Malic acid derived from fossil resources is currently applied in the food and beverage industries with a medium global production capacity. However, in the transition from a fossil‐based to a bio‐based economy, biotechnologically produced l‐malic acid may become an important platform chemical with many new applications, especially in the field of biopolymers. In this review, currently used petrochemical production routes to dl‐malic acid are outlined and insights into possible bio‐based alternatives for microbial l‐malic acid production are provided. Besides ecological reasons, the possibility to produce enantiopure l‐malic acid by microbial fermentation is the biggest advantage over chemical synthesis. State‐of‐the‐art and open challenges concerning production host engineering, substrate choice and downstream processing are addressed. With regard to production hosts, a literature overview is given covering the leading natural production strains of Aspergillus, Ustilago and Aureobasidium, as well as Escherichia coli as the most important engineered recombinant host. The utilization of renewable substrates as an alternative to glucose is emphasized in particular as a key aspect for a competitive bio‐based production. Out of the alternative substrates discussed in this review, the industrial side‐streams crude glycerol and molasses seem to be most promising for large‐scale l‐malic acid production. © 2019 The Authors. Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

https://onlinelibrary.wiley.com/doi/epdf/10.1002/jctb.6269

Malic acid production from renewables: A review

Dynamic control in metabolic engineering: Theories, tools, and applications.

Designer microbial consortia are an emerging frontier in synthetic biology that enable versatile microbiome engineering. However, the utilization of such consortia is hindered by our limited capacity in rapidly creating ecosystems with desired dynamics. Here we present the development of synthetic communities through social interaction engineering that combines modular pathway reconfiguration with model creation. Specifically, we created six two-strain consortia, each possessing a unique mode of interaction, including commensalism, amensalism, neutralism, cooperation, competition and predation. These consortia follow distinct population dynamics with characteristics determined by the underlying interaction modes. We showed that models derived from two-strain consortia can be used to design three- and four-strain ecosystems with predictable behaviors and further extended to provide insights into community dynamics in space. This work sheds light on the organization of interacting microbial species and provides a systematic framework-social interaction programming-to guide the development of synthetic ecosystems for diverse purposes.

Designing microbial consortia with defined social interactions

Cell division can perturb the metabolic performance of industrial microbes. The C period of cell division starts from the initiation to the termination of DNA replication, whereas the D period is the bacterial division process. Here, we first shorten the C and D periods of E. coli by controlling the expression of the ribonucleotide reductase NrdAB and division proteins FtsZA through blue light and near-infrared light activation, respectively. It increases the specific surface area to 3.7 μm−1 and acetoin titer to 67.2 g·L−1. Next, we prolong the C and D periods of E. coli by regulating the expression of the ribonucleotide reductase NrdA and division protein inhibitor SulA through blue light activation-repression and near-infrared (NIR) light activation, respectively. It improves the cell volume to 52.6 μm3 and poly(lactate-co-3-hydroxybutyrate) titer to 14.31 g·L−1. Thus, the optogenetic-based cell division regulation strategy can improve the efficiency of microbial cell factories. Manipulation of genes controlling microbial shapes can affect bio-production. Here, the authors employ an optogenetic method to realize dynamic morphological engineering of E. coli replication and division and show the increased production of acetoin and poly(lactate-co-3-hydroxybutyrate).

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Light-powered Escherichia coli cell division for chemical production.

CO2 sequestration engineering is an attractive strategy for achieving carbon- and energy-efficient bioproduction. However, the efficiency of heterotrophic CO2 sequestration is limited by bioproduct dependence and energy deficiency. Here, modular CO2 sequestration engineering was developed to produce target chemicals by integrating synthetic CO2 fixation and CO2 mitigation modules. A synthetic CO2 fixation pathway was designed, and then enhanced by light-driven reducing power using self-assembled cadmium sulfide nanoparticles. Next, a CO2 mitigation switch was designed, and then optimized by light-driven energy via proteorhodopsin. Finally, by integrating CO2 fixation and CO2 mitigation modules, the efficiency of CO2 sequestration was notably enhanced in Escherichia coli and the yields of l-malate and butyrate were increased to 1.48 and 0.79 mol/mol glucose, respectively, reaching theoretical yields. This CO2 sequestration system provides an efficient platform for channelling CO2 into value-added chemicals. Improving the efficiency of carbon yield in heterotrophic microorganisms is desired for biomanufacturing. Now, a product-independent and energy-efficient CO2 sequestration system that maximizes carbon conversion has been developed, as showcased by the production of chemicals reaching their theoretical yields.

Light-driven CO2 sequestration in Escherichia coli to achieve theoretical yield of chemicals

Industrial chemical production from renewable feedstocks by microbial cell factories provides a promising avenue towards sustainability. However, the small size of bacterial cells and environmental stress significantly affect microbial cell factory performance. Here, we engineered the Escherichia coli lifespan to improve the chemical production of poly(lactate-co-3-hydroxybutyrate) and butyrate. The replicative lifespan was shortened by deleting a carbon storage regulator, and the chronological lifespan was extended by deleting a response regulator and overexpressing sigma-38 in Escherichia coli. The replicative lifespan was fine-tuned using a two-output recombinase-based state machine, and the cell size was enlarged 13.4-fold. The highest poly(lactate-co-3-hydroxybutyrate) content of 52 wt% was achieved in a 5-l fermenter. The chronological lifespan was modulated through a multi-output recombinase-based state machine, resulting in the highest butyrate titre of 29.8 g l−1, by programming cell differentiation according to different fermentation stages. These results highlight the applicability of engineering the bacterial lifespan to increase microbial cell factory performance. To implement more sustainable processes in industry, a high efficiency of microbial biocatalytic systems for the production of industrial chemicals from renewable feedstocks is important. Now, engineering the lifespan of Escherichia coli is presented as a platform technology for improving the bioproduction of chemicals.

Engineering Escherichia coli lifespan for enhancing chemical production

Rewiring carbon flux in Escherichia coli using a bifunctional molecular switch

Microbial l-malate production from renewable feedstock is a promising alternative to petroleum-based chemical synthesis. However, high l-malate production of Aspergillus oryzae was achieved to date using organic nitrogen, with inorganic nitrogen still unable to meet industrial applications. In the current study, we constructed a screening system and nitrogen supply strategy to improve l-malate production with ammonium sulphate [(NH4)2SO4] as the sole nitrogen source. First, we generated and identified a high-producing mutant FMME218-37, which stably boosted l-malate production from 30.73 to 78.12 g/L, using a combined screening system with morphological characteristics. Then, by analyzing the fermentation parameters and physiological characteristics, we further speculated the key factor was the unbalance of carbon and nitrogen absorption. Finally, the titer and productivity of l-malate was increased to 95.2 g/L and 0.57 g/(L h) by regulating the nitrogen supply module to balance carbon and nitrogen absorption, which represented the highest level in A. oryzae with (NH4)2SO4 as nitrogen source achieved to date. Moreover, our findings using a low-cost substrate may lead to building an economical cell factory of A. oryzae for l-malate production.

Enhancing L-malate production of Aspergillus oryzae FMME218-37 by improving inorganic nitrogen utilization.

Aspergillus oryzae is a competitive natural producer for organic acids, but its production capacity is closely correlated with a specific morphological type. Here, morphology engineering was used for tailoring A. oryzae morphology to enhance l-malate production. Specifically, correlation between A. oryzae morphology and l-malate fermentation was first conducted, and the optimal range of the total volume of pellets in a unit volume of fermentation broth (V value) for l-malate production was 120-130 mm3 /ml. To achieve this range, A. oryzae morphology was improved by controlling the variation of operational parameters, such as agitation speed and aeration rate, and engineered by optimizing the expression of cell division cycle proteins such as tyrosine-protein phosphatase (CDC14), anaphase promoting complex/cyclosome activator protein (CDC20), and cell division control protein 45 (CDC45). By controlling the strength of CDC14 at a medium level, V value fell into the optimal range of V value and the final engineered strain A. oryzae CDC14(3) produced up to 142.5 g/L l-malate in a 30-L fermenter. This strategy described here lays a good foundation for industrial production of l-malate in the future, and opens a window to develop filamentous fungi as cell factories for production of other chemicals.

Qiang Ding

Papers

Light-powered Escherichia coli cell division for chemical production.

Engineering Escherichia coli lifespan for enhancing chemical production

Rewiring carbon flux in Escherichia coli using a bifunctional molecular switch

Enhancing L-malate production of Aspergillus oryzae FMME218-37 by improving inorganic nitrogen utilization.

Morphology engineering of Aspergillus oryzae for l-malate production.