issue by expressing a truncated H:gm variant (H:gmd) covering only the serotype-specific central region. This protein was also expressed in fusion to SefA (H:gmdSefA) to understand if the excellent translocation properties of SefA could be used to enhance the secretion and immunogenicity. Results: H:gmd and H:gmdSefA were both successfully translocated to the E. coli outer membrane as full-length proteins using the AIDA-I system. Whole-cell flow cytometric analysis confirmed that both antigens were displayed and accessible from the extracellular environment. In contrast to H:gm, the H:gmd protein was not only expressed as full-length protein, but it also seemed to promote the display of the protein fusion H:gmdSefA. Moreover, the epitopes appeared to be recognized by HT-29 intestinal cells, as measured by induction of the pro-inflammatory interleukin 8. Conclusions: We believe this study to be an important step towards a live bacterial vaccine against Salmonella due to the central role of the flagellar antigen H:gm and SefA in Salmonella infections and the corresponding immune responses against Salmonella.

  • 7.
    Gustavsson, Martin
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Hörnström, David
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Lundh, Susanna
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Belotserkovsky, Jaroslav
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Larsson, Gen
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Biocatalysis on the surface of Escherichia coli: melanin pigmentation of the cell exterior2016In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 6, 36117Article in journal (Refereed)
    Abstract [en]

    Today, it is considered state-of-the-art to engineer living organisms for various biotechnology applications. Even though this has led to numerous scientific breakthroughs, the enclosed interior of bacterial cells still restricts interactions with enzymes, pathways and products due to the mass-transfer barrier formed by the cell envelope. To promote accessibility, we propose engineering of biocatalytic reactions and subsequent product deposition directly on the bacterial surface. As a proof-of-concept, we used the AIDA autotransporter vehicle for Escherichia coli surface expression of tyrosinase and fully oxidized externally added tyrosine to the biopolymer melanin. This resulted in a color change and creation of a black cell exterior. The capture of ninety percent of a pharmaceutical wastewater pollutant followed by regeneration of the cell bound melanin matrix through a simple pH change, shows the superior function and facilitated processing provided by the surface methodology. The broad adsorption spectrum of melanin could also allow removal of other micropollutants.

  • 8.
    Gustavsson, Martin
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Jarmander, Johan
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Arvestad, Lars
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Larsson, Gen
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Extended signal peptides in autotransporters are associated with large passenger proteinsManuscript (preprint) (Other academic)
  • 9.
    Gustavsson, Martin
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Lee, Sang Yup
    Prospects of microbial cell factories developed through systems metabolic engineering2016In: Microbial Biotechnology, ISSN 1751-7907, E-ISSN 1751-7915, Vol. 9, no 5, 610-617 p.Article in journal (Refereed)
    Abstract [en]

    While academic-level studies on metabolic engineering of microorganisms for production of chemicals and fuels are ever growing, a significantly lower number of such production processes have reached commercial-scale. In this work, we review the challenges associated with moving from laboratory-scale demonstration of microbial chemical or fuel production to actual commercialization, focusing on key requirements on the production organism that need to be considered during the metabolic engineering process. Metabolic engineering strategies should take into account techno-economic factors such as the choice of feedstock, the product yield, productivity and titre, and the cost effectiveness of midstream and downstream processes. Also, it is important to develop an industrial strain through metabolic engineering for pathway construction and flux optimization together with increasing tolerance to products and inhibitors present in the feedstock, and ensuring genetic stability and strain robustness under actual fermentation conditions.

  • 10.
    Gustavsson, Martin
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Muraleedharan, Madhu Nair
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Larsson, Gen
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Surface Expression of omega-Transaminase in Escherichia coli2014In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 80, no 7, 2293-2298 p.Article in journal (Refereed)
    Abstract [en]

    Chiral amines are important for the chemical and pharmaceutical industries, and there is rapidly growing interest to use transaminases for their synthesis. Since the cost of the enzyme is an important factor for process economy, the use of whole-cell biocatalysts is attractive, since expensive purification and immobilization steps can be avoided. Display of the protein on the cell surface provides a possible way to reduce the mass transfer limitations of such biocatalysts. However, transaminases need to dimerize in order to become active, and furthermore, they require the cofactor pyridoxal phosphate; consequently, successful transaminase surface expression has not been reported thus far. In this work, we produced an Arthrobacter citreus omega-transaminase in Escherichia coli using a surface display vector based on the autotransporter adhesin involved in diffuse adherence (AIDA-I), which has previously been used for display of dimeric proteins. The correct localization of the transaminase in the E. coli outer membrane and its orientation toward the cell exterior were verified. Furthermore, transaminase activity was detected exclusively in the outer membrane protein fraction, showing that successful dimerization had occurred. The transaminase was found to be present in both full-length and proteolytically degraded forms. The removal of this proteolysis is considered to be the main obstacle to achieving sufficient whole-cell transaminase activity.

  • 11.
    Jarmander, Johan
    et al.
    KTH, School of Biotechnology (BIO), Bioprocess Technology (closed 20130101).
    Gustavsson, Martin
    KTH, School of Biotechnology (BIO), Bioprocess Technology (closed 20130101).
    Do, Thi-Huyen
    Samuelson, Patrik
    KTH, School of Biotechnology (BIO), Bioprocess Technology (closed 20130101).
    Larsson, Gen
    KTH, School of Biotechnology (BIO), Bioprocess Technology (closed 20130101).
    A dual tag system for facilitated detection of surface expressed proteins in Escherichia coli2012In: Microbial Cell Factories, ISSN 1475-2859, Vol. 11, 118Article in journal (Refereed)
    Abstract [en]

    Background: The discovery of the autotransporter family has provided a mechanism for surface expression of proteins in laboratory strains of Escherichia coli. We have previously reported the use of the AIDA-I autotransport system to express the Salmonella enterica serovar Enteritidis proteins SefA and H: gm. The SefA protein was successfully exposed to the medium, but the orientation of H:gm in the outer membrane could not be determined due to proteolytic cleavage of the N-terminal detection-tag. The goal of the present work was therefore to construct a vector containing elements that facilitates analysis of surface expression, especially for proteins that are sensitive to proteolysis or otherwise difficult to express. Results: The surface expression system pAIDA1 was created with two detection tags flanking the passenger protein. Successful expression of SefA and H:gm on the surface of E. coli was confirmed with fluorescently labeled antibodies specific for the N-terminal His(6)-tag and the C-terminal Myc-tag. While both tags were detected during SefA expression, only the Myc-tag could be detected for H: gm. The negative signal indicates a proteolytic cleavage of this protein that removes the His(6)-tag facing the medium. Conclusions: Expression levels from pAIDA1 were comparable to or higher than those achieved with the formerly used vector. The presence of the Myc- but not of the His(6)-tag on the cell surface during H:gm expression allowed us to confirm the hypothesis that this fusion protein was present on the surface and oriented towards the cell exterior. Western blot analysis revealed degradation products of the same molecular weight for SefA and H:gm. The size of these fragments suggests that both fusion proteins have been cleaved at a specific site close to the C-terminal end of the passenger. This proteolysis was concluded to take place either in the outer membrane or in the periplasm. Since H:gm was cleaved to a much greater extent then the three times smaller SefA, it is proposed that the longer translocation time for the larger H:gm makes it more susceptible to proteolysis.

  • 12.
    Jarmander, Johan
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Janoschek, Lars
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Lundh, Susanna
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Larsson, Gen
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Gustavsson, Martin
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Process optimization for increased yield of surface-expressed protein in Escherichia coli2014In: Bioprocess and biosystems engineering (Print), ISSN 1615-7591, E-ISSN 1615-7605, Vol. 37, no 8, 1685-1693 p.Article in journal (Refereed)
    Abstract [en]

    The autotransporter family of Gram-negative protein exporters has been exploited for surface expression of recombinant passenger proteins. While the passenger in some cases was successfully translocated, a major problem has been low levels of full-length protein on the surface due to proteolysis following export over the cytoplasmic membrane. The aim of the present study was to increase the surface expression yield of the model protein SefA, a Salmonella enterica fimbrial subunit with potential for use in vaccine applications, by reducing this proteolysis through process design using Design of Experiments methodology. Cultivation temperature and pH, hypothesized to influence periplasmic protease activity, as well as inducer concentration were the parameters selected for optimization. Through modification of these parameters, the total surface expression yield of SefA was increased by 200 %. At the same time, the yield of full-length protein was increased by 300 %, indicating a 33 % reduction in proteolysis.

  • 13. Nhan, Nguyen Thanh
    et al.
    Gonzalez de Valdivia, Ernesto
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Gustavsson, Martin
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Hai, Truong Nam
    Larsson, Gen
    KTH, School of Biotechnology (BIO), Bioprocess Technology.
    Surface display of Salmonella epitopes in Escherichia coli and Staphylococcus carnosus2011In: Microbial Cell Factories, ISSN 1475-2859, Vol. 10, 22- p.Article in journal (Refereed)
    Abstract [en]

    Background: Salmonella enterica serotype Enteritidis (SE) is considered to be one of the most potent pathogenic Salmonella serotypes causing food-borne disease in humans. Since a live bacterial vaccine based on surface display of antigens has many advantages over traditional vaccines, we have studied the surface display of the SE antigenic proteins, H: gm and SefA in Escherichia coli by the beta-autotransporter system, AIDA. This procedure was compared to protein translocation in Staphylococcus carnosus, using a staphylococci hybrid vector earlier developed for surface display of other vaccine epitopes. Results: Both SefA and H: gm were translocated to the outer membrane in Escherichia coli. SefA was expressed to full length but H: gm was shorter than expected, probably due to a proteolytic cleavage of the N-terminal during passage either through the periplasm or over the membrane. FACS analysis confirmed that SefA was facing the extracellular environment, but this could not be conclusively established for H: gm since the N-terminal detection tag (His(6)) was cleaved off. Polyclonal salmonella antibodies confirmed the sustained antibody-antigen binding towards both proteins. The surface expression data from Staphylococcus carnosus suggested that the H: gm and SefA proteins were transported to the cell wall since the detection marker was displayed by FACS analysis. Conclusion: Apart from the accumulated knowledge and the existence of a wealth of equipment and techniques, the results indicate the selection of E. coli for further studies for surface expression of salmonella antigens. Surface expression of the full length protein facing the cell environment was positively proven by standard analysis, and the FACS signal comparison to expression in Staphylococcus carnosus shows that the distribution of the surface protein on each cell was comparatively very narrow in E. coli, the E. coli outer membrane molecules can serve as an adjuvant for the surface antigenic proteins and multimeric forms of the SefA protein were detected which would probably be positive for the realisation of a strong antigenic property. The detection of specific and similar proteolytic cleavage patterns for both the proteins provides a further starting point for the investigation and development of the Escherichia coli AIDA autotransporter efficiency.

  • 14.
    Perez-Zabaleta, Mariel
    et al.
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Sjöberg, Gustav
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Guevara-Martinez, Monica
    KTH, School of Biotechnology (BIO), Industrial Biotechnology. Universidad Mayor de San Simón, Bolivia.
    Jarmander, Johan
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Gustavsson, Martin
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Quillaguaman, Jorge
    Larsson, Gen
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    Increasing the production of (R)-3-hydroxybutyrate in recombinant Escherichia coli by improved cofactor supply2016In: Microbial Cell Factories, ISSN 1475-2859, E-ISSN 1475-2859, Vol. 15, 91Article in journal (Refereed)
    Abstract [en]

    Background: In a recently discovered microorganism, Halomonas boliviensis, polyhydroxybutyrate production was extensive and in contrast to other PHB producers, contained a set of alleles for the enzymes of this pathway. Also the monomer, (R)-3-hydroxybutyrate (3HB), possesses features that are interesting for commercial production, in particular the synthesis of fine chemicals with chiral specificity. Production with a halophilic organism is however not without serious drawbacks, wherefore it was desirable to introduce the 3HB pathway into Escherichia coli. Results: The production of 3HB is a two-step process where the acetoacetyl-CoA reductase was shown to accept both NADH and NADPH, but where the V-max for the latter was eight times higher. It was hypothesized that NADPH could be limiting production due to less abundance than NADH, and two strategies were employed to increase the availability; (1) glutamate was chosen as nitrogen source to minimize the NADPH consumption associated with ammonium salts and (2) glucose-6-phosphate dehydrogenase was overexpressed to improve NADPH production from the pentose phosphate pathway. Supplementation of glutamate during batch cultivation gave the highest specific productivity (q(3HB) = 0.12 g g(-1) h(-1)), while nitrogen depletion/zwf overexpression gave the highest yield (Y-3HB/CDW = 0.53 g g(-1)) and a 3HB concentration of 1 g L-1, which was 50 % higher than the reference. A nitrogen-limited fedbatch process gave a concentration of 12.7 g L-1 and a productivity of 0.42 g L-1 h(-1), which is comparable to maximum values found in recombinant E. coli. Conclusions: Increased NADPH supply is a valuable tool to increase recombinant 3HB production in E. coli, and the inherent hydrolysis of CoA leads to a natural export of the product to the medium. Acetic acid production is still the dominating by-product and this needs attention in the future to increase the volumetric productivity further.

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