Strain engineering and bioprocessing strategies for biobased production of porphobilinogen in Escherichia coli.

Strain engineering and bioprocessing strategies were applied for biobased production of porphobilinogen (PBG) using Escherichia coli as the cell factory. The non-native Shemin/C4 pathway was first implemented by heterologous expression of hemA from Rhodopseudomonas spheroids to supply carbon flux from the natural tricarboxylic acid (TCA) pathways for PBG biosynthesis via succinyl-CoA. Metabolic strategies were then applied for carbon flux direction from the TCA pathways to the C4 pathway. To promote PBG stability and accumulation, Clustered Regularly Interspersed Short Palindromic Repeats interference (CRISPRi) was applied to repress hemC expression and, therefore, reduce carbon flowthrough toward porphyrin biosynthesis with minimal impact to cell physiology. To further enhance PBG biosynthesis and accumulation under the hemC-repressed genetic background, we further heterologously expressed native E. coli hemB. Using these engineered E. coli strains for bioreactor cultivation based on ~ 30 g L-1 glycerol, we achieved high PBG titers up to 209 mg L-1, representing 1.73% of the theoretical PBG yield, with improved PBG stability and accumulation. Potential biochemical, genetic, and metabolic factors limiting PBG production were systematically identified for characterization. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s40643-021-00482-3.

Strain engineering for high-level 5-aminolevulinic acid production in Escherichia coli.

Herein, we report the development of a microbial bioprocess for high-level production of 5-aminolevulinic acid (5-ALA), a valuable non-proteinogenic amino acid with multiple applications in medical, agricultural, and food industries, using Escherichia coli as a cell factory. We first implemented the Shemin (i.e., C4) pathway for heterologous 5-ALA biosynthesis in E. coli. To reduce, but not to abolish, the carbon flux toward essential tetrapyrrole/porphyrin biosynthesis, we applied clustered regularly interspersed short palindromic repeats interference (CRISPRi) to repress hemB expression, leading to extracellular 5-ALA accumulation. We then applied metabolic engineering strategies to direct more dissimilated carbon flux toward the key precursor of succinyl-CoA for enhanced 5-ALA biosynthesis. Using these engineered E. coli strains for bioreactor cultivation, we successfully demonstrated high-level 5-ALA biosynthesis from glycerol (~30 g L-1 ) under both microaerobic and aerobic conditions, achieving up to 5.95 g L-1 (36.9% of the theoretical maximum yield) and 6.93 g L-1 (50.9% of the theoretical maximum yield) 5-ALA, respectively. This study represents one of the most effective bio-based production of 5-ALA from a structurally unrelated carbon to date, highlighting the importance of integrated strain engineering and bioprocessing strategies to enhance bio-based production.

Engineering Escherichia coli for high-level production of propionate.

Mounting environmental concerns associated with the use of petroleum-based chemical manufacturing practices has generated significant interest in the development of biological alternatives for the production of propionate. However, biological platforms for propionate production have been limited to strict anaerobes, such as Propionibacteria and select Clostridia. In this work, we demonstrated high-level heterologous production of propionate under microaerobic conditions in engineered Escherichia coli. Activation of the native Sleeping beauty mutase (Sbm) operon not only transformed E. coli to be propionogenic (i.e., propionate-producing) but also introduced an intracellular "flux competition" between the traditional C2-fermentative pathway and the novel C3-fermentative pathway. Dissimilation of the major carbon source of glycerol was identified to critically affect such "flux competition" and, therefore, propionate synthesis. As a result, the propionogenic E. coli was further engineered by inactivation or overexpression of various genes involved in the glycerol dissimilation pathways and their individual genetic effects on propionate production were investigated. Generally, knocking out genes involved in glycerol dissimilation (except glpA) can minimize levels of solventogenesis and shift more dissimilated carbon flux toward the C3-fermentative pathway. For optimal propionate production with high C3:C2-fermentative product ratios, glycerol dissimilation should be channeled through the respiratory pathway and, upon suppressed solventogenesis with minimal production of highly reduced alcohols, the alternative NADH-consuming route associated with propionate synthesis can be critical for more flexible redox balancing. With the implementation of various biochemical and genetic strategies, high propionate titers of more than 11 g/L with high yields up to 0.4 g-propionate/g-glycerol (accounting for ~50 % of dissimilated glycerol) were achieved, demonstrating the potential for industrial application. To our knowledge, this represents the most effective engineered microbial system for propionate production with titers and yields comparable to those achieved by anaerobic batch cultivation of various native propionate-producing strains of Propionibacteria.

Biochemical and genetic engineering strategies to enhance hydrogen production in photosynthetic algae and cyanobacteria.

As an energy carrier, hydrogen gas is a promising substitute to carbonaceous fuels owing to its superb conversion efficiency, non-polluting nature, and high energy content. At present, hydrogen is predominately synthesized via chemical reformation of fossil fuels. While various biological methods have been extensively explored, none of them is justified as economically feasible. A sustainable platform for biological production of hydrogen will certainly impact the biofuel market. Among a selection of biological systems, algae and cyanobacteria have garnered major interests as potential cell factories for hydrogen production. In conjunction with photosynthesis, these organisms utilize inexpensive inorganic substrates and solar energy for simultaneous biosynthesis and hydrogen evolution. However, the hydrogen yield associated with these organisms remains far too low to compete with the existing chemical systems. This article reviews recent advances of biochemical, bioprocess, and genetic engineering strategies in circumventing technological limitations to hopefully improve the applicative potential of these photosynthetic hydrogen production systems.

Bioprocess development for production, purification, and structural characterization of recombinant hCD83ext as a potential therapeutic protein.

An effective bioprocess for the production of hCD83ext (i.e. the extracytoplasmic domain of human CD83) as a potential therapeutic protein was developed. It primarily consists of (1) cell cultivation for the production of recombinant glutathione-S-transferase-hCD83ext (GST-hCD83ext) fusion protein and (2) downstream processing for purification of hCD83ext. The developed bioprocess is robust, reproducible, easy to operate, and, most importantly, can generate hCD83ext with a high yield and purity. For cell cultivation, a high GST-hCD83ext expression level, estimated to be more than 10% of total cellular protein, with a cell density of 8 OD(600) was obtained by tuning several culture parameters, including medium recipe, host/vector system, induction condition, temperature, and aeration. For downstream processing, milligrams of very pure and low-endotoxin hCD83ext was obtained through simultaneous binding and cleavage of GST-hCD83ext in a GST affinity chromatographic column followed by a polishing step using anion exchange chromatography. To identify potential factors associated with bioactivity consistency, structural changes for the final product of hCD83ext were characterized and monitored. Formation of various hCD83ext multimeric forms, including dimer, trimer, and tetramer, via intermolecular disulfide bonds was observed.